BRG1-miR-302a-3p调控胰腺癌细胞化疗耐药及干细胞化的机制研究

The prognosis of pancreatic cancer remains very poor because of its chemoresistance due to the existence of cancer stem cells. Our previous study showed that BRG1 regulated the chemoresistance and the transcription of cancer stem cell related miRNA miR-302a-3p. Yet the molecular mechanisms and clinical significant of miR-302a-3p mediated by BRG1 on modulating chemoresistance and maintenance of stem-like cells in human pancreatic adenocarcinoma is still not clear. In this study, the expression level of BRG1 and miR-302a-3p will be increased and suppressed by expressional vector and siRNA technology, respectively, and the corresponding changes of chemoresistance and cancer stem cell transformation will be studied in order to show the role and the molecular mechanisms of BRG1-miR-302a-3p in these processes. Real time PCR and immunohistochemistry will be performed to inspect the expression patterns of BRG1 and miR-302a-3p both in resected pancreatic cancer specimen and in the serum of the patients, and the relationship of BRG1-miR-302a-3p pathway and the clinical data will be analyzed to figure out their clinical significance. Finally, studies in vivo, using both subcutaneously cancer cells injection model and primary pancreatic cancer tissue subcutaneously implantation model, will be proposed to validate these regulatory mechanisms, and to find out a more effective method which contributes to enhancing the chemosensitivity of pancreatic cancer.

肿瘤干细胞是胰腺癌化疗耐药等恶性表型的关键因素，前期研究中我们发现BRG1参与调控胰腺癌化疗耐药并与干细胞相关基因miR-302a-3p的转录调控相关，然而目前BRG1调控miR-302a-3p表达的具体机制及其介导胰腺癌化疗耐药及干细胞化的分子机制仍不清楚。为阐明其具体分子机制并明确其临床意义，本项目中我们将分别过表达及敲低BRG1及miR-302a-3p基因，探讨BRG1-miR-302a-3p路径对胰腺癌化疗耐药、干细胞维持等的调控作用，明确miR-302a-3p为其关键环节；明确BRG1通过调控miR-302a-3p介导胰腺癌细胞化疗耐药及干细胞化的分子机制；通过组织标本中BRG1蛋白和miR-302a-3p水平的检测，同时检测患者血清miR-302a-3p分泌水平，并应用裸鼠皮下移植瘤模型进行体内干预及联合治疗，探讨其异常表达与胰腺癌化疗耐药及肿瘤干细胞之间的相关性及临床意义。

BRM（Brahma）是染色质重塑调节复合物SWI/SNF的核心催化亚基，既往研究报道高表达BRM是胰腺癌患者预后不良的独立风险因子，但BRM在胰腺癌发生、发展中的生物学功能及其调控机制尚不明确。.本课题组研究发现BRM可通过介导JAK2/STAT3信号通路促进胰腺癌细胞增殖和化疗耐药，高表达BRM的患者预后较差。进一步研究发现在胰腺癌细胞系中沉默BRM的表达可以抑制胰腺癌细胞在体内体外的迁移和侵袭能力。沉默BRM的同时，细胞中miR-302a-3p也随之减少。通过CHIP实验证实，BRM可以直接结合在miR-302a-3p的promoter区并调控其在胰腺癌细胞中表达。对比35例来源于北京大学第一医院的胰腺癌患者肿瘤组织、癌旁组织以及血清中miR-302a-3p的表达水平，我们发现miR-302a-3p的表达水平与肿瘤组织的远处转移和血管侵袭相关。利用miR-302a-3p的模拟物和抑制物进行营救实验证实BRM通过调节miR-302a-3p表达增强胰腺癌细胞的迁移侵袭能力。机制研究中，qPCR及Western Blots发现细胞因子信号传导抑制蛋白5（Suppressor of cytokine signaling 5，SOCS5）可能是miR-302a-3p的潜在靶点。生物信息学分析明确在 SOCS5 mRNA 3’UTR 存在miR-302a-3p的结合位点，利用双荧光素酶实验证实miR-302a-3p可以直接结合在SOCS5的3’UTR区域，从而抑制SOCS5蛋白的翻译。体内实验发现沉默BRM联合miR-302a-3p抑制剂瘤内多点注射可以显著抑制肿瘤生长，增加SOCS5表达并降低STAT3靶基因表达，从而通过体内实验证实 BRM 通过靶向miR-302a-3p/SOCS5/STAT3 信号通路发挥生物学功能。.综上，BRM在胰腺癌细胞中促进miR-302a-3p转录，后者可与 SOCS5 mRNA 3’UTR直接结合从而抑制SOCS5表达，提高STAT3磷酸化水平从而激活下游靶分子（Cyclin D1、Survivin、MMP7、MMP9、VEGF）的表达，促进胰腺癌细胞增殖、迁移、侵袭和化疗耐药。这一研究揭示了BRM在胰腺癌中发挥生物学功能的分子机制，为研发新的胰腺癌治疗策略提供了潜在靶点，对高表达BRM 的胰腺癌患者具有重要的临床意义。