miR-139-5p通过靶向Notch1抑制上皮性卵巢癌细胞的增殖与侵袭

miR-139-5p inhibits proliferation and invasion of epithelial ovarian cancer cells by targeting Notch1

目的:探讨miR-139-5p靶向Notch1抑制上皮性卵巢癌(epithelial ovarian cancer,EOC)细胞增殖和侵袭的作用机制。方法:选取2018年1月至2018年12月在河南省南阳市中心医院妇科手术切除的24例EOC患者的癌和相应的癌旁组织标本,以及人卵巢癌细胞系SKOV3、ES2、HEY-T30和人卵巢上皮细胞株IOSE80,用qPCR检测EOC组织和细胞系中miR-139-5p和Notch1 mRNA的表达。将过表达miR-139-5p载体、重组质粒pLV-Notch1转染至SKOV3细胞,并设置空白对照组(Ctrl组)和阴性对照组(NC组),用双荧光素酶报告基因实验验证miR-139-5p与Notch13’-UTR靶向关系,用CCK-8、Transwell、划痕愈合实验分别检测细胞的增殖、侵袭和迁移能力,用Western blotting检测细胞中增殖和迁移相关蛋白的表达。结果:与癌旁组织和IOSE80细胞比较,EOC组织和细胞系中miR-139-5p表达显著降低、Notch1 m RNA表达显著升高(均P<0.01)。双荧光素酶报告基因实验结果证实,Notch1是miR-139-5p的靶基因。与NC组比较,miR-139-5p mimic组3 d时SKOV3细胞的增殖、侵袭、迁移能力和Notch1、NICD、Cyclin D1、Cyclin A1、Snail1、β-catenin及N-cadherin表达水平均明显降低(均P<0.01),E-cadherin表达水平明显升高(P<0.01);同时过表达Notch1可逆转miR-139-5p抑制SKOV3细胞增殖、侵袭与迁移的作用。结论:miR-139-5p可靶向Notch1抑制EOC细胞的增殖、侵袭和迁移能力,可能与其下调NICD、Cyclin D1、Cyclin A1、Snail1、β-catenin、N-cadherin而上调E-cadherin的表达水平有关。

Objective:To explore the action mechanism of miR-139-5 p inhibiting proliferation and invasion of epithelial ovarian cancer(EOC)cells by targetedly regulating Notch1.Methods:A total of 24 pairs of EOC tissues and its corresponding para-cancerous tissues from patients,who underwent surgical resection in the Department of Gynecology,Nanyang Central Hospital of Henan Province,were collected for this study;in addition,human ovarian cancer cell lines(SKOV3,ES2,HEY-T30)and human ovarian epithelial cell line IOSE80 were also collected.Real-time quantitative PCR(qPCR)was applied to detect mRNA expression of miR-139-5 p and Notch1 in EOC tissues and cell lines.The miR-139-5 p over-expression vector and recombinant plasmid pLV-Notch1 were transfected into SKOV3 cells.Blank control group(Ctrl group)and negative control group(NC group)were set up.Dual luciferase reporter gene assay was applied to verify the targeting relationship between miR-139-5 p and Notch13’-UTR.CCK-8,Transwell and Scratch healing experiments were applied to detect cell proliferation invasion and migration,respectively.Western blotting was applied to detect expressions of proliferation and migration related proteins in cells.Results:Compared with para-cancerous tissues and IOSE80 cells,the expression of miR-139-5 p was significantly decreased in EOC tissues and cell lines,while the expression of Notch1 mRNA was significantly increased(all P<0.01).The results of Dual luciferase reporter showed that Notch1 was the downstream target gene of miR-139-5 p.Compared with NC group,cell proliferation,invasion and migration ability,expression levels of Notch1,NICD,Cyclin D1,Cyclin A1,Snail1,β-catenin and N-cadherin were all significantly decreased on 3 d in miR-139-5 p mimic group(all P<0.01),while expression of E-cadherin was significantly increased(P<0.01);meanwhile,over-expression of Notch1 could reverse the inhibitory effect of miR-139-5 p on proliferation,invasion and migration of SKOV3 cells.Conclusion:miR-139-5 p can targetedly regulate Notch1 to inhibit proliferation,invasion and migration of EOC cells,which may be related to its down-regulation of NICD,Cyclin D1,Cyclin A1,Snail1,β-catenin and N-cadherin,and up-regulation of E-cadherin.