DNMT1 inhibits hepatocellular carcinoma cells by altering the epigenetic modification of Marveld1 gene
Objective to study the effect of DNA methyltransferase (DNMT1) on Marveld1 in hepatocellular Carcinoma (HCC), and explore its role in the molecular mechanism of HCC occurrence and development.Methods The expression level of Dnmt1 and Marveld1 genes was detected by RT-PCR in the intraoperative pathological specimens. The human hepatoma Bel7402 and SMMC7721 cell lines down-regulated by DNMT1 gene expression were constructed by SiRNA transfection. The methylation level of Marveld1 promoter region
... d1 promoter region was detected after RT-PCR and West-blot verification. The expression of Marveld1 gene, cell proliferation, invasion and change of cell cycle were detected by RT-PCR and West-blot. The expressions of P53, CyclinD1, P21 and P16 protein were detected by Westblot method.Results The expression of DNMT1 gene in human hepatocarcinoma tissues was higher than that in adjacent tissues. The methylation level of the Marveld1 gene promoter region in human hepatoma cell line down-regulated by DNMT1 gene expression was decreased, the expression level of Marveld1 gene was increased, and the proliferation and invasion ability of cells were weakened. The cell cycle was showed as G1-S phase arrest; P53 and P16 protein expressions were up-regulated, and CyclinD1 and P21 protein expressions were down-regulated.Conclusions Dnmt1 gene is highly expressed in human hepatocarcinoma tissues. Down-regulation of DNMT1 gene expression can decrease the methylation level of Marveld1 promoter region, up-regulate Marveld1 gene and human P53 and P16 proteins expressions, and down-regulate CyclinD1 and P21 proteins expressions, inhibit the proliferation and invasion of human hepatoma cells and can cause cell cycle G1-S arrest. Background Hepatocellular carcinoma (HCC) has become one of the most common malignant tumors worldwide. About 780,000 new cases of liver cancer occur each year, which accounting for 7.4% of all malignant tumors, and the mortality rate is about 9%. Statistics from IARC (International Agency for Research on Cancer) showed that HCC has become the fourth leading cause of cancer death in the world  . HCC has a high degree of malignancy, which is easy for recurrence after surgery, and has poor prognosis. Therefore, exploring the causes of HCC and the molecular mechanism of its pathogenesis is of great value for its early diagnosis, treatment and prognosis. Existing studies have found that the After transfection with Si-RNA, the RNA expression of DNMT3a and DNMT3b genes in the experimental group was significantly decreased (*p<0.05), Figure 5. However, the Marveld1 gene of human hepatocytes with down-regulated DNMT3a and DNMT3b gene expression was not significantly changed compared with the Marveld1 gene of human hepatocytes with down-regulated DNMT1 gene expression. Therefore, human hepatocellular carcinoma cells with down-regulated DNMT1 gene expression were selected as experimental subjects (#p>0.05) Discussion DNA methyltransferase (DNMT), as the key factor in the regulation of DNA methylation in tumor tissues, can affect the biological behavior of tumor cells by regulating the degree of methylation of gene promoters with different functional  . The DNMTs family has approximately five members: DNMT1, DNMT2, DNMT3a, DNMT3b, and DNMT3L, of which DNMT1 is the most important DNA methyltransferase and plays an important role in methylation status maintenance; deletion of DNMT1 may lead to decreasing of DNA methylation, affect the transcription of genes, and then regulate cell growth and differentiation. Proteins containing the MARVEL domain are evolutionarily conserved and widely expressed in normal adult tissues. They involve a variety of biological processes, including cell cycle progression, chemotactic activity, tight junctions, and clathrin-mediated endocytosis [9,     . As a member of this family, the research on the molecular function of Marbeld-1 is limited so far. Studies have shown that the expression of Marveld-1 gene in liver cancer and lung cancer tissues is lower than that in adjacent tissues. Overexpression of Marveld-1 gene can inhibit the proliferation of tumor cells, up-regulate P53 and P16 genes, and can down-regulate the expression of CyclinD1 gene, Marveld-1 can affect the G1-S phase checkpoint-associated proteins of the cell cycle mentioned above, and then lead to G1-S phase arrest of Marveld-1 overexpressing cells, thus exert the function of tumor suppressor gene   . The data in the TCGA database showed that the DNMT1 gene was highly expressed in HCC tissues 12 and low in Marveld1 gene, which was consistent with the clinical sample paired PCR results in this study. Marveld1 gene was hypermethylated in HCC tissues, and the survival of the samples with low expression of Dnmt1 gene was longer than that of the samples with high expression of Dnmt1 gene, suggesting that down-regulation of DNMT1 gene expression can up-regulate the expression of Marveld1 gene, and the results of inhibiting the malignant biological manifestations of HCC may have potential clinical application value. There is no data on the relationship between Marveld1 gene and DNMT1 gene in TCGA database. This study showed that the down-regulation of Dnmt1 gene can upregulate the expression of Marveld1 gene by down-regulating the methylation level of Marveld1 gene promoter region, and there is certain correlation between the expressions of two genes. This study found that in human HCC tissues, the expression of Dnmt1 gene in HCC tissues was higher than that in adjacent tissues. Down-regulation of the expression of Dnmt1 gene in HCC cells could down-regulate the methylation level of the promoter region of Marveld1 gene. The expression of Marveld1 gene can be up-regulated by the epigenetic modification, and can inhibit the proliferation and invasion of HCC cells that consistent with the existed study on the overexpression of Marveld1 gene  . Up-regulated P53, P21 and P16 genes, down-regulated the expression of CyclinD1 gene, and affecting the G1-S phase checkpoint-associated proteins in the cell cycle, thus leading to G1-S phase arrest in cells. The Dnmt1 gene can change pathway, cause the epigenetic modification of the Marveld1 gene through a promoter methylation level, thus resulting in up-regulation of its expression and enhancing the function of the tumor suppressor gene. Dnmt inhibitors have been used in clinical applications, and the main therapeutic areas are bladder cancer and myeloid leukemia. The results of this study suggest that Dnmt inhibitors may have some application prospects in the field of HCC at the in vitro cytological level, and related cells, animals and clinical trials may be a valuable research direction. Declarations Ethics approval and consent to participate Not applicable.