胃癌相关基因GCRG213真核表达载体的构建及其对胃癌细胞生长特性的影响

Construction of gastric cancer related gene GCRG213 eukaryotic expression vector and its effect on growth of gastric cancer cells

目的研究胃癌相关基因GCRG213正义、反义转染对胃癌细胞MKN45生长特性的影响。方法将从pGEM-T质粒上扩增出的胃癌相关基因GCRG213的DNA片段,按正向、反向克隆入真核表达载体pcDNA3·1(+)。测序正确的重组子pcDNA3·1-a(含GCRG213正向克隆)、pcDNA3·1-b(含GCRG213反向克隆)和空载体经脂质体转染人胃癌细胞系MKN45细胞,采用半定量RT-PCR及Westernblot方法比较转染不同质粒的MKN45细胞中GCRG213在mRNA和蛋白质水平上的表达差异。选取稳定转染不同质粒的MKN45细胞,采用细胞计数法绘制细胞生长曲线,流式细胞仪分析细胞的增殖状态,AnnexinVFITC/PI双标记法检测凋亡细胞。结果经测序证实,GCRG213正向克隆和反向克隆正确插入真核表达载体pcDNA3·1(+)。与对应的空载体比较,转染pcD-NA3·1-a的MKN45细胞中mRNA的表达上调35·4%,蛋白的表达上调49·4%,而转染pcDNA3·1-b的MKN45细胞中mRNA的表达下调32·1%,蛋白的表达下调50·3%。转染了GCRG213正向克隆的MKN45细胞生长增殖速度加快,凋亡率下降,而转染了GCRG213反向克隆的MKN45细胞生长增殖速度减慢,凋亡率增加。结论胃癌相关基因GCRG213可促进肿瘤细胞的生长,抑制肿瘤细胞凋亡,可能是恶性肿瘤癌变的促进因素之一。

Objective To investigate the effect of gene GCRG213 transfection （sense, anti-sense） on growth of gastric cancer cell MKN45. Methods The sense and anti-sense fragment of GCRG213 were obtained by PCR. They were cloned into eukaryotic expression vector pcDNA3. 1（＋）. The recombinant plasmid pcDNA3, 1-a, pcDNA3. 1-b and the vector pcDNA3. 1 were transfected separately into MKN45 cells conducted by lipofectamineTM 2000. Expression of GCRG213 was assessed with semi-quantitative RT-PCR and Western Blot. The growth graph was plotted by the methods of cell counting of three steady transfected cells. FACS was used to determine the cell cycle, and Annexin V FITC/PI bi-labeling method was used to determine the cell apoptosis. Results By sequencing, sense GCRG213 and antisense GCRG213 were proved to be successfully cloned into pcDNA3. 1. Transfecting the sense vector （pcDNA3. 1-a） into the MKN45 significantly increased the expression of GCRG213, both in mRNA level and protein level. Transfecting the anti-sense vector （pcDNA3. 1-b） into the MKN45 significantly decreased the expression of GCRG213, both in mRNA level and protein level. The growth of pcDNA3. 1-a transfected cells was faster than that of vector transfected cells, and the cell apoptosis decreased. But the growth of pcDNA3. 1-b transfected cells was slower in multiplication than that of vector transfected cells, and the cell apoptosis was increased. Conclusion Stable transfection showed that GCRG213 promoted cell to grow, inhibited the cell apoptosis. GCRG213 might be a new promoter to tumor.