| Epigenetic modification is an important pathway of molecular genetics the induction of tumorigenesis. DNA methylation is one of the key parts of epigenetics and the main ways to regulate gene expression. DNA's abnormal methylation implement by DNA methyltransferases (DNMTs). According to the function of enzyme, DNMTs can be divided to two types: de novo DNA methylation (DNMT3a, DNMT3b) and maintain DNA methylation(DNMTl). Among these, DNMT1 has an influential effect on the maintaining of DNA methylation. Now, it is necessary for normal growth and development that maintaining correct status of DNA methylation, many tumors's development is correlated with abnormal patterns of DNA methylation.In this study, we plan to suppress the expression of DNMT1 gene from the level of mRNA by RNA interference(RNAi). The activity of DNMT1 will be down regulation and inhibited in breast cancer cell lines. Many related genes can be relieved hypermethylation in their promoter regions, and the abnormal proliferation of breast cancer cell lines will be restrained. Our research works will establish considerable theoretical and experimental foundation for clinical molecular therapy of breast cancer.Objective: To construct the plasmid containing short hairpin RNA(shRNA) of DNA methyltransferase 1(DNMT1), and to suppress the expression of gene DNMT1 after transfected into human breast cancer cell lines MCF-7. To study the effect on proliferation and apoptosis of breast cancer cell lines MCF-7.Methods: Three 19bp reverse repeated motifs targeting coding sequence of DNMTl were synthesized, and inserted into pGCsi-H1 to generate the pGCsi-DNMT1 recombination plasmid, validated by DNA sequencing. After being purified, three siRNA-DNMT1 plasmids were transfected into human breast cancer cell lines MCF-7 by Lipofectamine 2000. Three groups were set: blank,vector and pGCsi-T3. After transfection, the effect on transcriptional level of mRNA of gene DNMT1 expression was detected by real-time quantitative PCR, simultaneously, the most efficient plasmid was determined.The most efficient plasmids was transfected into MCF-7 cells by Lipofectamine 2000. The cell morphous was observed under light microscope. By MTT assay, the cell growth status was detected and the cell growth curve was drawn. Apoptosis was analyzed by Annexin V/PI double-dyed, and cell cycle was detected with flow cytometry (FCM). The change of mRNA expression was analyzed by real-time quantitative PCR about many tumor-related genes, such as RASSF1A,P16,P21,P27 and ERβ.Results: Three recombinant plasmids pshRNA-DNMT1 were successfully constructed(pGCsi-T1,pGCsi-T2,pGCsi-T3), and confirmed by DNA sequencing. Relative to control, pGCsi-T2 and pGCsi-T3 induced a 64.2% and 74.7% inhibition of DNMT1 mRNA expression. Compared with blank and vector groups, pGCsi-T3 recombination plasmid has markedly inhibited the proliferation of breast cancer MCF-7 cells; A majority of cells gone apoptosis; and reduced the frequency of MCF-7 cells in S phase, while significantly increasing cells in G1/G0 phase; The level of mRNA expression of gene RASSF1A,P16,P21 and ERβobviously rised by the detection of real time PCR, but gene P27 was invariably.Conclusion: The results show that the short hairpin RNA of DNMT1 can efficiently and specifically inhibit the expression of gene DNMT1 in breast cancer MCF-7 cells. Furthermore, the pGCsi-T3 can obviously inhibit the proliferation of breast cancer cells, and stimulate the apoptosis, its mechanism is correlated with the up-regulation of gene expression, including anti-oncogene such as RASSF1A,P16,P21 and those genes maintaining normal growth of mammary gland cells such as ERβ, by relieving the hypermethylation in promoter regions through inhibiting the expression of gene DNMT1. |
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