The Effects Of 6-TG And Dmog On Growth Inhibition And Gene Expression Regulation Mechanism Of Breast Cancer Cells

Breast cancer is one of the leading causes of death among women worldwide.The occurrence of breast cancer is closely related to the abnormal gene expression in cells like other cancers.Including the silencing of tumor suppressor genes or the high activation of proto-oncogenes,and the activation of genes related to immune escape.The development of epigenetics provides a possible direction for the diagnosis,treatment and prognosis of cancer.DNA methylation as one of the regulation of gene expression has attracted much attention,but the mechanism remains to be further studied.6-TG(Thioguanine)is an inhibitor of DNMT1 which has made progresses in the treatment of leukemia.It also has a certain therapeutic effect in breast cancer cells,but the mechanism is still unclear.As an inhibitor of TET2,DMOG(Dimethyloxallyl Glycine)can prevent the formation of 5-methylated cytosine(5m C)catalyzed by 5-methylated cytosine(5hm C)on RNA.There were many studies focus on TET,but the regulation of tumor cell immune escape gene expression such as HLA-G by TET was rarely reported.Therefore,we used 6-TG to treat MDA-MB-231 cells,and explored its inhibitory effect on tumor cells in vivo and in vitro;Meanwhile,DMOG was used to treat MCF-7 cells,and gene expression and DNA methylation levels were detected to clarify the regulatory mechanism of HLA-G gene expression in MCF-7 cells.Finally revealing the potential targets of DNA methylation related enzymes in the occurrence,development and treatment of breast cancer by deeply studying the regulatory mechanism of DNA methylation related enzymes on the gene expression of breast cancer cells,and providing important theoretical basis for the diagnosis,treatment and prognosis of breast cancer.The following experiments were carried out in this study:1.The growth inhibition of 6-TG in MDA-MB-231 cells showed that:(1)We used different concentrations of 6-TG to treat MDA-MB-231 cell to determine the appropriate concentration,the result showed that the IC50 of 6-TG in 48 h was 2.5 μM;(2)The cloning formation and cell migration experiments showed that 6-TG significantly inhibited the cloning formation ability and migration ability of MDA-MB-231 cell,and promoted cell apoptosis;(3)The tumor bearing mice expriment showed that 6-TG significantly inhibited the growth of MDA-MB-231 tumor cell in NOD-SCID mice.2.The result of the study in mechanism 6-TG induced in MDA-MB-231 showed that:(1)6-TG inhibited the expressions of genes in cancer development pathway and virus infection pathway,and activated the apoptosis pathway by activating both m RNA and protein expressions of apoptosis-retated genes,such as DAXX and Casp8;(2)6-TG activated apoptosis pathway by inhibiting the expression of DNMT1 protein,and the DNA methylation levels of genes related to FAS,TRAIL and TNFR pathway such as DAXX,Casp8 were decreased;(3)6-TG reduced the methylation levels of tumor suppressor genes in MDA-MB-231 cell,such as TSC1 and RASSF1,which inhibited the abnormal activation of these pathways by interacting with nodal genes of tumorigenetic pathways and virus infection pathways.3.The study in the regulation of HLA-G showed that:(1)The HLA-G expression level in MCF-7 cell was higher than that in HBL-100 cell,and the DNA methylation level was lower;(2)The gene expression of DNMT1 and DNMT3 a in MCF-7 cell was lower than those in HBL-100 cell,and the expression of TET1 and TET3 was higher than those in HBL-100 cell;(3)100 μM DMOG significantly increased the DNA methylation level of HLA-G promoter by reducing the expression of TET1,TET2 and TET3 in MCF-7 cell,thereby reduced the m RNA expression level of HLA-G.