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目的 :探讨miR-199a对肝癌HepG2细胞增殖的影响及机制。方法 :使用DNA重组技术,将含有miR-199a的基因片段克隆入带有EGFP的慢病毒载体pLV-GFP-puro,DNA测序鉴定阳性克隆,并用脂质体介导法将慢病毒包装系统和带miR-199a的质粒共转染293T细胞包装病毒,纯化后感染肝癌HepG2细胞。用荧光显微镜观察感染效率;MTT法检测细胞增殖活性;Western blot检测HepG2细胞IκBα、p-NF-κBp65表达;并采用qRT-PCR分析感染Hep G2细胞miR-199a和NF-κB相关抗凋亡基因(TRAF2、c IAP2、Bfl-1和c FLIP)的表达情况。结果:成功构建了miR-199α重组慢病毒载体,并对其包装、纯化及浓缩后测定病毒滴度为1.08×10~8TU/m L;感染后HepG2细胞高表达miR-199a,抑制HepG2细胞增殖,并证实过表达miR-199a有效稳定IκBα表达,抑制NF-κB活性,进一步抑制NF-κB相关抗凋亡基因TRAF2、cIAP2、Bfl-1和c FLIP表达。结论:miR-199a重组慢病毒过表达载体有效感染HepG2细胞,并抑制Hep G2细胞增殖,其机制可能与miR-199a抑制NF-κB活性及其相关抗凋亡基因表达有关。
Objective: To investigate the effect of miR-199a on the proliferation of HepG2 cells and its mechanism. Methods: The gene fragment containing miR-199a was cloned into the lentiviral vector pLV-GFP-puro containing EGFP by DNA recombination technique. The positive clones were identified by DNA sequencing. The lentiviral packaging system and the lentivirus packaging system The plasmid of miR-199a was cotransfected into 293T cell packaging virus and purified to infect HepG2 cells. The infection efficiency was observed by fluorescence microscopy. The cell proliferation activity was detected by MTT assay. The expression of IκBα and p-NF-κBp65 in HepG2 cells was detected by Western blot. The anti-apoptotic genes of miR-199a and NF-κB in Hep G2 cells were analyzed by qRT-PCR (TRAF2, c IAP2, Bfl-1 and c FLIP). Results: The miR-199α recombinant lentiviral vector was successfully constructed and its viral titer was 1.08 × 10 ~ 8TU / m L after being packaged, purified and concentrated. The high expression of miR-199a in HepG2 cells inhibited the proliferation of HepG2 cells , And confirmed that overexpression of miR-199a could effectively stabilize IκBα expression and inhibit NF-κB activity and further inhibit the expression of TRAF2, cIAP2, Bfl-1 and c FLIP of NF-κB-associated anti-apoptotic genes. Conclusion: miR-199a recombinant lentivirus overexpression vector effectively infects HepG2 cells and inhibits the proliferation of Hep G2 cells. The mechanism may be related to the inhibition of NF-κB activity and the expression of anti-apoptotic genes by miR-199a.