反义miR-221/222上调Cx43恢复人胶质瘤细胞系U251细胞间缝隙连接通讯的体外研究  

Study on antisense miR-221/222 recovers GJIC of U251 cells through up-regulating Cx43 in vitro

作　　者：张春智[1,2] 康春生[1] 杨卫东[1] 王广秀[1] 张安玲[1] 浦佩玉[1] 

机构地区：[1]天津医科大学总医院神经外科天津市神经病学研究所神经肿瘤实验室,300052 [2]天津市环湖医院神经外科,300060

出　　处：《中国现代神经疾病杂志》2009年第6期578-582,共5页Chinese Journal of Contemporary Neurology and Neurosurgery

基　　金：国家自然科学基金资助项目(项目编号:30772231);国家自然科学基金资助项目(项目编号:30901772);教育部新世纪优秀人才支持计划资助项目(项目编号:NCET-07-0615);天津市科技计划项目(项目编号:07ZCGHHZ01000)

摘　　要：目的探讨反义miR-221/222上调缝隙连接蛋白43(Cx43)以恢复人胶质瘤细胞系U251细胞间缝隙连接通讯的作用机制。方法脂质体共转染反义寡核苷酸(AS-miR-221/222),下调U251细胞miR-221和miR-222表达水平,采用Northern blotting法检测U251细胞miR-221和miR-222表达水平;虫荧光素酶活性分析并获得靶基因;Western blotting法和免疫荧光法检测U251细胞Cx43表达水平;划痕标记染料示踪技术检测细胞间缝隙连接通讯。结果 AS-miR-221/222组U251细胞miR-221(t=1312.152,P=0.000)和miR-222(t=1226.031,P=0.000)表达水平明显降低;荧光活性明显高于其他各组(均P=0.000),证实Cx43基因为miR-221和miR-222靶基因;Cx43表达水平亦明显升高,且高于无义序列组(t=735.768,P=0.000)和对照组(t=686.252,P=0.000)。对照组和无义序列组U251细胞细胞间缝隙连接通讯缺失,罗氏黄仅传递划痕细胞边缘单列细胞;AS-miR-221/222组U251细胞细胞间缝隙连接通讯明显恢复,罗氏黄传递至划痕细胞邻近7～8列细胞。结论反义miR-221/222可通过上调Cx43表达水平恢复人胶质瘤细胞系U251细胞间缝隙连接通讯。Objective To explore the mechanism of antisense microRNA-221/222 （AS-miR-221/ 222） recovers gap junction intercellular communication （GJIC） of human glioma cell line U251 by up- regulating eonnexin 43 （Cx43）. Methods Co-transfect AS-miR-221/222 and Lipofectamine were used to down-regulate the miR-221 and miR-222 expressions of U251 cells in vitro. Northern blotting was conducted to detect the miR-221 and miR-222 expressions of U251 cells after transfection. Luciferase report was used in determining Cx43 to be a target gent of miR-221 and miR-222. Cx43 expression was detected by Western blotting and immunofluorescence. GJIC was detected by scrape loading dye transfer （SLDT）. Results The expressions of miR-221 （t = 1312.152, P = 0.000） and miR-222 （t = 1226.031, P= 0.000） of U251 cells in AS-miR-221/222 group were significantly lower than those in nonsense sequential group and control group. The fluorescence activity of U251 cells in AS- miR- 221/222 group was significantly higher than that in other groups （P = 0.000, for all）. Cx43 was confirmed to be the target gene of miR-221 and miR-222. Western blotting showed that Cx43 expression was significantly higher in AS-miR-221/222 group than that in nonsense sequential group （t = 735.768, P = 0.000） and control group （t = 686.252, P = 0.000）. Immunofluorescence showed that Cx43 expression significantly increased in AS-miR-221/ 222 transfected U251 cells. GJIC of U251 cells was deficient in control group and nonsense sequential group. Roche yellow fluorescence dye only transfered to monolayer cells adjacent to the scraped cells. After transfected AS-miR-221/222, GJIC was recovered and Roche yellow fluorescence dye transfered to 7-8 layer ceils adjacent to the scraped cells. Conclusion By up-regulating Cx43 expression, AS-miR-221/222 can recover GJIC of U251 cells.

关 键 词：RNA 反义 RNA 小分子干扰 连接蛋白43 脑肿瘤 神经胶质瘤 缝隙接合部 转染 细胞 培养的 

分 类 号：R739.4[医药卫生—肿瘤]