长链非编码RNA CASC11调控结直肠癌关键信号通路WNT及其作用的分子机制

lncRNA Cancer susceptibility candidate 11 (CASC 11) is a newly found long non coding RNA, located in 8q24.21 which is highly associated with colorectal cancer. While the function and mechanisms of CASC11 remain elusive. Our previous studies indicated that CASC11 was statistically upregulated in CRC tissues and CASC11 was significantly correlated with tumor size, TNM stage, invasion and lymph node metastasis; downregulated CASC11 could repress the proliferation and immigration capacity of CRC cells, while, up regulation of CASC11 had opposite effect. We also found that hnRNP-K could bind to CASC11 by RNA-pull-down assays, and through GSEA and western blot, we found that highly expressed CASC11 could up regulate the activity of WNT pathway, by dual fuciferase reporter gene assay, we found that c-Myc could bind to the promoter of CASC11. Based on these findings we propose the hypothesis that CASC11-hnRNP-K-β-catenin-WNT-c-Myc-CASC11 pathway and the positive feedback loop play an important role in CRC. In this study, we aim to further investigate the function of CASC11 in activating wnt pathway, and the mechanism of CASC11 in promoting CRC progression. This study could further provide a potential molecular marker and therapeutic target for diagnose and treatment of CRC.

lncRNA CASC11编码基因位于与结直肠癌高度相关的8q24.21区，但其作用及机制尚未见报道。我们前期工作发现：CASC11在人结直肠癌中的表达异常增高，其表达水平与肿瘤大小、TNM分期、浸润和淋巴结转移呈正相关。抑制结直肠癌细胞的CASC11的表达能显著降低其增殖和迁移力，过表达CASC11则能促进其增殖和迁移力。RNA-pull-down实验初步发现了其结合蛋白hnRNP-K，GSEA分析及WB实验我们发现CASC11能促进WNT通路上调，双荧光素酶报告基因技术初步验证了c-Myc能与CASC11启动子区结合。在此基础上我们提出CASC11-hnRNP-K-β-catenin-WNT-c-Myc-CASC11通路激活及正反馈环路假说。本项目将深入探讨CASC11在结直肠癌核心驱动信号通路WNT及发展演进中的作用及全新机制，为临床结直肠癌的诊断与靶向治疗提供新的实验依据。

LncRNA具有重要的生物学功能，其异常表达与人类疾病密切相关，多种肿瘤中发现lncRNA异常表达。癌症易感性基因11（cancer susceptibility candidate 11，CASC11）是新发现的一个LncRNA，其在结直肠癌中的功能及作用机制尚不清楚。我们通过qRT-PCR检测发现LncRNA CASC11在结直肠癌组织中的表达水平高于周围正常粘膜组织，且其表达水平与肿瘤大小、TNM分期、局部浸润和淋巴结转移呈显著相关性。抑制CASC11在结直肠癌细胞中的表达能显著降低结直肠癌细胞的增殖和迁移能力。过表达CASC11则能促进结直肠癌细胞的增殖和迁移。通过RNA-pull-down实验找到了CASC11的结合蛋白--hnRNP-K并发现CASC11可以通过增加hnRNP-K的稳定性，促进hnRNP-K蛋白的表达。通过GSEA生物信息学分析我们发现在CASC11高表达的结直肠癌细胞中WNT通路相关基因富集。WNT活性检测证实了CASC11能增强WNT通路活性，Western blot实验结果显示CASC11能促使β-catenin和hnRNP-K入核增多， WNT通路靶基因c-Myc、MMP7、CyclinD1表达增多，且CASC11对WNT通路的激活是通过hnRNP-K起作用。通过生物信息学分析我们在CASC11启动子找到了c-Myc结合位点，并用荧光素酶报告基因及CHIP实验验证了二者的结合。另外我们还发现c-Myc可以促进CASC11启动子结合位点的乙酰化。 通过系列研究，我们初步发现 LncRNA CASC11通过结合蛋白hnRNP-K激活WNT/β-catenin信号通路来促进结直肠癌的侵袭、转移和演进。C-Myc可以作为转录因子结合在CASC11启动子位置，并能使启动子乙酰化来促进CASC11表达。本项目深入探讨了CASC11在结直肠癌核心驱动信号通路WNT及发展演进中的作用及全新机制，为临床结直肠癌的诊断与靶向治疗提供了新的实验依据。本项目已发表论文6篇，其中SCI 7.0分以上2篇，ESI高被引论文1篇，JCR小类2区论文3篇，JCR小类3区论文3篇，被引次数已达173次。培养硕士研究生4人和博士研究生4人。