应用RNA干扰技术抑制人胃癌细胞环氧合酶-2基因的表达研究

Inhibitory Effects on the Expression of COX-2 in Human Gastric Cancer Cells by RNA Interference

目的研究应用RNA干扰技术进行抑制胃癌SGC-7901细胞COX-2的表达研究。方法依据siRNA设计原则，针对人COX-2的mRNA序列，设计并合成3条21bp的双链RNA，瞬时转染人胃癌细胞SGC-7901，Western印迹t验证基因沉默效率以筛选合适的siRNA序列；根据筛选出的有效序列，合成编码短发卡RNA的双链寡核苷酸，定向克隆到含有U6RNA聚合酶Ⅲ启动子的pSilencer^TM2．1-U6neo真核表达载体，稳定转染胃癌细胞SGC-7901中，经G418筛选，获得克隆细胞。结果筛选出一条有效的干扰序列，位于291—311bp，成功构建表达载体，COX-2在蛋白水平的表达明显被抑制。结论利用化学合成siRNA进行初筛，再构建shRNA表达载体，可快速实现目的基因的表达抑制。

Objective To inhibit the expression of COX-2 in gastric cell line SGC-7901 by RNAi method. Methods According to the mRNA sequence of COX-2, we synthesized 3 pieces of 21bp dsRNA and then instantly transfected them into gastric cell SGC-7901, and finally detected the silencing effect by westernblot method. Next, the shRNA of the most effect sequence was cloned-into the pSilencerTM 2.1-U6 neo eukarya expression vector, and then was stably transfected into SGC-7901 cells, and the clone cells were achieved by G418 screening. Results An effective silencing sequence was gained, which was located in the 291 - 311 bp. Besides,the express vector was successfully constructed and the protein expression level of COX- 2 was obviously decreased. Conclusion The expression of target gene could be rapidly inhibited by using the method of primary screening the effect of chemical synthesis siRNAs and then constructing the expression vector of the most effective one.