微小RNA-83a靶向作用对体外骨髓间充质干细胞诱导分化的影响及其机制

The effect and mechanism of microRNA-83a targeting on differentiation of bone marrow mesenchymal stem cells in vitro

目的观察微小RNA（miRNA，miR）-83a表达对体外骨髓间充质干细胞（BMSC）的诱导分化和生物学功能的影响。方法将实验分为3组：A组：PEF-miR-83a和PSV2neo共转染组；B组：未转染组；C组：PEF和PSV2neo共转染组。A组为实验组，B、C两组为对照组。按照说明书进行转染，加入400 μg PmlG419筛选，经3周后形成阳性克隆，扩增培养，应用基因转录方法检测miR-83a表达对体外BMSC的抗分化作用和生物学功能的影响；通过免疫组织化学法和实时定量聚合酶链反应（Real-time PCR）技术检测miR-83a在细胞中的表达，Real-time PCR测定转染后A549细胞中Ras、Raf、蛋白激酶B（Akt）和细胞外信号调节激酶（ERK） mRNA水平表达变化，并通过噻唑蓝（MTT）法、划痕实验、苏木素-伊红（HE）染色、显微镜等观察比较转染miR-83a的细胞的的迁移能力，诱导分化和形态学改变。结果Real-time PCR和免疫组织化学检查结果证实转染后细胞中miR-83a稳定表达；阿辛兰染色结果：培养第7天，诱导分化组细胞内充满了大量的棕色特异性染色颗粒，未诱导分化组未见特异性染色颗粒。Real-time PCR结果：A组细胞Ras、Raf、Akt mRNA相对表达量分别为0.97±0.27、0.54±0.18和0.99±0.31, B组细胞为1.79±0.36、1.70±0.33和2.06±0.42，C组细胞为1.68±0.30、1.58±0.25和1.89±0.31。和B、C组比较，A组细胞中Ras、Raf和Akt mRNA水平显著降低（t=1.531、1.838，P=0.016、0.011） 。MTT结果表明随着miR-83a与细胞效靶作用时间延长，抑制率明显增强，作用24、48、72 h后，细胞的凋亡率分别为（23.49±4.64）%、（45.73±8.72）%和（59.25±13.16）%，不同作用时间比较差异有统计学意义（χ^2=5.203、2.146，P=0.013、0.045）。划痕实验结果显示：miR-83a转染的细胞迁移能力低于正常细胞。miR-83a作用于细胞24 h后，形态学观察细胞发生凋亡或坏死。结论miR-83a转染对体外BMSC具有抑制分化和促进凋亡作用，它可能是通过抑制表皮生长因子受体（EGFR）和EGFRvⅢ信号传导通路中的RAS-RAF-丝裂原活化蛋白激酶（MAPK）和磷酸肌醇3激酶（PI3K）-Akt两条重要信号通路分子的活性来发挥作用。

Objective To study the anti-proliferation and inducing apoptosis effects of microRNA （miRNA, miR）-83a on bone marrow mesenchymal stem cells.Methods The experiment was divided into 3 groups： group A （PEF miR-83a and PSV2neo co-transfection group）, group B （non-transfection group）, and group C （PEF and PSV2neo co-transfection group）. Group A served as the experimental group, and groups B and C group as the control groups. Transfection according to instructions, effects of application of expression detected miR-83a transcription of bone marrow mesenchymal stem cells lines in vitro antiproliferative effect of and biological function; by detecting the expression level of miR-83a immunohistochemical method and real-time quantitative polymerase chain reaction （Real-time PCR） technology in cells, Real-time PCR was measured after transfection, bone marrow mesenchymal stem cells lines Ras, Raf, protein kinase B （Akt） and extracellular signal-regulated kinase （ERK）： mRNA expression changes, and through the methyl thiazol tetrazolium （MTT） method, the scratch test, hematoxylin and eosin （HE） staining, microscope observation and comparison of miR-83a transfection on the migration ability of cell proliferation, apoptosis and morphological changes. To analyse by using SPSS 14.0 statistical software.Results Real-time PCR and immunohistochemistry confirmed in miR-83a transfected cells with stable expression; Assingland staining results： after seventh days culture, the cells of the induction differentiation group were full of brown specific staining granules, and no specific staining granules were found in the undifferentiated differentiation group. Real-time PCR results： The expression of Ras, Raf and Akt mRNA in group A were 0.97±0.27, 0.54±0.18 and 0.99±0.31, in group B were 1.68±0.30, 1.58±0.25 and 2.06±0.42, in group C were 1.68±0.30, 1.58±0.25 and 1.89±0.31. Compared with group B and C, the levels of Ras, Raf and Akt mRNA in group A were significantly lower （t=1.531 and 1.838, P=0.016 and 0.011）. MTT assay showed that the apoptotic rates of the cells were （23.49±4.64）%, （45.73±8.72）% and （59.25±13.16）% after 24, 48 and 72 h, respectively. There was significant difference between the two groups （χ^2= 5.203 and 2.146, P =0.013 and 0.045）. The scratch test showed that miR-83a transfected cell migration ability is lower than the normal cells. The effect of miR-83a on lung cancer cell 24 h, morphological observation of lung cancer cell apoptosis or necrosis.Conclusion Transfection of miR-83a can inhibit proliferation and promote apoptosis effect on pituitary adenoma cells in vitro cells, which is likely to play a role through inhibition of epidermal growth factor receptor （EGFR） and EGFRvⅢ signal transduction pathway in the RAS-RAF-mitogen-activated protein kinase （MAPK） and phosphatidylinositol 3 kinase （PI3K）-Akt of two important signaling pathways activated.