miR-15b-5p affects the innate immune response through PAQR3 in patients with reflux esophagitis

Research object

All RE cases were from the gastroenterology department of gastroenterology. The patients was diagnosed with RE on gastroscopy according to Los Angeles criteria [12]. A total of 54 pathological cases were collected. Moreover, 33 healthy people who underwent physical examination in Jiaozhou Central Hospital of Qingdao were collected as controls.

As noted in earlier research, patients with RE exhibited classic symptoms including acid regurgitation, a burning sensation in the sternum, or intermittent chest pain. Additional diagnostic examinations revealed distal esophageal mucosal injury or sores, or the presence of esophageal stenosis. Histologically, these were defined by features such as lesions, necrosis, ulcerations, lengthening of papillae, increased intercellular spaces, basal cell hyperplasia, or infiltration of inflammatory cells [12], 13].

Pathological exclusions included: (1) endoscopic gastric and duodenal ulcers (active or inactive), erosive duodenitis; (2) endoscopic gastrointestinal tumors; (3) history of upper gastrointestinal surgery; (4) suspected or confirmed malignant tumor; (5) history of hypertension, heart disease, diabetes, cirrhosis, renal insufficiency, cerebrovascular disease; (6) chronic atrophic gastritis; (7) patients taking antibiotics, antacids, aspirin, proton pump inhibitors (PPI), mucosal protectants in the 4 weeks prior to the study.

Esophageal mucosal tissues and venous blood were taken from all patients. This study adheres to the tenets of the Declaration of Helsinki. All subjects provided written informed consent, and the study was authorized by the Medical Ethics Committee of Jiaozhou Central Hospital of Qingdao. All patients signed informed consent forms.

Cell culture and treatment

Telomerase-immortalized, nonneoplastic esophageal squamous (NES) cell line (NES-B3T), which was established from endoscopically obtained biopsy samples derived from the squamous mucosa of the lower esophagus in patients affected by gastroesophageal reflux disease [14] was used in this study.

The NES-B3T cells were cultivated in two different environments: cells at 70 % confluence were treated with acidic medium containing acidic deoxycholic acid (DAC, pH=5, 100 μmol/L), neutral medium (pH 7.0) without acidic medium was used in the control group. This acidic or neutral solution was administered to the cells for a 10-min duration, three times daily, to well seeded with an equal number of NES-B3T cells. This regimen was maintained for a span of 5 days, aiming to emulate the common physiological occurrences of gastroesophageal reflux [15].

Lipofectamine 2000 reagent was mixed with small interfering RNAs (siRNAs) specifically designed to target PAQR3 (si- PAQR3), a non-targeting siRNA control (si-NC), miR-15b-5p mimics, negative control mimics (mimic NC), miR-15b-5p inhibitor and negative control inhibitor (inhibitor NC) (GenePharma, Shanghai, China) for cultivation at 37 °C.

RNA extraction and qRT-PCR

TRIzol reagent (Invitrogen, USA) was employed to extract the total RNA from the esophageal mucosal tissues, blood samples and cells. Subsequently, the PrimeScript RT kit (Takara, Japan) was utilized for the conversion of RNA into complementary DNA (cDNA). The qRT-PCR reaction was then carried out utilizing the 7300 real-time PCR system (Applied Biosystems, USA) along with the SYBR Green I Master Mix kit (Invitrogen, USA). The relative mRNA expression levels were determined using the 2^−ΔΔCt method, with the cycle threshold (CT) values being normalized against the GAPDH, serving as the endogenous control.

Enzyme-linked immunosorbent assay (ELISA)

The ELISA kits (Bender MedSystems, Austria) (LOQ: 5 pg/mL, intra-assay CV 3.5 %, inter-assay CV 5 %) were employed to assess the alterations in the levels of interferon-β (IFN-β), interleukin (IL)-6, and IL-8. The optical density (OD) readings at 450 nm were deemed significant for indicating changes.

