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【目的】应用RNA干扰方法下调鞘氨醇激酶1(SPHK1)基因表达,研究SPHK1对胃癌细胞SGC-7901、MGC-803细胞增殖的影响及其可能的作用机制。【方法】免疫组织化学法检测5对胃癌及癌旁组织的SPHK1蛋白表达量;构建质粒、制备逆转录病毒并感染胃癌细胞株SGC-7901、MGC-803,筛选并鉴定SPHK1-RNAi和对照载体(RNAi-Vector)稳定表达细胞株;MTT法检测胃癌细胞的增殖变化;Western blot法检测SPHK1、p21、p27、Akt、p-Akt、Rb、p-Rb蛋白的表达水平。【结果】SPHK1蛋白在胃癌组织中过表达;与对照组比较,两株SPHK1-RNAi稳定表达的胃癌细胞SGC-7901、MGC-803中SPHK1表达显著下调,抑制率分别为81.2%、65.1%;SPHK1-RNAi胃癌细胞增殖能力较对照组显著减弱;SPHK1-RNAi胃癌细胞的磷酸化Akt及磷酸化Rb蛋白表达明显降低,而p21、p27的蛋白表达水平明显上调。【结论】下调SPHK1基因表达能抑制胃癌细胞的增殖,其机制可能与细胞内Akt信号被减弱,细胞周期抑制蛋白p21、p27的表达上调有关。
【Objective】 The purpose of this study was to investigate the effect of SPHK1 on the proliferation of gastric cancer cells SGC-7901 and MGC-803 by down-regulating the expression of SPHK1 gene by RNA interference. 【Methods】 The expression of SPHK1 protein in gastric cancer tissues and pericarcinoma tissues was detected by immunohistochemistry. The plasmids were constructed and the retroviral vector was constructed and infected into gastric cancer cell lines SGC-7901 and MGC-803. The SPHK1-RNAi and control vector The expression of SPHK1, p21, p27, Akt, p-Akt, Rb and p-Rb in gastric cancer cells were detected by MTT assay and Western blot. 【Results】 SPHK1 protein was overexpressed in gastric cancer tissues. Compared with the control group, SPHK1 expression was significantly down-regulated in two SPHK1-RNAi stable gastric cancer cells SGC-7901 and MGC-803 with the inhibitory rates of 81.2% and 65.1%, respectively. The proliferation ability of SPHK1-RNAi gastric cancer cells was significantly weaker than that of the control group. The expressions of phosphorylated Akt and phosphorylated Rb protein in SPHK1-RNAi gastric cancer cells were significantly decreased, while the expression levels of p21 and p27 were significantly increased. 【Conclusion】 Down-regulation of SPHK1 gene expression can inhibit the proliferation of gastric cancer cells. The mechanism may be related to the decrease of intracellular Akt signaling and the up-regulation of cell cycle arrest proteins p21 and p27.