中文摘要：

目的研究miR-429对人U87胶质瘤细胞增殖和凋亡的影响及可能机制。方法培养人U87胶质瘤细胞,应用LipofectamineLTX试剂将pre-miR-429质粒载体以及anti-miR-429质粒载体分别稳定转染人U87胶质瘤细胞;应用Real-time-PCR验证转染效率;应用CCK-8试剂盒检测miR-429对人U87胶质瘤细胞增殖能力的影响;应用Real-time PCR和Western blot检测人U87胶质瘤细胞中接头蛋白CRKL（v-crk avian sarcoma virus CT10oncogene homolog-like,CRKL）的mRNA和蛋白表达含量变化;将CRKL分别转染至U87胶质瘤细胞和miR-429过表达的U87胶质瘤细胞,应用CCK-8试剂盒检测人U87胶质瘤细胞增殖,应用流式细胞仪检测细胞凋亡的变化,应用Western blot法检测凋亡相关蛋白caspase-3和Bcl-2的蛋白表达变化。结果与对照组相比,miR-429过表达显著抑制了人U87胶质瘤细胞增殖,明显诱导了凋亡发生,显著降低了CRKLmRNA和蛋白在人U87胶质瘤细胞的表达水平,caspase-3的表达水平显著上调,Bcl-2的表达水平显著降低。CRKL过表达显著增强了人U87胶质瘤细胞增殖能力并且抑制了细胞凋亡,caspase-3的表达水平显著下调,Bcl-2的表达水平显著增强;CRKL过表达有效阻断了miR-429上调caspase-3、降低Bcl-2的作用,有效阻断了miR-429抑制U87胶质瘤细胞增殖和诱导凋亡的作用。结论 MiR-429抑制CRKL从而降低人U87胶质瘤细胞的增殖能力,诱导细胞凋亡。

英文摘要：

Objective To study the effect and potential mechanism of proliferation and apoptosis of human glioma U87 cells regulated by miR-429.Methods Human U87 glioma cells were cultured,LipofectamineLTX was applied to transfect U87 glioma cells with pre-miR-429 and anti-miR-429 plasmid vectors respectively,the efficiency of transfection was tested by real-time PCR,CCK-8 kit was applied to detect the proliferation of U87 glioma cells.Real-time PCR and Western blot were applied to measure the variation of CRKL（v-CRK avian sarcoma virus CT10-homologlike） mRNA and protein in U87 glioma cells.The normal U87 cells and miR-429 overexpression U87 cells were transfected with CRKL,CCK-8 kit was applied to detect the proliferation of U87 cells.The cell apoptosis was studied by flow cytometer,Western blot was applied to test the variation of apoptosis related protein caspase-3 and Bcl-2.Results Compared with control group,overexpression of miR-429 significantly inhibited the proliferation of U87 cells,and induced the apoptosis of U87 cells,obviously downregulated the expression level of CRKL mRNA and protein in U87 glioma cells,upregulated caspase-3 expression and reduced Bcl-2 expression greatly.The overexpressed CRKL dramaticaly promoted the proliferation of human U87 glioma cells and inhibited the apoptosis,largely reduced caspase-3 expression level,greatly increased Bcl-2 expression level.The overexpression of CRKL effectively blocked the efforts of miR-429 up-regulating caspase-3 and down-regulating Bcl-2,blocked the function of miR-429 inhibiting proliferation and inducing apoptosis.Conclusion MiR-429 inhibits proliferation and induces apoptosis of U87 glioma cells by down-regulating CRKL.