miR-145靶向调控KLF4参与人骨髓间充质干细胞向血管平滑肌细胞分化过程中的研究

MiR-145 targeting regulation of KLF4 involved in the differentiation of human bone marrow mesenchymal stem cells into vascular smooth muscle cells

目的探究miR-145靶向调控Krüppel样因子4(KLF4)参与人骨髓间充质干细胞(BMSCs)向血管平滑肌细胞(VSMC)分化的机制。方法将人BMSCs细胞分为阴性对照组、miR-145组、miR-145+KLF4组和KLF4组。miR-145组转染miR-145类似物以过表达miR-145;KLF4组转染KLF4 pcDNA;miR-145+KLF4组同时转染miR-145类似物和KLF4 pcDNA。培养48 h后,用实时荧光定量聚合酶链反应法检测KLF4 mRNA和miR-145的表达水平,用免疫荧光染色检测SMα-actin和SM calponin蛋白的表达水平。结果培养48 h后,阴性对照组、miR-145组、KLF4组和miR-145+KLF4组的miR-145表达量分别为1.49±0.12,5.76±0.24,1.54±0.15和5.68±0.25,KLF4 mRNA表达量分别为2.51±0.13,0.81±0.04,8.63±0.30和3.51±0.16,SMα-actin蛋白相对表达量分别为2.18±0.17,0.97±0.08,6.55±0.28和2.93±0.22,SM calponin蛋白相对表达量分别为1.22±0.13,0.56±0.04,3.91±0.25和1.93±0.10。miR-145组的上述指标与阴性对照组比较,差异均有统计学意义(均P<0.05)。结论在BMSCs中过表达miR-145会通过靶向KLF4,进而抑制SMα-actin和SM calponin蛋白的表达,从而抑制BMSCs向VSMC转化。

Objective To explore the mechanism of miR-145 targeting and regulating Krüppel-like factor 4(KLF4)involved in the differentiation of human bone marrow mesenchymal stem cells(BMSCs)into vascular smooth muscle cells(VSMC).Methods Human BMSCs cells were divided into negative control group,miR-145 group,miR-145+KLF4 group and KLF4 group.The miR-145 group was transfected with miR-145 mimic to overexpress miR-145.KLF4 group was transfected with KLF4 pcDNA.The miR-145+KLF4 group was transfected with miR-145 mimic and KLF4 pcDNA simultaneously.Real-time fluorescence quantitative polymerase chain reaction was used to detect the expression levels of KLF4 mRNA and miR-145.Immunofluorescence staining was used to detect the expression levels of SMα-actin and SM calponin proteins.Results After 48 h of culture,the expression levels of miR-145 in the negative control group,miR-145 group,KLF4 group and miR-145+KLF4 group were 1.49±0.12,5.76±0.24,1.54±0.15 and 5.68±0.25,the expression levels of KLF4 mRNA were 2.51±0.13,0.81±0.04,8.63±0.30 and 3.51±0.16,the expression levels of SMα-actin protein were 2.18±0.17,0.97±0.08,6.55±0.28 and 2.93±0.22,the expression levels of SM calponin protein were 1.22±0.13,0.56±0.04,3.91±0.25 and 1.93±0.10,respectively.There were significant differences in the above-mentioned indexes between the miR-145 and KLF4 group and the negative control group(all P<0.05).Conclusion Overexpression of miR-145 in BMSCs can inhibit the expressions of SMα-actin and SM calponin protein through targeting KLF4,and inhibit the transformation of BMSCs to VSMC.