miR-200b通过靶向CRKL抑制胶质瘤U251细胞增殖

MiR-200b inhibits the proliferation of glioma U251 cells by targeting CRKL

目的研究hsa-miR-200b对人U251胶质瘤细胞增殖的影响及相关作用机制。方法将miR-200b过表达和表达沉默质粒分别稳定转染至胶质瘤U251细胞,qRT-PCR验证转染效率,CCK-8法检测U251细胞增殖水平变化,qRTPCR以及Western blot方法检测的CRKL的m RNA和蛋白表达水平。双荧光素酶报告基因实验验证miR-200b与CRKL基因3’UTR的结合位点。将CRKL过表达和表达沉默质粒载体分别稳定转染至U251胶质瘤细胞,使用CCK-8法检测U251细胞增殖水平变化。分别建立miR-200b和CRKL的稳定双转染U251细胞系,使用CCK-8法检测对各组细胞增殖水平的变化。结果与对照组相比,miR-200b过表达显著抑制胶质瘤U251细胞的增殖并且抑制CRKL在U251胶质瘤细胞的m RNA和蛋白表达;双荧光素酶报告基因实验验证了miR-200b与CRKL基因3’UTR的结合位点;CRKL过表达显著增强胶质瘤U251细胞增殖能力;CRKL过表达有效阻断了miR-200b抑制胶质瘤U251细胞增殖的作用。结论 miR-200b能够通过靶向下调CRKL抑制胶质瘤U251细胞增殖。

Objective The effect and potential mechanism of proliferation of human glioma U251 cells regulated by miR- 20Oh. Methods Human glioma U251 cells were respectively transfected with miR-200b over-expression and silencing plasmid vec- tors, the efficience of transfeetion was tested by qRT-PCR, CCK-8 was applied to detect proliferation of glioma U251 cells. Dual-Lucif- erase Reporte Assay was used to verify the combination site of miR-200b and CRKL. qRT-PCR and Western blot assays were applied to measure the variation of CRKL in glioma U251 cells on mRNA and protein levels. Human glioma U251 cells were respectively trans- fected with CRKL over-expression and silencing plasmid vectors, CCK-8 was applied to detect proliferation of glioma U251 cells. Glio- ma U251 cell lines inversely coregulated by miR-200b and CRKL were established, CCK-8 was applied to detecting the variation of proliferation in glioma U251 cells. Results Compared with control group, over-expression of miR-200b significantly inhibited the pro- liferation, obviously brought down the expression level of CRKL on mRNA and protein levels in glioma U251 cells. Over-expression of CRKL improved the proliferation of human glioma U251 cells and effectively blocked the efforts of miR-200b inhibiting proliferation of U251 glioma cells. Conclusion MiR-200b inhibits proliferation of glioma U251 cells by targeting CRKL.