microRNA-372靶向SDC1抑制人胰腺癌上皮间质转化及侵袭

Effects of MicroRNA-372 and Its Target Gene SDC1 on Epithelial Mesenchymal Transition,Migration and Invasion in Human Pancreatic Cancer Cells

目的探讨microRNA-372 (miR-372)靶向调控SDC1基因表达抑制人胰腺癌上皮间质转化及侵袭的机制研究。方法收集人胰腺癌及癌旁组织手术标本;qRT-PCR和免疫组化法检测组织中miR-372和SDC1表达;筛选miR-372表达水平最高的人胰腺癌细胞系进行分组和转染,实验分成6组,即空白对照组、阴性对照组、miR-372 mimic组、miR-372 inhibitor组、siRNA-SDC1组和miR-372 inhibitor+siRNA-SDC1组;双荧光素酶报告基因试验验证miR-372和SDC1基因的靶向关系;CCK8法、流式细胞术、划痕试验及Transwell试验分别检测各组细胞转染后增殖能力、凋亡情况、迁移及侵袭能力;qRT-PCR和Western blot法检测各组细胞转染后SDC1和上皮细胞标志物E-cadherin及间质表型细胞标志物Vimentin的表达水平。结果SDC1蛋白在胰腺癌组织中的阳性表达率明显高于癌旁正常组织(P <0. 05)。胰腺癌中miR-372、SDC1表达与肿瘤分化程度、转移和TNM分期显著相关(P <0. 05)。双荧光素酶报告基因试验结果显示SDC1是miR-372的靶基因。与空白对照组和阴性对照组比较,miR-372 mimic组和siRNA-SDC1组细胞的生长、迁移和侵袭受到抑制,凋亡率增加,SDC1和Vimentin的表达水平明显降低,E-cadherin的表达水平明显升高(P均<0. 05);而miR-372 inhibitor组的趋势与miR-372 mimic组和siRNASDC1相反(P均<0. 05);然而,miR-372 inhibitor+siRNA-SDC1组与空白对照组和阴性对照组比较,各项指标差异均无统计学意义。结论 miR-372可能通过靶向抑制SDC1基因表达,抑制人胰腺癌细胞的迁移、侵袭和上皮间质转化。

Objective To explore the effects of microRNA-372(miR-372)and its target gene SDC1 on epithelial mesenchymal transition(EMT),migration and invasion in human pancreatic cancer(PC)cells.Methods Human PCtissues and theirparacancerous tissues were collected from PC patients.SDC1 expression in tissues was detected by qRT-PCR and immunohistochemistry.Human PC cell line with the highest expressions of miR-372 expression were selected,and then assigned into the blank,negative control(NC),miR-372 mimic,miR-372 inhibitor,siRNA-SDC1 and miR-372 inhibitor+siRNA-SDC1 groups.Double luciferase reporter gene assay was used to verify the targeting relationship between miR-372 and SDC1.The abilities of cell proliferation,apoptosis,migration and invasion were detected by CCK-8 assay,flow cytometry,scratch test and Transwell assay,respectively.Expression levels of SDC1 and EMT-related genes(E-cadherin and vimentin)after transfection were detected by qRT-PCR and Western blot.Results The positive expression rate of SDC1 protein in PC tissue was significantly higher than that in paracancerous tissues(P<0.05).The expressions of miR-372 and SDC1 in PC tissues were significantly related to tumor differentiation,metastasis and TNM staging(P all<0.05).Double luciferase reporter gene assay confirmed that SDC1 was the target gene of miR-372.Compared with the blank group and the NC group,the miR-372 mimic group and the siRNA-SDC1 group showed inhibited cell growth,migration and invasion,increased cell apoptosis,decreased expressions of SDC1 and vimentin,and increased expression of E-cadherin(P all<0.05).The miR-372 inhibitor group presented the opposite tendencies.However,there were no significant differences between the miR-372 inhibitor+siRNA-SDC1 group in comparison to the blank group and the NC group(P all>0.05).Conclusion Our study suggests thatmiR-372 could inhibit EMT,migration and invasion in human PC cells by inhibiting the expression of SDC1.