RORα miRNA转染对DADS抑制胶质瘤U251细胞增殖的影响  

Effect of RORα-miRNA transfection on proliferation inhibition of human glioma cell line U251 by diallyl disulfide

作　　者：凌晖[1] 李振丰[1] 曾铁兵[1] 文玲[1] 刘芳[1] 李艳兰[1] 朱亚平[1] 

机构地区：[1]南华大学病理学教研室病原生物学研究所,湖南衡阳421001

出　　处：《中国癌症杂志》2011年第2期99-102,共4页China Oncology

基　　金：国家自然科学基金资助项目(No:81071966);湖南省高校创新平台开放基金项目(No:09K074);湖南省高校重点实验室(肿瘤细胞与分子病理学)资助

摘　　要：背景与目的:维甲酸相关孤核受体α(retinoid acid receptor related orphan receptorα,RORα)可能参与肿瘤的调控。本实验室前期研究发现,二烯丙基二硫(diallyl disulfide,DADS)可抑制人胶质瘤U251细胞增殖,其抑制增殖作用可能与诱导RORα蛋白表达上调有关。为了明确RORα在DADS抑制人胶质瘤细胞增殖中的作用,本研究采用miRNA干扰技术抑制RORα表达,观察其对DADS抑制U251细胞增殖的影响。方法:首先将RORαmiRNA转染人胶质瘤U251细胞,运用Western blot检测转染前后RORα蛋白表达情况。实验分为未转染组、脂质体转染组和RORαmiRNA转染组及分别经30 mg/L DADS处理后的3组,共6组。MTT法检测RORα表达下调对DADS抑制胶质瘤U251细胞增殖的影响。结果:Western blot结果显示,转染RORαmiRNA细胞的RORα蛋白表达(0.09±0.05)明显低于未转染组(0.81±0.11)和脂质体转染组(0.89±0.15),其蛋白表达下调了89.5%(P<0.05)。MTT结果显示,U251细胞增殖活性在RORαmiRNA转染后48 h A570值为(0.98±0.15),高于未转染组(0.47±0.11)和脂质体转染组(0.45±0.10)(P<0.05),其增殖率高达108.5%。并且RORαmiRNA转染削弱了DADS对U251细胞的增殖抑制作用,转染RORαmiRNA细胞加入DADS后的细胞增殖率(A570值为0.69±0.20)明显高于DADS处理的未转染组(0.28±0.13)和脂质体转染组(0.25±0.12)(P<0.05),其抑制率由40.4%下降为29.6%。结论:miRNA干扰RORα表达可促进U251细胞增殖,并且削弱了DADS对U251细胞的增殖抑制作用,说明RORα参与了DADS抗胶质瘤U251细胞增殖的作用。Background and purpose： RORα protein may play a role in regulating tumor growth. In a previous study, growth inhibition was seen in glioblastoma cell line U251 treated with diallyl disulfide （DADS）, where involvement of up-regulated ROR alpha protein is suspected. In this study, miRNA that targeted the RORα gene was transfected into U251 cells in order to supply theoretical evidence for the role of RORα as a new target of malignant glioma in the DADS-induced inhibition effect. Methods： RORα miRNA plasmid was transfected into human brain glioblastoma U251 cells. Expression of RORα proteins were displayed through Western blot. The U251 cells were divided into 6 groups： untransfection, Lipofectamine^TM2000 and RORα-miRNA transfected group, and the same 3 groups treated with DADS. The impact on proliferation of DADS-induced U251 cells was investigated by MTT methods after the inhibition of RORα with miRNA. Results： Western blot showed that the U251 ceils transfected with RORα miRNA plasmid resulted in a dramatic down-regulation of RORα protein. The gray values of RORα protein in the RORα miRNA transfected group was （0.09±0.05）, which was lower than the untransfected group （0.81±0.11） and the liposome transfected group （0.89±0.15）. The RORα expression level in the RORα miRNA transfected group wasreduced by 89.5% as compared to that in the untransfected control group （P〈0.05）. The A570 value of the RORct miRNA 48 h transfected group （0.98~0.15） was higher than in the untransfected group （0.47~0.11） as well as the liposome transfected group （0.45i0.10）, as shown in results using the MTT method （P〈0.05）. After being treated with DADS, the A570 value in the RORct miRNA transfected group （0.69~0.20） was far lower than in the two other groups （0.28±0.13） and （0.25±0.12）, respectively （P〈0.05）, while the inhibition ratio of proliferation was reduced from 40.4% to 29.6%. Conelusion： Down regulation of RORct expression by miRNA interference

关 键 词：胶质瘤 维甲酸相关孤核受体α 微小RNA 二烯丙基二硫 增殖 U251细胞 

分 类 号：R730.264[医药卫生—肿瘤]