Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan.
Oncol Rep. 2010 Sep;24(3):701-7. doi: 10.3892/or_00000910.
ANRIL, a large antisense non-coding RNA, is in the proximity of CDKN2A and overlapped with CDKN2B at human chromosome 9p21, and has been strongly implicated in the association with high risk genetic markers of coronary artery disease (CAD). Mice model harboring large deletion of posterior part of ANRIL and CAD high risk genetic markers resulted in substantial suppression of both CDKN2A and CDKN2B; however, the mechanistic insights of regulation and function of ANRIL have remain elusive. To date multiple splice variants of ANRIL have been reported and expression of specific splice variant of ANRIL has been shown to be tightly associated with 9p21 CAD high risk markers. Here we identified a new splice variant of ANRIL and introduced it into HeLa cells to uncover functional aspects of ANRIL towards cellular function. For this purpose, we monitored global mRNA expressional changes and conducted gene ontology enrichment. The majority of mRNAs was down-regulated by ANRIL overexpression. Among them, a subset of mRNAs particularly involved in the regulation of nucleus and establishment or maintenance of chromatin architecture was significantly enriched. Such a circumstance was manifested after 48 h of ANRIL overexpression but no significant changes were seen after 24 h of ANRIL overexpression. Next we analyzed the sequences containing the intergenic region between ANRIL and CDKN2A (p14ARF) by introducing the sequences upstream of luciferase reporter gene. Based on the luciferase activity, the sequences tested were shown to act as promoter for ANRIL and p14ARF. Moreover, as well p14ARF, ANRIL promoter was responsive to transcription factor E2F1 in HeLa and A549 cells. Taken together, our present results indicate that co-regulation of ANRIL and p14ARF could be coupled by their unique intergenic region potentially through E2F1. Judged from the suppressive effect of ANRIL on a subset of mRNAs involved in the nuclear function suggests that ANRIL might have silencing effect on a specific gene set that accounts for a wide array of gene expression.
ANRIL 是一种长的反义非编码 RNA，位于人类 9p21 染色体上 CDKN2A 附近并与 CDKN2B 重叠，已被强烈提示与冠心病（CAD）的高风险遗传标记有关。携带 ANRIL 后段和 CAD 高风险遗传标记的大量缺失的小鼠模型导致 CDKN2A 和 CDKN2B 的表达明显受到抑制；然而，ANRIL 的调控和功能的机制仍不清楚。迄今为止，已经报道了多种 ANRIL 的剪接变体，并且已经表明特定的 ANRIL 剪接变体的表达与 9p21 CAD 高风险标记紧密相关。在这里，我们鉴定了 ANRIL 的一种新剪接变体，并将其引入 HeLa 细胞中，以揭示 ANRIL 对细胞功能的功能方面。为此，我们监测了全局 mRNA 表达变化并进行了基因本体富集分析。大多数 mRNAs 被 ANRIL 过表达下调。其中，一组特别参与核调控以及建立或维持染色质结构的 mRNAs 明显富集。这种情况在 ANRIL 过表达 48 小时后表现出来，但在 ANRIL 过表达 24 小时后没有观察到明显变化。接下来，我们通过引入荧光素酶报告基因上游的序列来分析包含 ANRIL 和 CDKN2A （p14ARF）之间的基因间区的序列。根据荧光素酶活性，测试的序列被证明可以作为 ANRIL 和 p14ARF 的启动子。此外，与 p14ARF 一样，ANRIL 启动子对 HeLa 和 A549 细胞中的转录因子 E2F1 有反应。总之，我们的研究结果表明，ANRIL 和 p14ARF 的共调控可能通过它们独特的基因间区通过 E2F1 耦合。从 ANRIL 对参与核功能的一组特定 mRNAs 的抑制作用判断，ANRIL 可能对一组特定基因具有沉默作用，该基因组涵盖了广泛的基因表达。
