Mechanism Of N6-methyladenosine Epigenetic Modification In Cancer Stem Cell-like Phenotype Acquisition Of Arsenite-induced Gastric Epithelial Cells

Purpose:In this study,we examined the characteristics of stemness in cells exposed to arsenic,and explored the regulatory effects of arsenic on N6-methyladenosine mRNA levels and its regulatory factors,as well as the regulatory effects of arsenic on pluripotency factors.Method:GES-1 cells were exposed to different concentrations of sodium arsenite（0.01μM,0.05μM,0.1μM,0.2μM）for 40 generations（about 5 months）.Sphere formation,colony formation,and Transwell experiments were performed to verify stem cell-like pheontype of cells exposed to arsenic.The levels of m~6A methylation,m~6A regulatory factors and key pluripotency factors in arsenic-exposed cells were detected by Dot blotting,Western blot（WB）,q RT-PCR respectively.Female Wistar rats were selected to construct precancerous animal models of gastric cancer.The rats were randomly divided into two groups.One group,serving as a control drank water free of contaminants,another group was treated with 150μg/m L MNNG supplied in the drinking water and 0.03%ranitidine in the fodder.After 14 weeks of continuous exposure,two rats in each group were sacrificed at random,and the pathological changes of gastric tissue were observed after hematoxylin-eosin（HE）staining.After successful modeling,the rats were divided into four groups（from control group and model group）:negative control group（NC-NC）,arsenic exposure group（NC-AS）,positive control group（MNNG-NC）,arsenic exposure group after atrophy（MNNG-AS）.After that,the negative control group and the positive control group were given normal diet and water,and the two arsenic exposure groups were given arsenic containing water with a concentration of 10 mg/L Na As O₂.After 12 weeks of exposure,the rats were sacrificed to collect gastric tissue,part of which was made into wax blocks,and the pathological changes of gastric tissues in each group were observed after HE staining.A part of total RNA was extracted from stomach tissue,and the m~6A modification level in stomach of rats in each group was detected.In the other part,total protein was extracted from gastric tissue and m~6A regulatory factors was detected.Results:CCK-8 results showed that sodium arsenite promoted GES-1 cells at low concentration while inhibited GES-1 cells at high concentration,suggesting a different dose-response relationship.The results showed that the sphere forming ability,colony-forming ability and motility of GES-1 cells were significantly improved after arsenic exposure,and the ability of GES-1 cells exposed to 0.1μM/L arsenic was the most significantly changed（P<0.001）.The m~6A level of GES-1 cells was significantly increased after arsenic exposure,and the highest level of GES-1 cells at 0.1μM/L exposure（P<0.001）.WB showed that after arsenic exposure,the expression of methyltransferase METTL3 increased,and the expression of demethylase FTO and ALKBH5 decreased（P<0.05）,which promoted the m~6A level.The expressions of pluripotency factors Sox2,Nanog and Klf4 were increased in different levels（P<0.05）.HE staining of rats 14 weeks after modeling showed that compared with the control group,gastric tissue glands of rats in the model group showed atrophy,and goblet cells appeared in some gastric tissues of rats with slightly irregular cell arrangement,indicating that the model was successfully constructed.After 12 weeks of arsenic exposure,the pathological changes of stomach of rats in each group were obvious.The rats in the arsenic exposure group all had different levels of precancerous lesions of gastric cancer.After the pathological changes,the level of malignant changes in the arsenic exposure group was obvious,and some of the rats had progressed to the stages of intestinal metaplasia and heteroplasia.Dot blotting showed that compared with NC-NC group,m6A level in the other three groups was significantly increased,and m~6A level in MNNG-As group was the highest（P<0.001）.WB showed that after arsenic exposure,the expression of METTL3 was up-regulated,the expression of FTO and ALKBH5 was down-regulated,and the expression of pluripotency factors Sox2 and Oct4 was up-regulated（P<0.05）.Conclusion:Arsenic exposure can induce normal cells to acquire tumor stem cell-like phenotype,which are manifested as enhanced self-renewal ability,colony formation ability and cell motility.This process may be related to cellular RNA methylation level and pluripotency factor.Arsenic can up-regulate methyltransferase and down-regulate demethylase to increase m~6A level,and positively regulate key pluripotent factors to promote cells to have tumor stem cell-like characteristics.In addition,it is speculated that arsenic can promote the development of precancerous lesions of gastric cancer,and even induce the formation of gastric cancer.Patients with precancerous lesions of gastric cancer who are exposed to arsenic have a greater risk of gastric cancer.