miR-199a对肝癌HepG2细胞增殖的影响及机制

Effects and mechanisms of miR-199a on proliferation of HepG2 cells

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 effects and underlying mechanisms of miR-199 a on proliferation of HepG2. Methods：The miR-199 a gene was cloned to lentiviral expression vector p LV-GFP-puro with EGFP by recombining DNA technology,and positive clones were identified by DNA sequencing. The 293 T cells were cotransfected with lentiviral packaged systems and miR-199 a gene plasmid by lipofectin regeant to package the lentiviral particles. The HepG2 cells were infected with purified recombinant lentivirus.The transfection efficiency was assessed under fluorescent microscope;MTT assay was used to detect cell proliferation;Western Blot was used to analyze expressions of IκBα and p-NF-κBp65;In addition,q RT-PCR was performed to detecte expressions of miR-199 a and NF-κB related anti-apoptotic genes（TRAF2,c IAP2,Bfl-1 and c FLIP）in transfected HepG2 cells. Results：The recombinant lentiviral vectors of miR-199 a gene were successfully constructed. The virus reached a titer of 1.08 ×108TU/m L after being packaging,purification and concentration. Interestingly,the transfected cells held high expression of miR-199 a,which inhibited the proliferation of HepG2 cells. In addition,the transfencted HepG2 cells stablely expressed IκBα,effectively inhibited NF-κB activity,and further suppressed expressions of NF-κB related anti-apoptotic genes（TRAF2,c IAP2,Bfl-1 and c FLIP）. Conclusion：The miR-199 a recombinant lentiviral vector had been successfully constructed and effectively transfected HepG2 cells,which inhibited the proliferation of HepG2 cells. The underlying mechanisms may be inhibit NF-κB activity,and further inhibit expressions of NF-κB related anti-apoptotic genes by miR-199 a.