Clinical value of lncRNA CARMN expression in threatened abortion and its effect on cell function of HTR-8/SVneo

Study subjects

A total of 105 patients aged 22–38 years old, 6–8 weeks pregnant and with early TA of single pregnancy treated in The 2nd Affiliated Hospital of Chengdu Medical College (Nuclear Industry 416 Hospital) were included as the TA group. In addition, 63 normal pregnant women who underwent normal labor examination in The 2nd Affiliated Hospital of Chengdu Medical College (Nuclear Industry 416 Hospital) were included as control group. All participants provided written consent for participation. This study was approved by the Ethics Committee of The 2nd Affiliated Hospital of Chengdu Medical College (Nuclear Industry 416 Hospital).

Inclusion and exclusion criteria

Inclusion criteria: 1) The patients in the TA group were accompanied by small amounts of vaginal bleeding and slight abdominal pain to varying degrees. The cervical opening was found to be closed by gynecological examination, and a small amount of bleeding was caused by vaginal bleeding in the uterine cavity. 2) Women with intrauterine single pregnancy. 3) Participants have complete medical records.

Exclusion criteria: 1) Patients with severe abnormalities of liver, kidney, heart, and lung function. 2) Patients with other serious endocrine system diseases. 3) People with immune system diseases. 4) Patients with abnormal coagulation function. 5) Patients with adverse pregnancy history. 6) Patients with gestational comorbidities, such as gestational diabetes, gestational hypertension. 7) People with mental illness, who are unable to cooperate with the completion of this study.

Collection of general information

General information was collected for all researchers, including age, gestational age, smoking history, and miscarriage history. In the morning, two tubes of 5 mL venous blood from the two groups of pregnant women were taken under a fasting state, and the serum was obtained after standing at room temperature for 30 min at 3,000 r/min and centrifuged for 10 min. The contents of serum β-human chorionic gonadotropin (β-hCG), progesterone and estradiol (E₂) in one tube were detected in the laboratory of The 2nd Affiliated Hospital of Chengdu Medical College (Nuclear Inoustry 416 Hospital), and the other tube was stored at −80 °C for reserve.

RNA extraction and qRT-PCR

Total RNA was isolated from the serum and transfected cells following the manufacturer’s guidelines provided with the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). The extracted RNA samples showed an acceptable quality, as indicated by their A260/A280 ratios, which fell within the range of 1.8–2.1. The reverse transcription and quantitative analysis were conducted with the PrimerScript RT Reagent Kit (TaKaRa, Otsu, Shiga, Japan). qRT-PCR was performed on the StepOne Plus Real-Time PCR System (Applied Biosystems). The expression level was determined using the 2^−ΔΔCt method. The primers used for qRT-PCR included:

CARMN forward: 5′-TGTGCTTGGCTGTCCTTTCT-3′.

CARMN reverse: 5′-AGGTGAATAGGGGGCAGACT-3′.

miR-515-5p forward:5′-TCGGCAGGUUCUCCAAAAGAAA-3′.

miR-515-5p reverse5′-CTCGCTTCGGCAGCACA-3′.

GAPDH forward: 5′-GTTAGGAAAGCCTGCCGGTG-3′.

GAPDH reverse: 5′-AGCATCGCCCCACTTGATTT-3′.

Cell culture and transfection

HTR-8/SVneo cells (Shaun Biotechnology, Wuhan, China) were cultured in DMEM medium containing 10 % FBS and incubated in a cell incubator with a CO₂ saturation of 5 % at 37 °C. The shRNA- CARMN (sh-CARMN) and negative control (sh-NC) were sourced from Transheep Bio (Shanghai, China). Furthermore, the pcDNA3.1-CARMN (pcDNA-CARMN), an empty pcDNA3.1 vector used for overexpression, as well as the miR-515-5p mimics/mimics NC and miR-515-5p inhibitor/inhibitor NC, were sourced from BeNa Bio (Beijing, China). HTR-8/SVneo cells were infected with the aforementioned agents via Lipofectamine 3,000 (Thermo Fisher Scientifc, Waltham, MA, USA).

