CircRNA-RAB23作为miR-532-3p海绵通过HMGA2促进肝内胆管癌生长及转移的作用和机制研究

Intrahepatic cholangiocarcinoma (ICC) is a highly fatal malignant tumor and the prognosis of patients with ICC is generally poor associated with early recurrence and distant metastasis. Intrahepatic cholangiocarcinoma represents the second most common primary liver cancer and is increasing in incidence. There has been clinical lack of effective treatments and specific biological targets. The circularRNA (circRNAs) have properties such as structural stability, high abundance, and tissue-specific expression. CircRNAs has been shown to be associated with the progression of a variety of tumors. Early, we screened the differential expression profiles of circRNAs in the ICC by the high throughput sequence method. In which, Circ-RAB23 was the most significantly increased circRNA. Further, we found that the expression of Circ-RAB23 was closely related to the tumor size, lymph node metastasis and survival of patients. Based on this, this project intends to establish the role of Circ-RAB23 on the growth and metastasis of ICC cells, in vivo and in vitro. FISH, RNA Pull down, and reporter gene detection methods will be applied to elucidate the mechanism by which Circ-RAB23 can be used as a sponge for mir-532-3p to relieve its inhibitory effect on HMGA2, thereby affecting the pathways related to epithelial mesenchymal transformation and promoting the metastasis of tumor cells. Through the establishment of CircRNA-key signaling pathway-cell transfer phenotype regulatory network, this project intends to provide new targets for the treatment and prognostic assessment of ICC.

肝内胆管癌（ICC）因进展迅速，易复发和远处转移，且对放化疗不敏感，预后极差。近30年来，全球ICC的发病率及死亡率有明显上升趋势，一直以来临床缺乏有效的治疗手段和特异性生物靶标。因此对于肝内胆管癌的研究必须从基础做起，结合临床，对肝内胆管癌的发生、转移以及预后影响因素等进行深入研究。环状RNA(circRNAs)具有组织与时空特异性、不易降解等特性，已经成为非编码RNA研究领域的又一全新研究热点。已证实circRNAs参与了多种肿瘤生物学功能的调节，与肿瘤的进展及预后密切相关，但是目前circRNAs在肝内胆管癌中是否起作用及其起作用的机制尚不十分清楚。前期我们通过高通量测序筛选出ICC特异性circRNAs表达谱，通过相关性分析及Kaplan-Meier生存分析，我们发现Circ-RAB23的表达与患者的肿瘤大小、病理性卫星灶、血管侵犯、分化程度以及淋巴结转移密切相关，并且高表达Circ-RAB23的患者总体生存期（OS）和无瘤生存期（DFS）更短，这提示我们，Circ-RAB23可能通过促进ICC肿瘤细胞的生长和转移从而影响患者的预后。体外实验也证实，ICC肿瘤细胞内过表达Circ-RAB23能够显著促进肿瘤细胞的恶性增殖以及侵袭、转移能力。本项目拟在此基础上，通过构建Circ-RAB23过表达与干扰细胞系，应用FISH、Pull down、报告基因检测等多种手段，从体内、体外两方面深入研究Circ-RAB23作为miR-532-3p海绵通过促进HMGA2表达进而影响ICC生长和转移的可能作用和机制。本项目拟希望通过此项研究，阐明Circ-RAB23通过miR-532-3p/HMGA2/EMT通路促进ICC生长及转移的作用机制，深化对circRNAs在ICC发展中所起作用的认识，为临床治疗ICC及改善患者的预后提供新的靶标。

环状RNA（circRNA）是区别于传统线性RNA的一类新型RNA，具有闭合环状结构，被证实在多种肿瘤的发生发展过程中起关键性的作用。近年来，肝内胆管癌（ICC）的发病率及死亡率上升明显，其进展迅速，容易转移，临床缺乏有效的治疗靶标，因此预后极差。前期，我们利用高通量测序的方法筛选了ICC患者癌与癌旁组织相比较的circRNAs表达差异，其中Circ-RAB23在癌中升高最显著。序列分析，以及一代Sanger测序、RNase R抗性实验、Oligo(dT)18引物PCR等体外实验证实，母基因的第二至六号外显子首尾相连环化是Circ-RAB23成环的主要形式。通过反向PCR方法，我们检测了Circ-RAB23在61对独立ICC以及癌旁样本中的表达，进一步验证了Circ-RAB23在ICC中呈现高表达。Circ-RAB23的表达高低与患者的肿瘤大小、病理性卫星灶、血管侵犯、分化状态以及淋巴结转移密切相关。同时，高表达Circ-RAB23的患者整体生存期（OS）和无瘤生存期（DFS）更短。通过慢病毒感染系统，我们利用肝内胆管癌细胞建立了Circ-RAB23稳定过表达或干扰的细胞系。体外实验表明，过表达Circ-RAB23能够显著促进ICC细胞的恶性增殖及侵袭、转移，而干扰则引起相反的效应；通过构建尾静脉肺转移模型，我们还进一步在体内水平证实了Circ-RAB23促转移的作用。机制方面，我们证实Circ-RAB23可以作为miR-532-3p的海绵，解除其对于HMGA2的抑制作用，进而影响上皮间质转化相关信号转导通路并促进了ICC细胞的转移。本项目从体内、体外两个方面探讨了Circ-RAB23影响ICC生长和转移的可能作用和机制，有望为临床治疗ICC及改善患者的预后提供新的靶标。