Abstract
BackgroundThe CDKN2A/B locus contains crucial tumor suppressors and a lncRNA gene ANRIL. However, the mechanisms that coordinately regulate their expression levels are not clear.MethodsNovel RNAs transcribed from the CDKN2A gene were screened by CDKN2A-specific RNA capture deep-sequencing and confirmed by Northern blotting and clone-sequencing. Long non-coding RNA (lncRNA) binding proteins were characterized by RNA pull-down combined with mass spectrometry and RNA immunoprecipitation. LncRNA functions in human cells were studied using a set of biological assays in vitro and in vivo.ResultsWe characterized a novel lncRNA, P14AS with its promoter in the antisense strand of the fragment near CDKN2A exon 1b in human cells. The mature P14AS is a three-exon linear cytoplasmic lncRNA (1043-nt), including an AU-rich element (ARE) in exon 1. P14AS decreases AUF1-ANRIL/P16 RNA interaction and then increases ANRIL/P16 expression by competitively binding to AUF1 P37 and P40 isoforms. Interestingly, P14AS significantly promoted the proliferation of cancer cells and tumor formation in NOD-SCID mice in a P16-independent pattern. Moreover, in human colon cancer tissues, the expression levels of P14AS and ANRIL lncRNAs were significantly upregulated compared with the paired normal tissues.ConclusionA novel lncRNA, P14AS, transcribed from the antisense strand of the CDKN2A/P14 gene, promotes colon cancer development by cis upregulating the expression of oncogenic ANRIL.
Highlights
The CDKN2A/B locus contains crucial tumor suppressors and a Long non-coding RNA (lncRNA) gene ANRIL
A three-exon 1043nt lncRNA transcribed from the antisense strand of the fragment around exon 1β of the CDKN2A locus was detected from the RNACap-Seq readouts (chr9: 21,989, 178-21,994,898 in the human genome GRCh37/hg19; (Additional file 2: Fig. S1A)
Coordinate overexpression of P14AS, ANRIL, and AUF1 in colon cancer tissues To determine whether P14AS, ANRIL, and P16 expression were coordinately upregulated in cancer development, we examined their expression status in colon cancer (CC) and their corresponding surgical margin (SM) tissue samples from 172 patients, and normal colon mucosa biopsy samples from 50 noncancer patients
Summary
The CDKN2A/B locus contains crucial tumor suppressors and a lncRNA gene ANRIL. The mechanisms that coordinately regulate their expression levels are not clear. Three important tumor suppressor genes (P16INK4A, P14ARF and P15INK4B) and the oncogenic lncRNA ANRIL (CDKN2B-AS1) resides in the human CDKN2A/B locus at chromosome 9p21 [1]. While ANRIL was reported to downregulate P15 and P16 expression by interacting with components of polycomb repressive complex-1/-2 [2,3,4,5], ANRIL was found to be coordinately transcribed with P16 in cancer cells and transcriptionally. For the first time, a novel lncRNA called P14AS (NCBI GenBank MK574077) transcribed from the antisense strand of the fragment around exon 1β of the CDKN2A gene. We found that AUF1 binds to P14AS and the AUF1 binding of the ARE (AU-rich element) in exon 1 of the P14AS gene increases ANRIL/P16 level
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