BRMS1shRNA表达载体的构建及对卵巢癌细胞转移的影响

目的 探讨乳腺癌转移抑制基因1( BRMS1)表达抑制对人卵巢癌细胞转移能力的影响及机制.方法 构建靶向BRMS1基因的短发夹状RNA(shRNA)真核表达载体pGPU6/GFP/Neo-BRMS1,并转染人卵巢癌OVCAR3细胞,经G418筛选获得稳定转染株.采用实时荧光定量PCR和Western blot法分别检测...

Construction of BRMS1 Short Hairpin RNA Vector and Its Effect on Metastasis of Human Ovarian Cancer Cells

Objective To investigate the effect of breast cancer metastasis suppressor 1(BRMS1) knockdown on the metastasis ability of human ovarian cancer cells. Methods Vector contained a short hairpin RNA(shRNA) against BRMS1 was constructed,and the positive clone was named pGPU6/GFP/Neo-BRMS1.BRMS1 shRNA was transfected into OVCAR3 cells,and the stably transfected cells were obtained after being screened with G418.Real time PCR and Western blot were used to detect the mRNA and protein of BRMS1.Cell adhesion was measured by cell adhesion assay.Matrigel invasion and Migration assay were measured by Transwell chamber method.Real time PCR was utilized to detect the expression of miR-146a. Results The accuracy of the constructs was confirmed by restriction endonuclease digesting and DNA sequencing.After the stable transfection of BRMS1 shRNA,the mRNA and protein expressions of BRMS1 in the OVCAR3 cells were efficiently down-regulated.After the down-regulation of BRMS1,the adhesion,invasion and migration of OVCAR3 cells were significantly promoted.Moreover,knockdown of BRMS1 obviously decreased the expressions of miR-146a mRNA by(44.69±4.60)%. Conclusion Knockdown of BRMS1 can promote adhesion,invasion and migration of ovarian cancer cells,and this phenomenon may be related with the down-regulation of miR-146a.