The Mechanism Of The Correlation Between DNMT2 And Gastric Cancer

Gastric cancer is a worldwide common malignant tumor and is a serious threat to human health.In 2015,there were about 1,000,000 new incidences and about 745,000 deaths caused by gastric cancer globally,accounting for 8.5%of the total cancer mortality in the world.The incidence and mortality of gastric cancer in China are more than two folds of the world average.Currently known factors relating to gastric cancer include Helicobacter pylori infection,intestinal microbiota,inflammatory microenvironment,genetics and epigenetics.Genetic and epigenetic changes are often caused by gene mutation,abnormal methylation or molecular polymorphismsDNA methyltransferase 2(DNMT2)belongs to the DNA methyltransferase family.It mainly methylates the carbon atom at the 5th position of the cytosine 38 in the anticodon loop of eukaryotic transfer RNA(tRNA),generating 5-methyl cytidine(m5C).The DNMT2 polymorphism rs 11254413 was reported to correlate with the incidence of gastric cancer in southern Chinese.This polymorphism leads to the variation of H101Y.Nevertheless,the molecular mechanism of the association of DNMT2 with gastric cancer is still unknown.Human gastric cancer and para-carcinoma tissues were used in this study.The DNMT2 coding sequences and the expression pattern in these tissues were analyzed.In addition,normal gastric epithelial celline GES-1 and gastric cancer cell line SGC7901 were used to investigate the influence and molecular mechanisms of wild type DNMT2 and its H101Y variant on cell proliferation,migration and invasion,with the aid of techniques in molecular biology and cell biologyThe results of this study are as follows:1.The sequence and distribution of DNMT2 in 23 pairs of gastric cancer and para-carcinoma tissuesThe sequencing results of DNMT2 coding sequences in 23 pairs of gastric cancer and para-carcinoma tissues showed that,H101Y variant accounted for 13.04%.This is consistent with the reported results;The IHC staining of these tissues revealed irregular structure of gastric cancer tissues in comparison with the para-carcinoma tissues.Large numbers of cancer cells,enhanced connective tissues,increased eosinophilic granulocytes and infiltrating lymphocytes were found in gastric cancer tissues.The gastric cancer tissues harbored multiple irregular gastric glands and many capillary blood vessels.In the para-carcinoma tissues containing H101Y variant,more infiltrating lymphocytes were found in interstitial tissues and more eosinophilic granulocytes were observed.This suggested that better immune response in patients with H101Y variant during gastric cancer progression and gastric cancer cells are more prone to migration.The H101Y variant influenced DNMT2 localization in gastric cancer tissues.DNMT2 was localized in both nucleus and cytoplasm,while more DNMT2 was in the nucleus than in the cytoplasm.In the gastric glands,more DNMT2 was found in para-carcinoma tissues than in gastric cancer tissues.In the connective tissues,more DNMT2 were observed in the gastric cancer tissues.In the gastric glands of gastric cancer patients with H101Y variant,more DNMT2 was found in para-carcinoma tissues than in gastric cancer tissues.In contrast,in the connective tissues,less DNMT2 was found in para-carcinoma tissues than in gastric cancer tissues with wild type DNMT22.Effect of DNMT2 on proliferation and migration of GES-1 and SGC7901 cellsUsing MTT assays to detect GES-1 and SGC7901 cell growth.We found that overexpression of DNMT2 or H101Y variant did not significantly affect cell viability or cell growth.The result suggested that the overexpression of DNMT2 or H101Y did not exert significant impact on cell proliferationTranswell and wound healing assays showed that overexpression of DNMT2 or H101Y variant promoted the migration of GES-1 cells;Overexpression of DNMT2 significantly inhibited the migration of SGC7901 cells,and overexpression of H101Y variant did not significantly influence SGC7901 cell migration3.RNA-seq of SGC7901 cells with overexpressed H101Y variantIn order to further explore the roles of H101Y variant in gastric cancer,we used RNA-seq to analyze transcriptome profiles of SGC7901 cells with overexpressed H101Y variant,with empty vector pcDNA3.1 as controlsThe results showed that:(1)the results of SNP analysis showed that the number of SNP in control group was slightly higher;(2)GO note:18277 functional genes were found,with 16546 genes functioning biological processes,18148 genes relating to cellular localization;KEGG note:Differential expression genes were enriched in the cell community,cell transport and metabolism,signal transduction,signaling molecules and interaction,cancer and so on.KEGG gene annotation quantity were enriched in signaling pathway of the interaction between cell metabolism,cancer,infectious diseases,endocrine and immune system;(3)Genes differential expression:The expression of 184 genes significantly altered.Among these genes,45 genes showed reduced expression,while 139 genes exhibited increased expression.