Lnc-ZKSCAN1介导胰腺癌外泌体miR-93/106b簇促进肝星状细胞自噬激活参与肿瘤转移前微环境形成的机制研究

Pancreatic ductal adenocarcinomas (PDACs) are highly liver metastatic with poor prognosis, mainly due to the delayed detection. Pre-metastatic niche (PMN) formation offers favorable “soil” for the liver metastasis in PDAC. Exosomes, intercellular communicator between cancer cells and their microenvironment, are emerging as core players in PMN formation mediated by hepatic stellate cells (HSCs) activation. Autophagy plays an essential role in HSCs activation. Our previous studies have demonstrated that long noncoding RNA (lncRNA) promoted pancreatic cancer metastasis via the stimulation of autophagy. However, the mechanism of lncRNA on inducing liver PMN initiation is not clear. The previous microarray data showed that higher lnc-ZKSCAN1 expression is correlated with early liver metastasis in PDAC tissues. Further study indicated that lnc-ZKSCAN1 derived exosomes miR-93/106b cluster is critical for liver PMN formation. In view of this, the clinicopathological studies, 3D culture models and various animal models will be performed to investigate the main molecular mechanisms. Lnc-ZKSCAN1 inhibits precursor miR-93/106b maturation and suppresses exosomes miR-93/106b expression in PDACs. These facilitate HSCs autophagic activation and eventually increase the PMN formation. Our study provides the new train of thought to reveal the main mechanism of liver metastasis, to find better indication for monitoring tumor metastasis and to develop molecular-targeted drugs in PDACs.

胰腺癌症状隐匿、早期即可出现肝转移，导致其预后极差。肿瘤转移前微环境(PMN)形成为胰腺癌肝转移提供有利“土壤”。外泌体作为细胞间通讯的媒介，其介导肝星状细胞(HSCs)活化是PMN形成的关键环节。自噬激活也是HSCs活化中的重要生物学过程。我们前期研究证实，lncRNA激活自噬促进胰腺癌转移，但其调控PMN形成的机制尚未阐明。基于前期芯片结果，我们筛选出lnc-ZKSCAN1与胰腺癌早期肝转移密切相关，进一步研究发现其通过抑制外泌体对miR-93/106b簇的呈递，促进肝脏PMN形成。有鉴于此，本项目借助临床病理学、3D共培养体系及多种胰腺癌动物模型，深入解析lnc-ZKSCAN1抑制pre-miR-93/106b成熟，减少外泌体对miR-93/106b簇呈递，激活HSCs自噬，促进PMN形成。为探究胰腺癌肝转移的分子机制、寻找监测肿瘤转移的有效指标及开发靶向治疗药物提供新的思路。

项目背景：胰腺导管腺癌(pancreatic ductal adenocarcinoma, PDAC)是一种恶性程度很高的腹腔实性肿瘤，它具有死亡率高、生存期短的特征。长链非编码RNA（lncRNA)在肿瘤的发生、发展中起着重要作用。本研究采用胰腺癌生存期不同的患者术后标本进行lncRNA测序，希望能够发现lncRNA在胰腺癌中的调控作用和机制。.研究内容：在生存期不同的胰腺癌患者标本测序基础上，通过qRT-PCR和生存分析，验证了lnc-FSD2-31:1在长生存期患者胰腺中高表达，采用RNA 荧光原位杂交(fluorescence in-situ hybridization, FISH)确定lnc-FSD2-31:1在胰腺癌细胞内的定位，通过EdU增殖实验、CCK-8实验、划痕实验、Transwell实验和免疫组化验证了lnc-FSD2-31:1对胰腺癌增殖和转移功能的影响。然后，在KEGG分析、3-D培养及体内、外功能实验的基础上，结合基因测序和网站预测，发现lnc-FSD2-31:1通过外泌体miR-4736作用于肿瘤成纤维细胞中的ATG7。最后，通过靶点预测、FISH、免疫荧光、免疫共沉淀和双荧光素酶实验确定并验证了lnc-FSD2-31:1，miR-4736和ATG7三者之间的相互结合位点和调控关系。.结果：1）生存期较长的患者中lnc-FSD2-31:1表达水平明显升高，与患者生存期呈正相关；（2）lnc-FSD2-31:1能够抑制胰腺癌的增殖、侵袭和转移；（3）Lnc-FSD2-31:1作为竞争性内源性RNA (ceRNA)抑制PDAC细胞中miR-4736的表达；（4）外泌体miR-4736运输至肿瘤成纤维细胞，并通过结合ATG7的3’-UTR区抑制ATG7的表达；（5）胰腺癌lnc-FSD2-31:1通过外泌体miR-4736调控ATG7介导肿瘤成纤维细胞的自噬、增殖及纤维化功能，同时肿瘤成纤维细胞能够释放细胞因子影响胰腺癌；（6）给予KPC转基因小鼠miR-4736抑制剂腹腔注射后，肿瘤生长明显受到抑制。.意义：Lnc-FSD2-31:1能够抑制胰腺癌增殖与转移。胰腺癌lnc-FSD2-31:1通过外泌体miR-4736调控ATG7介导肿瘤成纤维细胞的自噬、增殖及纤维化功能，同时肿瘤成纤维细胞能够释放细胞因子影响胰腺癌。