胃腺癌细胞中miR-23a靶基因PPP2R5E的鉴定  

Identification of mi R-23a targeted gene PPP2R5E in gastric adenocarcinoma cell line

作　　者：朱丽华[1] 李盖[2] 刘爱华[1] 张科[3] 李冀[1] 章广玲[1] 

机构地区：[1]河北联合大学基础医学院病原生物学科,河北唐山063000 [2]河北联合大学附属医院放射科,河北唐山063000 [3]河北联合大学继续教育学院,河北唐山063000

出　　处：《中国现代医学杂志》2015年第5期1-7,共7页China Journal of Modern Medicine

基　　金：河北省科学技术厅项目(No:132777138;10276158);国家自然科学青年基金(No:81201281/H1904);河北省自然科学基金(No:C2012401037;H2013209180)

摘　　要：目的利用双荧光蛋白报告基因分析系统验证mi R-23a的靶基因PPP2R5E。方法选取表达绿色荧光蛋白的质粒pc DNA3/EGFP,将PPP2R5E-3’UTR的特异性序列插入其中,并与红色荧光蛋白表达质粒p Ds Red2-N1及表达mi R-23a的质粒共同稳定转染胃腺癌细胞MGC803,稳定转染后提取细胞蛋白,荧光分光光度计进行定量检测。并通过半定量RT-PCR、实时定量PCR检测mi R-23a功能受抑制或过表达mi R-23a的MGC803细胞或胃腺癌组织中PPP2R5E基因m RNA的表达情况,验证mi R-23a对其调控作用。表达si RNA抑制MGC803中PPP2R5E m RNA的表达后,通过MTT和平板克隆形成实验观察其对胃腺癌细胞系MGC803细胞活性和克隆形成能力的影响。结果绿色荧光蛋白的表达量,稳定转染pc DNA3/EGFPPPP2R5E-3’UTR质粒和mi R-23a质粒组明显低于pc DNA3/EGFP-PPP2R5E-3’UTR和pc DNA3共同稳定转染组。mi R-23a功能受抑制的胃腺癌细胞MGC803中靶基因PPP2R5E的m RNA表达水平升高;相反,过表达mi R-23a的胃腺癌细胞MGC803及胃腺癌组织中靶基因PPP2R5E的m RNA表达水平下降。沉默MGC803细胞中的PPP2R5E后,细胞活性和克隆形成能力均加强。结论 PPP2R5E可能是mi R-23a的直接靶基因。【Objective】 To identify the mi R-23 a targeted gene PPP2R5 E with a dual fluorescent protein reporter assay system and then to detect the expression of PPP2R5 E in gastric adenocarcinoma cell line MGC803.【Methods】A sequence of PPP2R5E-3’UTR（untranslated region） was inserted into the plasmid which expressed green fluorescent protein（pc DNA3/EGFP）. This plasmid（pc DNA3/EGFP-PPP2R5E-3’UTR） and mi R-23 a and the plasmid expressing red fluorescent protein（p Ds Red2-N1） were co-transfected into MGC803 cells.The extracted protein was detected under the fluorescence spectrophotometer. Subsequently, the m RNA levels of the target gene in mi R-23a-inhibited, over-expressed gastric adenocarcinoma cells and tissues were detected with semi-quantitative RT-PCR and quantitative real-time PCR, in order to verify the regulating role of mi R-23 a in the target gene expression. After PPP2R5 E m RNA in MGC803 was inhibited with the corresponding si RNA, the changes of cell growth were detected with MTT assay and plate colony formation assay.【Results】 After mi R-23 a and the plasmid of pc DNA3/EGFP-PPP2R5E-3’UTR being co-transfected, the idensity of green fluorescent protein was significantly lower than in that group of co-transfected pc DNA3/EGFP-PPP2R5E-3’UTR with pc DNA3. In the mi R-23 a inhibited gastric adenocarcinoma cells, the m RNA level of PPP2R5 E was enhanced. And in the mi R-23 a over-expressed gastric adenocarcinoma cells or tissues, the m RNA level of PPP2R5 E was both inhibited. After PPP2R5 E m RNA in MGC803 was inhibited with the corresponding si RNA, the cell growth activity and colony formation activity were both promoted. 【Conclusion】PPP2R5E may be a direct target gene of mi R-23 a.

关 键 词：胃腺癌 MGC803 MI R-23a PPP2R5E 靶基因 

分 类 号：R735.2[医药卫生—肿瘤]