胆囊癌转移相关miRNAs的高内涵筛选与功能鉴定

Gallbladder carcinoma(GBC) is one of the most aggressive and lehal tumors,with extremely high metastasis activity and poor prognosis clinically. The dysfunction of miRNAs has been demonstrated participating in the regulation of various tumor development. However, whether miRNAs areinvolved in metastasis and progression of GBC remains obscure. We plan to thorougly explore critical miRNAs involved in metastasis and progression of GBC. A new designed high content miRNAs screening technology was applied for the first time to filter ou pro-metastatic miRNAs in GBC.The biological functions of miRNAs will be intensively studied by transwell, immunoblot, immunohistochemical and in situ hybridization assays. Tumorigenicity and liver metastasis will be further examined in nude mice. Previously, out of the miRNA library, the up-regulation of miR-20a in tumor tissues showed tight association with local invasion, distant metastasis and poor prognosis of GBCs clinically. Th ectopic expression of miR-20a could induce Epithelial-mesenchymal transition(EMT), and enhance the metastasis of GBC-SD cells in vitro and in vivo, through directly targeting 3'UTR of Smad7. Our project here, after analyzing the previous data, we discoved that miR-377, with low expression in GBC, which could downregulate the expression of F-actin, may be the target miRNA. We plan to further confirm the results of screening, explore the role of miR-377, and elucidate the molecular mechanism of miR-377-mediated metastasis repression of GBC. Taken together, we expect that our study will bring new clue on GBC therapy and prognosis in the future.

胆囊癌被认为是侵袭性和致死性最高的恶性肿瘤之一，因此寻找相关靶点进行干预成了胆囊癌治疗和预后的关键问题。miRNA通过转录后调控影响靶基因表达，在肿瘤的发生、发展、预后中具有重要的作用，但是目前关于miRNA对胆囊癌转移的调控未有系统报道。本项目拟通过高内涵筛选出发，筛选出转移抑制相关的miRNA，继而在胆囊癌细胞株、稳定表达细胞系和动物水平研究其生物学功能，运用生物信息学手段预测并验证其靶基因，并结合临床样本明确其在胆囊癌中的预后作用。申请人前期利用高内涵筛选检测F-actin明确了miR-20a在癌中高表达并通过靶向抑制Smad7促进了胆囊癌的侵袭转移，本项目对前期筛选结果进行分析并检测，初步发现miR-377在癌中低表达并下调了F-actin，可能是潜在的目标分子，拟进一步从细胞和动物水平明确miR-377是否调控了胆囊癌转移，明确其靶基因及作用机制，为胆囊癌的治疗提供新的理论依据。

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。miRNA通过转录后调控影响靶基因表达，在肿瘤的发生、发展、预后中具有重要的作用。肿瘤侵袭转移是一个多因素、多步骤的复杂过程，涉及信号转导、粘附相关分子、蛋白水解酶及众多细胞因子和调节因子等。miRNAs可以通过调控细胞迁移、侵袭、粘附、上皮细胞间质化参与肿瘤的转移。干预转移相关miRNA将会是应对肿瘤转移、复发的有效方式，这为阐明胆囊癌侵袭、转移特性的分子机制提供了方向。本研究通过系统性的研究，结合胆囊癌临床组织样本，从分子、细胞及动物等层面明确转移抑制miRNAs在胆囊癌发展中的重要作用，阐明其分子网络机制。基于此，利用高内涵筛选结合芯片检测等高通量手段，获得了胆囊癌转移相关miRNAs表达谱。通过功能试验初步明确了mir-7-2-3p和mir-29c-3p为研究靶标。进而在胆囊癌细胞系中过表达mir-7-2-3p和mir-29c-3p，发现细胞的转移能力明显下降，与细胞转移相关的分子呈现MET（间质细胞上皮化）的转变。进一步检测胆囊癌患者冰冻组织样本中mir-7-2-3p和mir-29c-3p的丰度，发现二者在胆囊癌组织中呈现明显的低表达。更重要的是，预后分析表明，mir-7-2-3p和mir-29c-3p在胆囊癌中的低表达或许介导了胆囊癌恶性高的生物学特性，影响了患者的整体生存。最终，为明确其靶基因及作用机制，将胆囊癌细胞系过表达mir-7-2-3p和mir-29c-3p后进行表达谱深度测序，数据分析明确了其发挥抑癌作用的潜在靶基因及共表达网络，为深入理解mir-7-2-3p、mir-29c-3p的作用机制，利用节点分子进行干预用于胆囊癌的基因治疗，提供了理论依据。总之，本课题筛选出了胆囊癌转移抑制相关miRNAs，为阐明胆囊癌的分子机制进而为胆囊癌的治疗提供了新的理论依据。