一种新的P16基因增强子反义链lncRNA P16AS的功能与肿瘤发生发展

It is well known that P16 gene is most frequently inactivated in the genome of many human cancers. It is deleted in about 10% of cancers and transcriptionally inactivated by DNA methylation in 30-40% of cancers. In our pilot studies, we have found that: (1) a novel multi-exonic lncRNA P16AS is transcribed from the antisense strand of P16 enhancer region; (2) There is a common deletion sequence (~4kb) within the P16 enhancer region in 98% of human cancer cell lines (n=318), in which at least one of the P16 alleles is deleted. Notably, exon-2 and –3 of P16AS are located within the common deletion region. (3) Methylation of P16 CpG islands around transcription start site represses P16AS transcription. These results suggest that P16AS inactivation may play crucial roles in carcinogenesis. In the present project, we will characterize the full length of P16AS, and detect the prevalence of P16AS inactivation by both allele deletion and epigenetic repression in common cancer tissues, including gastric carcinomas. Associations between P16AS inactivation and prognosis of cancers will also be analyzed. Using CRISPR/Cas9 and other biotechnologies to up-/down-regulate P16AS expression, we will entensively study effects of P16AS inactivation on immortalization or malignant transformation of normal cells. Furthermore, effects of P16AS on regulation of P16, P15, P14, ANRIL, related downstream signal pathways, and possible mechanisms will also be investigated. Results of these studies in the proposed projects will greatly improve our knowledge on function of this novel lncRNA in normal cells and cancer development.

已知P16基因在10%的肿瘤中存在拷贝缺失，在30-40%的肿瘤中存在DNA甲基化失活，是肿瘤基因组中失活频率最高的位点。我们在前期研究中发现：(1)P16基因增强子区反义链存在一个多外显子的lncRNA P16AS；(2)在98%的P16位点缺失肿瘤细胞系(n=318)中存在~4kb共同缺失序列，P16AS第2、3外显子亦处于其中；(3)P16启动子CpG岛甲基化能够抑制P16AS转录。这些结果提示P16AS缺失在肿瘤发生过程中可能发挥重要作用。在本研究中我们拟在鉴定P16AS全长的基础上，分析胃癌等常见肿瘤中P16AS位点缺失和转录变化及其与肿瘤临床病理特征的关系；利用CRISPR/Cas9和基因表达调控技术，研究P16AS缺失对细胞永生化/恶性转化的影响；探索P16AS对P16、P15、P14和ANRIL转录及其下游信号通路的影响及其分子机制。

CDKN2A基因座位是人类肿瘤组织中体细胞拷贝缺失频率最高的基因，由于体细胞基因拷贝缺失分析方法的普遍缺失，包括CDKN2A在内的各种体细胞基因拷贝缺失的用途及其作用机制有待研究。我们在前期发现肿瘤细胞中CDKN2A基因可能存在共同缺失区和该基因反义链存在一新的lncRNA线索的基础上，通过海量文献挖掘和肿瘤全基因组测序数据再分析，首次证实CDKN2A基因从启动子至第二外显子区存在1个5.1KB的共同缺失区（chr9: 21,970,277 - 21,975,386; hg19）；首次发现在90%以上的CDKN2A基因拷贝缺失肿瘤细胞中，该基因编码的P16蛋白和P14蛋白的DNA序列均发生了缺失；敲除共同缺失区中编码P16蛋白和P14蛋白所需的第二外显子促进细胞增殖的作用明显大于单独敲除P16蛋白或P14蛋白特异性第一外显子alpha或beta序列的作用；建立了灵敏的CDKN2A基因拷贝数测定方法，用该方法分析234例胃癌患者癌组织发现，CDKN2A拷贝缺失者胃癌远处转移风险高，患者生存时间短，提示CDKN2A拷贝缺失有重要应用前景。进一步建立了CDKN2A特异性RNA靶向捕获组合超深度2代测序技术，首次鉴定出了P16基因启动子上游反义链能够编码一个含3个外显子的lncRNA基因P14AS (1043nt; NCBI GenBank MK574077；chr9: 21,989,178-21,994,898；hg19)，其第一外显子存在AUF1结合序列ARE，P14AS通过竞争性结合AUF1来预防其下游的ANRIL等lncRNA的降解，具有癌基因的功能。P14AS和ANRIL在结肠癌组织中表达明显上调，能够增加结肠癌的转移风险。上述结果第一次揭示在90%以上的CDKN2A基因拷贝缺失肿瘤细胞中P16蛋白和P14蛋白同时失活，具有重要的科学价值和应用前景；同时还发现P16基因启动子上游反义链能够编码lncRNA P14AS，具有预防致癌性ANRIL降解的作用。P16启动子甲基化同时抑制P16、P14AS和ANRIL的转录。P14AS的工作2019年在线发表在Mol Cancer上，CDKN2A基因拷贝缺失的工作准备发表中，所建立的CDKN2A基因拷贝缺失测定方法申请了国际发明专利（PCT/CN2019/087172）。