Home Inhibition of miR-21 improves pulmonary vascular responses in bronchopulmonary dysplasia by targeting the DDAH1/ADMA/NO pathway
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Inhibition of miR-21 improves pulmonary vascular responses in bronchopulmonary dysplasia by targeting the DDAH1/ADMA/NO pathway

An erratum for this article can be found here: https://doi.org/10.1515/med-2023-0679
  • Ying Zhong EMAIL logo , Zhiqun Zhang and Xiaoqing Chen
Published/Copyright: December 9, 2022
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Open Medicine
From the journal Open Medicine Volume 17 Issue 1

Abstract

miR-21 has been confirmed to be overexpressed in neonatal rat lungs with hyperoxia-mediated bronchopulmonary dysplasia (BPD). The specific function of miR-21 in BPD is still unclear. We established the hyperoxia-induced BPD rat model in vivo and the hyperoxia-induced pulmonary microvascular endothelial cells (PMVECs) model in vitro. Transwell assay was utilized to detect the migratory capability of PMVECs. Tube formation assay was utilized to measure angiogenesis ability. ELISA was utilized to test nitric oxide (NO) production and the intracellular and extracellular Asymmetric Dimethylarginine (ADMA) concentration. Furthermore, the interaction between miR-21 and dimethylarginine dimethylaminohydrolase 1 (DDAH1) was evaluated using luciferase reporter assay. We found that miR-21 expression in PMVECs was increased by hyperoxia stimulation. Inhibition of miR-21 improved the migratory and angiogenic activities of PMVECs and overexpression of miR-21 exerted the opposite effects. Furthermore, knockdown of miR-21 increased NO production and decreased intracellular and extracellular ADMA concentration in hyperoxia-treated PMVECs. Next we proved that miR-21 could bind to DDAH1 and negatively regulate its expression. Rescues assays showed that DDAH1 knockdown reversed the effects of miR-21 depletion on hyperoxia-mediated PMVEC functions, NO production, and ADMA concentration. Importantly, miR-21 downregulation restored alveolarization and vascular density in BPD rats. This study demonstrates that inhibition of miR-21 improves pulmonary vascular responses in BPD by targeting the DDAH1/ADMA/NO pathway.

Keywords: bronchopulmonary dysplasia; miR-21; DDAH1; ADMA; NO

1 Introduction

Bronchopulmonary dysplasia (BPD) is a leading cause of chronic respiratory morbidity among survivors of preterm birth with the greatest risk for those born at 23–30 weeks gestational age [1,2]. The epidemiology of BPD continues to demonstrate that birth weight and gestational age are the most predictive risk factors for developing BPD, and the frequency of BPD has been approximately 40% in surviving infants born at ≤28 weeks gestational age and about 30% in infants with birth weight <1,000 g during the 20 years from 1993 to 2012 [3]. While many drugs have been tried to prevent or attenuate BPD [4,5], no specific and effective treatment is available; therefore, this disease is still associated with high mortality and morbidity [6]. Despite improved neonatal care, the number of BPD cases due to this condition have not decreased [7], secondary to increased survival of infants of lower gestational ages. Damaged intrauterine lung development and post-partum injury can harm angiogenic capability and alveolar formation, leading to distal alveolar simplification [8]. In recent years, studies have found that disrupted vascularization is the main cause for alveolar simplification in BPD [9,10]. Importantly, it may lead to the development of pulmonary hypertension (PH) [11]. The pathophysiology of BPD-PH includes decreased pulmonary blood vessels, endothelial cell dysfunction, and increased remodeling of resistance pulmonary arteries [10]. Therefore, the in-depth study of the pathogenesis of BPD will help to develop more effective therapeutic targets and improve the survival of BPD patients.

MicroRNAs (miRNAs) are small, endogenous, non-coding RNAs of ∼22–26 nucleotides in length that function primarily as post-transcriptional regulators [12]. A flow of studies have demonstrated that miRNAs can modulate lots of pathophysiological processes, including cell proliferation, metabolism, and organ development [13,14]. They have been considered as promising candidates for novel targeted therapeutic approaches to lung diseases [14]. For example, miR-27a relieves acute lung injury in mice by modulation of TLR4/MyD88/NF-κB pathway [15]. Adrenomedullin regulated by miRNA-574-3p protects premature infants with BPD [16]. MiR-21 was one of the first miRNAs to be identified as transcribed by RNA polymerase II [17]. It has been found to be upregulated in many pathological conditions including cancer and cardiovascular diseases [18]. A previous study reported that miR-21 was upregulated in neonatal rat lungs in response to hyperoxia exposure [19]. MiR-21 was detected as a common miRNA that changed in chronic lung disease patients and in the hyperoxia exposed mice [20]. Other studies have documented an increase in miR-21 in hypoxia-induced pulmonary hypertensive mice and localization of miR-21 to distal small arteries in the animal and human hypertensive lungs [21,22]. A hypoxia-induced increase in miR-21 levels contributes to endothelial dysfunction associated with a reduction in NO/cGMP signaling [23]. Additionally, elevated miR-21 expression induced by oscillatory shear stress or oxidized low-density lipoprotein resulted in endothelial cell activation [24]. Overexpression of miR-21 promotes endothelial dysfunction in HUVECs treated with TNF-α [25]. These reports suggest the close association between elevated miR-21 and pulmonary hypertension (PH) and endothelial dysfunction.

The main purpose of this study was the investigation of the biological function and mechanism of miR-21 in BPD. We hypothesized that inhibition of miR-21 could improve pulmonary vascular responses in BPD. We established the hyperoxia-induced BPD rat model in vivo and the hyperoxia-induced pulmonary microvascular endothelial cells (PMVECs) in vitro to estimate the influence of miR-21 on pulmonary vascular development.

2 Materials and methods

2.1 Cell culture and treatment

Human PMVECs obtained from BeNa Culture Collection Co., Ltd (Beijing, China) were incubated in DMEM (Gibco, NY, USA) added with 10% FBS (Gibco) and 100 U/mL Penicillin-Streptomycin solution (Gibco). PMVECs exposed to room air (RA) (21% O2/5% CO2/74% N2) served as the negative control (NC) group cells. PMVECs in the hyperoxia treatment group were exposed to high O2 gas (85% O2/5% CO2/10% N2). PMVECs were treated with hyperoxia (4, 16, and 24 h).

2.2 Cell transfection

Specific siRNAs against dimethylarginine dimethylaminohydrolase 1 (si-DDAH1) and its corresponding negative control (si-NC) (30 nM) were obtained from Genechem (Shanghai, China). miR-21 inhibitor, miR-21 mimic, NC inhibitor, and NC mimic (50 nM) were obtained from RiboBio (Guangzhou, China). PMVECs were cultured in RA or under hyperoxia for 24 h. Then, cell transfection was conducted for 48 h using Lipofectamine 3000 (Invitrogen, Carlsbad, CA) according to user guides. After 48 h, cells were collected, and the transfection efficiency was tested through RT-qPCR.

2.3 RT-qPCR

Total RNA was extracted from PMVECs utilizing TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) and cDNA was synthesized from 1 μg of total RNA by PrimeScriptTM RT reagent Kit with gDNA (Takara, Japan). Then, qPCR was performed on StepOnePlus Real-Time PCR System (Applied Biosystems, USA) with SYBR Premix ExTaqTM II (Takara). The expression of miR-21 or DDAH1 was calculated by the 2−ΔΔCt method normalized to U6 or GAPDH, respectively. The sequences of primers used are as follows:

miR-21 forward, 5′-GCGGCAACACCAGTCGATG-3′,

miR-21 reverse, 5′-GCGGCAACACCAGTCGATG-3′;

DDAH1 forward, 5′-ACGTCCTATTCACAGGCAG-3′,

DDAH1 reverse, 5′-TATCAGCCAAGATCTCAGCA-3′;

GAPDH forward, 5′-AACTCCCATTCTTCCACCT-3′,

GAPDH reverse, 5′-TTGTCATACCAGGAAATGAGC-3′;

U6 forward, 5′-CAGTTATGACGACCTAGACAG-3′,

U6 reverse, 5′-CAAATTTGCATGTCATCCTTGG-3′.

2.4 Transwell assay

PMVEC migration was estimated using 8.0 µm Transwell inserts (Corning Inc., Corning, NY, USA). The 1 × 105 PMVECs in 200 µL of serum-free medium were seeded on the upper chamber. Then, 900 µL of DMEM were filled in the lower chamber. After 24 h, cells in the top surface of the membrane were wiped off, and cells in the lower surface were fixed with 4% paraformaldehyde and dyed with crystal violet (Solarb, China) for 15 min. Next cells were washed with PBS three times, and examined by the microscope (Olympus, Japan).

2.5 Tube formation assay

For determining angiogenesis capacity in vitro, PMVECs (1 × 104 cells/well) were seeded in 96-well plates coated by Matrigel (Corning Inc.) and cultured in different conditions. The culture medium was changed every 24 h and cells were cultured for 72 h. The capillary-network formation was detected using an inverted microscope (Logos Biosystem, Villeneuve d’Ascq, France). The quantity of the nodes in three randomly chosen fields of each plate was examined.

2.6 ELISA

Cells were rinsed with pre-cold PBS and cultured with 200 μL of cell lysis buffer at 4°C for half an hour. Then, samples were subjected to centrifugation at 12,000 rpm for 10 min and the supernatant was collected and maintained at −80°C. The BCA kit was applied for determining cell lysate concentration. The concentration of ADMA in cell medium and lysates was evaluated using ELISA kits (Cusabio, Wuhan, China) in accordance with user guides. DDAH metabolic activity and the concentration of NO were measured using their corresponding ELISA kits (Cusabio) following the manufacturer’s instructions.

2.7 Bioinformatics analysis

The target genes of miR-21 were predicted from the websites including starBase (https://starbase.sysu.edu.cn/index.php), miRDB (http://www.mirdb.org/mirdb/index.html), and miRWalk (http://mirwalk.umm.uni-heidelberg.de/).

2.8 Luciferase reporter assay

The putative binding site of miR-21 in the DDAH1 3′UTR was predicted by starBase database. DDAH1 3′UTR fragments containing the binding site with miR-21 were inserted in the pmirGLO luciferase reporter vector (Promega, Madison, Wisconsin, USA). Cells treated with RA or 24 h of hyperoxia were subjected to co-transfection with pmirGLO OTX1 3′UTR luciferase reporter vector and miR-21 inhibitor or NC inhibitor with Lipofectamine 3000 (Invitrogen) for 48 h. Luciferase activity was measured by Dual-Luciferase Reporter Assay System (Promega). The luciferase activity of Firefly was normalized to that of Renilla.

