人源CerS2重组腺病毒构建及其对肝癌细胞HepG2细胞周期的作用

Construction of recombinant adenovirus vector with CerS2 gene and effect of Ceramide Synthasas 2 overexpression on cell cycle in hepatocarcinoma HepG2 cells

目的探讨神经酰胺合酶2(CerS2)过表达对肝癌细胞HepG2细胞周期的影响及可能机制。方法化学合成人源CerS2基因CDS区并构建重组穿梭质粒pDC315-CerS2-绿色荧光蛋白(GFP),采用Admax系统将成功构建的CerS2重组穿梭质粒与骨架质粒共转染HEK293细胞,通过Cre/loxP实现重组,并进行扩增、纯化及滴度测定。将正确构建的Adv-CerS2-GFP重组腺病毒与空载分别感染肝癌细胞HepG2(即对照组、空载组、实验组),激光共聚焦显微镜定位CerS2-GFP融合蛋白的表达,流式细胞术PI法检测各组细胞周期的分布;RT-qPCR、Western blot法检测各组细胞CerS2、p27mRNA及蛋白表达水平。结果经测序分析验证重组腺病毒构建成功。HepG2实验组CerS2、p27mRNA、蛋白相对表达水平较对照组、空载组明显增加(P<0.01)。对照组与空载组细胞周期时相分布差异无统计学意义(P>0.05),实验组细胞较对照组、空载组G_0/G_1期百分比明显增加(P<0.01);G_2/M、S期明显减少(P<0.01)。结论成功构建人源CerS2重组腺病毒且过表达的CerS2导致肝癌细胞HepG2细胞周期G_0/G_1期阻滞,其机制可能是通过上调p27从而抑制肝癌细胞的生长与增殖。

Objective To investigate the effect of ceramide synthase 2(CerS2)overexpression on HepG2 cell cycle and its possible mechanism.Methods CerS2 gene was synthesized by chemical method and inserted to the polyclonal sites of adenovirus shuttle plasmid pDC315-CerS2-green fluorescent protein(GFP)to generate a recombinant shuttle plasmid.HEK293 cells were cotransfected to the successfully constructed recombinant shuttle vector and framework plasmids using the Admax system and the Cre/loxP recombinant enzyme system,amplified,purified,and measured its titer.HepG2 cells were transfected with the correctly constructed recombinant adenovirus vector with CerS2 gene and the empty vector(the blank group,the empty vector group,the experimental group),subcellular localization of CerS2-GFP fusion protein was detected by confocal laser scanning,the changes of cell cycle was analyzed by flow cytometry propidiumiodide(PI)single staining;the expressions of mRNA and protein of CerS2 and p27 were measured by quantitative RT-qPCR and Western blot.Results The recombinant adenovirus vectors were successfully constructed.The mRNA and protein expressions of CerS2 and p27 significantly increased in the experimental group than the blank group and the empty vector group(P<0.01).There was no statistical significance in cell cycle between the blank group and the empty vector group(P>0.05),compared with the blank group and the empty vector group,G 0/G 1 phase of the experimental group increased(P<0.01),G 2/M and S phase significantly decreased(P<0.01).Conclusion The recombinant adenovirus vector was successfully constructed,and the overexpressed CerS2 leads to G 0/G 1 phase block in the HepG2 cell cycle of hepatoma cells,and the mechanism may be relative to inhibit the growth and proliferation of hepatoma cells by up-regulation of p27.