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 Ad5-miR-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.

国家重大传染病防治科技专项基金资助项目(No. 2008ZX10002-018)