Methylated+58CpG Site Decreases DCN MRNA Expression And Enhances TGF-β/Smad Signaling In High-metastatic NSCLC Cells

Background and Objective: Lung cancer is the leading cause of cancer deaths inmen and women worldwide. Non-small cell lung cancer (NSCLC) accounts forapproximately85%of all lung cancers. Since metastasis remains the cause of90%ofdeaths from solid tumors, elucidating the mechanisms underlying lung cancer metastasisappeared to be very important. Transforming growth factor β (TGF-β) is a ubiquitousand essential regulator of cellular and physiologic processes, such as cellularproliferation, migration, invasion, and immunosurveillance. Decorin (DCN) is amember of the extracellular matrix small leucine-rich proteoglycan family. It canspecifically binding to TGF-β and partially blocking TGF-β signaling pathway, leadingto growth suppression of distant tumors including lung cancer. However, themechanisms by which the reduced DCN expression causes lung cancer metastasisremains unclear.In our study we determinate to explore whether DNA methylation couldbe epigenetically responsible for inactivation of DCN and affect TGF-β/Smad signalingin metastatic NSCLC cells.Methods:(1) Transwell invasion and migration assays to determinate thematastasis ability of Low-(95C) and high-metastatic (95D) cells.(2) qRT-PCR analysiswas carried out to analysis the expression of DCN mRNA, and the expression levelswere calculated following normalization to β-actin mRNA levels. The goal is to confirmwhether the DCN expression level was reduced with the cell’s high matastasis ability.(3)95C and95D cell lines culture and DNA demethylating agent,5-aza-2’-deoxycitidine(5-Aza) treatments. The concentration of5-Aza was10μM and the cells were harvestedfor analysis the effects of hypomethylation on DCN gene expression.(4) A1,230-bpsequence, including DCN proximal promoter (-200~+1) and5’-UTR (+1~+1,030)regions was available for CpG sites selection. Methyl Primer Express Software v1.0 and TFSEARCH were used for the prediction of the putative functional CpG sites.(5)Clonal bisulfite sequencing and q-PCR were performed to verify the relationship ofhigh methylation level of+58CpG site with reduced expression level of DCN mRNA in95C and95D cell lines and in Lung carcinoma tissues with Stage III or IV NSCLC.(6)Western Blot analysis the expression level of putative transcription factor AHR in95Cand95D cell lines.(7) ChIP assay was performed in95C and95D cell lines to checkwhether AHR can bind to+58CpG site.(8) EMSA and luciferase reporter plasmidswere used to determinate the methylated+58CpG site will decrease the AHR bindingability.(9) ELISA was performed to determine expression levels of DCN protein fromcell culture supernatants.(10) The normalized p-Smad3level to total Smad3and theEMT mark E-cadherin were used to evaluate the activity of TGF-βsignal pathway.Results（1）DNAmethylation was involved in DCN mRNAexpression（.2）After in silicoprediction,+58CpG site was a candidate functional CpG site.(3) Methylation level of+58CpG was higher in95D than95C cells, and was associated with DCN mRNAexpression in metastatic NSCLC.(4) AhR was recruited to DCN5′-UTR regioncontaining+58CpG.(5) Methylation of+58CpG decreased binding ability oftranscriptional factors to DCN5’-UTR region.(6) Methylation of+58CpG significantlyinhibited luciferase reporter gene transcriptional activity.(7) DCN inhibitedphosphorylation of Smad3and enhanced E-cadherin expression in metastatic NSCLCcells.ConclusionIn this study, we have identified the methylated+58CpG in DCN5’-UTRassociated with reduced expression of DCN mRNA in high-metastatic NSCLC cells.Our findings reveal that+58CpG methylation may be one of mechanisms accountingfor reduced recruitment of transcriptional activator AhR to DCN5′-UTR, and suggestthat this mechanism promotes TGF-β/Smad signaling by enhancing phosphorylation ofSmad3, thereby down-regulates E-cadherin in high-metastatic NSCLC cells.