TGF-β1相关LncRNAs在胆囊癌转移中的作用及机制研究

Gallbladder carcinoma(GBC) is one of the most aggressive and lethal tumors in digestive system,with extremely high metastasis activity and poor prognosis clinically. New molecular therapeutic targets for GBC progression are still urgently needed. It is becoming increasingly clear that long noncoding RNAs are key regulators of biological function. Their emerging roles in the development and progression of human cancers may present novel diagnostic and therapeutic opportunities, also supply the prognostic information of tumor development. The role of TGF-β1-induced epithelial-mesenchymal transition (EMT) in cancer cell metastasis is well established. but the role of lncRNAs in TGF-β1 induced invasion and metastasis enhancement of GBC is still unknown. Our project will thoroughly explore critical LncRNAs involved in TGF-β1-induced metastasis and progression of GBC. Previously, we have carried out lncRNA microarray analysis of gallbladder carcinoma and adjacent gallbladder mucous membrane tissues. After verification via real-time PCR, RP11-537A6.9, which was found highly expressed in GBC, could be activated by TGF-β1 cytokine. Function study showed that inhibition of RP11-537A6.9by siRNA could notably suppress metastasis of GBC-SD cells in vitro. We will further investigate the role of RP11-537A6.9, explore its mechanism, and elucidate whether RP11-537A6.9 is a mediator of TGF-β1 signaling. Taken together, our study may provide a novel mechanism and potential therapeutic target for GBC in the future.

表观遗传与细胞微环境在肿瘤发生发展中至关重要。长链非编码RNA（lncRNA）通过表观遗传调控等方式参与了多种肿瘤的发生发展，但目前lncRNA在胆囊癌转移中的作用尚无系统性研究。本立项拟以发现胆囊癌转移相关lncRNA为目标，从胆囊癌炎症微环境LPS-TGF-β1为切入点，从分子、细胞及动物等层面研究lncRNA对胆囊癌生物学特性的调控及机制，明确相关lncRNA在TGF-β信号通路中的作用。申请人前期已对胆囊癌样本进行了lncRNA芯片检测获得了lncRNA差异表达谱，初步验证发现RP11-537A6.9等lncRNAs在胆囊癌中异常高表达，并且可被TGF-β1激活，干扰RP11-537A6.9后胆囊癌细胞的迁移及侵袭能力明显下降，可能是潜在靶标分子。本项目拟深入探索RP11-537A6.9的作用及机制，阐明其是否作为TGF-β通路的调节因子介导了胆囊癌的转移，为临床转化提供理论依据。

胆囊癌被认为是侵袭性和致死性最高的恶性肿瘤之一，发现时常已出现局部侵犯或肝脏转移，术后极易复发，整体预后差，因此寻找相关靶点进行干预成了胆囊癌治疗的关键问题。长链非编码RNA（lncRNA）通过表观遗传调控等方式调控肿瘤细胞的转移迁移、侵袭、粘附、上皮细胞间质化参与参与了多种肿瘤的发生发展，这为阐明胆囊癌侵袭、转移特性的分子机制提供了方向。本课题力求阐明炎症微环境下lncRNAs调控胆囊癌恶性表型的分子机制，探索对lncRNA进行临床转化用于胆囊癌临床治疗及预后的可行性。在前期工作的基础上，通过lncRNA芯片筛选出胆囊癌组织中差异性表达的lncRNAs，模拟胆囊癌病理生理微环境，从LPS-TGF-β1为切入点，寻找可以被TGF-β1激活并且可以促进胆囊癌细胞运动能力的lncRNAs，结合胆囊癌临床组织样本与临床信息，从分子、细胞及动物等多层面系统性研究相关lncRNAs对胆囊癌侵袭、转移特性的影响，明确其分子网络机制。课题实施中，结合多种生物学手段筛选并鉴定出了胆囊癌转移相关lncRNAs群，确定了LSAMP-AS3为靶标分子并进行深入研究。qPCR证实LSAMP-AS3等lncRNAs在胆囊癌中明显高表达。划痕、transwell、WB、细胞克隆形成、CCK8增殖实验等多种体外实验证实，抑制LSAMP-AS3后胆囊癌细胞的转移能力、增殖能力、肿瘤干细胞特性明显下降，而化疗敏感性性明显提高。慢病毒感染胆囊癌细胞后脾脏注射肝脏转移模型证实，干扰LSAMP-AS3水平后，胆囊癌肝转移灶明显减少。已经进行了自噬相关的测序，期望可以从自噬入手获得LSAMP-AS3在胆囊癌中发挥作用的相关机制。以上多种结果表明，LSAMP-AS3促进了胆囊癌的恶性表型，通过干预LSAMP-AS3的表达或其发挥作用的网络节点，或许为今后胆囊癌的治疗提供理论依据。