Methylation Of PTPRM And Its Role In Malignant Transformation Of BEAS-2B Cells Induced By Cigarette Smoke

Objective:To explore the epigenetic mechanism of malignant transformation of bronchus and lung induced by cigarette smoke,we screened the abnormal methylation genes by Illumina450 k methylation chip in the malignant transformation BEAS-2B cells model induced by cigarette smoke,which was established in the previous experiments.We selected the aberrant hypermethylated gene PTPRM to explore its expression in malignant transformed BEAS-2B cells and the mechanism of its influence on cell migration and invasion to provide a new theoretical basis for the diagnosis,treatment and prognosis of lung cancer.Methods:According to the results of Illumina450 k methylation chip and bioinformatics analysis,the abnormal changes of genes were enriched into different signal pathways.The abnormal hypermethylation gene PTPRM was screened by further literature query and Gene Ontology analysis.The PTPRM gene was thus identified as the study object by MSP experiments to verify its methylation in malignant transformation by cigarettes smoke exposure.The cell model of malignant transformation of cigarettes smoke exposure was established by comparing(a)the control group: the BEAS-2B cells passaged 40generations(2B-40),not exposed to cigarette smoke,and(b)the treatment groups: the BEAS-2B cells subcultured for 40 passages exposed to tobacco smoke 1 generation,10 generation,20 generation and 30(S1-40,S10-40,S20-40 and S30-40),as the treatment groups.The expression of PTPEM gene in the control group and the treatment group was detected by Q-PCR and western blot,with and without the methylation inhibitor 5-aza-2’-dC treated.The changes of cell migration and invasion were observed between the control group and the experimental group.In order to outline the mechanism of PTPRM on malignancy.The expression of CDH1 and the phosphorylation of β-catenin were traced efore and after knockdown of PTPRM gene to confirm the effect of PTPRM methylation and its impact on cell activities.Result:(1)Through methylation chip and KEGG bioinformatics analysis,it showed that genes showing DNA methylation changes,were enriched in NOD-like receptor signaling pathway,Cell adhesion molecules(CAMs)and Cytokine-cytokine receptor interaction.In particular,MSP results showed the DNA hypermethylation of PTPRM gene in malignant cells compared with controls.The mRNA expression and protein level of PTPRM gene decreased with the generation increase in the treatment group,while it showed varying degrees of recovery after 5-aza-2’-dC treated,indicating that the expression of PTPRM gene is regulated by abnormal methylation.(2)The results of transwell migration and invasion showed that the number of cells migrating and invading in the experimental group increased with the increase of malignant transformation,comparing with the control group.The migration and invasion function were enhanced after knocking down of PTPRM gene in cells,which indicated that PTPRM gene could affect cell migration and invasion function.(3)The results of Q-PCR and western blot showed that the expression of CDH1 decreased with the generation increasing in the exposed groups,while the phosphorylation level of β-catenin increased.The same changes were made in cells knocking out of the PTPRM gene,demonstrating that the expression of the PTPRM gene regulates the expression of the CDH1 gene and the phosphorylation level of the β-catenin,and thus regulates the cell migration and invasion function.Conclusion:1.The hypermethylation-specific PTPRM gene was screened out in malignant transformation cells.The expression of PTPRM gene was decreased gradually with the increasing of cigarette smoke exposure,which shows the dose-effect relationship.2.PTPRM gene can regulate the expression of CDH1 gene in the downstream,and the expression of CDH1 gene is decreased gradually with the decrease of PTPRM gene expression.Meanwhile,PTPRM gene can affect protein tyrosine phosphorylation of β-catenin,while the expression of PTPRM decreases,β-catenin increases phosphorylation.3.PTPRM gene can regulate the ability changes of cell migration and invasion by regulating the expression of CDH1 and β-catenin phosphorylation.