Dual-luciferase assay

A construct containing the PAQR3, and its various mutants was meticulously designed and inserted into the pmirGLO luciferase vector (Promega, Madison, WI, USA). This process led to the production of two separate gene constructs, namely PAQR3-wt and PAQR3-mut. Then these plasmids were transferred into the cells concurrently with the presence of either miR-15b-5p mimics or miR-15b-5p inhibitors. The relative luciferase activity was subsequently measured by employing the Dual Luciferase Assay Kit (Promega, USA).

Statistical analysis

GraphPad Prism version 9.0 and SPSS 26.0 were used to conduct statistical analysis. Data are presented as the mean±SD. The comparison of two groups of data was conducted using the independent samples t-test, while the comparison among multiple groups of data was performed using one-way or two-way analysis of variance (ANOVA). The chi-square test was used to analyze categorical variables. The diagnostic potential of miR-15b-5p in RE patients was evaluated using ROC curve. Chi-square test was used to analyze the correlation between miR-15b-5p expression and clinical features. A p-value of below 0.05 was statistically significant.

Expression and diagnostic role of miR-15b-5p in RE patients

No significant difference was found in age, gender, and BMI among the two groups (p>0.05) (Table 1). The level of miR-15b-5p in serum was higher in RE patients compared to healthy controls (Figure 1A). Figure 1B illustrated the diagnostic performance of miR-15b-5p for RE. The AUC of the ROC curve was 0.861, this corresponds to a sensitivity of 87.0 % and a specificity of 80.0 %, suggesting that miR-15b-5p potentially acts as a potent biomarker to differentiate individuals with RE from healthy subjects.

Figure 1:

Expression and diagnostic role of miR-15b-5p. (A) The expression of miR-15b-5p in serum of RE patients was detected by qRT-PCR. (B) ROC curve of miR-15b-5p for the diagnosis of RE. ***p<0.001 vs. control.

Correlation of miR-15b-5p expression with RE clinical characteristics

With the average level of miR-15b-5p in RE patients as the dividing line, RE patients were divided into high and low groups. As depicted in Table 2, a significant correlation was identified between miR-15b-5p and the Los Angeles Classification of the patients. A higher expression of miR-15b-5p was associated with a higher Los Angeles Classification, indicating a more severe clinical manifestation of the disease.

Expression of innate immune-related factors in RE patients

The levels of TLR4 and MyD88 are commonly elevated in patients experiencing RE in comparison to control group (Figure 2A and B). Additionally, the concentration of IFN-β, IL-6, and IL-8 were observed to be increased within the RE patient (Figure 2C–E). At the same time, we analyzed the correlation between these indicators and the level of miR-15b-5p. As shown in Figure 3, the levels of TLR4 (Figure 3A), MyD88 (Figure 3B), IFN-β (Figure 3C), IL-6 (Figure 3D), and IL-8 (Figure 3E) were positively correlated with miR-15b-5p.

Figure 2:

Levels of innate immune-related factors in RE patients. (A–B) The expression of TLR4 and MyD88 in serum of RE patients was detected by qRT-PCR. (C–E) The ELISA kit was used to detect IFN-β, IL-6 and IL-8 concentrations. ***p<0.001, **p<0.01 vs. control.

Figure 3:

Correlation between miR-15b-5P and innate immune-related factors in RE patients. (A–E) Correlation between miR-15b-5P and TLR4 (A), MyD88 (B), IFN-β (C), IL-6 (D), and IL-8 (E) in RE patients. ***p<0.001.

The changes of IFN-β, IL-6, and IL-8 after acidic medium treatment

To simulate the common physiological phenomena of reflux esophagus, the cells were exposed to an acidic medium containing DAC for 5 days and cells was collected to detect the levels of inflammatory factors after each day of intervention. After 3 days of exposure to the acidic medium, the cell secretion of IFN-β increased significantly and the secretion increased continuously to day 5 (Figure 4A). On days 3 and 4 of exposure to the acid medium, IL-6 secretion levels increased (Figure 4B). IL-8 secretion levels increased significantly on day 2 of exposure to the acid medium and continued to rise beginning on day 3 (Figure 4C).

Figure 4:

Results of exposing NES-B3T cells to acidic medium on the secretion of IFN-β, IL-6, and IL-8. (A–C) The levels of IFN-β, IL-6, and IL-8 were measured by ELISA from day 1 to day 5 after the acidic medium intervention. ***p<0.001, **p<0.01 vs. control; ###p<0.001, ##p<0.01, #p<0.05 vs. the previous day.

