miR-192-5p通过靶向ZEB2调控胰腺癌PANC-1细胞的恶性生物学行为

miR-192-5p regulates the malignant biological behaviors of pancreatic cancer PANC-1 cells by targeting ZEB2

目的:探讨miR-192-5p靶向E盒锌指结合同源框2(ZEB2)对胰腺癌PANC-1细胞增殖、迁移、侵袭和上皮间质转化(EMT)的影响及其作用机制。方法:利用TCGA数据库数据分析miR-192-5p和ZEB2在胰腺癌组织中的表达及两者的相关性。采用qPCR法和WB法分别检测人正常胰腺上皮细胞HPNE和胰腺癌PANC-1细胞中miR-192-5p和ZEB2的表达水平。利用脂质体转染技术转染PANC-1细胞,实验分为miR-192-5p mimic组、Mimic NC组、miR-192-5p inhibitor组、Inhibitor NC组、Mimic NC+pcDNA3.1组、miR-192-5p mimic+pcDNA3.1组、miR-192-5p mimic+pcDNA3.1-ZEB2组。CCK-8法、克隆形成、划痕愈合、Transwell实验分别检测转染PANC-1细胞的增殖、克隆形成、迁移和侵袭能力。qPCR法、WB法、双重免疫荧光实验检测PANC-1细胞中ZEB2、E-cadherin、vimentin的表达水平。通过生物信息学网站预测miR-192-5p的靶基因,并利用双荧光素酶报告基因实验验证miR-192-5p对靶基因的调控作用。结果:胰腺癌组织中ZEB2的表达显著高于正常胰腺组织(P<0.01),且胰腺癌组织中miR-192-5p和ZEB2表达呈负相关(r=-0.419,P<0.01)。与HPNE细胞比较,PANC-1细胞中miR-192-5p低表达、ZEB2蛋白高表达(均P<0.01)。miR-192-5p过表达后,PANC-1细胞增殖、克隆形成、迁移和侵袭能力均显著降低,E-cadherin的mRNA和蛋白表达均上调、vimentin和ZEB2 mRNA和蛋白表达均下调;而抑制miR-192-5p后得到了相反的结果(P<0.05或P<0.01)。miR-192-5p靶向调控ZEB2的表达,过表达ZEB2可部分逆转上调miR-192-5p对PANC-1细胞增殖、迁移、侵袭和EMT进程的抑制作用。结论:miR-192-5p通过靶向ZEB2调控胰腺癌PANC-1细胞的恶性生物学行为。

Objective:To investigate the effect and mechanism of miR-192-5p targeting ZEB2(zinc finger E-box binding homeobox 2)on the proliferation,migration,invasion and EMT(epithelial-mesenchymal transition)of pancreatic cancer PANC-1 cells.Methods:The TCGA database was used to analyze the expression of miR-192-5p and ZEB2 as well as their correlation in pancreatic cancer tissue.The expression levels of miR-192-5p and ZEB2 were detected by qPCR and WB in human normal pancreatic epithelial HPNE cells and pancreatic cancer PANC-1 cells.PANC-1 cells were transfected with liposome,and divided into miR-192-5p mimic group,Mimic NC group,miR-192-5p inhibitor group,Inhibitor NC group,Mimic NC+pcDNA3.1 group,miR-192-5p mimic+pcDNA3.1 group,and miR-192-5p mimic+pcDNA3.1-ZEB2 group.The proliferation,colony formation,migration and invasion abilities of transfected PANC-1 cells were detected by CCK-8,colony formation,scratch healing and Transwell experiments,respectively.The expression of ZEB2,E-cadherin and vimentin was detected by qPCR,WB method and double immunofluorescence assay.Bioinformatics was used to predict the target gene of miR-192-5p,and the regulatory effect of miR-192-5p on the target gene was verified by dual luciferase report gene experiment.Results:The expression of ZEB2 in pancreatic cancer tissue was significantly higher than that in normal pancreatic tissue(P<0.01),and the expression of miR-192-5p and ZEB2 in pancreatic cancer tissue was inversely correlated(r=-0.419,P<0.01).Compared with normal pancreatic epithelial HPNE cells,the expression level of miR-192-5p was significantly lower while the expression level of ZEB2 protein was higher in pancreatic cancer PANC-1 cells(all P<0.01).Overexpression of miR-192-5p significantly reduced the proliferation,colony formation,migration and invasion ability of PANC-1 cells,upregulated the mRNA and protein expression of E-cadherin,and downregulated the mRNA and protein expression of vimentin and ZEB2,while inhibition of miR-192-5p achieved the opposite results(P<0.05 or P<0.01).miR-192-5p targetedly regulated the expression of ZEB2,and overexpression of ZEB2 could reverse the inhibitory effects of miR-192-5p overexpression on the proliferation,colony formation,migration,invasion and EMT of PANC-1 cells.Conclusion:miR-192-5p regulates the malignant biological behaviors of pancreatic cancer PANC-1 cells by targeting ZEB2.