Establishment of an Acid-Induced Het-1A Cell Model and Functional Analysis of the miR-107/FGFRL1 Axis

摘要

Here, we present a protocol to establish an HCl-induced reflux esophagitis cell model using Het-1A cells and to investigate the miR-107/FGFRL1 axis using molecular and cellular assays.

摘要

Extensive evidence suggests that microRNAs (miRNAs) play a key role in gastrointestinal pathophysiological processes. However, their specific mechanisms in reflux esophagitis (RE) remain poorly understood. Here, we present a protocol to establish an acid-induced RE model and to investigate the miR-107/FGFRL1 axis using molecular and cellular assays. We conducted clinical sample analyses, including serum collection from patients with R E (n=94) and healthy controls (n=94), followed by quantitative detection via RT-qPCR. In vitro, an acid-induced injury model was established using Het-1A cells exposed to hydrochloric acid. Additionally, functional validation of the cells was performed using MTT cell viability assays and dual-luciferase reporter assays. Representative results indicate that miR-107 was downregulated in the serum of RE patients and in acid-treated Het-1A cells. miR-107 mediated hydrochloric acid-induced changes in Het-1A cell viability and inflammation levels by directly targeting FGFRL1 and is associated with changes in cell viability and inflammation through FGFRL1 targeting. This protocol enables investigation of miR-107/FGFRL1-mediated mechanisms in RE. This approach provides a reproducible platform for studying molecular mechanisms in RE.

引言

Reflux esophagitis (RE) is a digestive disease in which gastric acid, gastric contents repeatedly reflux into the esophagus, resulting in inflammation, erosion or even ulceration of the esophageal mucosa, the severity of which is related to the nature of the refluxed material, the frequency of reflux and the resistance of the esophageal mucosa^1,2. As a highly prevalent type of gastroesophageal reflux disease (GERD), RE exhibits a steadily rising incidence and progressively widening global impact³. The nonspecific clinical presentation of RE commonly leads to misdiagnosis or underdiagnosis. Moreover, the recurrent course of the disease frequently progresses to more serious gastrointestinal complications, imposing substantial physiological and socioeconomic burdens on affected individuals⁴. Standard clinical management of RE typically involves pharmacotherapy (such as proton pump inhibitors), minimally invasive endoscopic procedures, or surgical^5,6. However, therapeutic efficacy is often limited by medication-related adverse effects and procedural morbidity. Given the incomplete understanding of RE's molecular pathogenesis, elucidating its underlying mechanisms may reveal novel therapeutic targets and RE inform the development of more effective, patient-specific treatment strategies. This protocol aims to establish an in vitro RE model and to investigate the regulatory role of the miR-107/FGFRL1 axis using molecular and cellular assays.

MicroRNAs (miRNAs) have been extensively documented to participate in the pathogenesis of numerous human diseases through their ability to regulate mRNA translation via sequence-specific binding⁷. For example, miR-3682-3p affects the Wnt/β-catenin pathway by regulating FHL1 expression, leading to the progression of esophageal cancer⁸. miR-21-5p and miR-223-3p affect the course of eosinophilic esophagitis (EOE) by mediating the expression of downstream target genes⁹. miR-107 is a miRNA encoded on chromosome 10 in humans that has attracted significant research attention due to its implications in various tumorigenic processes^10,11,12. Current evidence indicates that miR-107 is a hotspot mediator in gastric cancer and esophageal cancer^13,14, but its pathophysiological functions and regulatory mechanisms in non-neoplastic digestive diseases need to be thoroughly investigated. However, appropriate in vitro models and integrated analytical approaches to study miR-107 function in RE remain limited.

Gastric acid reflux is widely recognized as the primary pathogenic factor responsible for esophageal mucosal injury in RE¹⁵. Recent studies propose that the pathogenic mechanism of RE involves not direct mucosal injury by gastric juices, but rather their stimulation of chemokine release from esophageal epithelial cells, initiating a cascade of inflammatory-mediated tissue damage¹⁶. To investigate the molecular mechanisms underlying this process, we treated Het-1A cells with hydrochloric acid to establish an in vitro acid injury model that replicates the key pathological features of epithelial damage caused by acid reflux. This approach provides a controlled and reproducible system to mimic acid-induced epithelial injury in vitro. We systematically explored the regulatory role of miR-107 in RE-associated inflammation and apoptosis, as well as its diagnostic potential. This protocol enables the investigation of miRNA-mediated regulation of inflammatory and apoptotic pathways in esophageal epithelial cells. Compared with traditional in vivo animal models and primary cell cultures, the HCl-induced Het-1A model combined with miR-107 functional assays offers exceptional stability, controllability, and reproducibility, making it particularly well-suited for studying acid-induced epithelial damage, miRNA-mediated post-transcriptional regulation, and inflammatory responses in esophageal cells¹⁷. This method is particularly suitable for studies investigating molecular mechanisms of acid-induced epithelial injury and miRNA-mediated regulation. When combined with clinical analyses of patients, this approach can be combined with clinical data to support mechanistic studies in RE.

