BRMS1 shRNA表达载体的构建及对卵巢癌细胞转移的影响

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

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BRMS1 mRNA和蛋白的表达,采用黏附实验检测细胞的黏附能力,采用Tr-answell小室法检测细胞的体外侵袭和迁移能力,采用实时荧光定量PCR法检测基因沉默后miR-146a的表达。结果经酶切和DNA测序证明重组载体构建成功。稳定转染BRMS1 shRNA后,OVCAR3细胞中BRMS1 mRNA和蛋白表达均受到明显抑制。BRMS1表达下调后,细胞的黏附、侵袭和迁移能力较对照组明显增强。干扰组细胞的miR-146a mRNA表达下调了(44.69±4.60)%。结论干扰BRMS1基因的表达可促进卵巢癌细胞的黏附、侵袭和迁移,其机制可能与下调miR-146a的表达有关。

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 arid 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.