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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 investigate the regulatory effect of miR-17 on transforming growth factor receptor beta 2 (TGFRβ2) in HEK293T cells. Methods The biological information prediction software was used to predict the target gene of miRNA-17. TGFRβ2 was selected as the target gene to construct pCDNA3.1 + miRNA-17 recombinant expression vector. HEK293T cells were divided into 3 groups: control group (no transfection), empty group (transfected with empty plasmid) and miR-17 group (transfected with pcDNA3.1-GW / EmGFP-miR- Real-time quantitative PCR was used to detect the expression of miRNA-17 and TGFRβ2 mRNA. Western blot was used to detect the expression of TGFRβ2 protein. HEK293T cells were further divided into 3 groups: CTL group (transfected with miR-17 non-sense oligonucleotide sequence), miR-17 group (transfected with pcDNA3.1-GW / EmGFP-miR- -miR-17 group (transfected with miR-17 antisense oligonucleotide sequence), and the fluorescence intensity of TGFRβ2 in each group was detected by immunofluorescence. Results HEK293 cells in no-load group and miR-17 group were in logarithmic phase after transfected with HEK293 cells for 24 hours. The number of cells in the HEK293 cells was normal and there was no significant difference compared with the control group (P> 0.05) (5.391 ± 0.053) were significantly higher than those in control group (1.436 ± 0.038), TGFβ1 mRNA and protein expression (2.449 ± 0.092, 1.030 ± 0.030) (1.144 ± 0.079) in ASO-miR-17 group was significantly higher than that in CTL group (0.776 ± 0.044), which was significantly higher than that in CTL group (6.135 ± 0.058,5.550 ± 0.040) and control group (6.862 ± 0.074,5.090 ± 0.030) And miR-17 group (0.917 ± 0.096) (P <0.05). Conclusion miR-17 can negatively regulate the expression of target gene TGFRβ2.