利用RNA干扰技术抑制环氧合酶-2表达对人胃癌细胞系SGC-7901增殖和凋亡影响的实验研究

目的研究环氧合酶(COX)-2表达与肿瘤细胞增殖和凋亡的关系,探讨利用RNA干扰技术作为肿瘤基因治疗的方法。方法构建靶向COX-2的短发夹状双链RNA(shRNA)的真核表达质粒WBH1转染人胃癌细胞系SGC-7901,采用逆转录聚合酶链反应(RT-PCR)和Western印迹分别从mRNA和蛋白质水平检测抑制效果。四甲基偶氮唑蓝(MTT)和流式细胞仪检测COX-2表达被抑制后细胞的增殖和凋亡情况。15只裸鼠随机分为3组,每组5只,皮下接种胃癌细胞。抑制组接种转染抑制质粒的胃癌细胞;正常对照组接种未转染的胃癌细胞;阴性对照组接种转染阴性对照质粒的胃癌细胞。4周后观察比较各组裸鼠皮下形成瘤体的大体和病理情况并计算抑瘤率。结果WBH1高效特异地抑制了人COX-2的表达,抑制率达70.42%。COX-2表达被抑制后,细胞增殖受到明显抑制(P=0.002),细胞凋亡率明显增高,与未转染和转染阴性对照质粒的胃癌细胞的凋亡率相比有统计学意义(52.28%±17.91%、0.52%±0.27%、0.54%±0.16%,P=0.009,实验重复5次)。正常对照组和阴性对照组所有裸鼠均有瘤体形成,平均瘤重分别为(0.490g±0.017g,5只)和(0.490g±0.013g,5只)。抑制组仅2只有瘤体形成平均瘤重0.050g±0.003g,与正常对照组相比差异有统计学意义(P<0.01),抑瘤率为89.8%。结论COX-2与肿瘤细胞的增殖和凋亡密切相关,抑制细胞中COX-2的表达可以抑制肿瘤细胞增殖,促进肿瘤细胞凋亡。通过构建靶向COX-2的shRNA真核表达载体导入细胞可以高效特异地抑制人胃癌细胞中COX-2的表达。

Effects of inhibition of cyclooxygenase-2 by RNA interference on proliferation and apoptosis of human gastric cancer cells: an experimental study with human gastric cancer cells and mice

OBJECTIVE: To study whether the expression level of cyclooxygenase-2 (COX-2) is correlated with the proliferation and apoptosis of cancer cells and to study whether the RNA interference technique can be used in anti-cancer gene therapy. METHODS: WBH1, a eukaryotic expression plasmid of shRNA targeting on COX-2, was constructed. Human gastric cancer cells of the line SGC-7901 were cultured and divided into 3 groups: to be transfected with WBH1 or negative control plasmid HK, or used as un-transfected control group. RT-PCR and Western blotting were used to detect the expression of COX-2 mRNA and protein. MTT method was used to detect the proliferation of the cells. The apoptosis of the cells was determined by flow cytometry. Fifteen nude mice were randomly divided into 3 equal groups: 10 to be inoculated subcutaneously with WBH1 plasmid transfected SGC-7901 cells (inhibition group) or negative control plasmid HK transfected SGC-7901 cells, and 5 were used as un-transfected controls. The mice were observed for 4 weeks to observe the survival and the tumorigenesis. Then the mice were killed to take out the tumors. The tumorigenic rate and tumor inhibition rate were evaluated. RESULTS: The proliferation of the SGC-7091 cells transfected with WHB1 plasmid did not changed significantly 24 and 48 hours after the transfection, however, decreased significantly 96 hours and 1 week after (both P < 0.01). The apoptotic rate of the SGC-7091 cells transfected with WHB1 plasmid was 52.28% +/- 17.91%, significantly higher than that of the cells transfected with the control plasmid HK (0.54% +/- 0.16%) and that of the un-transfected cells (0.52% +/- 0.27%, both P = 0.009) without a significant difference between the latter 2 groups (P = 0.998). Four weeks after inoculation the tumorigenic rate was 100% in both the un-transfected control mice and the mice inoculated with negative plasmid HK transfected SGC-7901 cells. There was no significant difference in tumor size between these 2 groups (P = 0.965). The tumorigenic rate of the mice in the inhibition group was 0.4 with an inhibition rate of 89.8%. The tumor weight of the inhibition group was 0.050 g +/- 0.003 g, significantly lighter than those of the control group and the group inoculated with negative plasmid transfected SGC cells (0.490 g +/- 0.017 g and 0.490 g +/- 0.013 g respectively, both P < 0.01). CONCLUSION: Construction of a eukaryotic expression vector expressing the specific shRNA targeting on COX-2, closely related to the proliferation and apoptosis of tumor cells, and transfection of it into the tumor cells helps inhibit the expression of COX-1, thus inhibiting the growth and proliferation of the tumor cells.