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目的探讨活化蛋白激酶C受体1C(RACK1,GNB2L1)在胃癌细胞HGC27中的表达及过表达RACK1对HGC27生长增殖的影响。方法体外培养胃癌未分化细胞HGC27和正常胃黏膜上皮细胞系GES-1,收集细胞48 h后,提取mRNA和蛋白,利用RT-PCR检测RACK1 mRNA在HGC27和GES-1细胞中的表达;利用Western blotting法检测RACK1蛋白在两种细胞中的表达;以人胚肾HEK293细胞c DNA为模板,构建pc DNA3.1Aflag-RACK1重组质粒,利用Lipo2000转染入HGC27细胞中,Western blotting法检测质粒的转染效率,MTT法检测过表达RACK1对HGC27胃癌细胞系生长增殖的影响。结果 HGC27胃癌细胞中RACK1的mRNA和蛋白水平表达低于GES-1细胞(P<0.01)。双酶切鉴定和测序分析表明,pc DNA3.1A-flag-RACK1重组质粒构建成功。将该质粒转入HGC27细胞后,与未转染组相比,空载转染组RACK1蛋白表达无明显区别(P>0.05),pc DNA3.1-RACK1转染组RACK1蛋白表达明显升高(P<0.01);转染pc DNA3.1A-flag-RACK1转染组细胞存活率在72 h和96 h明显少于pc DNA3.1空载对照组(P<0.01)。结论支架蛋白RACK1的mRNA和蛋白水平在HGC27细胞中低表达,上调RACK1的表达可明显抑制HGC27细胞增殖。
Objective To investigate the expression of activated protein kinase C receptor 1C (RACK1, GNB2L1) in gastric cancer cell HGC27 and the effect of overexpression of RACK1 on the growth and proliferation of HGC27. Methods Human gastric epithelial cell line HGC27 and normal gastric mucosal epithelial cell line GES-1 were cultured in vitro. After 48 h, the mRNA and protein were extracted. The expression of RACK1 mRNA in HGC27 and GES-1 cells was detected by RT-PCR. blotting method was used to detect the expression of RACK1 protein in two kinds of cells; pcDNA3.1Aflag-RACK1 recombinant plasmid was constructed by c DNA of human embryonic kidney HEK293 cells and transfected into HGC27 cells by Lipo2000; Staining efficiency and MTT assay were used to detect the effect of over-expression RACK1 on the growth and proliferation of HGC27 gastric cancer cell line. Results The mRNA and protein level of RACK1 in HGC27 gastric cancer cells was lower than that in GES-1 cells (P <0.01). Double enzyme digestion and sequencing analysis showed that pcDNA3.1A-flag-RACK1 recombinant plasmid was successfully constructed. Compared with untransfected group, the expression of RACK1 protein in no-transfected group had no significant difference (P> 0.05). The expression of RACK1 protein in pcDNA3.1-RACK1 transfected group was significantly increased P <0.01). The survival rate of transfected pcDNA3.1A-flag-RACK1 group was significantly lower than that of pcDNA3.1 control group at 72 h and 96 h (P <0.01). Conclusions The mRNA and protein level of scaffold protein RACK1 is low expressed in HGC27 cells. Up - regulation of RACK1 expression can significantly inhibit the proliferation of HGC27 cells.