Negative Regulation Of LncRNA MIR100HG By β-catenin And Its Biological Function

With the development of transcriptome sequencing,more and more evidence showed that there is a large number of non-coding RNA(ncRNA)in organisms.Previous studies suggested that ncRNA is "rubbish" of transcription,which has no practical effect.However,with the deepening of research,it has been found that ncRNA is not the by-product of transcription,but also an important regulator,which plays an important role in life.Therefore,more and more studies focus on the function of ncRNA,but the regulation mechanism of ncRNA expression is insufficient.Abnormal activation of Wnt/β-catenin signal is common in colorectal cancer,but there are few studies on the expression regulation of ncRNA.Therefore,the purpose of this study was to explore whether and how Wnt/β-catenin signaling involved in expression regulation of ncRNA in colorectal cancer.Stable overexpression of β-catenin could significantly inhibit the transcription of lncRNA MIR100HG.Dual luciferase reporter assay showed β-catenin could inhibit the promoter activity of lncRNA MIR100HG.And this inhibition could be released byβ-catenin inhibitors.In addition,DNA pull-down assay showed that β-catenin/TCF4 could bind the promoter of lncRNA MIR100HG.And then we validated that the binding site was 1120-1170 upstream of TSS by chromatin immunoprecipitation(ChIP)and mutant DNA pull-down assays.Further ChIP data showed that overexpression ofβ-catenin significantly decreased the enrichment of H3K27Ac,H3K4me3 and RNA polymerase Ⅱ,and increased the enrichment of H3K27me3.Activity inhibition ofβ-catenin could increase the enrichment of H3K27Ac.In order to explore this chromosome modification mechanism by β-catenin,we screened the histone modification related factors after β-catenin overexpression,and found that expression of histone deacetylase 6(HDAC6)was significantly upregulated by β-catenin,and inhibition of HDAC6 could relieve the transcriptional inhibition of lncRNA by β-catenin.ChIP data showed that HDAC6 could bind to the promoter of lncRNA MIR100HG.And co-immunoprecipitation assay showed that there was an interaction between β-catenin and HDAC6,which suggested that β-catenin could recruit HDAC6.Moreover,total level H3K27Ac and its enrichment on the promoter of lncRNA MIR100HG could be upregulated after inhibition of HDAC6.HDAC6 mediates the transcriptional inhibition of lncRNA MIR100HG by regulating H3K27Ac.In addition,we found that EZH2 could also be recruited by β-catenin to the promoter of lncRNA MIR100HG,and then participate in its transcriptional inhibition.Later,functional analysis showed that high expression of lncRNA MIR100HG could upregulate the cell cycle inhibitor p57,which could lead to G0/G1 phase arrest of cells and ultimately limited cell proliferation in vitro and in vivo.In conclusion,β-catenin could inhibit the transcription of lncRNA MIR100HG by recruiting histone modifying enzymes HDAC6 and EZH2 to the promoter of lncRNA MIR100HG in colorectal cancer.And lncRNA MIR100HG could induce cell cycle arrest and inhibit cell proliferation by upregulating p57.This study not only enriches the expression regulation mechanism of lncRNA,but also improves the mechanism of Wnt/β-catenin signaling,which provides a new idea for further exploration of the relationship between lncRNA and colorectal cancer.