2.9 Establishment of hyperoxia-induced BPD newborn rat models

All procedures and protocols of this study were approved by the Institutional Animal Care and Use Committee of Zhejiang University (Zhejiang, China) A total of 8 SPF pregnant Sprague–Dawley rats (gestational age of 15 days; Vital River Co. Ltd, Beijing, China) were housed in a light- and temperature-controlled room with free access to food and water. Pregnant rats were raised alone and they gave birth by themselves after 1 week. After delivery, 48 newborn rats born 12 h apart were randomly divided into hyperoxia treatment group (n = 36) and control group (n = 12). Rats in the hyperoxia group were divided into the BPD group (BPD rats) (n = 12), the BPD + antagomir NC group (n = 12), and the BPD + miR-21 antagomir group (n = 12). The 85% oxygen concentration was utilized for hyperoxia group. Rats in the control group were maintained in RA (21% oxygen). The newborn rats were raised for 1 week, and then injected with the antagomir NC and miR-21 antagomir (20 mg/kg/day; 0.2 mL/day; GenePharma, Shanghai, China) by the tail vein. The control rats (air group) were fed in a normal pressure room (21% oxygen), and the newborn rats used for BPD model in the hyperoxia group were raised in a plexiglass oxygen chamber (85% oxygen). The cage was opened for half an hour every day and water and food were provided. The maternal rats were utilized for feeding the newborn rats and were exchanged among cages every 24 h. On the 1st, 3rd, 7th, and 14th days, rats were separately anesthetized with intraperitoneal injection of 90 mg/kg pentobarbital sodium, and then they were euthanized by cervical dislocation. Next the abdominal cavity was opened quickly to remove the lungs. After rapid freezing with liquid nitrogen, the lungs were stored in a −80°C refrigerator for subsequent assays.

2.10 Histological analysis

After the rats were executed on the 14th day, lung tissues were collected and cut into 5-μm-thick sections, followed by fixing with 4% paraformaldehyde for 48 h. The slices were dehydrated with graded ethyl alcohol solutions. Following xylene dewaxing and hydration, the slices were dyed with hematoxylin (Solarbio, Beijing, China) and eosin for 5 min. The morphological changes were analyzed using a light microscope (Eclipse Ci; Nikon, Tokyo, Japan). In accordance with the previously described method [26], it was also necessary to detect the radial alveolar count and alveolar area/pulmonary septal area value.

2.11 Immunofluorescence staining

Pulmonary vessel density was quantified based on immunofluorescence staining of the endothelial-specific marker von Willebrand factor (vWF) in rat lung section. Briefly, 5 μm sized paraffin-embedded lung sections were stained overnight with a primary antibody against vWF (1:250, AB7356, Sigma-Aldrich) at 4°C. Sections were stained with an Alexa Fluor 594-conjugated secondary antibody (1:200; Jackson Immunoresearch) at room temperature for 2 h. After a final washing step, sections were mounted with DAPI (Sigma-Aldrich) and quantified for vessel density by a blinded observer. Using ImageJ, the vWF-positive vessels (<100 μm diameter) were counted in five 10× magnified fields per subject and averaged.

2.12 Western blot

The lung tissues or cells were collected and homogenized in RIPA lysis buffer (Roche Applied Science, Indianapolis, IN). The protein concentration was tested using a BCA protein assay kit (Beyotime, China). Then, 15 μg/lane samples were loaded onto a 4–20% gel, resolved by SDS-PAGE and then transferred to PVDF membranes (Millipore, USA). The membranes were blockaded with 5% skimmed milk for 2 h to block non-specific binding. The PVDF membranes were mixed with primary antibodies and incubated at 4°C overnight. The next day, after rinsing with TBST, the membranes were cultured with the HRP-conjugated secondary antibody (Abcam, ab6789, 1:2,000) for 2 h at room temperature. The band density was tested using Quantity One software with an ECL kit (BioRad Laboratories, Shanghai, China). The primary antibodies used were: p-eNOS (ser1177) (PA5-35879, 1:500, Thermo Fisher Scientific, USA), DDAH1 (sc-271337, 1:1,000, Santa Cruz Biotechnology, Inc., USA), and β-actin (ab8226, 1:1,000, Abcam) served as loading control.

2.13 Statistical analysis

Statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software, Inc., La Jolla, CA, USA). Group difference was analyzed by Student’s t-test or one-way ANOVA followed by Tukey’s post hoc analysis. Data are presented as mean value ± SD from three individual repeats. p < 0.05 was considered statistically significant.

3 Results

3.1 Inhibition of miR-21 improves the angiogenic activity of PMVECs

First, miR-21 expression in PMVECs with the treatment of RA or hyperoxia (4, 16, and 24 h) was tested by RT-qPCR. We found that, in comparison of RA treatment, miR-21 expression in PMVECs was notably increased after hyperoxia treatment in a time-dependent manner (p < 0.01) (Figure 1a). Then, we silenced miR-21 expression in PMVECs treated with hyperoxia-24 h by transfecting miR-21 inhibitor, and then found that miR-21 expression was downregulated after transfection (2.88 ± 0.23 vs 1.29 ± 0.11, p < 0.01) (Figure 1b). Transwell assay was conducted to measure the influence of miR-21 inhibition on the migration of PMVECs under hyperoxia. We discovered that hyperoxia inducement significantly repressed cell migration (285.00 ± 22.00 vs 89.00 ± 8.20, p < 0.01), while miR-21 depletion restored the migration of PMVECs (78.00 ± 7.00 vs 199.00 ± 12.00, p < 0.01) (Figure 1c). Next the tube formation assay was utilized to determine the angiogenic activity of PMVECs. The results demonstrated that miR-21 depletion restored the decreased number of nodes caused by hyperoxia inducement in PMVECs (255.00 ± 23.46 vs 573.00 ± 51.18, p < 0.01) (Figure 1d). These data suggested that miR-21 knockdown improves the migratory and angiogenic activity of PMVECs under hyperoxia treatment.

Figure 1 Inhibition of miR-21 improves the angiogenic activity of PMVECs. (a) RT-qPCR was used to measure miR-21 expression in PMVECs treated with RA or hyperoxia (4, 16 and 24 h). (b) The knockdown efficiency of miR-21 inhibitor in PMVECs treated with 24 h of hyperoxia was detected by RT-qPCR. (c) Transwell assay was applied for measuring the migration of hyperoxia-induced PMVECs when miR-21 was silenced. (d) Tube formation assay was performed to estimate the angiogenic activity of hyperoxia-induced PMVECs after miR-21 inhibition. Quantified values are mean values ± standard deviation of at least three independent experiments. ** p < 0.01.
Figure 1

Inhibition of miR-21 improves the angiogenic activity of PMVECs. (a) RT-qPCR was used to measure miR-21 expression in PMVECs treated with RA or hyperoxia (4, 16 and 24 h). (b) The knockdown efficiency of miR-21 inhibitor in PMVECs treated with 24 h of hyperoxia was detected by RT-qPCR. (c) Transwell assay was applied for measuring the migration of hyperoxia-induced PMVECs when miR-21 was silenced. (d) Tube formation assay was performed to estimate the angiogenic activity of hyperoxia-induced PMVECs after miR-21 inhibition. Quantified values are mean values ± standard deviation of at least three independent experiments. ** p < 0.01.

3.2 Knockdown of miR-21 increases NO production and decreases intracellular and extracellular ADMA concentration

Nitric oxide (NO) is an endogenous pulmonary vasodilator produced by endothelial NO synthase [27,28]. NO plays an important role in maintaining endothelial function, and its production is mainly mediated by Asymmetric Dimethylarginine (ADMA) [29,30]. Thus, the concentrations of NO and ADMA in PMVECs under hyperoxia were detected. According to western blot, we observed that hyperoxia treatment markedly inhibited eNOS (ser1177) phosphorylation in PMVECs (1.00 ± 0.13 vs 0.12 ± 0.02, p < 0.01), while it was increased by miR-21 knockdown (0.15 ± 0.02 vs 0.41 ± 0.03, p < 0.01) (Figure 2a). ELISA further presented that NO concentration was reduced by hyperoxia treatment and was elevated by miR-21 depletion (14.67 ± 1.35 vs 20.96 ± 2.11, p < 0.01) (Figure 2b). Additionally, we found that hyperoxia treatment elevated the intracellular and extracellular ADMA concentrations in PMVECs (0.38 ± 0.04 vs 0.55 ± 0.05, 1.23 ± 0.10 vs 2.10 ± 0.19, respectively, p < 0.01). However, miR-21 depletion decreased both intracellular and extracellular ADMA concentrations in hyperoxia-induced PMVECs (0.54 ± 0.06 vs 0.41 ± 0.03, 2.00 ± 0.22 vs 1.46 ± 0.12, respectively, p < 0.01) (Figure 2c and d). Overall, miR-21 knockdown improves hyperoxia-induced PMVECs dysfunction via repressing ADMA concentration and elevating NO production.

Figure 2 Knockdown of miR-21 increases NO production and decreases intracellular and extracellular ADMA concentration. (a) Western blot was utilized to test the p-eNOS (ser1177) level in PMVECs of different groups (RA group, hyperoxia-24 h group, hyperoxia-24 h + NC inhibitor group, hyperoxia-24 h + miR-21 inhibitor group). (b–d) ELISA was utilized to detect the NO production and the intracellular and extracellular ADMA concentration in PMVECs of different groups. Quantified values are mean values ± standard deviation of at least three independent experiments. ** p < 0.01.
Figure 2

Knockdown of miR-21 increases NO production and decreases intracellular and extracellular ADMA concentration. (a) Western blot was utilized to test the p-eNOS (ser1177) level in PMVECs of different groups (RA group, hyperoxia-24 h group, hyperoxia-24 h + NC inhibitor group, hyperoxia-24 h + miR-21 inhibitor group). (b–d) ELISA was utilized to detect the NO production and the intracellular and extracellular ADMA concentration in PMVECs of different groups. Quantified values are mean values ± standard deviation of at least three independent experiments. ** p < 0.01.

3.3 Overexpression of miR-21 strengthens the effects of hyperoxia in PMVECs

To obtain more reliable results, we used miR-21 mimic to detect the impact of miR-21 overexpression on PMVECs. PCR results showed that miR-21 expression was upregulated in hyperoxia-treated PMVECs after miR-21 mimic transfection (2.55 ± 0.23 vs 4.26 ± 0.38, p < 0.01) (Figure 3a). Transwell and tube formation assays demonstrated that miR-21 overexpression strengthened the inhibitory effects of hyperoxia on cell migration (45.27 ± 4.23 vs 16.19 ± 1.46, p < 0.001) and angiogenesis (255.96 ± 21.33 vs 76.80 ± 7.42, p < 0.001) (Figure 3b and c). Western blot revealed that miR-21 mimic inhibited eNOS (ser1177) phosphorylation in hyperoxia-treated PMVECs (0.55 ± 0.04 vs 0.19 ± 0.04, p < 0.001), (Figure 3d). NO concentration was further reduced by miR-21 depletion (15.63 ± 1.54 vs 6.26 ± 0.55, p < 0.001) (Figure 3e). Furthermore, miR-21 depletion decreased both intracellular and extracellular ADMA concentrations in hyperoxia-induced PMVECs (0.54 ± 0.05 vs 0.86 ± 0.07, 2.00 ± 0.19 vs 4.70 ± 0.45, respectively, p < 0.01) (Figure 3f and g). These results demonstrated that miR-21 overexpression promotes hyperoxia-induced PMVECs dysfunction.

