过表达miR-607通过下调TRPC5表达抑制肝细胞癌的生长和转移

Overexpression of miR-607 inhibits hepatocellular carcinoma cell growth and metastasis by down-regulating TRPC5

目的研究miR-607异常表达在肝细胞癌(HCC)中的临床意义及对肝癌细胞生长转移的影响。方法荧光定量PCR检测45对HCC及癌旁组织中miR-607表达,分析miR-607表达与患者临床病理特征间的相关性。通过转染miR-607 mimics提高HCC细胞(Huh-7和HCCLM3)内miR-607的表达水平。采用CCK-8、流式细胞仪、划痕愈合实验及transwell小室模型分别检测过表达miR-607对HCC细胞增殖、凋亡、迁移和侵袭的影响。双荧光素酶报告基因系统检测miR-607是否与潜在靶点TRPC5的mRNA 3’-非翻译区直接结合。Western blot检测过表达miR-607对HCC细胞内TRPC5、CCND1、MMP2表达及Akt磷酸化的影响。结果MiR-607在HCC组织及HCC细胞中的表达水平均降低(P<0.05);miR-607低表达与肿瘤直径>5 cm、血管侵犯及较晚TNM分期(Ⅲ+Ⅳ)密切相关(P<0.05)。过表达miR-607抑制HCC细胞增殖、迁移、侵袭并诱导细胞凋亡(P<0.05)。双荧光素酶报告基因系统证实miR-607与TRPC5 m RNA 3’-非翻译区直接结合(P<0.05)。过表达miR-607抑制HCC细胞内TRPC5、CCND1及MMP2表达并下调Akt的磷酸化水平(P<0.05)。结论miR-607低表达与肝细胞癌恶性临床特征密切相关,miR-607可能通过下调TRPC5表达进而抑制Akt通路激活来发挥抗HCC生长转移作用。

Objective To investigate the clinical implications of abnormal expression of miR-607 in hepatocellular carcinoma(HCC) and its influence on HCC cell proliferation and migration.Methods The expression of miR-607 in 45 pairs of HCC and adjacent tissues were detected with real-time PCR,and the correlation between miR-607 expression and clinicopathological features of the patients was analyzed.The effects of transfection with miR-607 mimics on proliferation,apoptosis,migration and invasion of two HCC cell lines (Huh-7 and HCCLM3) were evaluated using CCK-8 assay,flow cytometry,wound-healing assay and Transwell assay.A dual-luciferase reporter system was used to detect the direct binding between miR-607 and 3’-UTR of TRPC5 mRNA.Western blotting was used to measure the expressions of TRPC5,CCND1,MMP2 and phosphorylated Akt in the HCC cells.Results The expression of miR-607 was significantly decreased in HCC tissues (P=0.029) and HCC cell lines (P<0.05).In HCC patients,a low expression of miR-607 was associated with a larger tumor size (>5 cm,P=0.031),vascular invasion (P=0.027) and advanced TNM stages (Ⅲ+Ⅳ,P=0.015).In the two HCC cell line,overexpression of miR-607significantly inhibited cell proliferation,migration,and invasion and enhanced cell apoptosis (P<0.05).The results of dualluciferase reporter assay confirmed that TRPC5 was a direct target of miR-607 in HCC cells.Overexpression of miR-607significantly inhibited the expressions of TRPC5,CCND1,and MMP2 and suppressed Akt phosphorylation in HCC cells(P<0.05).Conclusion A low expression of miR-607 in HCC is associated with poor clinicopathological phenotypes of HCC.Overexpression of miR-607 inhibits HCC growth and metastasis possibly by down-regulating TRPC5,which causes Akt signaling inactivation.