miR-199a对肝癌HepG2细胞增殖的影响及机制
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江苏省六大人才高峰(2014-YY-008)


Effects and mechanisms of miR-199a on proli feration of HepG2cells
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    摘要:

    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的基因片段克隆人带有EGFP的慢病毒载体pLV-GFP-puro,DNA测序鉴定阳性克降,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;Westernblot检测HepG2细胞miR-199a和NF-kB相关抗凋亡基因(TRAF2、cIAP2、Bfl-1和cFLIP)的表达情况。结果:成功构建了miR-199a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1.08×108TU/mL;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IkBa表达,抑制NF-kB活性,进一步抑制NF-kB相关抗凋亡基因TRAF2、cIAP2\Bfl-1和cFLIP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HepG2细胞增殖,其机制可能与miR-199a抑制NF-kB活性及其相关抗凋亡基因表达有关。

    Abstract:

    Objective:To investigate effects and underlying me chanisms of miR-199a on prolifer ation of HepG2. Methods:The miR-199a gene was cloned to lentiviral expression vectorpLV-GFP-puro with EGFP by recombining DNA technology,and positive clones were i dentified by DNA sequencing. The 293T cells were cotransfected with lentiviral packaged systems and miR-199a gene plasmid by lipofectinregeant to package the lentiviral partic les.The HepG2 cells were infected with purified recombinant lentivirus. The transfec tion efficiency was assessed under fluores cent micros cope;MTT assay was used to detect cell proliferation;Western Blot was used to analyzae expressions of miR-199A and NF-kB related anti-apoptotic genes(TRAF2,cIAP2,Bfl-1 and cFLIP)in transfected HepG2 cells.Results:The recombinant lentiviral vectors of miR-199a gene were successfully constructed. The virus reached a titer of 1.0.8×108TU/mL after being packaging,purification and concentration. Interestingly,the transfected cells held high expression of miR-199a,which inhibited the pression of miR-199a,cells.ln addition,the transfencted HepG2 cells stablely expressed IkBa,effectively inhibited NF-kB activity,and further suppressed expressions of NF-kB related anti-apoptotic genes(TRAF2.c IAP2,Bfl-1 and cFLIP).Conclusion:The miR-199a recombinant lentiviral vector had been successfully constructed and effectively transfected HepG2 cells, which inhited the proliferation of HepG2 cells . The underlying me chanisms may beinhibit NF-kB activity,and further inhibitexpressions of NF-kB related anti-apoptotic genes by miR-199a.

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程顺舟,陈 欣,王 平,王吉荣. miR-199a对肝癌HepG2细胞增殖的影响及机制[J].南京医科大学学报(自然科学版),2017,(8):961-965

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  • 收稿日期:2016-05-29
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  • 在线发布日期: 2017-08-17
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