P14AS upregulates gene expression in the CDKN2A/2B locus through competitive binding to PcG protein CBX7

Abstract

Background: It is well known that P16INK4A, P14ARF, P15INK4B mRNAs, and ANRIL lncRNA are transcribed from the CDKN2A/2B locus. LncRNA P14AS is a lncRNA transcribed from antisense strand of P14ARF promoter to intron-1. Our previous study showed that P14AS could upregulate the expression level of ANRIL and P16INK4A and promote the proliferation of cancer cells. Because polycomb group protein CBX7 could repress P16INK4A expression and bind ANRIL, we wonder whether the P14AS-upregulated ANRIL and P16INK4A expression is mediated with CBX7.Results: In this study, we found that the upregulation of P16INK4A, P14ARF, P15INK4B and ANRIL expression was induced by P14AS overexpression only in HEK293T and HCT116 cells with active endogenous CBX7 expression, but not in MGC803 and HepG2 cells with weak CBX7 expression. Further studies showed that the stable shRNA-knockdown of CBX7 expression abolished the P14AS-induced upregulation of these P14AS target genes in HEK293T and HCT116 cells whereas enforced CBX7 overexpression enabled P14AS to upregulate expression of these target genes in MGC803 and HepG2 cells. Moreover, a significant association between the expression levels of P14AS and its target genes were observed only in human colon cancer tissue samples with high level of CBX7 expression (n = 38, p < 0.05), but not in samples (n = 37) with low level of CBX7 expression, nor in paired surgical margin tissues. In addition, the results of RNA immunoprecipitation (RIP)- and chromatin immunoprecipitation (ChIP)-PCR analyses revealed that lncRNA P14AS could competitively bind to CBX7 protein which prevented the bindings of CBX7 to both lncRNA ANRIL and the promoters of P16INK4A, P14ARF and P15INK4B genes. The amounts of repressive histone modification H3K9m3 was also significantly decreased at the promoters of these genes by P14AS in CBX7 actively expressing cells.Conclusions: CBX7 expression is essential for P14AS to upregulate the expression of P16INK4A, P14ARF, P15INK4B and ANRIL genes in the CDKN2A/2Blocus. P14AS may upregulate these genes’ expression through competitively blocking CBX7-binding to ANRIL lncRNA and target gene promoters.