高糖诱导肝细胞外泌体中miR-132差异表达促进EMT介导的胰腺癌转移的分子机制

Pancreatic cancer always presents infiltration and metastasis. Previous clinical findings have shown that hyperglycemia can accelerate its metastasis, but the mechanism is unknown. MiR-132 in exosomes of diabetic patients is significantly down-regulated. In previous studies, the expression of the target gene Sox4 of miR-132 in patients with type 2 diabetes combined with pancreatic cancer has been shown to increase significantly, and Sox4 can regulate the occurrence of EMT through multiple pathways. The liver is an organ metabolizing sugar, and its exosomes may be affected by blood glucose. Based on the above, we speculate that high glucose down-regulates miR-132 in exosomes of hepatocytes and promotes the expression of Sox4 which is a regulator of EMT, and thereby promoting the metastasis of pancreatic cancer. By detecting clinical samples, this study intends to analyze the relationship between miR-132 expression in exosomes in peripheral blood of patients with pancreatic cancer and the metastasis of pancreatic cancer, and clarify the source of miR-132 in exosomes. Under high glucose, the role and mechanism of miR-132 in exosomes of hepatocytes in regulating EMT of pancreatic cancer cells and then promoting pancreatic cancer metastasis were investigated at cellular level. By establishing a tumor-bearing diabetic model based on bioluminescence imaging in living animals, accurate space-time positioning was realized, providing an ideal model for the study of its molecular mechanism in the living body. This study may provide novel ideas and molecular targets for preventing and controlling the metastasis of pancreatic cancer based on hyperglycemia.

胰腺癌具有高度的浸润和转移能力，前期临床发现高血糖可加速其转移，但机制不明。糖尿病患者外泌体中miR-132显著下调，前期研究发现2型糖尿病合并胰腺癌患者miR-132的靶基因Sox4显著增加，而Sox4可通过多种途径调控EMT发生。肝脏为糖代谢器官，其外泌体极可能受血糖影响。基于以上，我们推测高糖下调肝细胞外泌体中的miR-132，促进EMT调控因子Sox4的表达，进而促进了胰腺癌的转移。本项目拟通过临床样本检测，分析胰腺癌患者外周血外泌体中miR-132表达量与胰腺癌转移之间的关联，明确外泌体miR-132的来源；通过细胞水平研究高糖条件下，肝细胞外泌体miR-132调控胰腺癌细胞EMT发生进而促进胰腺癌转移的作用与机制；通过建立基于生物发光活体成像技术的糖尿病荷瘤动物模型，实现精准的时空定位，在活体内精确地研究其分子机制。本项目可能为防控基于高血糖的胰腺癌转移提供新的思路和分子靶点。

随着疾病的进展，大部分胰腺癌最终会侵犯肝脏，形成肝脏转移，这也是胰腺癌患者最重要的死亡原因。因此，对胰腺癌转移分子机制的深入研究具有重要的理论和临床意义。主要研究内容如下：（1）选取胰腺癌转移组和胰腺癌未转移组各50例，收集临床血液标本，检测两组人群血浆外泌体中miR-132的表达，进行对比分析，通过相关和回归分析明确外泌体miR-132的表达量与胰腺癌分期、转移和预后的相关关系。（2）检测miR-132的来源，对外泌体中白蛋白、糖原和角蛋白18进行免疫荧光染色，证明外泌体来源。（3）在高糖环境条件与正常条件下分别培养肝脏细胞，提取其分泌的外泌体，检测miR-132的表达。同时将外泌体分别加入胰腺癌细胞中，检测胰腺癌细胞的增殖、迁移、侵袭和EMT相关标志（E-cadherin、N-cadherin和fibronectin）。模型动物水平：在胰腺癌细胞稳定过表达或者干扰miR-132，通过裸鼠荷瘤模型，HE检测肿瘤的转移和浸润能力，免疫组织化学检测E-cadherin、N-cadherin和fibronectin的表达。（4）机制研究。在胰腺癌过表达或者干扰miR-132，western-blot检测相关信号通路，包括TGF-β、Sox4和Erh2的变化。本研究的重要结果：（1）通过对患者的血液标本进行外泌体提取、电镜观察和Western blot检测，从而从临床角度证实高糖状态可能增强胰腺癌细胞的迁移和侵袭能力，且这一过程可能与肝细胞分泌的外泌体中miR-132的减少有关。（2）通过在高糖状态下进行细胞培养，从细胞学角度证实：高糖状态可能增强胰腺癌细胞的迁移和侵袭能力，且这一过程可能与肝细胞分泌的外泌体中miR-132的减少有关。（3）通过动物模型证明：高糖环境可能降低外泌体miR-132的含量，并促进胰腺癌的转移。这些结果将为临床防控胰腺癌转移提供理论依据和分子靶点。