腺病毒介导microRNA-99a过表达抑制肝癌细胞生长

Adenovirus mediated overexpression of miR-99a inhibits the growth of hepatocellular carcinoma cells

目的:利用腺病毒介导microrRNA-99a(miR-99a)的过表达,观察miR-99a对肝癌细胞生长的抑制作用,探讨一种腺病毒介导miRNA治疗肿瘤的新方法。方法:qRT-PCR检测正常肝细胞系L-02和肝癌细胞系HepG2、SMMC-7721、Hep3B和Huh7细胞中miR-99a的表达,构建含miR-99a的重组5型腺病毒Ad5-miR-99a。用空载腺病毒Ad-blank和Ad5-miR-99a分别感染Huh7细胞,MTT法和集落形成实验检测miR-99a过表达对Huh7细胞生长的抑制作用。结果:与正常肝细胞L02和其他肝癌细胞相比,miR-99a在肝癌Huh7细胞中表达量相对最低(P<0.01)。成功构建重组腺病毒Ad5-miR-99a,Ad5-miR-99a感染Huh7细胞后,miR-99a可在Huh7细胞中稳定高表达。集落形成实验和MTT实验显示,相对于Ad-blank组,Ad5-miR-99a可显著抑制Huh7细胞的生长和集落形成(P<0.01)。结论:成功构建重组腺病毒Ad5-miR-99a,miR-99a能有效抑制肝癌细胞的增殖,Ad5-miR-99a有可能成为治疗肝癌的新药物。

Objective： To study the inhibitory effect of adenovirus-mediated-miR-99a overexpression on proliferation of hepatocellular carcinoma cells（HCCs） and find a new kind of adenovirus mediated miRNA treatment for cancer.Methods： The expression of miR-99a was detected by quantitative real-time polymerase chain reaction（qRT-PCR） in human liver cell line L-02 and HCC cell lines HepG2,SMMC-7721,Hep3B and Huh7.The type 5 adenovirus vector containing miR-99（Ad5-miR-99a） was constructed.Huh7 cells were infected with empty adenovirus vector（Ad-blank） or Ad5miR-99a,and MTT and colony formation assay were used to examine the inhibitory effect of miR-99a on the growth of Huh7 cells.Results： miRNA-99a was markedly decreased in the Huh7 cell line compared with that in L02 cell line（P 0.01）.Ad5-miR-99a adenovirus vector was successfully constructed,and miR-99a was stably high expressed in Huh7 cells after infection with Ad5-miR-99a.MTT and colony formation assay showed that Ad5-miR-99a could inhibit the growth and colony formation of Huh7 cells compared with Ad-blank（P 0.01）.Conclusion： Adenovirus Ad5-miR-99a is successfully constructed,which can effectively suppress the growth of HCC.Ad5-miR-99a might be a prospective therapeutic approach for HCC in the near future.