CCK-8 assay

HTR-8/SVneo cells were inoculated into 96-well plates and allowed to proliferate for a 48-h period. 10 μL of CCK-8 solution (Procell Life Science & Technology) were added to each well. After a 2-h incubation at a temperature of 37 °C, the absorbance at 450 nm was determined using a microplate spectrophotometer.

Cell apoptosis assay

HTR-8/SVneo cells were inoculated into six-well plates. To assess apoptosis, the Annexin V-Fluos and Propidium Iodide (PI) Apoptosis Detection Kit (Sigma, Germany) was employed to detect the apoptosis rate. The examination was executed in line with the producer’s instructions, and the procedure was replicated three times independently.

Transwell assay

The cellular populations were re-dispersed within a medium devoid of serum, subsequent to which they were introduced into the upper compartment of a transwell plate. A DMEM medium with a high sugar content, which also included 20 % FBS, was poured into the lower chamber and allowed to incubate for a 24-h period. Following this, the medium was removed, the cells were immobilized through a 30-min immersion in methanol. Subsequently, the cells were colored using a 0.1 % crystal violet solution for an additional 20 min. Thereafter, the upper layer of cells that had not migrated was carefully removed using cotton swabs. These cells were then observed, and images were captured using an optical microscope for documentation purposes.

Dual-luciferase reporter assay

The CARMN sequence was inserted into the pmirGLO vector (Promega, Shanghai, China), leading to the production of two distinct luciferase reporter constructs: CARMN-WT (for wild-type) and CARMN-MUT (for mutant). Subsequently, these vectors were coinjected into HTR-8/SVneo cells employing Lipofectamine 2000. After a 48-h incubation period, the Promega Dual-Luciferase Reporter Assay Kit was utilized in compliance with the producer’s instructions.

Statistical analysis

Data statistical analysis was conducted using SPSS version 26.0 and GraphPad Prism version 9. The data was displayed as mean ± standard deviation (SD) and examined using an independent sample t-test or one-way ANOVA. The diagnostic utility of CARMN in TA was evaluated using a ROC curve. Additionally, logistic regression was utilized to determine the risk factors associated with the onset of TA. p<0.05 were deemed as being statistically significant differences.

Comparison of general data from different study groups

Individuals experiencing TA did not show a variation from those with uncomplicated pregnancies in terms of age, gestational age, BMI, or smoking history. However, significant disparities were noted between the two groups in terms of a prior history of abortion and serum levels of β-hCG, progesterone, and E₂ (Table 1).

Differential expression of CARMN in serum of TA patients and predicted value of TA

Reduced expression of CARMN was observed in the serum of individuals experiencing TA (Figure 1A). In these patients with TA, CARMN expression was lower in women who terminated pregnancy than in women who continued pregnancy (Figure 1B). The ROC curve analysis revealed that the CARMN biomarker yielded an AUC of 0.891 for predicting TA, exhibiting a sensitivity of 85.71 % and a specificity of 82.54 % (Figure 1C). In addition, CARMN predicted the pregnancy outcome of TA patients with an AUC of 0.814, a sensitivity of 82.93 %, and a specificity of 78.13 % (Figure 1D).

Figure 1:

The level of CARMN in patients with TA and has diagnostic value for TA. (A) The expression level of CARMN in serum of TA patients was detected by qRT-PCR. (B) The level of CARMN in patients with continuation pregnancy and those with termination pregnancy was detected by qRT-PCR. (C) ROC curve of serum CARMN for predicting TA. ∗∗∗P<0.001. (D) ROC curve of serum CARMN for predicting continuation and termination in TA patients.

Logistic regression analysis of risk factors for TA

With the occurrence of TA as the dependent variable and age, gestatory week, BMI, smoking history, abortion history, serum β-hCG, progesterone, E₂ levels, and CARMN expression as the independent variables, binary logistic regression analysis showed that abortion history, serum β-hCG, progesterone, E₂ levels, and the expression of CARMN were risk factors for TA (Table 2).