Inhibition of miR-15b-5p suppressed innate immunity

Taking into account the alterations in inflammatory markers, subsequent experiments involved subjecting the cells to an acidic environment for a duration of 3 days. An increase of miR-15b-5p was observed in cells that were exposed to acidic medium. The level of miR-15b-5p was reduced following miR-15b-5p inhibitor transfection (Figure 5A). Additionally, the expression of TLR4 and MyD88 was upregulated after the intervention of acid and downregulated after the inhibition of miR-5b-5p (Figure 5B and C). Similarly, the production of IFN-β, IL-6, and IL-8 by cells was elevated upon the addition of acidic medium, and this increase was reversed when miR-15b-5p was inhibited (Figure 5D–F).

Figure 5:

Inhibition of miR-15b-5p suppressed innate immunity. (A) The expression of miR-15b-5p in NES-B3T cells was detected by qRT-PCR. (B–C) The expression of TLR4 and MyD88 in NES-B3T cells was detected by qRT-PCR. (D–F) The ELISA kit was used to detect IFN-β, IL-6 and IL-8 concentrations in NES-B3T cells. ***p<0.001, **p<0.01, *p<0.05 vs. control; ###p<0.001, ##p<0.01, #p<0.05 vs. acidic medium and inhibitor-NC.

PAQR3 was a target for miR-15b-5p

Reduced expression of PAQR3 was observed in the serum of RE patients (Figure 6A), and this was inversely associated with the levels of miR-15b-5p (Figure 6B). Concurrently, treatment of NES-B3T cells with acidic medium led to a reduction in the expression of PAQR3 (Figure 6C). Through the starBase database, it was discovered that miR-15b-5p possessed potential binding sites for PAQR3, as depicted in Figure 6D. After the administration of miR-15b-5p mimics, there was a notable decrease in luciferase activity in the PAQR3-wt vector. In contrast, the application of miR-15b-5p inhibitors resulted in an enhancement of luciferase activity in the PAQR3-wt vector. However, the PAQR3-mut vector exhibited negligible alterations in luciferase activity (Figure 6E).

Figure 6:

PAQR3 was a target for miR-15b-5p. (A) The expression of PAQR3 in serum of RE patients was detected by qRT-PCR. (B) The expression of PAQR3 and miR-15b-5p in serum of RE patients was negatively correlated. (C) The expression of PAQR3 in NES-B3T cells was detected by qRT-PCR. (D) The binding site sequence between PAQR3 and miR-15b-5p was forecasted utilizing the starBase database. (E) The interaction between PAQR3 and miR-15b-5p was verified by dual-luciferase reporter gene assay. ***p<0.001, **p<0.01 vs. control.

miR-15b-5p modulated the innate immune of RE by regulating PAQR3

To better understand the underlying molecular mechanism of miR-15b-5p mediating innate immunity, we transfected NES-B3T cells with miR-15b-5p and si-PAQR3. As depicted in Figure 7A, the level of PAQR3 was upregulated in cells following the miR-15b-5p inhibitor transfection, whereas it was downregulated upon transfection with si-PAQR3. The si-PAQR3 vector was capable of counteracting the reduction in TLR4 and MyD88 mRNA levels induced by the miR-15b-5p inhibitor (Figure 7B and C). Furthermore, the suppression of PAQR3 mitigated the reduced levels of IFN-β, IL-6, and IL-8 resulting from miR-15b-5p inhibition (Figure 7D–F).

Figure 7:

miR-15b-5p modulated the innate immune system of RE by regulating PAQR3. (A) The expression of PAQR3 was detected by qRT-PCR. (B–C) The expression of TLR4 and MyD88 in NES-B3T cells was detected by qRT-PCR. (D–F) The ELISA kit was used to detect IFN-β, IL-6 and IL-8 concentrations in NES-B3T cells. ***p<0.001, **p<0.01 vs. acidic medium and inhibitor-NC; ###p<0.001, ##p<0.01, #p<0.05 vs. acidic medium, miR-15b-5p inhibitor and si-NC.