Figure 3 Overexpression of miR-21 strengthens the effects of hyperoxia in PMVECs. (a) The overexpression efficiency of miR-21 mimic in PMVECs treated with 24 h of hyperoxia was detected by RT-qPCR. (b) Transwell assay was applied to detect the migration of hyperoxia-induced PMVECs after miR-21 overexpression. (c) Tube formation assay was performed to estimate the angiogenic activity of hyperoxia-induced PMVECs after miR-21 overexpression. (d) Western blot was utilized to test the p-eNOS (ser1177) level in PMVECs of different groups (RA group, hyperoxia-24 h group, hyperoxia-24 h + NC mimic group, hyperoxia-24 h + miR-21 mimic group). (e–g) ELISA was utilized to detect the NO production and the intracellular and extracellular ADMA concentration in PMVECs of different groups. Quantified values are mean values ± standard deviation of at least three independent experiments. ** p < 0.01, *** p < 0.001.
Figure 3

Overexpression of miR-21 strengthens the effects of hyperoxia in PMVECs. (a) The overexpression efficiency of miR-21 mimic in PMVECs treated with 24 h of hyperoxia was detected by RT-qPCR. (b) Transwell assay was applied to detect the migration of hyperoxia-induced PMVECs after miR-21 overexpression. (c) Tube formation assay was performed to estimate the angiogenic activity of hyperoxia-induced PMVECs after miR-21 overexpression. (d) Western blot was utilized to test the p-eNOS (ser1177) level in PMVECs of different groups (RA group, hyperoxia-24 h group, hyperoxia-24 h + NC mimic group, hyperoxia-24 h + miR-21 mimic group). (e–g) ELISA was utilized to detect the NO production and the intracellular and extracellular ADMA concentration in PMVECs of different groups. Quantified values are mean values ± standard deviation of at least three independent experiments. ** p < 0.01, *** p < 0.001.

3.4 miR-21 negatively regulates DDAH1

The targets of miR-21 were identified by utilizing bioinformatics prediction websites including starBase, miRDB, and miRWalk. Venn diagram shows 15 common target genes (PBRM1, GID4, HSD17B4, PDZD2, STAG2, MTMR12, SATB1, SC5D, POLR3B, PIK3R1, LYRM7, PHF14, HIC2, AIF1L, and DDAH1) predicted by these databases (Figure 4a). RT-qPCR showed that only DDAH1 was significantly downregulated in PMVECs transfected with miR-21 mimic (1.00 ± 0.04 vs 0.61 ± 0.05, p < 0.01) (Figure 4b). However, the other genes had no significant change. Studies have indicated that DDAH depletion is closely correlated with endothelial dysfunction and it is believed to be the mechanism responsible for ADMA-mediated eNOS impairments [31,32]. DDAH1 has been indicated to exert the vital function in regulating NO‐mediated apoptosis and angiogenesis in PMVECs [33]. Thus, we detected the interaction of DDAH1 and miR-21. The protein expression of DDAH1 was downregulated by miR-21 inhibitor and was upregulated by miR-21 mimic compared to control (1.00 ± 0.03 vs 0.26 ± 0.03, 1.00 ± 0.05 vs 2.25 ± 0.21, respectively, p < 0.001) (Figure 4c). RT-qPCR and western blot results illustrated that the mRNA and protein levels of DDAH1 were decreased in hyperoxia-induced PMVECs in a time-dependent manner (p < 0.01) (Figure 4d and e). Additionally, DDAH1 levels inhibited by hyperoxia treatment were increased by miR-21 knockdown (0.32 ± 0.04 vs 0.82 ± 0.07, 0.34 ± 0.03 vs 0.92 ± 0.08, respectively, p < 0.01) (Figure 4f and g). Furthermore, through starBase database, we obtained the binding site of miR-21 and DDAH1 (Figure 4h). The luciferase activity of DDAH1 3'UTR was almost unchanged in RA-treated PMVECs, but it reduced in hyperoxia-induced PMVECs (1.00 ± 0.11 vs 0.28 ± 0.03, p < 0.01). However, miR-21 knockdown elevated its luciferase activity in hyperoxia-induced PMVECs (0.28 ± 0.03 vs 0.97 ± 0.08, p < 0.01) (Figure 4i). Overall, miR-21 negatively regulates DDAH1 in hyperoxia-induced PMVECs.

Figure 4 MiR-21 negatively regulates DDAH1. (a) Venn diagram showing the prediction results of target genes from starBase, miRDB, and miRWalk databases. (b) The mRNA expression of candidates in miR-21 mimic-transfected PMVECs was measured by RT-qPCR. (c) DDAH1 protein expression in PMVECs transfected with miR-21 mimic or inhibitor was measured by western blot. (d and e) RT-qPCR and western blot were used to measure DDAH1 expression in PMVECs treated with RA or hyperoxia (4, 16 and 24 h). (f and g) DDAH1 expression in PMVECs of different groups (RA group, hyperoxia-24 h group, hyperoxia-24 h + NC inhibitor group, hyperoxia-24 h + miR-21 inhibitor group). (h) The binding site of DDAH1 and miR-21. (i) Luciferase reporter assay was performed to verify the interaction of DDAH1 and miR-21 in PMVECs with RA or 24 h of hyperoxia. Quantified values are mean values ± standard deviation of at least three independent experiments. ** p < 0.01, *** p s< 0.001.
Figure 4

MiR-21 negatively regulates DDAH1. (a) Venn diagram showing the prediction results of target genes from starBase, miRDB, and miRWalk databases. (b) The mRNA expression of candidates in miR-21 mimic-transfected PMVECs was measured by RT-qPCR. (c) DDAH1 protein expression in PMVECs transfected with miR-21 mimic or inhibitor was measured by western blot. (d and e) RT-qPCR and western blot were used to measure DDAH1 expression in PMVECs treated with RA or hyperoxia (4, 16 and 24 h). (f and g) DDAH1 expression in PMVECs of different groups (RA group, hyperoxia-24 h group, hyperoxia-24 h + NC inhibitor group, hyperoxia-24 h + miR-21 inhibitor group). (h) The binding site of DDAH1 and miR-21. (i) Luciferase reporter assay was performed to verify the interaction of DDAH1 and miR-21 in PMVECs with RA or 24 h of hyperoxia. Quantified values are mean values ± standard deviation of at least three independent experiments. ** p < 0.01, *** p s< 0.001.

3.5 DDAH1 knockdown reverses the effects of miR-21 depletion on hyperoxia-induced PMVEC behaviors, NO production, and ADMA concentration

For the sake of validating the interaction between DDAH1 and miR-21 in hyperoxia-induced PMVECs, we performed a series of rescue assays. DDAH1 protein expression was inhibited in hyperoxia-induced PMVECs by transfecting with si-DDAH1 (1.00 ± 0.15 vs 0.34 ± 0.04, p < 0.01) (Figure 5a). Then, it was illustrated from transwell assays that cell migration promoted by miR-21 depletion was reduced by si-DDAH1 transfection (221.67 ± 20.25 vs 102.43 ± 8.76, p < 0.01) (Figure 5b). Furthermore, tube formation assay indicated that cell angiogenic activity enhanced by miR-21 depletion was weakened by DDAH1 silencing (612.33 ± 50.29 vs 398.64 ± 37.16, p < 0.01) (Figure 5c). Next we discovered that miR-21 knockdown markedly elevated p-eNOS level in cells, but DDAH1 silencing did not affect its level (2.87 ± 0.25 vs 2.96 ± 0.22) (Figure 5d). Moreover, ELISA results illustrated that NO concentration elevated by miR-21 depletion was reduced by DDAH1 downregulation (21.41 ± 2.03 vs 16.17 ± 1.31, p < 0.05) (Figure 5e). Additionally, the extracellular ADMA concentration decreased by miR-21 depletion was recovered by DDAH1 inhibition (1.36 ± 0.12 vs 2.08 ± 0.14, p < 0.05) (Figure 5f). Overall, DDAH1 knockdown reverses the effects of miR-21 depletion on hyperoxia-induced PMVEC behaviors, NO production, and ADMA concentration.

Figure 5 DDAH1 knockdown reverses the effects of miR-21 depletion on hyperoxia-induced PMVEC behaviors, NO production, and ADMA concentration. (a) Western blot was performed to test the knockdown efficiency of si-DDAH1 in hyperoxia-induced PMVECs. (b) Transwell assay was applied for measuring the migration of hyperoxia-induced PMVECs in different groups (NC inhibitor group, miR-21 inhibitor group, miR-21 inhibitor + si-NC group, miR-21 inhibitor + si-DDAH1 group). (c) Tube formation assay was performed to estimate the angiogenic activity of hyperoxia-induced PMVECs in different groups. (d) Western blot was utilized to test the p-eNOS (ser1177) level in hyperoxia-induced PMVECs of different groups. (e and f) ELISA was utilized to detect the NO production and the extracellular ADMA concentration in hyperoxia-induced PMVECs of different groups. Quantified values are mean values ± standard deviation of at least three independent experiments. * p < 0.05, ** p < 0.01.
Figure 5

DDAH1 knockdown reverses the effects of miR-21 depletion on hyperoxia-induced PMVEC behaviors, NO production, and ADMA concentration. (a) Western blot was performed to test the knockdown efficiency of si-DDAH1 in hyperoxia-induced PMVECs. (b) Transwell assay was applied for measuring the migration of hyperoxia-induced PMVECs in different groups (NC inhibitor group, miR-21 inhibitor group, miR-21 inhibitor + si-NC group, miR-21 inhibitor + si-DDAH1 group). (c) Tube formation assay was performed to estimate the angiogenic activity of hyperoxia-induced PMVECs in different groups. (d) Western blot was utilized to test the p-eNOS (ser1177) level in hyperoxia-induced PMVECs of different groups. (e and f) ELISA was utilized to detect the NO production and the extracellular ADMA concentration in hyperoxia-induced PMVECs of different groups. Quantified values are mean values ± standard deviation of at least three independent experiments. * p < 0.05, ** p < 0.01.

3.6 miR-21 is upregulated and DDAH1 is downregulated in the lung of BPD rats

We performed the animal assays to further verify the correlation of miR-21 and DDAH1 in vivo. We established the BPD rat model by treating newborn rats with hyperoxia and obtained the lung tissues of rats on the indicated day and detected miR-21 and DDAH1 expression in tissues. RT-qPCR results indicated that, in comparison to air group, miR-21 expression was upregulated and DDAH1 expression was downregulated in lung tissues of BPD rats (1.00 ± 0.03 vs 2.26 ± 0.21, 1.00 ± 0.03 vs 0.39 ± 0.03, respectively, p < 0.01) (Figure 6a and b). Then, western blot further illustrated the downregulation of DDAH1 protein level in lung tissues of BPD rats (1.00 ± 0.08 vs 0.46 ± 0.94, p < 0.01) (Figure 6c). Furthermore, Pearson’s correlation analysis indicated that DDAH1 expression was negatively correlated with miR-21 expression in lung tissues of BPD rats (Figure 6d).

Figure 6 MiR-21 is upregulated and DDAH1 is downregulated in lung tissues of BPD rats. (a) MiR-21 expression was tested by RT-qPCR in lung tissues of rats treated with air or hyperoxia. (b and c) DDAH1 mRNA and protein expression in lung tissues of rats of different groups. (d) Pearson’s correlation analysis showing the correlation of miR-21 expression and DDAH1 expression in lung tissues of BPD rats. N = 12. Quantified values are mean values ± standard deviation of at least three independent experiments. * p < 0.05, ** p < 0.01.
Figure 6

MiR-21 is upregulated and DDAH1 is downregulated in lung tissues of BPD rats. (a) MiR-21 expression was tested by RT-qPCR in lung tissues of rats treated with air or hyperoxia. (b and c) DDAH1 mRNA and protein expression in lung tissues of rats of different groups. (d) Pearson’s correlation analysis showing the correlation of miR-21 expression and DDAH1 expression in lung tissues of BPD rats. N = 12. Quantified values are mean values ± standard deviation of at least three independent experiments. * p < 0.05, ** p < 0.01.

