miR-597在肝细胞性肝癌中表达及其对癌细胞增殖、迁移和侵袭的影响与机制

Expression of miR-597 in hepatocellular carcinoma and its effects on proliferation,migration,and invasion of cancer cells

目的观察肝细胞性肝癌(HCC)组织及细胞系中mi R-597的表达情况,以及上调mi R-597表达对HCC癌细胞增殖、迁移和侵袭能力的影响,并探讨其作用机制。方法选择20例HCC患者,其中男性12例,女性8例;年龄21~69岁,中位年龄46岁;临床分期T1N0M0期10例,T2N0M0期3例,T3N0M0期3例,T4N0M0期4例;14例有乙型肝炎病史,1例有乙型肝炎、丙型肝炎重叠感染史,5例无肝炎病史。取癌组织及对应的癌旁组织,采用实时荧光定量聚合酶链反应(RT-qPCR)检测其mi R-597 mRNA表达水平。采用RT-qPCR检测不同肝癌细胞系及正常肝上皮细胞中mi R-597mRNA表达水平。将体外传代培养人HCC细胞系Hep3B,分为对照组和mi R-597过表达组,分别转染对照(mi R-NC)及mi R-597模拟物(mi R-597 mimics),用RT-qPCR检测细胞中mi R-597 mRNA表达水平;用MTT实验检测Hep3B细胞的增殖能力;划痕实验和Transwell实验检测Hep3B细胞的迁移和侵袭能力;荧光素酶报告实验确定FOSL2是否是mi R-597的靶基因;通过Western blot实验检测Hep3B细胞FOSL2蛋白的表达水平。采用RT-qPCR检测癌组织及对应的癌旁组织中FOSL2 mRNA表达水平,分析mi R-597与FOSL2表达的相关性。结果HCC组织中mi R-597 mRNA表达水平明显低于癌旁组织(P<0.01)。HCC细胞系中mi R-597 mRNA表达水平明显低于肝正常上皮细胞(P<0.05)。mi R-597过表达组miR-597 mRNA表达水平显著高于对照组(P<0.05),且细胞增殖、迁移和侵袭能力均显著低于对照组(P<0.05)。mi R-597直接靶定FOSL2的3′非翻译区(3′-UTR),同时mi R-597过表达组FOSL2蛋白的表达水平相对于对照组明显降低(P<0.05),HCC组织中FOSL2 mRNA表达水平明显高于癌旁组织(P<0.01),且FOSL2与mi R-597的表达呈显著负相关(r=-0.784,P<0.001)。结论mi R-597在HCC组织中低表达而FOSL2高表达,上调mi R-597的表达能够抑制HCC细胞的增殖、迁移和侵袭,其机制可能与下调FOSL2蛋白的表达有关。

Abastract:Objective To observe the expression of mi R-597 in hepatocellular carcinoma(HCC)tissues,and investigate the effect of up-regulating mi R-597 expression on HCC cells proliferation,migration and invasion ability,and explore its mecha-nism.Methods A total of 20 patients with HCC were enrollted,which included 12 males and 8 females,aged 21-69 years old with median age of 46 years old.There were 10 patients at clinical stage T1 N0 M0,3 at stage T2 N0 M0,3 at stage T3 N0 M0 and 4 at stage T4 N0 M0;14 cases with history of hepatitis B,1 case with history of overlapping infection of hepatitis B and hepatitis C,and 5 without history of hepatitis.The tumor tissues and corresponding adjacent tissues were taken,and the expression level of mi R-597 m RNA was detected by real-time quantitative polymerase chain reaction(RT-q PCR).The levels of mi R-597 m RNA in different HCC cell lines and normal liver epithelial cells was detected by RT-q PCR.The in vitro subcultured human HCC cell line Hep3 B was divided into control group(transfected control mi R-NC)and mi R-597 overexpression group(transfected mi R-597 mimics),and detected by RT-q PCR the expression level of mi R-597 m RNA in cells.The MTT ex-periment was used to detect proliferation ability of Hep3 B cells.The scratch test and Transwell test was used to detect migra-tion and invasion ability of Hep3 B cells.The luciferase reporter experiment was used to test whether FOSL2 was target gene ofmi R-597;Western blot experiment was performed to detect expression level of FOSL2 protein in Hep3 B cell.The RT-q PCR was used to detect the expression level of FOSL2 m RNA in carcinoma tissues and corresponding adjacent tissues,and analyze the correlation between mi R-597 and FOSL2 expression.Results The expression level of mi R-597 m RNA in HCC tissues was significantly lower than that in adjacent tissues(P<0.01).The expression level of mi R-597 m RNA in HCC cell lines was significantly lower than that of normal liver epithelial cells(P<0.05).The expression level of mi R-597 m RNA in mi R-597 overexpression group was significantly higher than that in control group(P<0.05),and cell proliferation,migration and invasion abilities were significantly lower than those in control group(P<0.05).Test results showed that mi R-597 directly targeted 3′-UTR of FOSL2,and protein expression level of FOSL2 in mi R-597 overexpression group was significantly lower than that in control group(P<0.05).The expression level of FOSL2 m RNA in HCC tissues was significantly higher than that in adjacent tissues(P<0.01),and the expression of FOSL2 and mi R-597 were significantly negatively correlated(r=-0.784,P<0.001).Conclusion It is demonstrated that mi R-597 shows low expression in HCC tissues while FOSL2 shows high expression,upregulating expression of mi R-597 can inhibit proliferation,migration and invasion of HCC cells,of which mechanism may be related to down-regulation of FOSL2 protein expression.