RNA干扰下调SPHK1基因表达抑制胃癌细胞增殖  

RNA Interference Down-regurates SPHK1 Gene and Inhibits Proliferation of Gastric Cancer Cell

作　　者：张娈景[1] 夏金堂[2] 张珑涓[1] 简卫华[2] 赖越元[2] 陈连周[1] 黄晓卉[1] 李雯[1] 

机构地区：[1]中山大学附属第一医院普通外科实验室,广东广州510080 [2]广州医学院附属广州市第一人民医院肝胆外科,广东广州510180

出　　处：《中山大学学报（医学科学版）》2013年第3期321-325,共5页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(30973395;81172337);广州市医药卫生科技计项目(201102A212012)

摘　　要：【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] To investigate the effects and possible mechanism of SPHK1 on the proliferation of human gastric cancer cell lines SGC-7901,MGC-803 by using RNA interfering methods.[Methods] The expression of SPHK1 protein of five pairs of gastric tumor tissues and adjacent non-tumor tissues was detected by immunohistochemistry.The plasmids were constructed and retroviruses were transfected into SGC-7901 and MGC-803 cells respectively.The stable SPHK1-RNAi and RNAi-Vector cell lines were obtained after screening and identification.Cell proliferation rate was determined by MTT assay.The protein expression levels of SPHK1,p21,p27,Akt,p-Akt,Rb,p-Rb were tested by Western blotting.[Results] SPHK1 protein was overexpressed in gastric tumor tissues.In the SPHK1-RNAi stable cell lines SGC-7901 and MGC-803,the expression of SPHK1 were significantly decreased for 81.2% and 65.1% compared with that in RNAi-Vector control group.The cell proliferation of both SPHK1-RNAi gastric cancer cell lines were also markedly reduced.The phosphorylation levels of Akt and Rb protein were significantly decreased,p21 and p27 protein were increased in SPHK1-RNAi group compared with that in RNAi-Vector group.[Conclusion] The proliferation of gastric cancer cells can be obviously inhibited by down-regulating the expression of SPHK1.The mechanism may be partly related with the Akt-dependent cell cycle checkpoint pathway.

关 键 词：鞘氨醇激酶1 胃癌细胞 细胞增殖 AKT 

分 类 号：R735.2[医药卫生—肿瘤]