Role and mechanism of microRNA-579-3p/zinc finger-containing and BTB domain-containing 4 pathway in epithelial-mesenchymal transition of gastric cancer
Objective To investigate the role of microRNA-579-3p(miR-579-3p)/zinc finger-con-taining and BTB domain-containing 4(ZBTB4)pathway in epithelial-mesenchymal transition(EMT)of gastric cancer.Methods Differentially expressed miRNAs between gastric cancer and adjacent tissues were screened by bioinformatics.Real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of miR-579-3p and ZBTB4 in gastric cancer tissues and adjacent tissues of 50 patients.The expression of miR-579-3p in gastric epithelial cell line(GES-1)and gastric cancer cell lines(HGC27,AGS and MKN45)was detected,and the relationship between miR-579-3p and clinico-pathological features was analyzed.Bioinformatics analysis was used to analyze the target gene of miR-579-3p.Dual luciferase reporter gene was used to verify the regulatory relationship between miR-579-3p and ZBTB4.miR-579-3p inhibitor was used to interfere the expression of miR-579-3p in HGC27 cells.The cell counting kit-8(CCK-8)assay,scratch test and Transwell chamber test were used to detect cell proliferation,migration and invasion.RT-qPCR and Western blotting were used to detect the ex-pression levels of Vimentin,matrix metalloproteinase(MMP)-2,Nectin-4 and E-cadherin in the cells.T test and chi-square test were used to analyze the data.P＜0.05 was considered statistically significant.Results After bioinformatics screening,RT-qPCR showed that the expression of miR-579-3p in gastric cancer tissues was significantly higher than that in adjacent tissues(3.259±1.307 vs.1.326±0.419,t=9.959,P＜0.01).The expression of miR-579-3p was correlated with gastric cancer differentiation and lymph node metastasis(X2=4.500,7.714,P＜0.05).The expression of miR-579-3p in gastric cancer cell lines was higher than that in GES-1 cell lines(P＜0.01).HGC-27 cells transfected with miR-579-3p inhibitors significantly decreased their activity(0.500±0.068 vs.1.223±0.089,t=15.780,P＜0.01),migration rate(0.228±0.039 vs.0.533±0.056,t=7.707,P＜0.01)and number of invasive cells[(89.000±7.550)cells vs.(206.700±8.505)cells,t=17.920,P＜0.01].RT-qPCR showed that the mRNA expression of Vimentin(0.248±0.030 vs.1.061±0.150,t=9.258,P＜0.01),MMP-2(0.178±0.030 vs.1.013±0.195,t=3.320,P＜0.01)and Nectin-4(0.465±0.065 vs.1.042± 0.154,t=5.990,P＜0.01)in the miR-579-3p inhibitors group was significantly decreased as compared with the control group,while the mRNA expression of E-cadherin(3.249±0.460 vs.1.000±0.175,t=7.990,P＜0.01)was significantly increased.Western blotting showed that the expression of Vimentin(0.420±0.271 vs.1.004±0.095,t=3.528,P＜0.05),MMP-2(0.663±0.119 vs.0.922±0.068,t=3.283,P＜0.05)and Nectin-4(0.660±0.130 vs.0.919±0.080,t=2.982,P＜0.05)decreased,while the expression of E-cadherin protein increased(1.352±0.113 vs.0.994±0.005,t=5.483,P＜0.01)as compared with the control group after inhibiting the expression of miR-579-3p.Bioinformatics a-nalysis showed that ZBTB4 was the target gene of miR-579-3p.Dual luciferase reporter gene analysis showed that the activity of ZBTB4 gene in the wild group after overexpression of miR-579-3p was signifi-cantly lower than that in the control group(0.323±0.020 vs.1.095±0.131,t=11.690,P＜0.01).RT-qPCR showed that the expression of ZBTB4 in gastric cancer tissues was lower than that in adjacent tis-sues(t=7.713,P＜0.01).Pearson correlation analysis showed that ZBTB4 was negatively correlated with miR-579-3p(r=-0.470,P＜0.01).RT-qPCR and Western blotting showed that ZBTB4 expression was significantly enhanced after inhibiting miR-579-3p(P＜0.01).Conclusion Activation of miR-579-3p/ZBTB4 pathway promotes EMT in gastric cancer.
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