3.7 miR-21 antagomir restores alveolarization and vascular density in neonatal rats with BPD

H&E staining was performed to observe the histopathological alterations of lung tissues of rats. The results showed that, in comparison to the neonatal rats treated with air, the number of alveoli in neonatal rats with BPD was decreased and the structure was simplified. Alveolar wall rupture combined with pulmonary bullae, and the ratio of alveolar area/pulmonary septal area increased. After miR-21 antagomir transfection, the number of alveoli in neonatal rats with BPD increased, the lung tissue structure was mature, the alveolar structure was regular, the septal area decreased, and the proportion of alveolar area/pulmonary septal area decreased (Figure 7a–c). Then, to detect vascular development, vWF-positive small blood vessels were tested using immunofluorescence staining. We discovered that the vessel density was reduced in the BPD group and BPD + antagomir NC group (28.32 ± 2.11 vs 12.09 ± 1.03, p < 0.01), whereas it was then elevated in the BPD + miR-21 antagomir group (11.54 ± 0.83 vs 20.36 ± 1.46, p < 0.05) (Figure 7d and e), indicating that miR-21 depletion could alleviate hyperoxia-induced obstruction of pulmonary vascular development.

Figure 7 MiR-21 antagomir restores alveolarization and vascular density in neonatal rats with BPD. (a) H&E staining assay was utilized to detect the histopathological alterations of rats in different groups (air group, BPD group, BPD + NC antagomir group, BPD + miR-21 antagomir group). (b and c) The radial alveolar count and the alveolar area/pulmonary septal area value were detected. (d and e) Immunofluorescence staining was applied for measuring the vWF-positive vessels in different groups. N = 12. Quantified values are mean values ± standard deviation of at least three independent experiments. * p < 0.05, ** p < 0.01.
Figure 7

MiR-21 antagomir restores alveolarization and vascular density in neonatal rats with BPD. (a) H&E staining assay was utilized to detect the histopathological alterations of rats in different groups (air group, BPD group, BPD + NC antagomir group, BPD + miR-21 antagomir group). (b and c) The radial alveolar count and the alveolar area/pulmonary septal area value were detected. (d and e) Immunofluorescence staining was applied for measuring the vWF-positive vessels in different groups. N = 12. Quantified values are mean values ± standard deviation of at least three independent experiments. * p < 0.05, ** p < 0.01.

4 Discussion

The present study reported the hyperoxia-induced increase in miR-21 expression in the pulmonary vasculature in vitro and in vivo and evaluated the effect of miR-21 on pulmonary vascular responses in BPD. We showed that miR-21 contributed to the hyperoxia-induced degradation of DDAH1, increased ADMA concentration, and decreased eNOS phosphorylation and NO production in human PMVECs, thereby inhibiting PMVEC migration and angiogenesis. Most importantly, we found that miR-21 blockade significantly attenuated the development of hyperoxia-induced BPD in neonatal rats.

Long term exposure to hyperoxia can change the development of the lung and vascular bed, leading to BPD in premature infants [34]. Hyperoxia injury has been confirmed as one of the most common causes of BPD [35]. The pulmonary capillary endothelium is the major target site that is severely damaged by oxygen toxicity [36]. As human infants who die of BPD receive oxygen for more days, their pulmonary vasculature is damaged, and angiogenesis related factors are reduced [37]. Thus, hyperoxia exposure is extensively applied to induce BPD model, leading to the inhibition of distal microvascular formation and the imbalance of pro-angiogenic and anti-angiogenic cytokines in lung development [38]. It is reported that newborn mice treated with hyperoxia display greater arrest in lung alveolarization and angiogenesis [39]. In this study, we established the hyperoxia-induced PMVEC dysfunction in vitro, and found that hyperoxia treatment repressed the migratory ability and angiogenesis of PMVECs. Accumulating studies have suggested that miRNAs are promising candidates for novel targeted therapeutic approaches to lung diseases, including BPD [40]. In this study, we found that miR-21 expression in PMVECs was markedly increased by hyperoxia stimulation. MiR-21 is reported to be overexpressed in hyperoxia-mediated BPD [19], which is consistent with our findings. Furthermore, knockdown of miR-21 in hyperoxia-induced PMVECs promoted the migratory ability and angiogenesis ability. Previously, it was reported that miR-21 knockdown could inhibit cell proliferation, migration, and angiogenesis of hypoxic cardiac microvascular endothelial cells [41]. In this study, we believe that knockdown of miR-21 plays a crucial role in promoting angiogenesis in BPD.

During normal lung development, angiogenesis is coordinated accurately, maintaining a balance between cytokines promoting expansion and cytokines promoting the stability of endothelial barrier [42]. Normal alveolar development depends on angiogenesis [43]. Therefore, vascular growth disorder has become a recognized feature of BPD [44]. NO is a signaling molecule involved in modulating vascular tone, vascular remodeling, endothelial permeability, and angiogenesis [45]. eNOS is the main source of vascular NO, expressed in vascular endothelial cells and exerts the crucial function in regulating vascular tone [46]. Its activity is regulated via the phosphorylation of Ser1177 and dephosphorylation of Ser113 [47]. Accumulating studies have confirmed that ADMA is the endogenous NOS inhibitor, and it can repress NOS isoforms, causing diminished bioavailability of NO and endothelial dysfunction [48,49]. In this study, we found that hyperoxia treatment markedly decreased eNOS (ser1177) phosphorylation in PMVECs, while it was then increased by miR-21 knockdown. NO concentration was reduced by hyperoxia treatment and then elevated by miR-21 depletion. Furthermore, hyperoxia treatment elevated the intracellular and extracellular ADMA concentrations in PMVECs. However, miR-21 depletion decreased this effect. Thus, this study confirmed that miR-21 knockdown improved hyperoxia-induced PMVECs dysfunction and promoted angiogenesis via repressing ADMA concentration and elevating NO production. Dihydromyricetin attenuates TNF-α-induced endothelial dysfunction via miR-21-mediated DDAH1/ADMA/NO pathway [50], which further supports our findings.

DDAH has two isoforms, DDAH1 and DDAH2. Most of the ADMA are degraded to citrulline via DDAH or excreted by the kidneys in vivo [51]. DDAH1 is the vital enzyme in mediating ADMA in vascular endothelium [52]. Studies have indicated that DDAH depletion is closely correlated with endothelial dysfunction, and it is believed to be the mechanism responsible for ADMA-mediated eNOS impairments [53]. Blueberry anthocyanin‑enriched extract ameliorates transverse aortic constriction‑induced myocardial dysfunction through the DDAH1/ADMA/NO signaling pathway in mice [54]. DDAH1 mediates renal tissue protection in diabetic nephropathy via the ADMA-NOS3-interaction [55]. In this study, we found that DDAH1 was expressed at a low level in hyperoxia-induced PMVECs. Importantly, through prediction of bioinformatics tools and verification of assays, we confirmed that DDAH1 could bind to miR-21 in PMVECs. Furthermore, rescue assays illustrated that DDAH1 knockdown reversed the effects of miR-21 depletion on hyperoxia-induced PMVEC migration, angiogenesis, eNOS phosphorylation, NO production, and ADMA concentration. Previous studies suggested that DDAH1 is vital for regulating NO‐mediated apoptosis and angiogenesis in PMVECs [33]. DDAH1 is conducive to exercise-induced cardiac angiogenesis [56]. DDAH1 may be protective against the development of PH in BPD patients [57]. Furthermore, miR-21- facilitates the pathogenesis of atherosclerosis by regulating DDAH1-ADMA-eNOS-NO pathway [58]. Thus, we concluded that miR-21 knockdown promoted pulmonary angiogenesis in BPD via regulating DDAH/ADMA/NO pathway.

In animal assays, we observed that miR-21 expression was upregulated and DDAH1 expression was downregulated in lung tissues of BPD rats in a time-dependent manner. In addition, miR-21 antagomir restored alveolarization and vascular density in neonatal rats with BPD. Furthermore, Pearson’s correlation analysis indicated that DDAH1 was negatively correlated with miR-21 in lung tissues of BPD rats. It further suggested that miR-21 depletion repressed the progression of BPD.

There are limitations to this study. As miR-21 acts on multiple targets, at this stage we are not yet able to ascribe the benefits of miR-21 inhibition exclusively to upregulation of a specific target or pathway. Although the relative improvements seen in alveolarization and angiogenesis are impressive, more work needs to be performed to define the specific pathways that contribute to these findings. Additionally, although the in vivo model we used correlates with the pathological phenotype and long term consequences of neonatal lung disease [59,60], these experiments cannot fully replicate the combination of factors that contribute to the development of BPD. Thus, verifying the efficacy of miR-21 inhibition on BPD treatment is needed in a variety of animal models for future clinical trial. Additionally, our study was carried out to seek answers for acute pathological changes during BPD. Whether miR-21 inhibition has a wide therapeutic time window and exerts long-lasting effects in BPD is the next research direction.

5 Conclusion

Taken together, this study proved that inhibition of miR-21 improves pulmonary vascular responses in BPD by targeting the DDAH1/ADMA/NO pathway (Figure 8). Therefore, exploring the contributing mechanisms of miR-21-inhibition might help identify potential therapeutic strategies to improve lung structure and function of patients with BPD.

Figure 8 Scientific concept map of miR-21 mediated BPD development through DDAH1/ADMA/NO pathway. Under hyperoxia treatment, miR-21 is upregulated in PMVECs, and miR-21 inhibits DDAH1 expression. It increases ADMA concentration, leading to the decrease in eNOS phosphorylation and NO production. Finally, lung vascular function is impaired and BPD progression is deteriorated.
Figure 8

Scientific concept map of miR-21 mediated BPD development through DDAH1/ADMA/NO pathway. Under hyperoxia treatment, miR-21 is upregulated in PMVECs, and miR-21 inhibits DDAH1 expression. It increases ADMA concentration, leading to the decrease in eNOS phosphorylation and NO production. Finally, lung vascular function is impaired and BPD progression is deteriorated.

Acknowledgements

We appreciate the supports of our experimenters.

  1. Funding information: The work was supported by National Natural Science Foundation of China Grants (Approval number: 82001588) and Natural Science Foundation of Zhejiang Province Grant (Approval number: LY20H040008).