方案

1. Patient recruitment

1. Conduct the study in accordance with the Declaration of Helsinki.
2. Obtain approval for the research protocol from the Ethics Committee of Jiaozhou Central Hospital of Qingdao (Approval Number: 2023033-01).
3. Ensure all participants provided signed informed consent prior to study initiation.
4. Identify and enroll 94 patients with reflux esophagitis (RE) from Jiaozhou Central Hospital of Qingdao.
5. Include 94 healthy volunteers as the control group.
6. Diagnose all RE patients via gastroscopy to confirm esophageal mucosal lesions and erosions. Group the patients according to the Los Angeles classification criteria¹⁸.
7. Included RE patients who have digestive system discomfort lasting over 3 months, no history of upper gastrointestinal surgery, and provided signed informed consent for this study.
8. Exclude patients with Barrett's esophagus, esophageal varices, esophageal stenosis, deep ulcers, or malignant tumor lesions via gastroscopy.

2. Serum samples collection

1. Collect 5 mL of fasting venous blood from each participant between 8:00 and 10:00 AM after an overnight fast of at least 8 hours.
2. Centrifuge the collected blood at 4 °C 3000 × g for 15 min.
3. Collect the supernatant carefully after centrifugation.
4. Store the separated serum at -80 °C in a freezer until subsequent measurement.
   NOTE: Samples can be stored at −80 °C for long-term storage and processed at a later time

3. Cell culture

1. Purchase human esophageal squamous cell lines Het-1A from a commercial cell bank. Quickly thaw cells in a 37 °C water bath within 1–2 min.
2. Resuscitate Het-1A cells and culture them in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.
3. Set the incubator parameters to 37°C and 5% CO₂ for cell culture maintenance.
4. Replace medium every 2 days and passage cells at 80–90% confluence using 0.25% trypsin‑EDTA.

4. Cell induction

1. Inoculate Het-1A cells into 6-well plates and adjust the cell density to 2×10⁵cells·well^-1 in 2 mL complete medium.
2. Dilute concentrated hydrochloric acid (HCl) with phosphate-buffered saline (PBS) and adjust the pH to 4.0.
   CAUTION: Hydrochloric acid is corrosive and irritating. Handle hydrochloric acid using appropriate personal protective equipment (including safety goggles, gloves, and a lab coat) and work in a chemical fume hood
3. Add 2 mL of the prepared HCl solution to each well to induce the cells. 
4. Discard HCl solution and wash cells twice with pre‑warmed PBS before assays at each time point.
5. Measure cell viability using an MTT assay at 0, 10, 20, and 30 min.

5. Real-time quantitative polymerase chain reaction (RT-qPCR)

1. Extract total RNA from Het-1A cells using a phenol-chloroform reagent.
2. Assess RNA purity and concentration using a NanoDrop spectrophotometer, confirming an A260/A280 ratio between 1.8 and 2.0.
3. Configure the reverse transcription amplification system using the reverse transcription kit.
4. Perform reverse transcription in a PCR machine at 37°C for 15 min, 85°C for 5 s, then hold at 4°C.
5. Generate circular DNA (cDNA) via reverse transcription.
6. Perform RT-qPCR reactions in a 20 µL reaction volume using the SYBR Green PCR master mix.
7. Set the PCR cycling conditions as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. 
8. Use glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and small nuclear RNA (U6) as internal controls for miR-107 and FGFRL1.
9. Calculate data using the 2-ΔΔCt method.
10. Quantify the mRNA content of inflammatory factors (IL-6, IL-8) and apoptosis‑related regulatory molecules (Fas, p53) by RT-qPCR.

