紫杉醇调节TIMP-1启动子活性及TIMP-1降低紫杉醇对乳腺癌细胞增殖抑制作用的研究

Regulation of TIMP-1 Promoter by Paclitaxel and the Inhibitory Effect of TIMP-1 on the Paclitaxel-Induced Inhibition of Cell Proliferation in Breast Carcinoma Cell Line

目的探讨TIMP-1在降低MCF-7对紫杉醇敏感性中的作用。方法采用DNA重组技术构建含timp-1基因启动子的荧光素酶报告质粒并瞬时转染MCF-7细胞,在撤血清条件下测定荧光素酶的相对活性,以验证重组质粒的有效性;重组质粒瞬时转染入MCF-7细胞中,经紫杉醇处理后,测定荧光素酶的相对表达活性;检测在紫杉醇作用下,TIMP-1过表达克隆与对照克隆细胞增殖的情况。结果酶切、PCR及测序鉴定结果证实成功构建带有TIMP-1启动子的报告基因表达质粒;且撤血清条件下TIMP-1启动子转录活性显著增强,1000nmol/L紫杉醇处理的MCF-7细胞中TIMP-1启动子调控的荧光素酶活性显著降低;紫杉醇对TIMP-1过表达克隆的细胞增殖抑制作用显著低于对照克隆。结论成功地构建了含timp-1基因启动子的荧光素酶报告质粒,准确地评价TIMP-1启动子在紫杉醇作用过程中的活性变化,并进一步阐明TIMP-1在降低乳腺癌细胞对紫杉醇敏感性中的作用。

Objective To explore if TIMP-1 can decrease the sensitivity of MCF-7 cells to paclitaxel. Methods DNA recombination technique was used to construct the plasmid luciferase expressing vector containing full-length sequence of timp-1 gene promoter and the plasmid was transfected into MCF-7 cells and the luciferase activity was detected in the cells cultured in serum-free medium to verify the efficacy of the vector. The vector was then transfected into MCF-7 cells and after the cells were treated with paclitaxel, the activity of luciferase was measured. The cell proliferation was examined by MTT in clones over-expressing TIMP-1 and control clones in the presence of paclitaxel. Results Restriction enzyme digestion, PCR and DNA sequencing confirmed that TIMP- 1 promoter luciferase reporter gene expression vector was successfully constructed. The activity of TIMP-1 promoter was significantly enhanced in the medium without serum and the findings were coincident with the previously reported results. The activity of TIMP-1 promoter was remarkably decreased after MCF-7 cells were subjected to paclitaxel treatment. The inhibition of cell-proliferation induced by paclitaxel was significantly decreased in clones with TIMP-1-overexpression. Conclusions The luciferase expression vector containing full-length sequence of timp-1 gene promoter was constructed successfully and the activity changes of TIMP-1 promoter were correctly analyzed after MCF-7 cells was treated by paclitaxel. TIMP-1 decreased the sensitivity to paclitaxel in breast carcinoma cell line.