  2. Conflict of interest: No conflicts of interest, financial or otherwise, are declared by the authors.

  3. Data availability statement: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Received: 2022-05-22
Revised: 2022-09-07
Accepted: 2022-10-02
Published Online: 2022-12-09

© 2022 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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  15. Pericentric inversion of chromosome 6 and male fertility problems
  16. Staple line reinforcement with nebulized cyanoacrylate glue in laparoscopic sleeve gastrectomy: A propensity score-matched study
  17. Retrospective analysis of crescent score in clinical prognosis of IgA nephropathy
  18. Expression of DNM3 is associated with good outcome in colorectal cancer
  19. Activation of SphK2 contributes to adipocyte-induced EOC cell proliferation
  20. CRRT influences PICCO measurements in febrile critically ill patients
  21. SLCO4A1-AS1 mediates pancreatic cancer development via miR-4673/KIF21B axis
  22. lncRNA ACTA2-AS1 inhibits malignant phenotypes of gastric cancer cells
  23. circ_AKT3 knockdown suppresses cisplatin resistance in gastric cancer
  24. Prognostic value of nicotinamide N-methyltransferase in human cancers: Evidence from a meta-analysis and database validation
  25. GPC2 deficiency inhibits cell growth and metastasis in colon adenocarcinoma
  26. A pan-cancer analysis of the oncogenic role of Holliday junction recognition protein in human tumors
  27. Radiation increases COL1A1, COL3A1, and COL1A2 expression in breast cancer
  28. Association between preventable risk factors and metabolic syndrome
  29. miR-29c-5p knockdown reduces inflammation and blood–brain barrier disruption by upregulating LRP6
  30. Cardiac contractility modulation ameliorates myocardial metabolic remodeling in a rabbit model of chronic heart failure through activation of AMPK and PPAR-α pathway
  31. Quercitrin protects human bronchial epithelial cells from oxidative damage
  32. Smurf2 suppresses the metastasis of hepatocellular carcinoma via ubiquitin degradation of Smad2
  33. circRNA_0001679/miR-338-3p/DUSP16 axis aggravates acute lung injury
  34. Sonoclot’s usefulness in prediction of cardiopulmonary arrest prognosis: A proof of concept study
  35. Four drug metabolism-related subgroups of pancreatic adenocarcinoma in prognosis, immune infiltration, and gene mutation
  36. Decreased expression of miR-195 mediated by hypermethylation promotes osteosarcoma
  37. LMO3 promotes proliferation and metastasis of papillary thyroid carcinoma cells by regulating LIMK1-mediated cofilin and the β-catenin pathway
  38. Cx43 upregulation in HUVECs under stretch via TGF-β1 and cytoskeletal network
  39. Evaluation of menstrual irregularities after COVID-19 vaccination: Results of the MECOVAC survey
  40. Histopathologic findings on removed stomach after sleeve gastrectomy. Do they influence the outcome?
  41. Analysis of the expression and prognostic value of MT1-MMP, β1-integrin and YAP1 in glioma
  42. Optimal diagnosis of the skin cancer using a hybrid deep neural network and grasshopper optimization algorithm
  43. miR-223-3p alleviates TGF-β-induced epithelial-mesenchymal transition and extracellular matrix deposition by targeting SP3 in endometrial epithelial cells
  44. Clinical value of SIRT1 as a prognostic biomarker in esophageal squamous cell carcinoma, a systematic meta-analysis
  45. circ_0020123 promotes cell proliferation and migration in lung adenocarcinoma via PDZD8
  46. miR-22-5p regulates the self-renewal of spermatogonial stem cells by targeting EZH2
  47. hsa-miR-340-5p inhibits epithelial–mesenchymal transition in endometriosis by targeting MAP3K2 and inactivating MAPK/ERK signaling
  48. circ_0085296 inhibits the biological functions of trophoblast cells to promote the progression of preeclampsia via the miR-942-5p/THBS2 network
  49. TCD hemodynamics findings in the subacute phase of anterior circulation stroke patients treated with mechanical thrombectomy
  50. Development of a risk-stratification scoring system for predicting risk of breast cancer based on non-alcoholic fatty liver disease, non-alcoholic fatty pancreas disease, and uric acid
  51. Tollip promotes hepatocellular carcinoma progression via PI3K/AKT pathway
  52. circ_0062491 alleviates periodontitis via the miR-142-5p/IGF1 axis
  53. Human amniotic fluid as a source of stem cells
  54. lncRNA NONRATT013819.2 promotes transforming growth factor-β1-induced myofibroblastic transition of hepatic stellate cells by miR24-3p/lox
  55. NORAD modulates miR-30c-5p-LDHA to protect lung endothelial cells damage
  56. Idiopathic pulmonary fibrosis telemedicine management during COVID-19 outbreak
  57. Risk factors for adverse drug reactions associated with clopidogrel therapy
  58. Serum zinc associated with immunity and inflammatory markers in Covid-19
  59. The relationship between night shift work and breast cancer incidence: A systematic review and meta-analysis of observational studies
  60. LncRNA expression in idiopathic achalasia: New insight and preliminary exploration into pathogenesis
  61. Notoginsenoside R1 alleviates spinal cord injury through the miR-301a/KLF7 axis to activate Wnt/β-catenin pathway
  62. Moscatilin suppresses the inflammation from macrophages and T cells
  63. Zoledronate promotes ECM degradation and apoptosis via Wnt/β-catenin
  64. Epithelial-mesenchymal transition-related genes in coronary artery disease
  65. The effect evaluation of traditional vaginal surgery and transvaginal mesh surgery for severe pelvic organ prolapse: 5 years follow-up
  66. Repeated partial splenic artery embolization for hypersplenism improves platelet count
  67. Low expression of miR-27b in serum exosomes of non-small cell lung cancer facilitates its progression by affecting EGFR
  68. Exosomal hsa_circ_0000519 modulates the NSCLC cell growth and metastasis via miR-1258/RHOV axis
  69. miR-455-5p enhances 5-fluorouracil sensitivity in colorectal cancer cells by targeting PIK3R1 and DEPDC1
  70. The effect of tranexamic acid on the reduction of intraoperative and postoperative blood loss and thromboembolic risk in patients with hip fracture
  71. Isocitrate dehydrogenase 1 mutation in cholangiocarcinoma impairs tumor progression by sensitizing cells to ferroptosis
  72. Artemisinin protects against cerebral ischemia and reperfusion injury via inhibiting the NF-κB pathway
  73. A 16-gene signature associated with homologous recombination deficiency for prognosis prediction in patients with triple-negative breast cancer
  74. Lidocaine ameliorates chronic constriction injury-induced neuropathic pain through regulating M1/M2 microglia polarization
  75. MicroRNA 322-5p reduced neuronal inflammation via the TLR4/TRAF6/NF-κB axis in a rat epilepsy model
  76. miR-1273h-5p suppresses CXCL12 expression and inhibits gastric cancer cell invasion and metastasis
  77. Clinical characteristics of pneumonia patients of long course of illness infected with SARS-CoV-2
  78. circRNF20 aggravates the malignancy of retinoblastoma depending on the regulation of miR-132-3p/PAX6 axis
  79. Linezolid for resistant Gram-positive bacterial infections in children under 12 years: A meta-analysis
  80. Rack1 regulates pro-inflammatory cytokines by NF-κB in diabetic nephropathy
  81. Comprehensive analysis of molecular mechanism and a novel prognostic signature based on small nuclear RNA biomarkers in gastric cancer patients
  82. Smog and risk of maternal and fetal birth outcomes: A retrospective study in Baoding, China
  83. Let-7i-3p inhibits the cell cycle, proliferation, invasion, and migration of colorectal cancer cells via downregulating CCND1
  84. β2-Adrenergic receptor expression in subchondral bone of patients with varus knee osteoarthritis
  85. Possible impact of COVID-19 pandemic and lockdown on suicide behavior among patients in Southeast Serbia
  86. In vitro antimicrobial activity of ozonated oil in liposome eyedrop against multidrug-resistant bacteria
  87. Potential biomarkers for inflammatory response in acute lung injury
  88. A low serum uric acid concentration predicts a poor prognosis in adult patients with candidemia
  89. Antitumor activity of recombinant oncolytic vaccinia virus with human IL2
  90. ALKBH5 inhibits TNF-α-induced apoptosis of HUVECs through Bcl-2 pathway
  91. Risk prediction of cardiovascular disease using machine learning classifiers
  92. Value of ultrasonography parameters in diagnosing polycystic ovary syndrome
  93. Bioinformatics analysis reveals three key genes and four survival genes associated with youth-onset NSCLC
  94. Identification of autophagy-related biomarkers in patients with pulmonary arterial hypertension based on bioinformatics analysis
  95. Protective effects of glaucocalyxin A on the airway of asthmatic mice
  96. Overexpression of miR-100-5p inhibits papillary thyroid cancer progression via targeting FZD8
  97. Bioinformatics-based analysis of SUMOylation-related genes in hepatocellular carcinoma reveals a role of upregulated SAE1 in promoting cell proliferation
  98. Effectiveness and clinical benefits of new anti-diabetic drugs: A real life experience
  99. Identification of osteoporosis based on gene biomarkers using support vector machine
  100. Tanshinone IIA reverses oxaliplatin resistance in colorectal cancer through microRNA-30b-5p/AVEN axis
  101. miR-212-5p inhibits nasopharyngeal carcinoma progression by targeting METTL3
  102. Association of ST-T changes with all-cause mortality among patients with peripheral T-cell lymphomas
  103. LINC00665/miRNAs axis-mediated collagen type XI alpha 1 correlates with immune infiltration and malignant phenotypes in lung adenocarcinoma
  104. The perinatal factors that influence the excretion of fecal calprotectin in premature-born children
  105. Effect of femoral head necrosis cystic area on femoral head collapse and stress distribution in femoral head: A clinical and finite element study
  106. Does the use of 3D-printed cones give a chance to postpone the use of megaprostheses in patients with large bone defects in the knee joint?
  107. lncRNA HAGLR modulates myocardial ischemia–reperfusion injury in mice through regulating miR-133a-3p/MAPK1 axis
  108. Protective effect of ghrelin on intestinal I/R injury in rats
  109. In vivo knee kinematics of an innovative prosthesis design
  110. Relationship between the height of fibular head and the incidence and severity of knee osteoarthritis
  111. lncRNA WT1-AS attenuates hypoxia/ischemia-induced neuronal injury during cerebral ischemic stroke via miR-186-5p/XIAP axis
  112. Correlation of cardiac troponin T and APACHE III score with all-cause in-hospital mortality in critically ill patients with acute pulmonary embolism
  113. LncRNA LINC01857 reduces metastasis and angiogenesis in breast cancer cells via regulating miR-2052/CENPQ axis
  114. Endothelial cell-specific molecule 1 (ESM1) promoted by transcription factor SPI1 acts as an oncogene to modulate the malignant phenotype of endometrial cancer
  115. SELENBP1 inhibits progression of colorectal cancer by suppressing epithelial–mesenchymal transition
  116. Visfatin is negatively associated with coronary artery lesions in subjects with impaired fasting glucose
