RNAi沉默CD14基因对人胃癌细胞生物学行为的影响

Effect of CD14 gene silenced by RNAi on the biological behavior in human gastric cancer cells

目的以胃癌SGC-7901细胞为研究对象,在细胞水平观察沉默CD14基因对胃癌细胞增殖、凋亡、周期及下游靶因子TNF-α、IL-8的影响,探讨CD14基因在胃癌中的发病地位。方法重组CD14shRNA慢病毒为载体转染胃癌SGC-7901细胞。实验分3组,正常组、慢病毒阴性对照组(NCshRNA)、CD14沉默组(CD14shRNA)。采用荧光显微镜观察转染效率,确定最佳转染时间;Western blotting检测CD14蛋白的沉默效率;MTT法检测细胞增殖,流式细胞仪检测细胞周期及凋亡,Real-time PCR技术检测TNF-α、IL-8表达。结果成功转染慢病毒CD14shRNA的SGC-7901人胃腺癌细胞可表达绿色荧光蛋白,测得在病毒MOI=10时,转染72 h后,慢病毒的转染效率最佳。转染后24 h,正常组细胞增殖率为218.18%,阴性对照组为216.27%,CD14shRNA组为211.63%,各组间相比,差异无统计学意义(P>0.05);转染后48 h、72 h,正常组细胞增殖率为427.49%、479.22%,阴性对照组为417.84%、473.37%,CD14shRNA组为345.54%、362.64%,经比较,CD14沉默后细胞增殖率下降(P<0.05)。CD14shRNA组细胞早期凋亡、晚期凋亡及总凋亡率均高于正常组,差异有统计学意义(P<0.05)。CD14shRNA组细胞G1期42.57%、S期38.65%、G2期13.22%,正常组细胞G1期35.91%、S期52.60%、G2期11.49%,差异有统计学意义(P<0.05)。Western blotting结果显示,在转染胃癌细胞后,CD14shRNA慢病毒载体组细胞CD14蛋白表达相对于正常组及阴性对照组明显减少(P<0.05);Real-time PCR结果显示,CD14shRNA转染胃癌细胞后,TNF-α、IL-8 mRNA表达较正常组、阴性对照组明显下降。结论 CD14基因沉默后,胃癌细胞增殖能力减弱,细胞周期阻滞在S期,细胞合成减少,凋亡率增加,TNF-α、IL-8表达下降,这说明CD14基因在胃癌细胞增殖、发展过程中占据重要地位。

Objective To investigate the cell proliferation,cell apoptosis,cell cycle and the variation of downstream target factor TNF-α and IL-8 by silencing CD14 gene at cell level in gastric cancer cell SGC-7901 in order to evaluate the role of CD14 gene in gastric cancer. Methods The gastric cancer cell SGC-7901 was transfected by recombinant chronic virus CD14 shRNA. The SGC-7901 cells were divided into three groups： normal group,chronic virus negative control group（ NCshRNA） and CD14-silenced group（ CD14shRNA）. Fluorescence microscope was used to observe transfection efficiency to ensure the optimal transfection time; silence efficiency of protein CD14 and the expression of gene TNF-α and IL-8 were detected by Western blotting and Real-time PCR separately. Cell proliferation was detected by MTT,cell cycle and apoptosis was detected by flow cytometry. Results SGC-7901 cell successfully transfected by CD14 shRNA which can express green fluorescent protein. Transfected after 72 hours,the efficiency of transfection reach the peak when MOI = 10. Transfected after 24 hours,there was no significant difference among three groups（ P〉 0. 05）. Whereas,CD14 shRNA group had significant inhibiting effect on cell proliferation when transfected after 48 hours and 72 hours（ P 〈0. 05）. Early apoptosis,late apoptosis and total apoptosis rate were increased significantly in CD14 shRNA group than normal group（ P 〈0. 05）. There were 42. 57% cells in G1 phase,38. 65% cells in S phase and 13. 22% cells in G2 phase in CD14 shRNA group,normal group had 35. 91% cells in G1 phase,52. 60% cells in S phase,11. 49% cells in G2 phase. There was significant difference between two groups（ P 〈0. 05）. Western blotting showed that the expression of CD14 protein in CD14 shRNA group were decreased significantly was on the decrease compared with normal group and negative control group（ P 〈0. 05）. The changes of TNF-α and IL-8 gene in CD14 shRNA group was on the decline. Conclusion After silencing CD14 gene,the proliferation ability of gastric cancer cell is decreased,cell cycle arrest in S phase,cell synthesis is reduced,cell apoptosis rate is in the increasment and the expression of TNF-α,IL-8 gene is on the decline. CD14 gene plays an important role in proliferation and development of gastric cancer cells.