Relevance of CARMN to clinical features of TA patients

According to the average level of serum CARMN expression in patients with TA, the participants were categorized into groups with low and high expression levels, and the Chi-square test was performed. Patients with a history of miscarriage and low serum levels of β-hCG, progesterone, and E2 had lower serum CARMN expression (Table 3).

Effect of CARMN on HTR-8/SVneo cells

In order to assess the impact of CARMN on HTR-8/SVneo cells, we introduced pcDNA-CARMN and sh-CARMN into these cells and determined the functional role of CARMN under vitro conditions. Transfection of pcDNA-CARMN markedly increased the levels of CARMN expression, while the transfection with sh-CARMN noticeably decreased CARMN expression (Figure 2A). Subsequently, the influence of CARMN overexpression and knockdown on the cell viability, apoptosis, and migration was investigated. Figure 2B–D demonstrated that pcDNA-CARMN transfection significantly enhanced the cell viability, and migration, and conversely, suppressed the apoptosis rate. sh-CARMN markedly suppressed the cell viability, and migration, while enhancing the apoptotic ratio.

Figure 2:

Effects of overexpression or knockdown of CARMN on the function of HTR-8/SVneo cells. (A) The transfection efficiency of overexpression or knockdown of CARMN was detected by qRT-PCR. (B) The changes in cell viability after CARMN overexpression or knockdown were detected by CCK-8. (C) Annexin V/PI assay was used to detect apoptosis after CARMN overexpression or knockdown. (D) The transwell assay was employed to assess the capacity of cell migration following CARMN overexpression or knockdown. ^**p<0.01. ^***p<0.001.

miR-515–5p is a target of CARMN

Figure 3A illustrated that the miR-515-5p level was elevated in TA compared to normal pregnant women, and this upregulation was inversely associated with the levels of CARMN expression (Figure 3B). Figure 3C showed the binding sites of CARMN and miR-515-5p. The relative luciferase activity in cells was substantially augmented following the co-transfection of CARMN-WT with miR-515-5p inhibitors. Conversely, when CARMN-WT and miR-515-5p mimics were co-inoculated, a marked decrease in the relative luciferase activity was observed.

Figure 3:

Expression of miR-515-5p in TA patients and miR-515-5p is a target of CARMN. (A) The expression level of miR-515-5p in serum of TA was detected by qRT-PCR. (B) Pearson correlation analysis revealed a negative association between CARMN and miR-515–5p. (C) The sequence of the binding site between CARMN and miR-515-5p was forecasted utilizing the starbase database and the specific interaction between CARMN and miR-515-5p was investigated using a dual luciferase reporter gene assay. ^**p<0.01. ^***p<0.001.

CARMN regulates HTR-8/SVneo cell viability, migration, and apoptosis by regulating miR-515-5p

CARMN overexpression significantly decreased the level of miR-515-5p, this trend was reversed after transfection with miR-515-5p mimic (Figure 4A). miR-515-5p overexpression reversed the promoting effect of CARMN overexpression on the cell viability (Figure 4B). miR-515-5p mimics partially offset the inhibitory effect of CARMN overexpression on cell apoptosis (Figure 4C). Transwell experiments showed that miR-515-5p mimics partially reversed the promotional impacts of elevated CARMN expression on the migration of HTR-8/SVneo cells (Figure 4D).

Figure 4:

miR-515-5p partially counteracts the effects of sh-CARMN on cellular function. (A) miR-515-5p level was assessed by qRT-PCR after sh-CARMN and miR-515-5p inhibitor treatment. (B) Cell viability was assessed using the CCK-8 assay. (C) Cell apoptosis was quantified by the annexin V/PI assay. (D) The migration capability of cells was assessed by the transwell assay. ^*p<0.05, ^**p<0.01, ^***p<0.001.