shRNA靶向抑制COX-2基因表达对胃癌细胞SGC-7901增殖的影响

Effects of COX-2 gene expression inhibited by shRNA on proliferation of gastric carcinoma SGC-7901 cells

目的探讨环氧化酶-2（COX-2）短发夹RNA（shRNA）对胃癌细胞SGC-7901的生长和细胞周期的影响。方法构建COX-2基因的特异性小RNA干扰质粒,构建靶向抑制COX-2基因的重组表达载体pshRNA-COX-2。实验分3组：未转染胃癌细胞组,阴性对照HK组,pshRNA-COX-2转染组。用质脂体lipofectamine^TM2000转染胃癌细胞SGC-7901,RT-PCR和Western blot分别检测COX-2基因mRNA和蛋白表达情况,流式细胞术检测细胞周期,细胞计数检测细胞的生长变化。结果与阴性对照组相比,pshRNA-COX-2重组表达载体抑制COX-2 mRNA及蛋白表达的抑制率分别为70.1%和43.2%;G0～G1期细胞由61.5%上升至70.2%,S期细胞由27.3%下降至21.7%;细胞生长明显减慢。结论pshRNA-COX-2重组表达载体能显著抑制COX-2的表达,从而抑制胃癌细胞生长。

Objective To investigate the inhibiting effects of shRNA（short hairpin RNA,shRNA） on COX-2 gene expression,the cell cycle and growth of gastric carcinoma SGC-7901 cells.Methods Specific shRNA plasmid to COX-2 were constructed,and then transfected into SGC-7901 cells by lipofectamine methods.Tests were divided into three groups： untransfected gastric carcinoma SGC-7901 cells group,negative control HK group and pshRNA-COX-2 group.Gastric carcinoma SGC-7901 cells were transfected with LipofectamineTM 2000.Expression of COX-2mRNA and protein were detected with reverse transcriptional polymerase chain reaction（RT-PCR） and Western blot respectively.Cell cycle analysis and cell growth chart were detected with flow cytometry and cell count respectively.Results Compared with negative control HK group,recombinant expression vector pshRNA-COX-2 resulted in the reduction of COX-2mRNA and protein expresion by 70.1% and 43.2% respectively;cells in G0-G1 phase increased from 61.5%to 70.2%,cells in S phase decreased from 27.3% to 21.7%,and the growth of SGC-7901 cells cells was slowed significantly.Conclusions Recombinant expression vector pshRNA-COX-2 can significantly inhibit the expression of COX-2 gene,result in the increase of cells in G0-G1 phase and decrease of cells in S phase,and suppress proliferation of gastric carcinoma SGC-7901 cells.