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目的:构建DEC1真核表达载体pEGFP-Nl-DEC1,探讨DEC1基因对乳腺癌细胞增殖的影响及可能机制。方法:提取人乳腺癌癌细胞MCF-7总RNA,扩增获得DEC1基因,将DEC1基因插入真核表达质粒pEGFP-Nl,将成功构建的pEGFPNl-DEC1及pEGFP-Nl质粒利用PEI转染MCF-7细胞,并通过G418筛选得到稳定转染细胞系。通过Western blot检测转染前后细胞DEC1、CyclinD1蛋白表达水平;MTT法检测转染前后MCF-7细胞增殖的变化。结果:成功构建DEC1真核表达载体pEGFP-Nl-DEC1;与pEGFP-Nl组和空白对照组相比,pEGFP-Nl-DEC1组DEC1表达明显增高(P<0.05);cyclin D1的表达明显降低(P<0.05);pEGFP-Nl-DEC1组细胞增殖能力明显受到抑制。结论:过表达DEC1可抑制乳腺癌MCF-7细胞的增殖,DEC1可能通过下调cyclin D1基因的表达从而阻止细胞周期及TGF-β信号通路而发挥作用。
OBJECTIVE: To construct DEC1 eukaryotic expression vector pEGFP-Nl-DEC1 to investigate the effect of DEC1 gene on the proliferation of breast cancer cells and its possible mechanism. METHODS: Total RNA was extracted from human breast cancer cell line MCF-7. The DEC1 gene was amplified and inserted into the eukaryotic expression vector pEGFP-Nl. The recombinant plasmid pEGFPN1-DEC1 and pEGFP-N1 were transfected into MCF- 7 cells, and stable transfected cell lines were obtained by G418 screening. Western blot was used to detect the expression of DEC1 and CyclinD1 protein before and after transfection. The proliferation of MCF-7 cells was detected by MTT assay. Results: The DEC1 eukaryotic expression vector pEGFP-Nl-DEC1 was successfully constructed. Compared with pEGFP-Nl group and blank control group, the expression of DEC1 in pEGFP-Nl-DEC1 group was significantly increased (P <0.05) P <0.05). The proliferation of pEGFP-Nl-DEC1 group was significantly inhibited. CONCLUSION: DEC1 can inhibit the proliferation of breast cancer MCF-7 cells. DEC1 may play a role in inhibiting the cell cycle and TGF-β signaling pathway by down-regulating the expression of cyclin D1 gene.