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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 investigate the inhibitory effect of miR-99a on the growth of hepatocellular carcinoma cells by adenovirus-mediated overexpression of microrRNA-99a (miR-99a), and to explore a new method of adenovirus-mediated miRNA therapy for tumors. Methods: The expression of miR-99a in normal liver cell line L-02 and HepG2, SMMC-7721, Hep3B and Huh7 cells was detected by qRT-PCR and the recombinant adenovirus Ad5-miR-99a . Huh7 cells were infected with empty vector Ad-blank and Ad5-miR-99a respectively. The inhibitory effect of miR-99a overexpression on the growth of Huh7 cells was detected by MTT assay and colony formation assay. Results: The expression of miR-99a in HCC Huh7 cells was the lowest (P <0.01) compared with normal L02 cells and other hepatoma cells. After successful construction of recombinant adenovirus Ad5-miR-99a and Ad5-miR-99a infected Huh7 cells, miR-99a was stably overexpressed in Huh7 cells. Colony formation assay and MTT assay showed that Ad5-miR-99a significantly inhibited Huh7 cell growth and colony formation (P <0.01) compared with Ad-blank group. Conclusion: The successful construction of recombinant adenovirus Ad5-miR-99a, miR-99a can effectively inhibit the proliferation of liver cancer cells, Ad5-miR-99a may become a new drug for the treatment of liver cancer.