Research Of DNMT1, Promoter Methylation Status And Expression Of Tumor Suppressor Genes In Non-small Cell Lung Cancer(NSCLC): A Study Of Tumor Cell Lines And Tumor Growth In Nude Mice

Objective: DNA methylation is an epigenetic DNA modification catalyzed by DNA methyltransferase1(DNMT1).The purpose of this study was to investigate DNMT1 gene and protein expression and the effects of methylation statu on tumor suppressor genes in human non-small cell lung cancer(NSCLC)cell lines grown in vitro and in vivo. Methods: Human lung adenocarcinoma cell lines, A549, and H838 were grown in vitro and inoculated subcutaneously into nude mice to form tumors, and were then treated with the DNA methylation inhibitor, 5-aza-2’-deoxycytidine(5-AZA-Cd R), with and without treatment with the benzamide histone deacetylase inhibitor, entinostat(MS-275). The cells were divided into a control group, 5-aza-2’-deoxycytidine(5-AZA-Cd R) treated group, and a MS-275 treated group and combination treatment group. After epigenetic treatment, cells were collected, DNA, total RNA, protein were extracted, respectively. DNMT1 and tumor suppressor genes(RASSF1A, ASC,APC, MGMT, CDH13, DAPK, ECAD, P16, GATA4) protein expression were quantified by Western blotting. DNMT1 and tumor suppressor genes(RASSF1A,ASC, APC, MGMT, CDH13, DAPK, ECAD, P16, GATA4) m RNA expression were quantified by RT-PCR. Promoter methylation status of tumor suppressor genes(RASSF1A, ASC, APC, MGMT, CDH13, DAPK, ECAD, P16, and GATA4) was evaluated by methylation-specific PCR(MSP). Cell growth was studied using the CCK8 kit. Results: Part One: 1. Treatment of non-small cell lung cancer(NSCLC) cells with inhibitors of DNA methyltransferase(DNMT)and histone deacetylase(HDAC) reduced DNMT1 transcription expression. 2.Treatment with 5-AZA-Cd R and MS-275 inhibited DNMT1 expression in NSCLC cells. 3. Treatment with 5-AZA-Cd R and MS-275 resulted in tumor suppressor gene demethylation in NSCLC cells. 4. Treatment with 5-AZA-Cd R and MS-275 induced tumor suppressor gene expression in NSCLC cells. 5.Treatment with 5-AZA-Cd R and MS-275 inhibited proliferation in NSCLC cells.Part Two:1. Treatment with 5-AZA-Cd R and MS-275 inhibited tumor growth in vivo. 2. Treatment with 5-AZA-Cd R and MS-275 restored expression of RASSF1 A and APC in vivo. 3. Treatment with 5-AZA-Cd R and MS-275 resulted in the demethylation of RASSF1 A and APC in vivo. Conclusions:Methylation status of the tumor suppressor genes was regulated by the DNMT1 gene, with the decrease of DNMT1 expression following DNA methylation treatment. Demethylation of tumor suppressor genes(APC, ASC, and RASSF1A)inhibited tumor growth in nude mice. The results of this study support a role for methylation of DNA as a potential epigenetic clinical biomarker of prognosis or response to therapy and for DNMT1 as a potential therapeutic target in NSCLC.