Hedgehog信号通路调控BRG1表达在胰腺癌细胞干细胞化及化疗耐药中的机制研究

The prognosis of pancreatic cancer is very poor because of its resistance to chemotherapy. Our previous study showed that BRG1 modulated by Hedgehog (Hh) signaling pathway regulated the acquired chemoresistance in pancreatic cancer cells. Inhibition of Hh signaling pathway led to a significant up-regulation of BRG1 expression. Moreover, the abnormal expression of BRG1 resulted in the significant changes of cancer stem cell related miRNAs transcription. The mechanisms of these regulations are not clear. In this study, the activity of Hh pathway will be changed to find out the modulation of BRG1 expression and chemosensitivity in pancreatic cancer cells, as well as in chemoresistant strains. The expression level of BRG1 will be increased and suppressed by expressional vector and siRNA technology respectively, and the corresponding changes of cancer stem cell transformation and chemoresistance will be studied in order to show the role of Hh-BRG1 in these processes. In pancreatic cancer cell lines with different BRG1 expression levels, the altered miRNA transcription patterns will be screened in order to find out the crucial role of miRNAs on the Hh-BRG1 modulated cancer stem cell transformation and acquired chemoresistance in pancreatic cancer cells. Finally, studies in vivo 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 cells.

胰腺癌化疗效果不佳，预后极差，化疗耐药为根本性原因。本课题组前期研究发现： Hedgehog（Hh）信号通路介导BRG1参与调控胰腺癌细胞获得性化疗耐药，抑制Hh活性后BRG1显著上调，且BRG1的异常表达导致与肿瘤干细胞相关的miRNA转录水平改变，但其调控机制仍不明确。本课题拟通过调控Hh信号通路活性，观察BRG1表达变化及其对胰腺癌细胞化疗药物敏感性的影响，并在耐药株中进行验证；通过改变BRG1表达水平，证明Hh-BRG1途径对胰腺癌肿瘤干细胞及化疗药物敏感性的调控作用；在不同BRG1表达水平的胰腺癌细胞系中，对前期工作中筛选出的与肿瘤干细胞相关的miRNA进一步检测及功能性验证，以明确miRNA对上述变化有调控作用；建立动物模型，体内条件下进一步验证Hh-BRG1及miRNA介导肿瘤细胞干细胞化并产生化疗耐药，探寻改变胰腺癌细胞耐药行为的治疗靶点。

作为SWI/SNF染色质重塑调节复合物的核心催化亚基， BRG1和BRM参与核小体动员过程，并可与多种转录因子、共激活因子、共抑制因子相互作用调节基因转录，与细胞增殖、个体发育、细胞粘附、细胞因子信号应答以及多种类型的DNA修复过程密切相关。近年来，基因组测序和蛋白组学分析发现BRG1和BRM等多个SWI/SNF复合物相关基因在约20%的人类肿瘤中广泛突变，但其相关的分子机制尚待进一步研究。.本项目通过调控Hedgehog信号通路活性，观察BRG1表达变化及其对胰腺癌细胞化疗药物敏感性的影响并在耐药株中进行验证，发现BRG1作为染色质重塑调节复合物SWI/SNF的核心催化亚基通过上调pSer473-AKT及其下游分子pThr145-p21cip/waf表达水平活化AKT信号通路促进细胞存活，从而介导的胰腺癌细胞本体耐药和获得性耐药；通过miRNA芯片发现BRG1敲除后miRNA表达谱发生明显变化，在此基础上，进一步通过生物信息学技术分析与化疗耐药相关的miRNA的变化情况；临床研究表明BRG1表达水平与胰腺癌患者分期临界相关，高表达BRG1的胰腺癌患者总体预后更差，胰腺癌患者血清中miR-302a-3p表达明显上调且与胰腺癌患者肿瘤大小、分化程度以及血管浸润显著相关。此外，本项目明确SWI/SNF复合物的另一核心催化亚基BRM通过调控miR-302a-3p/SOCS5/STAT3信号通路，促进胰腺癌细胞迁移和侵袭。最后，本项目通过体内外研究证实来源于人胰腺癌PANC-1细胞的肿瘤细胞球具有自我更新、分化潜能并高表达干细胞标志物，这些细胞具有典型的上皮间质转化表型、Hedgehog信号通路高度活化；通过沉默SMO抑制Hedgehog信号通路活性可以逆转细胞上皮间质转化、增加吉西他滨敏感性并在体内外抑制细胞迁移、侵袭。.综上，本项目现已按照计划完成研究内容并取得重要进展，发表SCI论文7篇、中文核心期刊论文10篇，并培养4名博士研究生毕业，另有4名博士研究生仍在进行后续研究。该项目所提供材料详实，数据真实可靠，研究成果有助于解析胰腺癌发生发展和化疗耐药的分子机制，有望为靶向胰腺癌化疗耐药和肿瘤干细胞的肿瘤分子治疗提供新的靶点和理论依据。