脂质体介导Cyclin D1干扰RNA对人肝癌细胞株HepG2全细胞蛋白质组表达的影响

目的探讨CyclinD1干扰RNA对人肝癌细胞株HepG2细胞中CyclinD1mRNA的表达、对细胞周期和增殖以及蛋白质组表达变化的影响。方法构建CyclinD1干扰RNA的重组质粒pU6-CyclinD1-siRNA，并设立阴性对照空载体转染组和空白对照组。采用脂质体介导转染肝癌细胞株HepG2细胞，经G418筛选阳性转染细胞克隆，流式细胞术、MTT法检测其细胞周期、细胞生长曲线的改变；反转录PCR法及双向凝胶电泳．质谱技术比较转染前后CyclinD1mRNA的表达和蛋白质组表达的变化。结果（1）成功构建了CyclinD1干扰RNA的重组质粒pU6．CyclinD1．siRNA。并获得稳定转染组细胞HepG2-CyclinD1siRNA。（2）稳定转染组细胞与阴性对照组细胞和空白对照组相比，CyclinD1mRNA表达水平降低。细胞周期明显改变，从G1期到s期发生阻滞。（3）通过双向电泳-图像分析-质谱技术得到稳定转染组低表达蛋白6个，经数据库搜索分别是锌指蛋白ZFP-36、角蛋白19、热休克蛋白伴侣素10、波形蛋白、肿瘤拒绝抗原gp96、未知蛋白。这些蛋白与细胞的增殖、细胞凋亡的信号转导调节、以及肿瘤的发生、发展等相关。结论CyclinD1干扰RNA可明显降低肝癌HepG2细胞CyclinD1mRNA的表达，亦可阻滞肝癌HepG2的细胞周期，抑制细胞增殖；CyclinDI干扰RNA干扰后差异表达的蛋白质涉及细胞的增殖、细胞凋亡的信号转导调节、以及肿瘤的发生、发展。

Effects of lipofectamine-mediated cyclin DI-siRNA on the total proteome of HepG2 cells

Objective To explore the mRNA expression of cyclin D1, cell cycle, proliferation, and the changes of proteome expression in human liver cancer cells HepG2 after cyclin D1 was interfered. Methods The recombinant plasmid pU6-cyclin DI-siRNA was constructed to inhibit Cyclin D1. The control group and negative group （without insert sequence plasmid） were constructed. The plasmids pU6-Cyclin DI-siRNA and pU6 were transfected into HepG2 cells with liposome transfection, respectively. The positive clones named HepG2-cyclin D1 siRNA cells, HepG2-neo cells were selected by G418. The cell cycle and cell growth curve were compared with flow cytometry （FCM）, and methyl thiazolyl tetrazolium （MTr）. The mRNA expression of cyclin D1 was compared with real-time polymerase chain reaction （RT-PCR）. The different proteins before and after transfected cells were identified with two-dimensional gel electrophoresis and mass spectrometry. Results （1）The recombinant plasmid pU6-cyclin DI-siRNA was constructed suc- cessfully. The stable transfection cells were obtained. （2）Compared with the negative control group （HepG2- neo） and the blank group, the level of eyclin D1 mRNA expression in the stable HepG2-Cyclin DI-siRNA- transfected HepG2-neo cells was lower. Compared with the negative control group （HepG2-neo） and the blank group, the growth in the stable HepG2-Cyclin D1 cells was slower, more cells in the cyclin in G0/G1 phase, and less cells in G2/M and S phases. （3）The six downregulated poteins similar to zinc finger protein ZFP-36, keratinl9, chaperonin 10-related protein （Hsp10,HSPE1 ,CPN10）, vimentin, tumor rejection antigen （gp96）, unknown] were found with two-dimensional gel electrophoresis and mass spectrometry. Conclusions The mRNA expression level of Cyclin D1 in HepG2 cells was down - regulated with cyclin D1 siRNA. The growth of the HepG2 was inhibited with cyclin D1 siRNA. The differentially expressed pro- teins derived from inhibition of cyclin D1 were mainly related to the signal transduction regulation of cell proliferation and apoptosis, and occurrence and progression of a tumor.