BCSG1基因RNAi表达载体的构建与鉴定

Construction and Identification of Recombinant Plasmid of RNA Interference of BCSG1

目的:构建乳腺癌特异性基因(BCSG1)RNAi重组质粒,并检测其在三阴乳腺癌细胞株MDA-MB-321中对BCSG1基因表达的干扰效果和对细胞增殖的影响。方法:将两组干扰片段BCSG1-R1、BCSG1-R2及阴性对照片段BCSG1-C分别克隆入pGenesil-1穿梭质粒,经酶切鉴定及测序后,将测序正确的克隆转染入大肠杆菌DH5α中进行扩增Real-time PCR检测重组质粒转染MDA-MB-321细胞后,各组细胞内BCSG1-mRNA表达水平,Transwell细胞侵袭实验检测重组质粒对MDA-MB-321细胞侵袭能力的影响。结果:成功将干扰片段和阴性对照片段克隆入pGenesil-1质粒,构建出pGenesil-1-BCSG1-R1、pGenesil-1-BCSG1-R2和pGenesil-1-BCSG1-C重组质粒。与对照组相比,BCSG1-R1和BCSG1-R2组的BCSG1-mRNA表达水平明显下调(P<0.05);干扰后,MDA-MB-321细胞侵袭能力明显下降(P<0.05)。结论:成功构建BCSG1-RNAi重组质粒,可有效下调三阴乳腺癌细胞株MDA-MB-321内BCSG1的表达及细胞的侵袭能力。

Objective To construct the recombinant plasmid of RNA interference of breast cancer-specific gene 1（BCSG1） and detect the interference effect on the tipple negative breast cancer cell line MDA-MB-321. Methods Two couples of interference DNA fragment named BCSG1-R1 and BCSG1-R2, and one negative control fragment named BCSG1-C were cloned into pGenesil-1 plasmid vector. After being digested and identified correctly by restriction enzyme analysis and sequencing, the clones were propagated in Escherichia coli DH5α. Real-time PCR was used to test BCSG1-mRNA in MDA-MB-321 cells infected by plasmid vector; Transwell was employed to detect the effect of BCSG1-RNAi recombination on the invasion of MDA-MB-321cells. Results Two interference fragments and one negative control fragment were cloned into pGenesil-1 plasmid vector correctly, and the recombinant vectors named pGenesil-1-BCSG1-R1、pGenesil-1-BCSG1-R2 and pGenesil-1- BCSG1-C wereafter knockdown of BCSG1（P〈0.05）. Conclusion The plasmid vector recombination of BCSG1-RNAi could inhibit the expression of BCSG1 and reduce the invasive ability in triple negative breast cancer cell line MDA-MB-321.