Functional And Mechanistic Study Of Long Non-coding RNA DMDRMR In Clear Cell Renal Cell Carcinoma

DNA methylation plays an important role in regulation of gene expression and mediating tumor initiation and progression.Long non-coding RNAs(lncRNAs)are involved in multi-level regulation of gene expression and modulate numerous hallmarks of cancer.DNA methylation-regulated protein-coding genes have been extensively studied,which revealed some molecular drivers in tumor initiation and progression.However,DNA methylation-regulated lncRNAs and their consequences remain poor understanding.Therefore,this study aims to explore DNA methylation-deregulated lncRNAs,as well as its consequent molecular function and mechanism in cancer.In the study,lncRNA profile that are potentially deregulated by DNA methylation was comprehensively constructed based on Huamn illumina 450K methylation chip data of 12 cancer types from The Cancer Genome Atlas(TCGA)database.A DNA methylation-deregulated and RNA m6A reader-cooperating lncRNA(DMDRMR)was further studied.DMDRMR is higher expression by its hypomethylated promotor in tumors compared to adjacent normal tissues.Subsequently,in vitro,cell proliferation and transwell assays show that,both knockdown and knockout of DMDRMR inhibit tumor growth,metastasis and invasion in clear cell renal cell carcinoma(ccRCC)cell lines,in contrast,overexpression of DMDRMR promotes tumor growth,metastasis and invasion in ccRCC cell lines.In vivo mouse model shows that knockdown ofDMDRMR inhibits subcutaneous xenograft tumor growth and metastasis of ccRCC cells.Mechanically,using RNA pull down,transcriptome sequencing and RNA binding protein immunoprecipitation assays,I demonstrated that DMDRMR binds to insulin like growth factor 2 mRNA binding protein 3(IGF2BP3)to stabilize their target genes,including the cyclin dependent kinase 4(CDK4)and three extracellular matrix components,fibronectin 1(FN1),collagen type VI alpha 1 chain(COL6A1)and laminin subunit alpha 5(LAMA5),by specifically enhancing IGF2BP3's reading on them in an N6-methyladenosine(m6A)-dependent manner.The complex of DMDRMR/IGF2BP3 binds to 5' untranslated region(UTR)of CDK4 transcript,which is modified by m6A,and increase the expression of CDK4.Consequently,the G1/S transition and cell proliferation are accelerated,and the resistance to CDK4/6 inhibitor palbociclib is enhanced.In addition,the complex of DMDRMR/IGF2BP3 binds to the exon 20 of FN1 transcript,which is modified by m6A,and elevates the FN1 expression level.Consequently,the metastasis and invasion of ccRCC cell lines are enhanced.Using chi-square test,spearman's correlation and Kaplan-Meier analysis from multiple clinical cohorts further reveal that both DMDRMR and IGF2BP3 are high expression and positive association in ccRCC,and a high coexpression of DMDRMR and IGF2BP3 is associated with poor outcomes for ccRCC patients.In summary,this study demonstrates a key role of DMDRMR in ccRCC and reveals a novel DMDRMR/IGF2BP3/CDK4(FN1)axis that mediates the initiation and progression of ccRCC.Furthermore,this study reveals that DMDRMR is a cofactor for m6A reader IGF2BP3 to stabilize target genes and promote carcinogenesis.This study provides a potential therapeutic target and clinical application in ccRCC.