The Study Of The Pathway Of Metallothionein 1M In Non-small Cell Lung Cancer

?Objectives?1?To analyze the differential expression of Metallothionein 1M(MT1M)through quantitative Real-time PCR and Western Blot.2?To elucidate the roles of MT1 M in cell viability,wound healing,Transwell migration.To investigate molecular mechanisms of MT1 M in NSCLC.It may provide the new targeting therapy for lung cancer.3?To select transcriptional regulation factor of MT1 M using bioinformatics analysis.To investigate the expression pattern of MT1 M in NSCLC.Whether the expression pattern affect the regulation of lung adenocarcinoma cell viability and migration and its underlying cellular and molecular mechanisms.?Methods?Our analysis of differentially expressed m RNA array data(GSE81292)also showed lost MT1 M expression in lung adenocarcinoma(LUAD).We collected 6 lung carcinoma tissues and adjacent tissues to assess the expression levels of MT1 M using q RT-PCR and Western Blot.Then we chose A549 cell to detect cell viability,migration,and gene expression in vitro and explore the underlying molecular events.We first stably infected A549 cells with lentivirus carrying MT1 M c DNA.Afterward,we assessed the changes in cell viability,wound healing,and Transwell migration and used a Luciferase reporter assay to evaluate p53 binding to the MT1 M promoter.We next assessed whether MDM2 knockdown can synergize with the inhibitory effect of MT1 M overexpression on A549 cells.The MDM2 si RNA was able to knockdown MDM2 expression and reduce MT1 M expression in A549 cells.The results indicated that MDM2 regulated MT1 M expression.We then assessed cell viability and migration as well as protein expression and found that MDM2 knockdown synergizes with the inhibitory effect of MT1 M overexpression on A549 cell viability and migration as well as protein expression.Lastly,we confirmed the regulation among MT1M?p53?MDM2 using immunohistochemistry and immunoprecipitation.?Results?1?the roles of MT1 M in NSCLCWe overexpressed MT1 M protein in A549 cells using lentivirus carrying MT1 M coding sequences and our cell viability assay showed that A549 cell viability was significantly reduced compared to the vector-only control(NC)-infected cells.Wound healing and Transwell cell migration assays revealed that the migration ability of A549 cells after MT1 M overexpression was attenuated.Furthermore,the expression of cell migration-related proteins,including MMP2,MMP9,AND MMP14,assayed using Western blot was significantly decreased after MT1 M overexpression.2?the regulation in p53 to MT1 M expression levelOur data showed that luciferase activity dramatically increased in p53 and MT1 M promoter co-transfected cells,indicating that p53 was able to bind to the MT1 M promoter to upregulate MT1 M expression.Cell viability and migration assays revealed that the p53 inhibitor was able to antagonize the inhibitory effects of MT1 M on A549 cell viability and migration capacity.In addition,the expression of cell mobility-related proteins like MMP2,MMP9,and MMP14 was also partially recovered after treatment with the p53 inhibitor.3?the regulation in MDM2 to p53-MT1MThe cell viability and migration assay showed that after MDM2 knockdown,the viability and migration capacity of A549 cells were significantly slower than those of control cells.We then assessed cell viability and migration as well as protein expression and found that MDM2 knockdown synergizes with the inhibitory effect of MT1 M overexpression on A549 cell viability and migration as well as protein expression.?Conclusions?1? MT1 M was lower expression level in NSCLC cells.It may act as a tumorsuppressor and inhibit NSCLC cell viability,migration.This report provided the new targeting therapy for lung cancer.2?p53 was able to bind to the MT1 M promoter to upregulate MT1 M expression.p53 was able to regulate MT1 M expression level on A549 resulting in affecting cell viability and migration capacity.3?We revealed a novel MDM2-p53 regulation of MT1 M expression in A549 cells to inhibit MT1 M expression and subsequently suppress MT1 M antitumor activity in vitro.