The mechanism of microRNA-124-3p in regulating proliferation and invasion of gastric cancer cell by aryl hydrocarbon receptor-activated β-catenin pathway / 中华消化杂志

ABSTRACT

Objective:To explore the value of microRNA(miR)-124-3p and its target gene aryl hydrocarbon receptor (AHR) in the diagnosis and prognostic evaluation of gastric cancer, and the related molecular mechanisms in regulating proliferation and invasion of gastric cancer cell.Methods:The clinical and prognostic characteristics of patients with gastric adenocarcinoma expressing miR-124-3p were obtained from The Cancer Genome Atlas database and Genotype-Tissue Expression database. The correlation between miR-124-3p expression level and pathological stage, TNM stage, overall survival (OS), disease-specific survival (DSS) and progression-free interval (PFI) in patients with gastric adenocarcinoma were studied by bioinformatics analysis. The interaction sites between miR-124-3p and AHR mRNA were predicted by Target Scan 7.1 online tool. The target binding sites of miR-124-3p in AHR mRNA were verified by subcutaneous tumorigenesis experiment in mice, immunohistochemistry, dual luciferase assay, quantitative real time-polymerase chain reaction (RT-qPCR) and Western blotting. Nine male Balb/c nude mice, aged 4 to 6 weeks with weight of (18.43±0.29) g were injected with miR-124-3p simulant (miR-124-3p group), negative control simulant (negative control group) and 0.9% sodium chloride solution (sodium chloride control group) through the tail vein. Gastric cancer cell lines (MKN-45, AGS) were transfected with RNA simulants (including miR-124-3p simulant, negative control simulant and 0.9% sodium chloride solution). The expression of AHR and Catenin β 1 gene ( CTNNB1) at mRNA level, the expression of AHR and β-catenin at protein level in 3 mice groups and the effects of miR-124-3p transfection on the proliferation and invasion of transfected gastric cancer cells were analyzed. Pearson correlation analysis and Holm-Sidak corrected multiple t test were used for statistical analysis. Results:Low expression of miR-124-3p was positively correlated with severe pathological stages and TNM stages in patients with gastric adenocarcinoma ( R2=0.83 and 0.86, P=0.031 and 0.023). High expression of miR-124-3p was positively correlated with OS, DSS and PFI ( R2=1.00, 0.99 and 0.99, P=0.029, 0.044 and 0.049). The results of subcutaneous tumorigenesis experiment in mice demonstrated that the number of apoptotic cells in the tumor of miR-124-3p group was more than that of negative control group and sodium chloride control group ((43.33±1.86)/high power field (HPF) vs. (20.00±1.73)/HPF and (18.67±1.76)/HPF), and the differences were statistically significant ( t=8.55 and 8.33, P=0.013 and 0.014). The results of immunohistochemistry showed that the optical density of AHR protein in mice tumor tissue of miR-124-3p group was lower than that of negative control group and sodium chloride control group (0.081±0.008 vs. 0.276±0.019 and 0.273±0.018), and the differences were statistically significant ( t=9.06 and 7.51, P=0.012 and 0.017). The results of dual luciferase assay indicated that the fluorescence intensity in wild-type AHR MKN-45 cells transfected with miR-124-3p simulant was lower than that of negative control group (0.293±0.020 vs. 1.000±0.032), and the difference was statistically significant ( t=18.56, P<0.001). The results of RT-qPCR demonstrated that the mRNA levels of AHR and CTNNB1 in MKN-45 cells transfected with miR-124-3p simulant were both lower than those in untreated MKN-45 cells (0.51±0.09 vs. 1.02±0.02, 0.46±0.03 vs. 1.03±0.01), and the differences were statistically significant ( t=4.51 and 16.60, P=0.046 and 0.004). The results of Western blotting experiments showed that the relative protein expression levels of AHR and β-catenin of MKN-45 cells transfected with miR-124-3p simulant were lower than those of transfected with 0.9% sodium chloride solution and negative control simulant (3 332.94±81.25 vs. 9 041.60±439.79 and 8 276.54±562.52, 2 725.79±167.57 vs. 9 701.94±410.02 and 8 081.66±275.84), and the differences were statistically significant ( t=15.49, 7.91, 17.35 and 19.42, P=0.004, 0.016, 0.003 and 0.003). Conclusions:MiR-124-3p is correlated with diagnosis and prognosis of gastric cancer. MiR-124-3p induces apoptosis of gastric cancer cells in vitro and vivo by negatively regulating AHR expression at mRNA and protein level, thereby down-regulating the expression of CTNNB1 mRNA and β-catenin pathway-related protein. Therefore, miR-124-3p may become a potential diagnostic and prognostic marker of gastric cancer.