(4)KEGG enrichment analysis:Compared with the control group,most genes with differential expression in H101Y group was enriched in HIF-1 signaling pathway;glucose metabolism and glycolysis pathway;metabolic pathways;cancer related signaling pathways.4.Effect of DNMT2 and H101Y variant on extracellular matrix receptor(ECM-receptor)signaling pathway in GES-1 cellsIn order to further validate the transcriptome analysis and to reveal the molecular mechanism underlying the effect of DNMT2 and H101Y variant on cell migration,the expression of genes relating to ECM-receptor signaling pathway was detected in GES-1 cells with over expressed DNMT2 or H101Y variant,using GES-1 harboring empty pcDNA3.1 vector as control.Q-PCR and Western blot were used to monitor the expression of these genes relating to ECM-receptor signaling pathway.siRNAs were used to knock down DNMT2.The result showed that DNMT2 and H101Y in GES-1 cells might also affect cell migration through ITGB1,ITGB4 and MMP2.In these groups,the expression of ITGB1 decreased first and then increased;the expression of ITGB4 increased first and then decreased;the expression of MMP2 showed no significant change in DNMT2 group but increased significantly in H101Y group.5.Effects of DNMT2 and H101Y variant on ECM-receptor signaling pathway in SGC7901 cellsThe same method was used to analyze the changes of migration factors in SGC7901 cells.We found that DNMT2 and H101Y may affect SGC7901 cell migration through ITGB3,MMP9 and MMP12.In SGC7901,the expression of ITGB3 decreased in cells with overexpressed DNMT2,while increased in cells with overexpressed H101Y variant.The expression of MMP9 and MMP12 decreased in these groups.Thus,DNMT2 may inhibit the migration of gastric cancer cells by reducing the expression of ITGB3,MMP9 and MMP12.6.The effect of other cancerous variants of DNMT2 on the proliferation and migration of GES-1In this study,the effect of another 15 cancerous DNMT2 variants on the proliferation and migration of gastric cancer cell line was examined.These variants include E63K,G155S,G155C,G155V,E185D,E185K,E202V,E202Q,D226Y,D226H,L257I,L257V,E317G,E317D,R371H.They were constructed using site-directed mutagenesis.Cell count,MTT,Transwell assays and scratch healing assays were used to monitor the effect of DNMT2 and 15 variants on the proliferation and migration of cells.The wild type DNMT2 or each of 15 cancerous variants was transfected into GES-1 cells.The results showed similar cell counts and MTT assay data among all cell groups.Overexpression of DNMT2 significantly promoted the migration of GES-1 cells,while overexpression of cancerous variants of DNMT2 inhibited GES-1 migration,as shown by transwell assays.In consistence with this finding,wound healing assays also revealed inhibited migration of GES-1 by most cancerous DNMT2 variants.Only wild type DNMT2,E63K and E185D variants promoted cell migration.The wound healing data of most variants,with the exception of E202Q/V and L257I,were consistent with their enzyme activities(Li HR,2018,unpublished data).7.The effect of other cancerous variants of DNMT2 on the proliferation and migration of SGC7901The wild type DNMT2 or each of 15 cancerous variants was transfected into SGC7901 cells.The results showed similar cell counts and MTT assay data among all cell groups.Overexpression of each of G155S/C/V、E185K、D226Y/H、L2571/V、E317G/D and R371H variants significantly promoted the migration of SGC7901 cells,though these variants exhibited decreased tRNA methylation activities.E185D increased enzyme activity of DNMT2,but inhibited SGC7901 migration.Cell scratch experiments showed that,while G155C/V,D226H and E317G/D decreased DNMT2 activity,they promoted migration of SGC7901 cells;E63K,E202V and L257I increased the enzyme activity of DNMT2,but inhibit SGC7901 cell migration;R371H and D226Y can significantly decreased the activity of DNMT2,while over expression of R371H and D226Y in cell death of SGC7901 48h after transfection.Conclusions:(1)H101Y variant of DNMT2 accounted for 13.04%among the 23 pairs of gastric cancer samples collected in this study;H101Y alterations affects the localization of DNMT2 protein in cells and resulted in increased lymphocyte infiltration in gastric cancer tissues,which may be related to the metastasis and prognosis of gastric cancer.(2)H101Y variant of DNMT2 is involved in the signaling pathway changes of gastric cancer cells line SGC7901.The changes of ECM-receptor signaling pathway affect the migration of gastric cancer cells.DNMT2 and H101Y can promote or inhibit the migration of GES-1 cells by regulating the expression of MMP2,ITGB1 and ITGB4 and interacting with ECM.DNMT2 and H101Y variant also affect SGC7901 migration by modulating the expression of MMP9,MMP12 and ITGB3(3)The tested DNMT2 variants can affect growth and migration of GES-1 and SGC7901 cell lines.The variants with decreased enzyme activity can promote gastric cancer cell migration,while the variants with increased enzyme activity inhibited gastric cancer cell migration.The data showed that DNMT2 influenced the growth and movement to regulate cell migrationThe results of this study revealed the functional mechanism of H101Y variant of DNMT2 and other cancerous variants in cancer,providing a new potential target for early diagnosis and intervention of gastric cancer.