Long Non-coding RNA CARMN Suppress Epithelial-mesenchymal Transition Through MMP2 In Breast Cancer

Objective Breast cancer is the most commonly diagnosis cancer among women worldwide.Early detection and intervention may be the most effective method to improve breast cancer outcome.Mounting evidence provided valuable insights into cancer-related driver genes in early stage breast cancer.Current studies suggest that epithelial-mesenchymal transition(EMT)process involves in the progression of early breast cancer.This study explored the possible driver long non-coding RNA in early stage breast cancer,and investigated the underlying mechanism of lncRNA CARMN in breast cancer EMT process.Methods Bioinformation analysis of samples from TCGA and GEO database and q RT-PCR were used to identify and validate potential driver lncRNAs in early stage breast cancer.Significantly differentially expressed lncRNA CARMN was selected for further prognostic analysis.CCK-8 assay,colony formation assay,scratch assay,Transwell assay and chemosensitivity assay were conducted to explore the functions relative to proliferation,migration,invasion and chemosensitivity of CARMN in breast cancer cells.A series of potential downstream targets and relative functional pathways of CARMN were identified by analyzing GEO samples.q RT-PCR and western blot were used to detect the expression of predicted target MMP2 in m RNA and protein level,and the changes of EMT markers were also detected.After co-transfecting MMP2 and CARMN in breast cancer cell,the expression of MMP2 and relative pathways were detected.We further used metastatic model to investigate the effect of CARMN on breast cancer metastasis in vivo.To further investigate the mechanism of MMP2 regulation by CARMN,RNA pull-down followed by mass spectrometry was used to identify CARMN binding protein,and DHX9 was chose to subsequent RIP assay to verify the combination.DHX9 was knocked down by transfection of sh-DHX9 plasmid,and changes in MMP2 at m RNA and protein level were detected.Ch IP assay was used to determine the direct binding of DHX9 to the MMP2 promoter region and the effect of over-expressing CARMN on the combination.Results Lnc RNA CARMN was significantly down-regulated in breast cancer samples and cell lines,and a higher CARMN expression in cancer related to a better prognosis in breast cancer.In vitro,overexpression of CARMN inhibited cell proliferation and metastasis,and enhanced the sensitivity of breast cancer cells to doxorubicin.The biological function of CARMN was reconfirmed that overexpression of CARMN inhibited the metastasis of breast cancer in vivo.Over-expression MMP2 could rescue CARMN inducing changes of metastasis ability of breast cancer cells and EMT suppression.In addition,we proved that MMP2 is a direct target of DHX9,which directly binds to MMP2 promoter and activate its transcription.CARMN functioned as a decoy by binding to DHX9 leading to the transcriptional inhibition of MMP2,thereby inhibiting the metastasis and EMT of breast cancer cells.Conclusions Our results demonstrate that CARMN acts as a tumor suppressor by inhibiting tumor proliferation and migration,and enhance the sensitivity of breast cancer cells to doxorubicin.CARMN suppresses MMP2 mediated EMT by transcriptional inhibiting MMP2 via competitively binding to DHX9 and blocking the binding of DHX9 to MMP2 promoter region,thereby playing an important role in breast cancer metastasis.Moreover,down-regulation of CARMN is an early event in breast cancer progression,which might provide potential biomarker for progression and metastasis in early stage breast tumor.