miR-17对靶基因转化生长因子受体β_2的负性调控表达  

Negative regulation of miR-17 to target genes transforming growth factor receptor beta 2

作　　者：赵洋[1] 张魁[1] 董然[1] 

机构地区：[1]首都医科大学附属北京安贞医院北京市心肺血管疾病研究所心脏外科,北京100029

出　　处：《中华实用诊断与治疗杂志》2015年第2期129-131,135,共4页Journal of Chinese Practical Diagnosis and Therapy

基　　金：国家自然科学基金(81270300)

摘　　要：目的探讨miR-17在HEK293T细胞中对转化生长因子受体β2(transforming growth factor receptor beta 2,TGFRβ2)的调控作用。方法采用生物学信息预测软件对miRNA-17的靶基因进行预测,选定TGFRβ2为靶基因,构建pCDNA3.1+miRNA-17重组表达载体。将HEK293T细胞分为3组,分别为对照组(无转染)、空载组(转染空质粒)和miR-17组(转染pcDNA3.1-GW/EmGFP-miR-17质粒),3组采用实时定量PCR检测miRNA-17和TGFRβ2mRNA表达水平,采用Western blot检测TGFRβ2蛋白表达水平。将HEK293T细胞再分为3组,分别为CTL组(转染miR-17无义寡核苷酸序列)、miR-17组(转染pcDNA3.1-GW/EmGFP-miR-17质粒)和ASO-miR-17组(转染miR-17反义寡核苷酸序列),采用免疫荧光法检测各组TGFRβ2荧光强度。结果转染24h后,空载组和miR-17组HEK293细胞处于对数生长期,细胞突起和包体均正常,与对照组比较差异无统计学意义(P>0.05);miR-17组miR-17表达量(5.391±0.053)明显高于空载组(1.654±0.075)与对照组(1.436±0.038),TGFRβ2mRNA和蛋白表达量(2.449±0.092、1.030±0.030)明显低于空载组(6.135±0.058、5.550±0.040)和对照组(6.862±0.074、5.090±0.030)(P<0.05);ASO-miR-17组TGFRβ2荧光强度(1.144±0.079)明显高于CTL组(0.776±0.044)和miR-17组(0.917±0.096)(P<0.05)。结论 miR-17可负性调节靶基因TGFRβ2的表达。Objective To discuss the regulatory role of miR-17 in transforming growth factor receptor beta 2（TGFRβ2）in HEK293 cell.Methods Biological information prediction software was used to predict target gene of miRNA-17,and TGFRβ2was designated as target gene to establish pCDNA3.1＋miRNA-17 recombinant expression vector.HEK293 T cells were divided into three groups：control group with no transfection,empty retrovirous group transfected by BC-V,and miR-17 group transfected by pcDNA3.1-GW/EmGFP-miR-17 plasmid.The expressions of miRNA-17 and TGFRβ2mRNA were detected by real-time PCR,and the expression of TGFRβ2 protein was detected by Western blot in three groups.HEK293 Tcells were redivided into three groups：CTL group transfected by miR-17 nonsense oligonucleotide sequence,miR-17 group transfected by pcDNA3.1-GW/EmGFP-miR-17 plasmid and ASO-miR-17 group transfected by miR-17 antisense oligonucleotides sequence.The TGFRβ2fluorescence intensity was detected by immunofluorescence in three groups.Results After transfection for 24 hours,empty retrovirus group and miR-17 group were in logarithmic phase,and both cell ecptomas and enclosed object were normal,showing no significant differences in comparison with control group（P〉0.05）.The miR-17 expression was significantly higher in miR-17group（5.391±0.053）than that in empty retrovirous group（1.654±0.075）and control group（1.436±0.038）,the TGFRβ2mRNA and protein expressions were significantly lower in miR-17group（2.449±0.092,1.030±0.030）than those in empty retrovirous group（6.135±0.058,5.550±0.040）and control group（6.862±0.074,5.090±0.030）（P〈0.05）.The TGFRβ2fluorescence intensity was significant higher in ASO-miR-17group（1.144±0.079）than that in CTL group（0.776±0.044）and miR-17group（0.917±0.096）（P〈0.05）.Conclusion miR-17 can negatively regulate the expression of target gene TGFRβ2.

关 键 词：miR-17 转化生长因子受体β2 HEK293T细胞 靶基因 

分 类 号：R346[医药卫生—基础医学]