  117. Treatment and outcomes of mechanical complications of acute myocardial infarction during the Covid-19 era: A comparison with the pre-Covid-19 period. A systematic review and meta-analysis
  118. Neonatal stroke surveillance study protocol in the United Kingdom and Republic of Ireland
  119. Oncogenic role of TWF2 in human tumors: A pan-cancer analysis
  120. Mean corpuscular hemoglobin predicts the length of hospital stay independent of severity classification in patients with acute pancreatitis
  121. Association of gallstone and polymorphisms of UGT1A1*27 and UGT1A1*28 in patients with hepatitis B virus-related liver failure
  122. TGF-β1 upregulates Sar1a expression and induces procollagen-I secretion in hypertrophic scarring fibroblasts
  123. Antisense lncRNA PCNA-AS1 promotes esophageal squamous cell carcinoma progression through the miR-2467-3p/PCNA axis
  124. NK-cell dysfunction of acute myeloid leukemia in relation to the renin–angiotensin system and neurotransmitter genes
  125. The effect of dilution with glucose and prolonged injection time on dexamethasone-induced perineal irritation – A randomized controlled trial
  126. miR-146-5p restrains calcification of vascular smooth muscle cells by suppressing TRAF6
  127. Role of lncRNA MIAT/miR-361-3p/CCAR2 in prostate cancer cells
  128. lncRNA NORAD promotes lung cancer progression by competitively binding to miR-28-3p with E2F2
  129. Noninvasive diagnosis of AIH/PBC overlap syndrome based on prediction models
  130. lncRNA FAM230B is highly expressed in colorectal cancer and suppresses the maturation of miR-1182 to increase cell proliferation
  131. circ-LIMK1 regulates cisplatin resistance in lung adenocarcinoma by targeting miR-512-5p/HMGA1 axis
  132. LncRNA SNHG3 promoted cell proliferation, migration, and metastasis of esophageal squamous cell carcinoma via regulating miR-151a-3p/PFN2 axis
  133. Risk perception and affective state on work exhaustion in obstetrics during the COVID-19 pandemic
  134. lncRNA-AC130710/miR-129-5p/mGluR1 axis promote migration and invasion by activating PKCα-MAPK signal pathway in melanoma
  135. SNRPB promotes cell cycle progression in thyroid carcinoma via inhibiting p53
  136. Xylooligosaccharides and aerobic training regulate metabolism and behavior in rats with streptozotocin-induced type 1 diabetes
  137. Serpin family A member 1 is an oncogene in glioma and its translation is enhanced by NAD(P)H quinone dehydrogenase 1 through RNA-binding activity
  138. Silencing of CPSF7 inhibits the proliferation, migration, and invasion of lung adenocarcinoma cells by blocking the AKT/mTOR signaling pathway
  139. Ultrasound-guided lumbar plexus block versus transversus abdominis plane block for analgesia in children with hip dislocation: A double-blind, randomized trial
  140. Relationship of plasma MBP and 8-oxo-dG with brain damage in preterm
  141. Identification of a novel necroptosis-associated miRNA signature for predicting the prognosis in head and neck squamous cell carcinoma
  142. Delayed femoral vein ligation reduces operative time and blood loss during hip disarticulation in patients with extremity tumors
  143. The expression of ASAP3 and NOTCH3 and the clinicopathological characteristics of adult glioma patients
  144. Longitudinal analysis of factors related to Helicobacter pylori infection in Chinese adults
  145. HOXA10 enhances cell proliferation and suppresses apoptosis in esophageal cancer via activating p38/ERK signaling pathway
  146. Meta-analysis of early-life antibiotic use and allergic rhinitis
  147. Marital status and its correlation with age, race, and gender in prognosis of tonsil squamous cell carcinomas
  148. HPV16 E6E7 up-regulates KIF2A expression by activating JNK/c-Jun signal, is beneficial to migration and invasion of cervical cancer cells
  149. Amino acid profiles in the tissue and serum of patients with liver cancer
  150. Pain in critically ill COVID-19 patients: An Italian retrospective study
  151. Immunohistochemical distribution of Bcl-2 and p53 apoptotic markers in acetamiprid-induced nephrotoxicity
  152. Estradiol pretreatment in GnRH antagonist protocol for IVF/ICSI treatment
  153. Long non-coding RNAs LINC00689 inhibits the apoptosis of human nucleus pulposus cells via miR-3127-5p/ATG7 axis-mediated autophagy
  154. The relationship between oxygen therapy, drug therapy, and COVID-19 mortality
  155. Monitoring hypertensive disorders in pregnancy to prevent preeclampsia in pregnant women of advanced maternal age: Trial mimicking with retrospective data
  156. SETD1A promotes the proliferation and glycolysis of nasopharyngeal carcinoma cells by activating the PI3K/Akt pathway
  157. The role of Shunaoxin pills in the treatment of chronic cerebral hypoperfusion and its main pharmacodynamic components
  158. TET3 governs malignant behaviors and unfavorable prognosis of esophageal squamous cell carcinoma by activating the PI3K/AKT/GSK3β/β-catenin pathway
  159. Associations between morphokinetic parameters of temporary-arrest embryos and the clinical prognosis in FET cycles
  160. Long noncoding RNA WT1-AS regulates trophoblast proliferation, migration, and invasion via the microRNA-186-5p/CADM2 axis
  161. The incidence of bronchiectasis in chronic obstructive pulmonary disease
  162. Integrated bioinformatics analysis shows integrin alpha 3 is a prognostic biomarker for pancreatic cancer
  163. Inhibition of miR-21 improves pulmonary vascular responses in bronchopulmonary dysplasia by targeting the DDAH1/ADMA/NO pathway
  164. Comparison of hospitalized patients with severe pneumonia caused by COVID-19 and influenza A (H7N9 and H1N1): A retrospective study from a designated hospital
  165. lncRNA ZFAS1 promotes intervertebral disc degeneration by upregulating AAK1
  166. Pathological characteristics of liver injury induced by N,N-dimethylformamide: From humans to animal models
  167. lncRNA ELFN1-AS1 enhances the progression of colon cancer by targeting miR-4270 to upregulate AURKB
  168. DARS-AS1 modulates cell proliferation and migration of gastric cancer cells by regulating miR-330-3p/NAT10 axis
  169. Dezocine inhibits cell proliferation, migration, and invasion by targeting CRABP2 in ovarian cancer
  170. MGST1 alleviates the oxidative stress of trophoblast cells induced by hypoxia/reoxygenation and promotes cell proliferation, migration, and invasion by activating the PI3K/AKT/mTOR pathway
  171. Bifidobacterium lactis Probio-M8 ameliorated the symptoms of type 2 diabetes mellitus mice by changing ileum FXR-CYP7A1
  172. circRNA DENND1B inhibits tumorigenicity of clear cell renal cell carcinoma via miR-122-5p/TIMP2 axis
  173. EphA3 targeted by miR-3666 contributes to melanoma malignancy via activating ERK1/2 and p38 MAPK pathways
  174. Pacemakers and methylprednisolone pulse therapy in immune-related myocarditis concomitant with complete heart block
  175. miRNA-130a-3p targets sphingosine-1-phosphate receptor 1 to activate the microglial and astrocytes and to promote neural injury under the high glucose condition
  176. Review Articles
  177. Current management of cancer pain in Italy: Expert opinion paper
  178. Hearing loss and brain disorders: A review of multiple pathologies
  179. The rationale for using low-molecular weight heparin in the therapy of symptomatic COVID-19 patients
  180. Amyotrophic lateral sclerosis and delayed onset muscle soreness in light of the impaired blink and stretch reflexes – watch out for Piezo2
  181. Interleukin-35 in autoimmune dermatoses: Current concepts
  182. Recent discoveries in microbiota dysbiosis, cholangiocytic factors, and models for studying the pathogenesis of primary sclerosing cholangitis
  183. Advantages of ketamine in pediatric anesthesia
  184. Congenital adrenal hyperplasia. Role of dentist in early diagnosis
  185. Migraine management: Non-pharmacological points for patients and health care professionals
  186. Atherogenic index of plasma and coronary artery disease: A systematic review
  187. Physiological and modulatory role of thioredoxins in the cellular function
  188. Case Reports
  189. Intrauterine Bakri balloon tamponade plus cervical cerclage for the prevention and treatment of postpartum haemorrhage in late pregnancy complicated with acute aortic dissection: Case series
  190. A case of successful pembrolizumab monotherapy in a patient with advanced lung adenocarcinoma: Use of multiple biomarkers in combination for clinical practice
  191. Unusual neurological manifestations of bilateral medial medullary infarction: A case report
  192. Atypical symptoms of malignant hyperthermia: A rare causative mutation in the RYR1 gene
  193. A case report of dermatomyositis with the missed diagnosis of non-small cell lung cancer and concurrence of pulmonary tuberculosis
  194. A rare case of endometrial polyp complicated with uterine inversion: A case report and clinical management
  195. Spontaneous rupturing of splenic artery aneurysm: Another reason for fatal syncope and shock (Case report and literature review)
  196. Fungal infection mimicking COVID-19 infection – A case report
  197. Concurrent aspergillosis and cystic pulmonary metastases in a patient with tongue squamous cell carcinoma
  198. Paraganglioma-induced inverted takotsubo-like cardiomyopathy leading to cardiogenic shock successfully treated with extracorporeal membrane oxygenation
  199. Lineage switch from lymphoma to myeloid neoplasms: First case series from a single institution
  200. Trismus during tracheal extubation as a complication of general anaesthesia – A case report
  201. Simultaneous treatment of a pubovesical fistula and lymph node metastasis secondary to multimodal treatment for prostate cancer: Case report and review of the literature
  202. Two case reports of skin vasculitis following the COVID-19 immunization
  203. Ureteroiliac fistula after oncological surgery: Case report and review of the literature
  204. Synchronous triple primary malignant tumours in the bladder, prostate, and lung harbouring TP53 and MEK1 mutations accompanied with severe cardiovascular diseases: A case report
  205. Huge mucinous cystic neoplasms with adhesion to the left colon: A case report and literature review
  206. Commentary
  207. Commentary on “Clinicopathological features of programmed cell death-ligand 1 expression in patients with oral squamous cell carcinoma”
  208. Rapid Communication
  209. COVID-19 fear, post-traumatic stress, growth, and the role of resilience
  210. Erratum
  211. Erratum to “Tollip promotes hepatocellular carcinoma progression via PI3K/AKT pathway”
  212. Erratum to “Effect of femoral head necrosis cystic area on femoral head collapse and stress distribution in femoral head: A clinical and finite element study”
  213. Erratum to “lncRNA NORAD promotes lung cancer progression by competitively binding to miR-28-3p with E2F2”
  214. Retraction
  215. Expression and role of ABIN1 in sepsis: In vitro and in vivo studies
  216. Retraction to “miR-519d downregulates LEP expression to inhibit preeclampsia development”
  217. Special Issue Computational Intelligence Methodologies Meets Recurrent Cancers - Part II
  218. Usefulness of close surveillance for rectal cancer patients after neoadjuvant chemoradiotherapy
https://doi.org/10.1515/med-2022-0584
Keywords for this article
bronchopulmonary dysplasia; miR-21; DDAH1; ADMA; NO
Creative Commons
BY 4.0

