【摘要】 目的探讨乳腺癌转移抑制基因1(BRMS1)对c13*细胞转移侵袭、粘附的影响及机制。方法由质粒PC-MV-HA-BRMS1中扩增目的基因BRMS1,将其插入pcDNA3.1(+)质粒中,构建pcDNA3.1(+)-BRMS1质粒。经酶切及测序鉴定正确后,转染c13*细胞,PCR、Real-time PCR、Western blot检测BRMS1的表达;划痕实验、Transwell实验、粘附实验检测c13*细胞运动、侵袭、粘附能力;Western blot检测整合素β1、p-AKT、AKT表达。结果成功构建了pcDNA3.1(+)-BRMS1质粒;转染目的基因的c13*细胞中,BRMS1的mRNA和蛋白表达均显著增加,细胞运动、侵袭、粘附能力降低,整合素β1及p-AKT表达均下降,AKT表达无改变。结论成功构建pcDNA3.1(+)-BRMS1质粒。BRMS1可能通过抑制整合素β1/AKT通路,抑制卵巢癌c13*细胞的运动、侵袭和粘附能力。更多还原

【Abstract】 Objective To construct and express the recombinant plasmid of pcDNA3.1(+)-BRMS1 in ovarian cancer cells(c13*),and observe the effects of BRMS1 on migration,invasion and adhesion of c13* cells.Methods BRMS1 cDNA was obtained from the plasmid PCMV-HA-BRMS1 by PCR,and inserted into pcDNA3.1(+)plasmid to generate a recombinant plasmid pcDNA3.1(+)-BRMS1.The recombinant plasmid was verified by sequencing and enzyme digestion analysis,and transfected into c13* cells by using liposome.The BRMS1 expression was detected by PCR,real-time PCR,and Western blot after transfection.The effects of BRMS1 overexpression on the migration,invasion and adhesion of c13* were investigated by wound-healing,Transwell,and adhesion assay respectively.At last,the expression of integrin beta 1,p-AKT,and AKT was detected by Western blot after transfection.Results pcDNA3.1(+)-BRMS1 recombinant plasmid was constructed and transfected into c13* cells successfully,and BRMS1 was over-expressed.The migration,invasion,and adhesion capability of c13* cells was decreased after pcDNA3.1(+)-BRMS1 transfection.The expression of integrin beta 1 and p-AKT was down-regulated after transfection,but AKT expression had no significant change.Conclusion pcDNA3.1(+)-BRMS1 recombinant plasmid was successfully constructed and transfected.The decreased migration,invasion and adhesion capability by enhancing the BRMS1 expression in c13* cells may be mediated through the altered expression of integrin beta 1/AKT signal pathway.更多还原