靶向Mcl-1的shRNA重组质粒的构建及其对HepG2细胞增殖和凋亡的影响

Construction of Recombinant Plasmids Containing shRNA Targeting Mcl-1 Gene and Its Effect on Proliferation and Apoptosis of HepG2 Cells

目的设计以髓细胞白血病基因-1(Mcl-1)为靶点的短发夹状RNA(shRNA),构建携带此shRNA的重组质粒,探讨脂质体转染该重组质粒对肝癌细胞HepG2增殖及凋亡的影响。方法设计3对针对Mcl-1基因不同位点的shRNA片段的真核表达载体psi RNA-hH1neo-Mcl,用阳离子脂质体法将重组质粒转染至人肝癌细胞株HepG2中,通过RT-PCR及Western blot方法检测转染前后Mcl-1 mRNA及蛋白表达情况,筛选能高效抑制Mcl-1基因表达的shRNA重组质粒并应用MTT和流式细胞仪分析转染后细胞增殖和凋亡变化的情况。结果成功构建含shRNA片段的重组质粒,依次命名为pMclsi-1、pMclsi-2、pMclsi-3。经测序证实,插入的DNA片段的序列与设计序列完全一致。重组质粒转染HepG2细胞后,Mcl-1基因的mRNA水平及蛋白水平明显下调,其中以pMclsi-1重组质粒下调效应最强。MTT检测显示pMclsi-1可明显抑制HepG2细胞增殖,流式细胞仪测定转染后24 h细胞凋亡率为6.5%,48 h细胞凋亡率为15.6%,显著高于对照组(3.9%,P<0.05)。结论采用RNAi技术可特异阻断Mcl-1基因的表达,Mcl-1基因有促进细胞增生及抑制凋亡的作用。

Objective To construct recombinant plasmids containing short hairpin RNA（shRNA） that targets the myeloid cell leukemia-1 （Mcl-1） gene, and to assay its effects on proliferation and apoptosis of HepG2 cells transfected with recombinant plasmids. Methods Three shRNAs were designed according to the coding sequence of Mcl-1 gene,and cloned into the downstream of HI promoter of psiRNA-hHlneo. Then the recombinant plasmids were transfected into HepG2 cells with lipofectamine2000. The expression of Mel-1 mRNA and protein was detected by using RT-PCR and Western blot respectively. The best effective silencing plasmid was screened, the cellular proliferation capacity measured by using MTT assay, and the apoptosis state analyzed by flow cytometry. Results Three recombinant plasmids were successfully constructed and confirmed through DNA sequencing,then designated as pMclsi-1,pMclsi-2 and pMclsi-3 respectively. The expression of Mcl-1 mRNA and protein was obviously decreased in HepG2 cells transfeeted with the plasmids,and the pMclsi-1 had the best silencing effect. MTT assay indicated pMclsi-1 could inhibit the cellular proliferation,and the apoptosis rate was increased from 6.5% at the 24th h to 15.6% 48th h after transfection,whieh was obviously higher than control siRNA（3.9% ,P〈0.05）. Conclusion The constructed recombinant plasmids containing Mcl-1 shRNA can specifically block the Mcl-1 expression,and Mel-1 gene may promote proliferation and inhibit apoptosis of HepG2 cells.