Articles in the same Issue

  1. Research Articles
  2. AMBRA1 attenuates the proliferation of uveal melanoma cells
  3. A ceRNA network mediated by LINC00475 in papillary thyroid carcinoma
  4. Differences in complications between hepatitis B-related cirrhosis and alcohol-related cirrhosis
  5. Effect of gestational diabetes mellitus on lipid profile: A systematic review and meta-analysis
  6. Long noncoding RNA NR2F1-AS1 stimulates the tumorigenic behavior of non-small cell lung cancer cells by sponging miR-363-3p to increase SOX4
  7. Promising novel biomarkers and candidate small-molecule drugs for lung adenocarcinoma: Evidence from bioinformatics analysis of high-throughput data
  8. Plasmapheresis: Is it a potential alternative treatment for chronic urticaria?
  9. The biomarkers of key miRNAs and gene targets associated with extranodal NK/T-cell lymphoma
  10. Gene signature to predict prognostic survival of hepatocellular carcinoma
  11. Effects of miRNA-199a-5p on cell proliferation and apoptosis of uterine leiomyoma by targeting MED12
  12. Does diabetes affect paraneoplastic thrombocytosis in colorectal cancer?
  13. Is there any effect on imprinted genes H19, PEG3, and SNRPN during AOA?
  14. Leptin and PCSK9 concentrations are associated with vascular endothelial cytokines in patients with stable coronary heart disease
  15. Pericentric inversion of chromosome 6 and male fertility problems
  16. Staple line reinforcement with nebulized cyanoacrylate glue in laparoscopic sleeve gastrectomy: A propensity score-matched study
  17. Retrospective analysis of crescent score in clinical prognosis of IgA nephropathy
  18. Expression of DNM3 is associated with good outcome in colorectal cancer
  19. Activation of SphK2 contributes to adipocyte-induced EOC cell proliferation
  20. CRRT influences PICCO measurements in febrile critically ill patients
  21. SLCO4A1-AS1 mediates pancreatic cancer development via miR-4673/KIF21B axis
  22. lncRNA ACTA2-AS1 inhibits malignant phenotypes of gastric cancer cells
  23. circ_AKT3 knockdown suppresses cisplatin resistance in gastric cancer
  24. Prognostic value of nicotinamide N-methyltransferase in human cancers: Evidence from a meta-analysis and database validation
  25. GPC2 deficiency inhibits cell growth and metastasis in colon adenocarcinoma
  26. A pan-cancer analysis of the oncogenic role of Holliday junction recognition protein in human tumors
  27. Radiation increases COL1A1, COL3A1, and COL1A2 expression in breast cancer
  28. Association between preventable risk factors and metabolic syndrome
  29. miR-29c-5p knockdown reduces inflammation and blood–brain barrier disruption by upregulating LRP6
  30. Cardiac contractility modulation ameliorates myocardial metabolic remodeling in a rabbit model of chronic heart failure through activation of AMPK and PPAR-α pathway
  31. Quercitrin protects human bronchial epithelial cells from oxidative damage
  32. Smurf2 suppresses the metastasis of hepatocellular carcinoma via ubiquitin degradation of Smad2
  33. circRNA_0001679/miR-338-3p/DUSP16 axis aggravates acute lung injury
  34. Sonoclot’s usefulness in prediction of cardiopulmonary arrest prognosis: A proof of concept study
  35. Four drug metabolism-related subgroups of pancreatic adenocarcinoma in prognosis, immune infiltration, and gene mutation
  36. Decreased expression of miR-195 mediated by hypermethylation promotes osteosarcoma
  37. LMO3 promotes proliferation and metastasis of papillary thyroid carcinoma cells by regulating LIMK1-mediated cofilin and the β-catenin pathway
  38. Cx43 upregulation in HUVECs under stretch via TGF-β1 and cytoskeletal network
  39. Evaluation of menstrual irregularities after COVID-19 vaccination: Results of the MECOVAC survey
  40. Histopathologic findings on removed stomach after sleeve gastrectomy. Do they influence the outcome?
  41. Analysis of the expression and prognostic value of MT1-MMP, β1-integrin and YAP1 in glioma
  42. Optimal diagnosis of the skin cancer using a hybrid deep neural network and grasshopper optimization algorithm
  43. miR-223-3p alleviates TGF-β-induced epithelial-mesenchymal transition and extracellular matrix deposition by targeting SP3 in endometrial epithelial cells
  44. Clinical value of SIRT1 as a prognostic biomarker in esophageal squamous cell carcinoma, a systematic meta-analysis
  45. circ_0020123 promotes cell proliferation and migration in lung adenocarcinoma via PDZD8
  46. miR-22-5p regulates the self-renewal of spermatogonial stem cells by targeting EZH2
  47. hsa-miR-340-5p inhibits epithelial–mesenchymal transition in endometriosis by targeting MAP3K2 and inactivating MAPK/ERK signaling
  48. circ_0085296 inhibits the biological functions of trophoblast cells to promote the progression of preeclampsia via the miR-942-5p/THBS2 network
  49. TCD hemodynamics findings in the subacute phase of anterior circulation stroke patients treated with mechanical thrombectomy
  50. Development of a risk-stratification scoring system for predicting risk of breast cancer based on non-alcoholic fatty liver disease, non-alcoholic fatty pancreas disease, and uric acid
  51. Tollip promotes hepatocellular carcinoma progression via PI3K/AKT pathway
  52. circ_0062491 alleviates periodontitis via the miR-142-5p/IGF1 axis
  53. Human amniotic fluid as a source of stem cells
  54. lncRNA NONRATT013819.2 promotes transforming growth factor-β1-induced myofibroblastic transition of hepatic stellate cells by miR24-3p/lox
  55. NORAD modulates miR-30c-5p-LDHA to protect lung endothelial cells damage
  56. Idiopathic pulmonary fibrosis telemedicine management during COVID-19 outbreak
  57. Risk factors for adverse drug reactions associated with clopidogrel therapy
  58. Serum zinc associated with immunity and inflammatory markers in Covid-19
  59. The relationship between night shift work and breast cancer incidence: A systematic review and meta-analysis of observational studies
  60. LncRNA expression in idiopathic achalasia: New insight and preliminary exploration into pathogenesis
  61. Notoginsenoside R1 alleviates spinal cord injury through the miR-301a/KLF7 axis to activate Wnt/β-catenin pathway
  62. Moscatilin suppresses the inflammation from macrophages and T cells
  63. Zoledronate promotes ECM degradation and apoptosis via Wnt/β-catenin
  64. Epithelial-mesenchymal transition-related genes in coronary artery disease
  65. The effect evaluation of traditional vaginal surgery and transvaginal mesh surgery for severe pelvic organ prolapse: 5 years follow-up
  66. Repeated partial splenic artery embolization for hypersplenism improves platelet count
  67. Low expression of miR-27b in serum exosomes of non-small cell lung cancer facilitates its progression by affecting EGFR
  68. Exosomal hsa_circ_0000519 modulates the NSCLC cell growth and metastasis via miR-1258/RHOV axis
  69. miR-455-5p enhances 5-fluorouracil sensitivity in colorectal cancer cells by targeting PIK3R1 and DEPDC1
  70. The effect of tranexamic acid on the reduction of intraoperative and postoperative blood loss and thromboembolic risk in patients with hip fracture
  71. Isocitrate dehydrogenase 1 mutation in cholangiocarcinoma impairs tumor progression by sensitizing cells to ferroptosis
  72. Artemisinin protects against cerebral ischemia and reperfusion injury via inhibiting the NF-κB pathway
  73. A 16-gene signature associated with homologous recombination deficiency for prognosis prediction in patients with triple-negative breast cancer
  74. Lidocaine ameliorates chronic constriction injury-induced neuropathic pain through regulating M1/M2 microglia polarization
  75. MicroRNA 322-5p reduced neuronal inflammation via the TLR4/TRAF6/NF-κB axis in a rat epilepsy model
  76. miR-1273h-5p suppresses CXCL12 expression and inhibits gastric cancer cell invasion and metastasis
  77. Clinical characteristics of pneumonia patients of long course of illness infected with SARS-CoV-2
  78. circRNF20 aggravates the malignancy of retinoblastoma depending on the regulation of miR-132-3p/PAX6 axis
  79. Linezolid for resistant Gram-positive bacterial infections in children under 12 years: A meta-analysis
  80. Rack1 regulates pro-inflammatory cytokines by NF-κB in diabetic nephropathy
  81. Comprehensive analysis of molecular mechanism and a novel prognostic signature based on small nuclear RNA biomarkers in gastric cancer patients
  82. Smog and risk of maternal and fetal birth outcomes: A retrospective study in Baoding, China
  83. Let-7i-3p inhibits the cell cycle, proliferation, invasion, and migration of colorectal cancer cells via downregulating CCND1
  84. β2-Adrenergic receptor expression in subchondral bone of patients with varus knee osteoarthritis
  85. Possible impact of COVID-19 pandemic and lockdown on suicide behavior among patients in Southeast Serbia
  86. In vitro antimicrobial activity of ozonated oil in liposome eyedrop against multidrug-resistant bacteria
  87. Potential biomarkers for inflammatory response in acute lung injury
  88. A low serum uric acid concentration predicts a poor prognosis in adult patients with candidemia
  89. Antitumor activity of recombinant oncolytic vaccinia virus with human IL2
  90. ALKBH5 inhibits TNF-α-induced apoptosis of HUVECs through Bcl-2 pathway
  91. Risk prediction of cardiovascular disease using machine learning classifiers
  92. Value of ultrasonography parameters in diagnosing polycystic ovary syndrome
  93. Bioinformatics analysis reveals three key genes and four survival genes associated with youth-onset NSCLC
  94. Identification of autophagy-related biomarkers in patients with pulmonary arterial hypertension based on bioinformatics analysis
  95. Protective effects of glaucocalyxin A on the airway of asthmatic mice
  96. Overexpression of miR-100-5p inhibits papillary thyroid cancer progression via targeting FZD8
  97. Bioinformatics-based analysis of SUMOylation-related genes in hepatocellular carcinoma reveals a role of upregulated SAE1 in promoting cell proliferation
  98. Effectiveness and clinical benefits of new anti-diabetic drugs: A real life experience
  99. Identification of osteoporosis based on gene biomarkers using support vector machine
  100. Tanshinone IIA reverses oxaliplatin resistance in colorectal cancer through microRNA-30b-5p/AVEN axis
  101. miR-212-5p inhibits nasopharyngeal carcinoma progression by targeting METTL3
  102. Association of ST-T changes with all-cause mortality among patients with peripheral T-cell lymphomas
  103. LINC00665/miRNAs axis-mediated collagen type XI alpha 1 correlates with immune infiltration and malignant phenotypes in lung adenocarcinoma
  104. The perinatal factors that influence the excretion of fecal calprotectin in premature-born children
  105. Effect of femoral head necrosis cystic area on femoral head collapse and stress distribution in femoral head: A clinical and finite element study
  106. Does the use of 3D-printed cones give a chance to postpone the use of megaprostheses in patients with large bone defects in the knee joint?
  107. lncRNA HAGLR modulates myocardial ischemia–reperfusion injury in mice through regulating miR-133a-3p/MAPK1 axis
  108. Protective effect of ghrelin on intestinal I/R injury in rats
  109. In vivo knee kinematics of an innovative prosthesis design
  110. Relationship between the height of fibular head and the incidence and severity of knee osteoarthritis
  111. lncRNA WT1-AS attenuates hypoxia/ischemia-induced neuronal injury during cerebral ischemic stroke via miR-186-5p/XIAP axis
  112. Correlation of cardiac troponin T and APACHE III score with all-cause in-hospital mortality in critically ill patients with acute pulmonary embolism
