摘要
目的:研究miR-99a对肝癌的细胞的抑制作用和机制,提供一种腺病毒介导的miRNA治疗肿瘤的新方法。
方法:qRT-PCR检测肝癌和对应正常肝组织中miR-99a的表达;通过统计分析检验构miR-99a与肝癌患者预后的关系;在miR-99a作用机制的研究方面,重组5型腺病毒Ad5-miR-99a,通过体外和体内实验验证miR-99a对肝癌细胞的抑制作用;通过双荧光素酶报告基因系统验证miR-99a调控的新靶基因EIF2C2;在体内、体外实验和临床组织中,通过qRT-PCR、Western blot、免疫荧光和免疫组化检测EIF2C2及其下游基因PTEN的表达。
结果:miR-99a在肝癌组织中低表达,与肝癌患者的预后相关;体内体外实验显示过表达miR-99a可抑制HCC的生长;miR-99a靶向抑制EIF2C2的表达,从而促进PTEN的表达。
结论:miR-99a与肝癌患者的预后相关,通过靶向调控EIF2C2/PTEN信号通路抑制HCC的生长,在将来很有可能成为治疗肝癌的新方法。
Objective:
In order to further research the suppression of has-miR-99a on proliferation ofhepatocellular carcinoma cells through constructing adenovirus vector.
Methods:
Expression of miR-99a was detected by quantitative real-time polymerase chainreaction (qRT-PCR) in HCC. The adenovirus vector Ad5-miR-99a were constructed.Through the Luciferase Reporter Assay found that miR-99a regulation of the new target genesEIF2C2. Then we detected EIF2C2and downstream PTEN gene expression by qRT-PCR、Western blot、immunofluorescence and Immunohistochemistry in vitro and in vivo.
Results: MicroRNA-99a markedly decreases in HCC.miR-99a expression correlateswith HCC patients’ survival, and miR-99a restoration suppresses HCC growth invitro and in vivo by targeting EIF2C2/PTENpathway. MiR-99a could stable highexpression in HCC after transfected Ad5-miR-99a for48hours.
Conclusion:Ad5-miR-99a successfully suppresses HCC growth and could be aprospective therapeutic approach for HCC in the near future.
引文
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