Abstract 999: P16 DNA methylation inactivates transcription of IncRNA ANRIL

Abstract The exonic ANRIL (P15AS) is a 3.8-kb lncRNA transcribed from the antisense strand of the P14 promoter and flanking regions. Recently, we have constructed a P16 promoter DNA methyltransferase (P16-Dnmt) using the pTRIPZ vector that can specifically methylate human P16 CpG islands and found that P16 methylation directly leads to gene transcription silence [Cui et al. Genome Biology 2015, 16:252]. However, whether ANRIL transcription is affected by P16 DNA methylation has not previously been reported. In the present study, correlation between endogenous P16, P15, P14 mRNA and ANRIL levels in a panel of cell lines were determined using quantitative RT-PCR. Unexpectedly, the results showed that ANRIL transcriptional level was positively and significantly correlated with the P16 mRNA level (r = 0.774, P = 0.003), but not correlated with the P15 or P14 mRNA levels (r = 0.09 or 0.30, P = 0.78 or 0.35). These phenomena were confirmed using the public available transcriptome data in the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE). Furthermore, transcription of both ANRIL and P16 mRNA were observed only in cancer cell lines in which the P16 alleles are unmethylated (Caski, SGC7901, BGC823, GES1, Siha and HeLa), but not in these cell lines in which the P16 alleles are fully methylated (Colo205, PC3, MHCC97H, RKO, SW480 and AGS). These suggest the possibility that methylation of the P16 promoter may inactivate transcription of ANRIL as well as P16. To study the possible causality between P16 DNA methylation and transcriptional silence of ANRIL, we employed P16-Dnmt to induce P16-specific DNA methylation in gastric cancer BGC823 cells, and found that transcription levels of both P16 and ANRIL were significantly decreased by the induced P16 DNA methylation (P<0.001 and 0.024, respectively). Besides, transcription level of P14 was also reduced (P = 0.043) while the P14 promoter CpG islands remained to be unmethylated. In contrast, P16 DNA demethylation induced by an artificial P16-specific transcription factor [Zhang et al. Human Gene Therapy 2012, 23:1071-81] re-activated transcription of both ANRIL and P16 in H1299 cells (P<0.001). The mechanism of inactivation of ANRIL expression by P16 DNA methylation is under investigation. In conclusion, transcription of ANRIL is regulated by P16 DNA methylation. Citation Format: Ying Gan, Baozhen Zhang, Chenghua Cui, Dajun Deng. P16 DNA methylation inactivates transcription of IncRNA ANRIL. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 999.