SNHG6-003调控外泌体促进肝癌细胞侵袭转移的分子机制

We have demonstrated that the expression of SNHG6-003 has been dysregulated in hepatocellular carcinoma (HCC) and may serve as novel prognostic markers and therapeutic targets (Oncogene, 2017). Recently, our preliminary data showed that SNHG6-003 promoted HCC cell invasion and migration by inducing exosomal secretion. The exosomes secreted by SNHG6-003-overexpressed cells activated hepatic stellate cell (HSC), suppressed glycolytic metabolism and glucose consumption in normal cells, and induced endothelial-mesenchymal transition (EndoMT). SNHG6-003 may also play a role in the establishment of a “pre-metastatic niche” and in turn facilitate cancer cell proliferation and metastasis. Thus, based on our data, we will elucidate the exact role and reliability of SNHG6-003-induced exosomes in tumor invasion and metastasis. Firstly, we will investigate the molecular mechanism by which SNHG6-003 control exosome synthesis and release. Secondly, we will explore how SNHG6-003-induced exosomes modulate tumor environment and pre-metastatic niche. We also explore the positive feedback of the stromal cells on tumor cells. Lastly, we will investigate the effect of blocking SNHG6-003 in HCC cells on tumor environment and metastasis. In conclusion, in this project, we aim to clarify the biological function and molecular mechanism in which SNHG6-003-induced exosomes promote HCC metastasis, which might provide new biomarkers and targets for the treatment of cancer.

我们前期研究结果发现SNHG6-003在肝癌发生发展中发挥重要作用（Oncogene. 2017）。近期，我们预实验结果表明：SNHG6-003促进肝癌细胞外泌体释放而增强细胞侵袭和迁移能力；SNHG6-003调控的外泌体可诱导肝星状细胞增殖活化，降低正常肝细胞对葡萄糖的利用，诱导正常内皮细胞间质化，从而改造肿瘤微环境。基于前期研究结果，本项目拟深入探讨SNHG6-003调控外泌体促进肝癌细胞转移的分子机制。首先，阐明SNHG6-003促进外泌体释放的分子机制；其次，探讨SNHG6-003调控的外泌体对肿瘤微环境和转移前微环境的影响，以及微环境中的间质细胞对肿瘤细胞的正反馈作用；最后，探讨阻断肝癌细胞SNHG6-003对肿瘤微环境和转移前微环境的影响。据此，阐明SNHG6-003调控外泌体促进肝癌侵袭转移的分子机制并为肝癌治疗提供新的靶点。

我们在既往的研究中发现长链非编码 RNA（lncRNA）SNHG6-003在肝癌发生、发展过程中发挥重要的调控作用，在本研究中我们进一步探讨其在肝癌中的生物学功能及相应分子机制。我们首先通过在肝癌细胞株中过表达或干扰SNHG6-003后行体外迁移和侵袭实验分析SNHG6-003在肝癌中的生物学功能。随后提取细胞培养基中外泌体通过 western blot、NTA(Nanoparticle Tracking Analysis)、电镜检测以及miRNA芯片，探讨SNHG6-003对肝癌细胞释放外泌体的调控作用及分子机制。接着，利用体内、体外实验以及miRNA芯片等技术结合生物信息学探讨SNHG6-003调控的外泌体对肿瘤微环境和转移前微环境的影响，以及微环境中的间质细胞对肿瘤细胞的正反馈作用。最后，利用双荧光素酶报告实验、NTA实验、小鼠肝癌肺转移模型和肝转移模型、小鼠皮下成瘤实验等探讨阻断肝癌细胞SNHG6-003对肿瘤微环境和转移前微环境的影响，并探讨其联合PD-1抑制剂的治疗效果。结果表明SNHG6通过吸附miR-30a-3p调控肝癌细胞侵袭转移，并且miR-30a-3p可以通过SNAP23发挥调控肝癌细胞侵袭转移和释放外泌体的作用。发现肝癌组织SNHG6与SNAP23的表达呈明显的正相关关系，miR-30a-3p在肝癌细胞外泌体中表达较高，并且在肿瘤患者的血和尿的外泌体中表达均较正常人高，提示SNHG6-003及其调控基因/miRNA可作为预测肝癌转移及治疗效果新的标志物。装载miR-30a-3p的肝星状细胞外泌体可以抑制肝癌的侵袭转移，并且我们通过体内及体外实验进一步证实了该疗效；靶向SNHG6-003通过调节免疫微环境增强PD-1抑制剂的治疗效果并进行了临床样本验证。总之，SNHG6通过吸附miR-30a-3p进而调控SNAP23发挥调控肝癌细胞侵袭转移和释放外泌体的作用，SNHG6-003及其调控基因/miRNA可作为预测肝癌转移及治疗效果新的标志物，装载miR-30a-3p的肝星状细胞外泌体可抑制肝癌的侵袭转移并能与免疫治疗发挥协同作用。