6. Cell transfection

1. Culture Het-1A cells statically in 6-well plates at a density of 2×10⁵ cells·well-1 until they reach the logarithmic growth phase.
2. Obtain synthetic oligonucleotides (miR-107 mimic/ov-FGFRL1; 50 nM), downregulated plasmids (miR-107 inhibitor; 100 nM), and their corresponding negative control fragments from a commercial supplier.
3. Transfect the above-mentioned fragments into cells using lipofectamine reagent for 48 h.

7. Cell viability

1. Seed Het-1A cells into 96-well plates at a density of 5×10³ cells·well^-1  in 100 µL medium per well and culture overnight.
2. Add methyl thiazolyl tetrazolium (MTT) solution (20 µL, 5 mg·mL^-1, final concentration 0.5 mg·mL^-1) to the Het-1A cells and incubate for 4 h at 37 °C.
3. Add dimethyl sulfoxide (DMSO, 150 µL) and vortex the mixture for 10 min.
4. Measure the absorbance value (490 nm) of each well by a microplate reader.
5. Set 3 replicate wells per group and repeat 3 independent times.

8. ELISA assay

1. Detect IL-6 and IL-8 in the cells using ELISA kit following the manufacturer’s standard operating procedure.
2. Repeat the procedure three independent times.

9. Target prediction and luciferase activity assay

1. Predict potential targets of miR-107 using miRDB and ENCORI databases. Identify overlapping targets and verify complementary binding sites.
2. Identify complementary binding sites between miR-107 and FGFRL1.
3. Clone and obtain the wild-type FGFRL1 3'UTR containing miR-107 binding sites (wt-FGFRL1). Generate specific binding site mutants (mut-FGFRL1).
4. Co-transfect Het-1A cells with wt/mut-FGFRL1 and miR-107 mimic/inhibitor (or NC controls).
5. Measure luciferase activity using a dual-luciferase reporter assay system.

10. Statistical analysis

1. Perform ROC analysis using GraphPad Prism by inputting the expression data, selecting the ROC curve function, and exporting the results.
2. Assess intergroup differences by Student's t-test (for two groups) or one-way analysis of variance (ANOVA; for multiple groups) followed by Tukey’s post hoc test.
3. Consider p < 0.05 as statistically significant.
4. The detailed information of original data was summarized in supplementary Table 1.

结果

Patient recruitment and baseline characteristics These results describe the expression and diagnostic performance of miR-107 in RE patients. Table 1 records the general characteristics of healthy controls and RE patients. The results show no statistically significant differences between the two groups in terms of gender, age, and underlying medical history (p > 0.05), indicating comparability.

Serum miR-107 expression and diagnostic perform...

讨论

RE is an inflammatory disease of the esophageal mucosa caused by gastroesophageal reflux with a wide range of symptoms that may affect quality of life and carry the risk of developing serious complications¹⁹. Under normal physiological conditions, the stomach remains protected against gastric acid and bile due to its inherent defense mechanisms. In contrast, the esophagus, lacking a robust mucus barrier, is more susceptible to damage from these agents²⁰. Therefore, we are e...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
美国类型文化收藏	美国马纳萨斯		商业细胞银行
二甲基亚硫酸盐	Sigma，德国	D4540
双荧光素酶报告检测套件	Promega，美国	E1910	双荧光素酶报告测定系统
FastStart 通用 SYBR 绿色主套件	德国罗什	4913850	SYBR Green PCR主混合
胎儿牛血清	Gibco，纽约，美国	10100147C
基因制药公司	中国上海
氯化氢	美国密苏里州西格玛-奥尔德里奇	1099730001
IL-6 ELISA套件	R&D Systems，美国	D6050	ELISA套件
IL-8 ELISA套件	R&D Systems，美国	D8000C	ELISA套件
孵化器	Forma Scientific，美国马萨诸塞州	311 S/N29035
脂氧胺3000	Invitrogen，美国	L3000015	脂蛋白试剂
甲基噻唑基四唑	Sigma，德国	M5655
微板读器	BioTek，佛蒙特州，美国	ELx808
纳米滴光度计	美国马萨诸塞州热力学	ND-1000
青霉素/链霉素	Gibco，纽约，美国	15070063
PrimeScript 光线追踪套件	高原，日本	DRR047A	逆转录套件
TRIzol	美国马萨诸塞州热力学	10296010	苯酚-氯仿试剂
超低温冷冻柜及nbsp;	中国青岛海尔	DW-86W100