MicroRNA-340在肝细胞肝癌中抑制细胞增殖并促进凋亡

目的 探讨微RNA-340(miR-340)在肝细胞肝癌中的表达特点及其对细胞生物学行为的影响.方法 收集重庆医科大学附属第一医院肝胆外科2015年3月至2016年9月收治的40例肝细胞肝癌患者手术后切除的癌组织和癌旁组织标本.通过qPCR检测组织标本中miR-340的表达,并分析miR-340表达与临床病理学指标的关系.分别培养肝癌细胞株Hep3B、Bel-7402、HepG2和SMMC-7721以及正常肝细胞株HL-7702,48 h后使用qPCR检测5种细胞中miR-340的表达水平.通过转染增加或抑制SMMC-7721细胞中miR-340的表达,然后分别于细胞培养24、48、72 h后采用CCK-8法检测SMMC-7721细胞增殖,流式细胞术检测细胞凋亡.通过生物信息学软件预测miR-340的靶基因,并使用qPCR和蛋白质印迹法进一步验证miR-340对靶基因的作用.结果 癌组织中miR-340的表达低于癌旁组织(P＜0.01),并且miR-340的表达与乙肝表面抗原、HBV DNA载量、肿瘤大小以及临床TNM分期有关(P＜0.01).正常肝细胞HL-7702内miR-340的表达高于4种肝癌细胞Hep3B、Bel-7402、HepG2和SMMC-7721(P＜0.05,P＜0.01).增加miR-340表达可抑制SMMC-7721细胞的增殖(P＜0.05,P＜0.01),而抑制miR-340的表达则促进SMMC-7721细胞的增殖(P＜0.05,P＜0.01);增加miR-340的表达可促进SMMC-7721细胞的凋亡(P＜0.01),而抑制miR-340则减少凋亡(P＜0.01).生物信息学分析显示S期激酶相关蛋白2(SKP2)基因是miR-340下游的一个靶基因.qPCR和蛋白质印迹分析结果显示增加SMMC-7721细胞中miR-340的表达可抑制SKP2的mRNA和蛋白表达,抑制miR-340表达则增加SKP2的mRNA和蛋白表达.结论 miR-340的异常表达可能与乙肝病毒的感染有关,其异常表达有助于评价病情以及预后.在肝癌细胞系SMMC-7721细胞中,miR-340能够抑制细胞增殖并促进凋亡,这一作用结果可能是通过miR-340对SKP2的抑制而实现的.

MicroRNA-340 suppresses cell proliferation and induces cell apoptosis in hepatocellular carcinoma

Objective To investigate the expression of microRNA-340 (miR-340) in hepatocellular carcinoma (HCC) and its effect on cell biological behavior. Methods We collected 40 frozen HCC tissues and adjacent non-tumor tissues from patients undergoing hepatectomy of HCC at The First Affiliated Hospital of Chongqing Medical University from Mar. 2015 to Sep. 2016. The expression of miR-340 in all tissues was detected by qPCR and the relationship between miR-340 expression and clinicopathological parameters was analyzed. Simultaneously, the expression of miR-340 in normal hepatocyte (HL-7702) and four hepatoma cells lines (Hep3B, Bel-7402, HepG2, SMMC-7721) was detected by qPCR after incubation for 48 h. The eukaryotic expression vector with miR-340 or control reagent was transfected into SMMC-7721 cells using EndoFection^TM-Max to increase or inhibit the expression of miR-340, and then the cells were cultured for 24 h, 48 h and 72 h. The proliferation of SMMC-7721 cells was detected by CCK-8 assay, and the apoptosis was detected by flow cytometry. The target gene of miR-340 was predicted by bioinformatics software, and the effect of miR-340 on target gene was further verified by qPCR and Western blotting. Results The expression of miR-340 in HCC tissues was significantly lower than that in the adjacent non-tumor tissues (P<0.01), and was correlated with hepatitis B surface antigen, HBV DNA, tumor size and TNM stage (all P<0.01). Besides, the expression of miR-340 in HL-7702 cells was significantly higher than that in Hep3B, Bel-7402, HepG2 and SMMC-7721 cells (P<0.05, P<0.01). CCK-8 assay results showed that overexpression of miR-340 inhibited proliferation of SMMC-7721 cells, while inhibition of miR-340 promoted cell proliferation (P<0.05, P<0.01). Overexpression of miR-340 significantly promoted SMMC-7721 cells apoptosis, while suppression of miR-340 significantly inhibited cells apoptosis (all P<0.01). S-phase kinase-associated protein 2 (SKP2) was a target gene of miR-340 as indicated by bioinformatics software. Further, qPCR and Western blotting results showed that overexpression of miR-340 inhibited the mRNA and protein expression of SKP2, while inhibition of miR-340 increased the mRNA and protein expression of SKP2. Conclusion The abnormal expression of miR-340 may be associated with the HBV infection, and miR-340 may be an indicator to evaluate the progression and prognosis of HCC. MiR-340 can inhibit proliferation and promote apoptosis of SMMC-7721 cells, which may be effected by inhibiting the SKP2 expression.