  113. LncRNA LINC01857 reduces metastasis and angiogenesis in breast cancer cells via regulating miR-2052/CENPQ axis
  114. Endothelial cell-specific molecule 1 (ESM1) promoted by transcription factor SPI1 acts as an oncogene to modulate the malignant phenotype of endometrial cancer
  115. SELENBP1 inhibits progression of colorectal cancer by suppressing epithelial–mesenchymal transition
  116. Visfatin is negatively associated with coronary artery lesions in subjects with impaired fasting glucose
  117. Treatment and outcomes of mechanical complications of acute myocardial infarction during the Covid-19 era: A comparison with the pre-Covid-19 period. A systematic review and meta-analysis
  118. Neonatal stroke surveillance study protocol in the United Kingdom and Republic of Ireland
  119. Oncogenic role of TWF2 in human tumors: A pan-cancer analysis
  120. Mean corpuscular hemoglobin predicts the length of hospital stay independent of severity classification in patients with acute pancreatitis
  121. Association of gallstone and polymorphisms of UGT1A1*27 and UGT1A1*28 in patients with hepatitis B virus-related liver failure
  122. TGF-β1 upregulates Sar1a expression and induces procollagen-I secretion in hypertrophic scarring fibroblasts
  123. Antisense lncRNA PCNA-AS1 promotes esophageal squamous cell carcinoma progression through the miR-2467-3p/PCNA axis
  124. NK-cell dysfunction of acute myeloid leukemia in relation to the renin–angiotensin system and neurotransmitter genes
  125. The effect of dilution with glucose and prolonged injection time on dexamethasone-induced perineal irritation – A randomized controlled trial
  126. miR-146-5p restrains calcification of vascular smooth muscle cells by suppressing TRAF6
  127. Role of lncRNA MIAT/miR-361-3p/CCAR2 in prostate cancer cells
  128. lncRNA NORAD promotes lung cancer progression by competitively binding to miR-28-3p with E2F2
  129. Noninvasive diagnosis of AIH/PBC overlap syndrome based on prediction models
  130. lncRNA FAM230B is highly expressed in colorectal cancer and suppresses the maturation of miR-1182 to increase cell proliferation
  131. circ-LIMK1 regulates cisplatin resistance in lung adenocarcinoma by targeting miR-512-5p/HMGA1 axis
  132. LncRNA SNHG3 promoted cell proliferation, migration, and metastasis of esophageal squamous cell carcinoma via regulating miR-151a-3p/PFN2 axis
  133. Risk perception and affective state on work exhaustion in obstetrics during the COVID-19 pandemic
  134. lncRNA-AC130710/miR-129-5p/mGluR1 axis promote migration and invasion by activating PKCα-MAPK signal pathway in melanoma
  135. SNRPB promotes cell cycle progression in thyroid carcinoma via inhibiting p53
  136. Xylooligosaccharides and aerobic training regulate metabolism and behavior in rats with streptozotocin-induced type 1 diabetes
  137. Serpin family A member 1 is an oncogene in glioma and its translation is enhanced by NAD(P)H quinone dehydrogenase 1 through RNA-binding activity
  138. Silencing of CPSF7 inhibits the proliferation, migration, and invasion of lung adenocarcinoma cells by blocking the AKT/mTOR signaling pathway
  139. Ultrasound-guided lumbar plexus block versus transversus abdominis plane block for analgesia in children with hip dislocation: A double-blind, randomized trial
  140. Relationship of plasma MBP and 8-oxo-dG with brain damage in preterm
  141. Identification of a novel necroptosis-associated miRNA signature for predicting the prognosis in head and neck squamous cell carcinoma
  142. Delayed femoral vein ligation reduces operative time and blood loss during hip disarticulation in patients with extremity tumors
  143. The expression of ASAP3 and NOTCH3 and the clinicopathological characteristics of adult glioma patients
  144. Longitudinal analysis of factors related to Helicobacter pylori infection in Chinese adults
  145. HOXA10 enhances cell proliferation and suppresses apoptosis in esophageal cancer via activating p38/ERK signaling pathway
  146. Meta-analysis of early-life antibiotic use and allergic rhinitis
  147. Marital status and its correlation with age, race, and gender in prognosis of tonsil squamous cell carcinomas
  148. HPV16 E6E7 up-regulates KIF2A expression by activating JNK/c-Jun signal, is beneficial to migration and invasion of cervical cancer cells
  149. Amino acid profiles in the tissue and serum of patients with liver cancer
  150. Pain in critically ill COVID-19 patients: An Italian retrospective study
  151. Immunohistochemical distribution of Bcl-2 and p53 apoptotic markers in acetamiprid-induced nephrotoxicity
  152. Estradiol pretreatment in GnRH antagonist protocol for IVF/ICSI treatment
  153. Long non-coding RNAs LINC00689 inhibits the apoptosis of human nucleus pulposus cells via miR-3127-5p/ATG7 axis-mediated autophagy
  154. The relationship between oxygen therapy, drug therapy, and COVID-19 mortality
  155. Monitoring hypertensive disorders in pregnancy to prevent preeclampsia in pregnant women of advanced maternal age: Trial mimicking with retrospective data
  156. SETD1A promotes the proliferation and glycolysis of nasopharyngeal carcinoma cells by activating the PI3K/Akt pathway
  157. The role of Shunaoxin pills in the treatment of chronic cerebral hypoperfusion and its main pharmacodynamic components
  158. TET3 governs malignant behaviors and unfavorable prognosis of esophageal squamous cell carcinoma by activating the PI3K/AKT/GSK3β/β-catenin pathway
  159. Associations between morphokinetic parameters of temporary-arrest embryos and the clinical prognosis in FET cycles
  160. Long noncoding RNA WT1-AS regulates trophoblast proliferation, migration, and invasion via the microRNA-186-5p/CADM2 axis
  161. The incidence of bronchiectasis in chronic obstructive pulmonary disease
  162. Integrated bioinformatics analysis shows integrin alpha 3 is a prognostic biomarker for pancreatic cancer
  163. Inhibition of miR-21 improves pulmonary vascular responses in bronchopulmonary dysplasia by targeting the DDAH1/ADMA/NO pathway
  164. Comparison of hospitalized patients with severe pneumonia caused by COVID-19 and influenza A (H7N9 and H1N1): A retrospective study from a designated hospital
  165. lncRNA ZFAS1 promotes intervertebral disc degeneration by upregulating AAK1
  166. Pathological characteristics of liver injury induced by N,N-dimethylformamide: From humans to animal models
  167. lncRNA ELFN1-AS1 enhances the progression of colon cancer by targeting miR-4270 to upregulate AURKB
  168. DARS-AS1 modulates cell proliferation and migration of gastric cancer cells by regulating miR-330-3p/NAT10 axis
  169. Dezocine inhibits cell proliferation, migration, and invasion by targeting CRABP2 in ovarian cancer
  170. MGST1 alleviates the oxidative stress of trophoblast cells induced by hypoxia/reoxygenation and promotes cell proliferation, migration, and invasion by activating the PI3K/AKT/mTOR pathway
  171. Bifidobacterium lactis Probio-M8 ameliorated the symptoms of type 2 diabetes mellitus mice by changing ileum FXR-CYP7A1
  172. circRNA DENND1B inhibits tumorigenicity of clear cell renal cell carcinoma via miR-122-5p/TIMP2 axis
  173. EphA3 targeted by miR-3666 contributes to melanoma malignancy via activating ERK1/2 and p38 MAPK pathways
  174. Pacemakers and methylprednisolone pulse therapy in immune-related myocarditis concomitant with complete heart block
  175. miRNA-130a-3p targets sphingosine-1-phosphate receptor 1 to activate the microglial and astrocytes and to promote neural injury under the high glucose condition
  176. Review Articles
  177. Current management of cancer pain in Italy: Expert opinion paper
  178. Hearing loss and brain disorders: A review of multiple pathologies
  179. The rationale for using low-molecular weight heparin in the therapy of symptomatic COVID-19 patients
  180. Amyotrophic lateral sclerosis and delayed onset muscle soreness in light of the impaired blink and stretch reflexes – watch out for Piezo2
  181. Interleukin-35 in autoimmune dermatoses: Current concepts
  182. Recent discoveries in microbiota dysbiosis, cholangiocytic factors, and models for studying the pathogenesis of primary sclerosing cholangitis
  183. Advantages of ketamine in pediatric anesthesia
  184. Congenital adrenal hyperplasia. Role of dentist in early diagnosis
  185. Migraine management: Non-pharmacological points for patients and health care professionals
  186. Atherogenic index of plasma and coronary artery disease: A systematic review
  187. Physiological and modulatory role of thioredoxins in the cellular function
  188. Case Reports
  189. Intrauterine Bakri balloon tamponade plus cervical cerclage for the prevention and treatment of postpartum haemorrhage in late pregnancy complicated with acute aortic dissection: Case series
  190. A case of successful pembrolizumab monotherapy in a patient with advanced lung adenocarcinoma: Use of multiple biomarkers in combination for clinical practice
  191. Unusual neurological manifestations of bilateral medial medullary infarction: A case report
  192. Atypical symptoms of malignant hyperthermia: A rare causative mutation in the RYR1 gene
  193. A case report of dermatomyositis with the missed diagnosis of non-small cell lung cancer and concurrence of pulmonary tuberculosis
  194. A rare case of endometrial polyp complicated with uterine inversion: A case report and clinical management
  195. Spontaneous rupturing of splenic artery aneurysm: Another reason for fatal syncope and shock (Case report and literature review)
  196. Fungal infection mimicking COVID-19 infection – A case report
  197. Concurrent aspergillosis and cystic pulmonary metastases in a patient with tongue squamous cell carcinoma
  198. Paraganglioma-induced inverted takotsubo-like cardiomyopathy leading to cardiogenic shock successfully treated with extracorporeal membrane oxygenation
  199. Lineage switch from lymphoma to myeloid neoplasms: First case series from a single institution
  200. Trismus during tracheal extubation as a complication of general anaesthesia – A case report
  201. Simultaneous treatment of a pubovesical fistula and lymph node metastasis secondary to multimodal treatment for prostate cancer: Case report and review of the literature
  202. Two case reports of skin vasculitis following the COVID-19 immunization
  203. Ureteroiliac fistula after oncological surgery: Case report and review of the literature
  204. Synchronous triple primary malignant tumours in the bladder, prostate, and lung harbouring TP53 and MEK1 mutations accompanied with severe cardiovascular diseases: A case report
  205. Huge mucinous cystic neoplasms with adhesion to the left colon: A case report and literature review
  206. Commentary
  207. Commentary on “Clinicopathological features of programmed cell death-ligand 1 expression in patients with oral squamous cell carcinoma”
  208. Rapid Communication
  209. COVID-19 fear, post-traumatic stress, growth, and the role of resilience
  210. Erratum
  211. Erratum to “Tollip promotes hepatocellular carcinoma progression via PI3K/AKT pathway”
  212. Erratum to “Effect of femoral head necrosis cystic area on femoral head collapse and stress distribution in femoral head: A clinical and finite element study”
  213. Erratum to “lncRNA NORAD promotes lung cancer progression by competitively binding to miR-28-3p with E2F2”
  214. Retraction
  215. Expression and role of ABIN1 in sepsis: In vitro and in vivo studies
  216. Retraction to “miR-519d downregulates LEP expression to inhibit preeclampsia development”
  217. Special Issue Computational Intelligence Methodologies Meets Recurrent Cancers - Part II
  218. Usefulness of close surveillance for rectal cancer patients after neoadjuvant chemoradiotherapy
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