MiR-29a通过靶向调控CLDN1抑制肝癌细胞的增殖及侵袭功能

Effect of MiR-29a targeted-regulating CLDN1 expression on the proliferation and invasion of hepatocellular carcinoma cells

[目的]探索miR-29a在肝癌组织与正常肝组织中的基因表达水平差异,并进一步探讨其对人肝癌细胞增殖及转移能力的影响及相关作用机制。[方法]通过实时荧光定量PCR测定肝癌细胞及正常肝细胞中miR-29a的基因表达水平;将分别携带miR-29a的过表达miR-29a mimic、空载体miR-NC以及抑制体anti-miR-29a的脂质体转染入肝癌细胞系HepG2中,并采用实时荧光定量PCR验证转染后的HepG2细胞中miR-29a的基因表达。MTT及Transwell试验观察转染后的HepG2细胞的增殖和侵袭能力的改变;采用生物学信息软件预测miR-29a靶基因,并采用实时荧光定量PCR与Western blot在基因与蛋白水平进行验证。[结果]与正常肝细胞比较,miR-29a在肝癌细胞中基因表达下降(P<0.05);通过脂质体转染后,较miR-NC组比较miR-29a在miR-29a mimic中miR-29a基因显著升高(P<0.01),anti-miR-29a组中miR-29a基因显著降低(P<0.05);MTT和Transwell试验提示较miR-NC组比较miR-29a mimic组的肝癌细胞增殖能力和侵袭能力均减弱(P<0.05);anti-miR-29a组肝癌细胞的增殖和侵袭能力增加(P<0.01),且差异均有统计学意义。生物学信息软件提示紧密连接蛋白1(CLDN1)可能为miR-29a靶基因,实时荧光定量PCR及western blot提示与miR-NC组相比,miR-29a mimic组内CLDN1表达下降(P<0.05),相反anti-miR-29a组内CLDN1表达增加(P<0.05),差异均具有统计学意义。[结论]肝癌细胞可能通过抑制miR-29a基因的表达以提高CLDN1蛋白的转录与翻译从而促进肝癌细胞增殖及转移能力。

[Objective]To explore the differences in gene expression of miR-29 a between hepatocellular carcinoma cells and normal liver cells,and further to investigate the effects of miR-29 a on the proliferation and invasion of hepatocellular carcinoma cell line HepG2 and the potential mechanisms.[Methods]The gene expression level of miR-29 a of hepatocellular carcinoma cells and normal liver cells was detected by qRT-PCR;miR-29 a mimic,empty vector miR-NC and miR-29 a inhibitor were transfected into HepG2 by lipofectamine method,namely overexpression group,control group and inhabitation group,and then we evaluated the gene expression of miR-29 a by qRT-PCR.The effects of miR-29 a on the proliferation and invasion of HepG2 cells were observed by MTT and Transwell assay.Using biological information forecast software to predict the potential target of miR-29 a.qRT-PCR and Western blot were used to validate the target relationship between miR-29 a and CLDN1 in transfected HepG2 with miR-29 a mimic、empty vector miR-NC and miR-29 a inhibitor.[Results]qRT-PCR results presented that the expression of miR-29 a in normal liver cells was obviously higher than that in the hepatocellular carcinoma cells(P<0.05);After transfection with liposome,compared with the microRNA-NC group,the microRNA-29 a gene in the microRNA-29 a mimic was significantly higher(P<0.01),while the microRNA-29 a gene in the anti-microRNA-29 a group was significantly lower(P<0.05).MTT and Transwell experiments showed that,compared with control group,the proliferation and invasion ability of HepG2 transfected with miR-29 a mimic was obviously decreased(P<0.05),and these abilities in anti-miR-29 a group were increased(P<0.01).The biological information software suggested that CLDN1 might be a target of miR-29 a.qRT-PCR and western blot showed that the expression of CLDN1 in miR-29 a mimic group was decreased compared with the miR-NC group(P<0.05),and the expression of CLDN1 in anti-miR-29 a group was increased(P<0.01).[Conclusion]Hepatocellular carcinoma cells may enhance the proliferation and invasion by inhibiting the gene expression of miR-29 a and increasing the transcription and translation of CLDN1.