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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 RNAi recombinant plasmid of breast cancer-specific gene (BCSG1) and to detect its interference effect on BCSG1 gene expression and cell proliferation in triple negative breast cancer cell line MDA-MB-321. Methods: Two pairs of interference fragments BCSG1-R1, BCSG1-R2 and BCSG1-C were cloned into pGenesil-1 shuttle plasmid respectively. After identification and sequencing, the correct clones were transfected into E. coli DH5α Real-time PCR was used to detect the expression of BCSG1-mRNA in each group after transfected with recombinant plasmid. The effect of recombinant plasmid on the invasiveness of MDA-MB-321 cells was detected by Transwell invasion assay. Results: The interference fragment and the negative control fragment were successfully cloned into pGenesil-1 plasmid to construct recombinant plasmids pGenesil-1-BCSG1-R1, pGenesil-1-BCSG1-R2 and pGenesil-1-BCSG1-C. Compared with the control group, the expression of BCSG1-mRNA in BCSG1-R1 and BCSG1-R2 groups was significantly decreased (P <0.05). The invasion ability of MDA-MB-321 cells was significantly decreased after interference (P <0.05). CONCLUSION: The constructed recombinant plasmid BCSG1-RNAi can effectively down-regulate the expression of BCSG1 and the invasiveness of cells in the triple-negative breast cancer cell line MDA-MB-321.