摘要
目的探讨miR-378对肝癌细胞生长增殖的影响及其机制。方法用miR-378过表达慢病毒感染肝癌细胞株HepG2和稳定表达HBV的肝癌细胞株HepG2．2．15，采用四甲基偶氮唑盐和克隆形成实验研究其在肝癌发生和发展中的作用。生物信息学方法预测miR-378的靶基因，荧光素酶报告实验验证靶基因，并用实时定量PCR方法和Westernblot方法研究上调miR-378表达对其靶基因表达的影响，分析miR-378在肝癌中作用的机制。采用SPSS13．0软件进行统计分析，两组间比较采用t检验。结果与空载体感染组比较，miR-378过表达慢病毒感染组肝癌细胞的增殖活性降低和克隆形成减少，差异有统计学意义沪〈0．01或尸〈0．05）。用生物信息学方法预测胰岛素样生长因子1类受体（IGF1R）为miR-378的靶基因，荧光素酶报告实验结果显示，与对照组比，miR-378与IGF1R3’-UTR共转染组荧光酶活性下降了41．8％，差异有统计学意义（P〈0．01）。上调肝癌细胞中miR-378表达后，IGF1RmRNA表达与对照组差异无统计学意义垆〉0．05），而IGFlR蛋白表达水平显著降低垆〈0．01）。结论miR-378可抑制肝癌细胞的增殖和克隆形成，起抑癌基因的作用，其作用是通过抑制其下游靶基因IGF1R发挥的。HBV导致的miR-378下调表达可能是HBV相关性肝癌发生和发展的分子机制之一。
Objective To investigate the impact and mechanism of microRNA （miR）-378 on hepatocellular carcinoma （HCC） cell growth. Methods The human hepatoma cell line HepG2 and its derivative HepG2.2.15 （stably expressing hepatitis B virus （HBV）） were transduced with lentiviruses expressing miR-378 or non-expressing controls （nc-Lv）. Effects on cell proliferation were assessed by the MTT assay and on colony-formation efficiency by clonogenic assay. Targets of miR-378 were predicted by bioinformatic analysis and validated by luciferase reporter assay in the human embryonic kidney cell line HEK293. Real-time polymerase chain reaction and western blotting were used to monitor expression of the endogenous targets in miR-378- overexpressing HepG2 and HepG2.2.15 cells. Results The HepG2 and HepG2.2.15 cells transduced with lentivirus expressing miR-378 showed significantly lower cell proliferation and colony formation than the control cells transduced with nc-Lv （P 〈 0.01 and P 〈 0.05, respectively）. The insulin-like growth factor 1 receptor （IGF1R） was predicted as a potential target of miR-378, and luciferase reporter activity of IGF1R was significantly decreased in the HEK293cells co-transfected with miR-378 （by 41.8% vs. the control cells, P 〈 0.01）. Moreover, the miRNA-378- mediated effect was narrowed down to the 3＇-untranslated region （UTR） of IGF1R. The miRNA-378- mediated reduction of IGF1R specifically involved its protein expression （P 〈 0.01） and not its mRNA expression （P 〉 0.05）. Conclusion miR-378 may suppress growth characteristics ofHBV-related HCC by directly targeting the IGF1R 3＇-UTR and inhibiting its protein expression.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2013年第8期609-613,共5页
Chinese Journal of Hepatology
基金
基金项目：无锡市医院管理中心项目（YGZ1016,YGZ1106）
