胃癌上皮-间充质转化过程中微小RNA-579-3p/含锌指和BTB域4通路的作用及其机制

Role and mechanism of microRNA-579-3p/zinc finger-containing and BTB domain-containing 4 pathway in epithelial-mesenchymal transition of gastric cancer

目的探讨微小RNA-579-3p(miR-579-3p)/含锌指和BTB域4(ZBTB4)通路在胃癌上皮-间充质转化(EMT)中的作用机制。方法生物信息学筛选胃癌与癌旁组织差异表达的微小RNA(miRNA);实时荧光定量聚合酶链反应(RT-qPCR)法检测50例患者胃癌和癌旁癌旁组织中miR-579-3p、ZBTB4表达;检测胃上皮细胞株GES-1和胃癌细胞株HGC27、AGS、MKN45中miR-579-3p的表达, 分析miR-579-3p与临床病理特征的关系。生物信息学分析miR-579-3p靶基因。双荧光素酶报告基因验证miR-579-3p与ZBTB4的调控关系。应用miR-579-3p抑制物(inhibitor)干扰HGC27细胞株miR-579-3p表达, 细胞计数试剂盒(CCK-8)法、划痕实验、Transwell小室实验检测细胞增殖、迁移和侵袭能力;RT-qPCR和蛋白质印迹法(Western blot)检测mRNA和蛋白表达水平。采用t检验、χ^(2)检验分析数据。结果生物信息学筛选后RT-qPCR检测结果发现miR-579-3p在胃癌组织中表达显著高于癌旁组织(3.259±1.307比1.326±0.419, t=9.959, P<0.01), miR-579-3p表达与胃癌分化和淋巴结转移明显相关(χ^(2)=4.500、7.714, P<0.05);miR-579-3p在胃癌细胞株表达均高于GES-1(P<0.01)。转染miR-579-3p抑制剂的HGC-27细胞活性(0.500±0.068比1.223±0.089, t=15.780, P<0.01)、迁移率(0.228±0.039比0.533±0.056, t=7.707, P<0.01)和侵袭细胞数[(89.000±7.550)个比(206.700±8.505)个, t=17.92, P<0.01]显著低于对照组。RT-qPCR结果显示miR-579-3p inhibitors组波形蛋白(Vimentin)(0.248±0.030比1.061±0.150, t=9.258, P<0.01)、基质金属蛋白酶-2(MMP-2)(0.178±0.030比1.013±0.195, t=3.320, P<0.01)和连接蛋白细胞黏附分子(Nectin-4)(0.465±0.065比1.042±0.154, t=5.990, P<0.01)的mRNA表达明显低于对照组, 而E-钙黏蛋白(E-cadherin)mRNA(3.249±0.460比1.000±0.175, t=7.990, P<0.01)的表达明显高于对照组, Western blot检测显示抑制miR-579-3p表达后Vimentin(0.420±0.271比1.004±0.095, t=3.528, P<0.05)、MMP-2(0.663±0.119比0.922±0.068, t=3.283, P<0.05)、Nectin-4表达(0.660±0.130比0.919±0.080, t=2.982, P<0.05)低于对照组, 而E-cadherin蛋白表达(1.352±0.113比0.994±0.005, t=5.483, P<0.01)高于对照组。生信分析结果显示ZBTB4是miR-579-3p靶基因;双荧光素酶报告基因分析表明过表达miR-579-3p后野生组中ZBTB4基因活性明显低于对照组(0.323±0.020比1.095±0.131, t=11.690, P<0.01)。RT-qPCR显示ZBTB4在胃癌组织中表达低于癌旁组织(t=7.713, P<0.01);皮尔逊相关分析结果显示ZBTB4与miR-579-3p呈负相关(r=-0.470, P<0.01)。RT-qPCR和Western blot显示抑制miR-579-3p后ZBTB4表达明显增强(P<0.01)。结论 miR-579-3p/ZBTB4通路激活促进胃癌的EMT。

Objective To investigate the role of microRNA-579-3p(miR-579-3p)/zinc finger-containing 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 clinicopathological 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 expression 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(χ^(2)=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 analysis 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 significantly 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 tissues(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.