BRG1/miR-485-3p/PAK1轴调控胰腺癌细胞迁移、侵袭及干性维持的机制研究

Recent studies have shown that BRG1, a core catalytic subunit of chromatin remodeling complex SWI/SNF, plays an important role in cancer metastasis and stem cell phenotype maintenance through regulating miRNA transcription. However, its molecular mechanisms remain unclear and no relevant study on pancreatic cancer (PC) has been reported. Our previous study have found that BRG1 induced miR-485-3p promoter methylation and suppressd miR-485-3p expression. Re-expression of miR-485-3p inhibited PC cell mobility, migration, invasion, and decreased the proportion of CD24+/CD44+/ESA+ cancer stem cell. In this study, we intend to silence or overexpress BRG1, miR-485-3p and its predicted target molecule (PAK1) in PC cells, following observation of their effects on cellular migration, invasion and stem cell phenotype maintenance in vivo and in vitro. Then the exact molecular mechanisms of BRG1/miR-485-3p/PAK1 signaling axis in potentiating PC metastasis and maintaining stem cell phenotype would be explored. Emphatically, the key role of BRG1 on hypermethylation of miR-485-3p promoter is to be elucidated. Moreover, the clinical significance of the key genes in BRG1/miR-485-3p/PAK1 signaling axis would be determined using samples from PC patients who have received surgical treatment. The results are expected to reveal the prelimenary regulatory network of PC metastasis and stem cell phenotype maintenance from the perspective of epigenetics.

染色质重塑调节复合物SWI/SNF核心催化亚基BRG1参与miRNA转录，对肿瘤细胞迁移、侵袭等生物学行为有重要调控作用，但具体机制尚不明确，与胰腺癌的相关性研究未见报道。本研究前期工作发现：BRG1可通过DNA甲基化抑制miR-485-3p转录；过表达miR-485-3p可抑制胰腺癌细胞迁移、侵袭并降低肿瘤干细胞比例。本项目拟进一步沉默或过表达BRG1、miR-485-3p及其预测靶分子PAK1，观察BRG1/miR-485-3p/PAK1轴对胰腺癌细胞EMT、迁移、侵袭及干细胞表型的影响并探讨其调控机制，阐明BRG1对miR-485-3p基因启动子高甲基化修饰的关键作用；由表观遗传学角度初步揭示胰腺癌细胞迁移、侵袭和干性维持的调控网络；通过临床标本检测及动物模型体内研究，评价BRG1/miR-485-3p/PAK1轴关键基因的临床意义及作为干预靶点的可能性。

MicroRNA （miRNA）是长度为19-24 核苷酸的内源性小分子非编码RNA，主要通过与靶基因mRNA 3’UTR 完全或部分互补配对结合，抑制mRNA 翻译或直接导致其降解从而在转录后水平调控基因表达。miRNA 已被证明对胰腺癌等多种肿瘤的转移有重要的调控作用，课题组前期在研究Hedgehog通路中通过二代测序筛选出miR-485-3p在胰腺癌组织中表达显著下调。本项目进一步通过分析miR-485-3p在胰腺癌组织中的差异表达情况，发现胰腺癌组织中低表达且与胰腺癌患者预后相关；在胰腺癌细胞中过表达miR-485-3p可抑制胰腺癌细胞迁移侵袭、干性维持以及提高对吉西他滨的化疗敏感性。通过生物信息学分析，课题组发现miR-485-3p可能靶向PAK1以及转录因子SOX9和铁死亡负性调节因子SLC7A11。一方面明确了miR-485-3p通过体内外实验明确了PAK1参与调控胰腺癌细胞迁移侵袭。另一方面证实了miR-485-3p通过SLC7A11介导的细胞铁死亡途径参与调控胰腺癌细胞干性维持和化疗敏感性。同时课题组进一步探究了miR-485-3p的上游调控机制，明确了胰腺癌细胞中miR-485-3p的启动子区域，该区域存在CpG岛且在低氧环境下可被DNMT3B甲基化。经后续实验证实了DNMT3B/miR-485-3p/SLC7A11通路诱导胰腺癌细胞干性转化及化疗耐药等恶性表型。这两部分内容协同证实了miR-485-3p在胰腺癌转移、干性转化及化疗耐药中的作用及其潜在的分子机制。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。.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 23篇。中文核心期刊论文12篇，并培养6名博士研究生毕业以及1名硕士研究生毕业，另有3名博士研究生和1名硕士研究生进行后续研究。