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目的靶向人DNMT1(DNA methyltransferase 1,DNMT1)构建RNA干扰载体,研究其对乳腺癌细胞周期、增殖及凋亡的影响。方法靶向DNMT1基因设计三条短发夹状RNA(short hairpin RNA,shRNA)的寡核苷酸片段,构建重组体pGCsi-DNMT1,转染至乳腺癌细胞株MCF-7,定量PCR法检测DNMT1mRNA表达水平;流式细胞技术分析细胞周期的变化;MTT法检测细胞生长情况;Annexin V/PI双染法观察细胞凋亡情况;定量PCR法分析沉默DNMT1基因后对RASSF1A、p16、p21、p27及ERβ基因表达的影响。结果在构建的靶向DNMT1的shRNA重组质粒pGCsi-DNMT1中,成功筛选到pGCsi-T3能显著下调DNMT1的表达。实时定量PCR检测结果证实重组质粒pGCsi-DNMT1对乳腺癌MCF-7细胞中DNMT1基因的转录有明显的抑制作用。MCF-7细胞转染后,pGCsi-DNMT1可明显抑制乳腺癌MCF-7细胞的增殖;大量细胞发生凋亡;细胞周期分析可见S期明显减少,而G1/G0期细胞显著增加;定量PCR检测到乳腺癌细胞中RASSF1A、p16、p21及ERβ基因mRNA表达水平明显升高,而p27基因表达水平未见明显变化。结论重组质粒pGCsi-DNMT1能特异有效地抑制MCF-7细胞内DNMT1的表达、抑制细胞增殖、促进细胞凋亡,并可通过抑制DNMT1的活性来解除相关基因启动子区的高度甲基化状态,从而促进肿瘤相关基因的表达,提示DNMT1可作为乳腺癌基因治疗的新靶标。
Objective To construct RNA interference vector targeting human DNA transferase 1 (DNMT1) and study its effect on cell cycle, proliferation and apoptosis of breast cancer. Methods Three short hairpin RNA (shRNA) oligonucleotide fragments targeting DNMT1 gene were designed to construct recombinant pGCsi-DNMT1 and transfected into breast cancer cell line MCF-7. Quantitative PCR was used to detect DNMT1 mRNA expression. Level; flow cytometry analysis of changes in cell cycle; MTT assay for cell growth; Annexin V/PI double staining method for observation of cell apoptosis; Quantitative PCR analysis of DNMT1 gene silencing for RASSF1A, p16, p21, p27, and ERβ The effect of gene expression. Results The pGCsi-T3 was successfully screened in the shRNA recombinant plasmid pGCsi-DNMT1 targeting DNMT1. The expression of DNMT1 was significantly down-regulated. Real-time quantitative PCR results confirmed that the recombinant plasmid pGCsi-DNMT1 had a significant inhibitory effect on the transcription of DNMT1 gene in breast cancer MCF-7 cells. After MCF-7 cells were transfected, pGCsi-DNMT1 significantly inhibited the proliferation of breast cancer MCF-7 cells; a large number of cells apoptosis; cell cycle analysis showed that the S phase significantly reduced, while the G1/G0 phase cells significantly increased; quantitative PCR detection The expression levels of RASSF1A, p16, p21 and ERβ mRNA were significantly increased in breast cancer cells, but no significant changes in the expression level of p27 gene were observed. Conclusion The recombinant plasmid pGCsi-DNMT1 can specifically inhibit the expression of DNMT1 in MCF-7 cells, inhibit cell proliferation and promote cell apoptosis, and can relieve the hypermethylation status of the promoter region of related genes by inhibiting the activity of DNMT1. This promotes the expression of tumor-associated genes, suggesting that DNMT1 may serve as a new target for gene therapy of breast cancer.