摘要
目的:探讨miR-377-5p与缺氧诱导因子-1α(hypoxia inducible factor-1,HIF-1α)的靶向关系以及通过控血管内皮生长因子(vascular endothelial growth factor, VEGF)信号通路对肝细胞癌(hepatocellular carcinoma,HCC)细胞增殖、侵袭和EMT的调控作用。方法:qPCR检测35例人HCC组织及癌旁组织标本中miR-377-5p的表达水平。将HepG2细胞分为对照组、mimic NC组、miR-377-5p mimic组,qPCR检测转染效率;EdU染色、Transwell和Western blotting(WB)检测miR-377-5p过表达对HepG2细胞增殖、侵袭及其增殖相关蛋白Ki-67、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)及上皮间质转化(epithelial-mesenchymal transition,EMT)标志蛋白E-cadherin、N-cadherin表达的影响;qPCR、WB检测miR-377-5p过表达对HepG2细胞中,缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)表达的影响。荧光素酶报告基因实验验证miR-377-5p与HIF-1α基因的靶向关系。结果:HCC组织中miR-377-5p表达水平较癌旁组织降低(P<0.01)。与对照组相比,miR-377-5p mimic组HepG2细胞中miR-377-5p水平明显升高,细胞的增殖、侵袭能力降低(均P<0.01),EMT、N-cadherin表达水平降低(均P<0.01)而E-cadherin表达水平显著升高(P<0.01);miR-377-5p mimic组中HIF-1αmRNA和蛋白表达水平均降低(P<0.01或P<0.05)。miR-377-5p靶向抑制HIF-1α基因表达,并抑制VEGF通路的激活(均P<0.05)。结论:miR-377-5p通过靶向抑制HIF-1α基因表达和下调VEGF信号通路从而抑制HepG2细胞的增殖、侵袭和EMT。
Objective:To explore the targeting relationship between miR-377-5p and hypoxia inducible factor-1(HIF-1α), and investigate the regulatory effect of miR-377-5p on proliferation, invasion and epithelial-mesenchymal transition(EMT) of hepatocellular carcinoma(HCC) cells through vascular endothelial growth factor(VEGF) signaling pathway. Methods:The expression of miR-377-5p in35 pairs of human HCC tissues and para-cancerous tissues was detected by qPCR. Then, HepG2 cells were divided into control group, mimic-NC group and miR-377-5p mimic group. q PCR was used to detect the transfection efficiency;the effects of miR-377-5p over-expression on proliferation and invasion of HepG2 cells were examined by EdU staining and Transwell assay, respectively;and the effect of miR-377-5p over-expression on the expressions of proliferation-related protein Ki-67, proliferating cell nuclear antigen(PCNA) and epithelial-mesenchymal transition(EMT) markers(E-cadherin and N-cadherin) were detected by Western blotting(WB);the effect of miR-377-5p over-expression on the expression of hypoxia inducible factor-1α(HIF-1α) in HepG2 cells was detected by qPCR and WB;and the targeting relationship between miR-377-5p and HIF-1α gene was determined by Luciferase reporter gene assay.Results: The expression of miR-377-5p in HCC tissues was significantly lower than that in para-cancerous tissues(P<0.01). Compared with the control group, the expression of miR-377-5p in HepG2 cells of miR-377-5p mimic group elevated significantly, and the proliferation, invasion and the expression of N-caderin proteins decreased, significantly(all P<0.01), while the expression of E-caderin increased significantly(P<0.01). At the same time, the mRNA and protein expressions of HIF-1α in miR-377-5p mimic group decreased significantly(P<0.01 or P<0.05). miR-377-5p targetedly inhibited the expression of HIF-1α gene and suppressed the activation of VEGF pathway(all P<0.05). Conclusion: miR-377-5p inhibits the proliferation, invasion and EMT of HepG2 cells via targeted
作者
杨晋
贺凯
张孟瑜
吴力乐
秦蜀
冯春红
罗鸣
夏先明
YANG Jin;HE Kai;ZHANG Mengyu;WU Lile;QIN Shu;FENG Chunhong;LUO Ming;XIA Xianming(Department of Hepatobiliary Surgery,the Affiliated Hospital of Southwest Medical University,Luzhou 646000,Sichuan,China)
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2020年第3期248-254,共7页
Chinese Journal of Cancer Biotherapy
基金
四川省卫生和计划生育委员会科研资助项目(No.16PJ149)。
