外泌体介导linc-ROR调控胰腺癌细胞-脂肪细胞对话促进胰腺癌侵袭转移的机制研究

Exosome plays a pivotal role in modulation of crosstalk between cancer cells and stromal cells, including adipocytes in pancreatic cancer (PC), which further promotes the invasion and metastasis of PC, but the molecular mechanism still remains unclear.Our previous study confirmed that linc-ROR promoted cell proliferation, invasion and metastasis via HIF-1α/ZEB1 pathway in PC, level of tumor-secreted exosomal linc-ROR was elevated in PC cells and resulted in higher expression of IL-1β and promoted the invasion of PC after co-culture with adipocytes, but the molecular mechanisms should be further investigated. Based on the literature review and our previous study, we hypothesize that tumor-secreted linc-ROR could be incorporated into exosomes and transmitted to adipocytes, they can activate the HIF-1α pathway, promote adipocytes to secrete IL-1β, IL-1β can further promote expreesion of HIF-1α and linc-ROR in PC cells, induce the epithelial-mesenchymal transition (EMT), form a positive feedback loop between cancer cells and adipocytes, and finally enhance the invasion and metastasis of PC. To test the hypothesis, we will investigate role and mechanism of exosome-transmitted linc-ROR in regulation of crosstalk between PC cells and adopocytes, dedifferentiation of adipocytes, EMT process and invasion and metastasis in PC, the combination analysis of expression level of linc-ROR in serum exosome extracted from patients with PC, in vitro cell co-culture and in vivo tumor growth will be applied in the research. Our study will provide the new insights for understanding mechanism of PC metastasis and also lead to identify a new molecular target for early detection and targeted therapy in patients with PC.

外泌体参与调控胰腺癌细胞与脂肪细胞等间质细胞对话，促进胰腺癌转移，但机制不明。本课题组前期研究发现：① linc-ROR可激活HIF-1α/ZEB1通路，促进胰腺癌转移；②胰腺癌细胞分泌的外泌体中linc-ROR高表达，与脂肪细胞共培养后IL-1β水平升高，增强胰腺癌细胞侵袭能力。基于此，我们提出如下假说：胰腺癌细胞来源外泌体linc-ROR进入脂肪细胞后上调HIF-1α活性，刺激脂肪细胞分泌IL-1β，后者可上调胰腺癌细胞HIF-1α和linc-ROR表达，形成正反馈环路，介导胰腺癌EMT，进而促进胰腺癌侵袭转移。本课题将临床标本检测和体内外实验相结合，采用体外细胞共培养、裸鼠体内成瘤等多种实验方法，从临床标本、细胞及动物等不同层面，探索胰腺癌细胞来源外泌体linc-ROR参与胰腺癌细胞-脂肪细胞对话，调控脂肪细胞去分化，促进胰腺癌侵袭转移的机制，为研究胰腺癌转移机制及临床诊治提供思路。

胰腺癌是恶性程度最高的消化系统肿瘤，预后极差。外泌体参与调控胰腺癌细胞与脂肪细胞等间质细胞对话，促进胰腺癌转移，但机制不明。本课题首先通过收集临床胰腺癌患者和健康人群血清外泌体样本，分析外泌体linc-ROR表达水平的差异；超速离心法提取提取多种胰腺癌细胞系培养液上清中外泌体，使用透射电子显微镜观察外泌体镜下微观形态，粒径分析技术测算外泌体直径和浓度范围，免疫印迹法检测外泌体表面蛋白标记物；RNase A和Triton X-100检测外泌体结构的稳定性。在胰腺癌细胞系PANC-1/BxPC-3中分别上调/下调linc-ROR表达水平，构建linc-ROR慢病毒质粒转染的稳转胰腺癌细胞系。采用Transwell培养小室将各组胰腺癌细胞与脂肪细胞共培养，通过荧光标记外泌体后，观察脂肪细胞摄取外泌体情况及细胞形态改变、发育成熟能力，检测胰腺癌细胞在linc-ROR/HIF-1α/ZEB1正反馈环路中各相关因子的表达水平差异、成熟脂肪细胞分子标记物和成纤维样细胞分子标记物表达水平变化。划痕实验、Transwell、克隆形成、实时无标记细胞分析技术、Edu等功能实验检测共培养对胰腺癌细胞增殖、侵袭、迁移能力的影响。最后进行动物实验体内水平验证linc-ROR相关的共培养微环境对胰腺癌细胞增殖能力的影响。研究结果表明：linc-ROR在胰腺癌患者血清外泌体表达水平明显高于健康人；脂肪细胞在摄取外泌体linc-ROR后形态学发生明显改变，且成熟脂肪细胞分子标记物表达水平降低，成纤维样细胞分子标记物表达水平增高。胰腺癌细胞与脂肪细胞共培养后，促进脂肪细胞的去分化，分泌IL-1β，进而激活胰腺癌细胞HIF-1α/ZEB1通路表达，促进胰腺癌细胞EMT发生，促进胰腺癌细胞增殖、侵袭以及迁移。本研究系统探索胰腺癌细胞来源外泌体linc-ROR参与胰腺癌细胞-脂肪细胞对话，调控脂肪细胞去分化，促进胰腺癌侵袭转移的机制，为研究胰腺癌侵袭转移机制及临床诊治提供新的思路。