功能未知的长链非编码RNA: AC114812.8调控膀胱癌恶性进展的分子机制

A long non-coding RNA (lncRNA) with unknown function, named as AC114812.8, which is identified by our lncRNA sequencing screening,is suggested to be associated with bladder cancer . Our previous work is described as follow: quantitative RT-PCR demonstrated that AC114812.8 was significantly over-expressed in high stage bladder cancer tissues compared with low stage bladder. Silencing AC114812.8 by shRNA significantly suppressed the invasion and migration ability of bladder cancer cell lines. Moreover, bioinformatic analysis and RIP-PCR experiment revealed that AC114812.8 could bind with the chromosome repressive complex 2 (PRC2) so as to gain an epigenetically regulating potential. Expression microarray and in vitro validation suggested that silencing AC114812.8 significantly up-regulated the expression of a metastatic suppressor gene of bladder cancer, WIF-1, which is also a target gene of PRC2. Based on above results, we therefore hypothesized that AC114812.8 might suppress the bladder cancer metastasis by recuiting PRC2 and subsequently decreasing the expression of WIF-1. In this proposal, firstly, we plan to clarify the functional role of AC114812.8 in the invasion and metastasis of bladder cancer. To achieve this goal, we design to over-express and knock-down AC114812.8 both in vivo (in both passitive and initiative metastatic NOD/SCID mice model) and in vitro (in two separated bladder cancer cell lines) and evaluate the impact of AC114812.8 on the abilities of migration and invasion. Secondly, we aim to explore how AC114812.8 exerts its regulatory function. Highlighted by the relationship between WIF-1 gene and AC114812.8, we design to investigate the mutual interaction between WIF-1 and AC114812.8 via CHIP, ChIRP, and rescue experiments. Finally, we design to evaluate the expression level of AC114812.8, EZH2, and WIF-1 in a high through-put human bladder cancer tissue microarray, as well as their correlation with clinical profiles. Our study is the original source of innovation and can provide new potential biomarkers for the prevention and treatment of bladder cancer.

AC114812.8(简称AC)是通过测序获得、差异表达于不同级别膀胱癌、功能未知的长链非编码RNA。前期研究发现AC在膀胱癌进展过程中，表达显著升高。沉默AC可显著抑制膀胱癌细胞株增殖侵袭等恶性行为；生信分析及预实验提示AC与组蛋白复合体PRC2结合，而PRC2可结合膀胱癌分级相关基因WIF-1启动子；表达谱芯片及预实验提示AC过表达WIF-1表达显著下调。从而提出AC114812.8可通过招募PRC2表观调控WIF-1促进膀胱癌恶性进展的科学假说。本研究拟采用过表达/沉默策略，结合体内体外实验，论证AC在膀胱癌恶性进展的作用；应用ChIP、ChIRP等技术，结合拯救实验，深入阐明AC招募 PRC2表观调控WIF-1的分子机制；最后扩大样本评价AC、PRC2及 WIF-1表达在膀胱癌诊疗中的临床价值。国内外尚未见有关AC促进膀胱癌恶性进展的研究，本研究有望为膀胱癌的防治提供新靶点。

长链非编码 RNA (lncRNA)是一类特殊的 RNA 分子，其长度大于 200bp，在转录调控中具有重要意义。 lncRNA—AC114812.8 在膀胱癌中显著高表达，本研究旨在揭示其在膀胱中的生物学功能及其机制，为膀胱癌的诊疗提供潜在的治疗靶标。 通过 qRT-PCR 实验，我们发现 AC114812.8 在膀胱癌中的表达水平显著升高。克隆形成实验和 CCK-8 实验结果表明，AC114812.8 沉默后膀胱癌细胞的增殖能力显著减弱；相反，AC114812.8 的过表达显著促进膀胱癌细胞的增殖。 AC114812.8 沉默后可抑制膀胱癌细胞的迁移和侵袭能力；相反，过表达 AC114812.8 后膀胱癌细胞的迁移和侵袭能力显著增强。动物实验结果显示AC114812.8 过表达组的小鼠肿瘤体积和重量明显高于对照组。研究发现 miR-371b-5p 可能是潜在的与 AC114812.8结合的miRNA。荧光素酶基因报告实验和 RNA pull down 进一步证明了 AC114812.8 和 miR-371b-5p 之间的直接结合。推测 miR-371b-5p 潜在的下游靶基因，我们发现 miR 371b-5p 和 FUT4 的 3’UTR 区域结合的可能性较高。为了验证这一预测，我们进行了荧光素酶报告基因实验，结果表明 miR-371b-5p 可以结合到 FUT4 3’UTR 区域发挥调控功能。为了验证我们的结果，我们进行了挽救实验。结果表明， miR 371b-5p 可以挽救 AC114812.8 对 FUT4 表达水平和膀胱癌细胞增殖、迁移能力的增强效果。最后，用 Western Blot 分析了 AC114812.8 对膀胱癌细胞中 EMT相关基因表达的影响。结果表明，AC114812.8 过表达 在膀胱癌中促进了EMT 进程。结论：AC114812.8 可以通过充当 miR-371b-5p 海绵，抑制 miR-371b 5p 对靶基因 FUT4 的沉默作用，从而调控 FUT4 促进膀胱癌的进展。