Hsa_circ_0001922调控外泌体miRNAs分选促进前列腺癌转移的机制研究

Exosomal miRNAs play an important role in the metastasis of PCa. However, it is unclear how miRNAs involved with PCa metastasis are loaded into exosomes. In the preliminary experiments, we found by transcriptome sequencing that hsa_circ_0001922 was associated with PCa metastasis, and confirmed that compared with PC-3M-2B4, the expression of hsa_circ_0001922 in high metastatic potential PC-3M-1E8 was increased significantly. In addition, we observed that exosomes from PC-3M-1E8 significantly promoted the migration potential of PC-3M-2B4, which involved with the different expressions of their exosomal miRNAs, and the high expression of hnRNPA1 was observed in PC-3M-1E8. These results indicate that has_circ_0001922 may affect PCa metastasis through regulating the sorting of exosomal miRNAs, but how the hsa_circ_0001922 works in the sorting of PCa exosomal miRNAs is unkown at present. In this study, we will use the PCa metastatic cell model to analyze the molecular mechanism of hsa_circ_0001922 regulating the sorting of exosomal miRNAs, confirm the expression profile of exosomal PCa metastasis-associated miRNAs and investigate their role in prostate cancer metastasis, and then validate the role of hsa_circ_0001922 in the PCa metastasis through nude mice experiments in vivo, and finally explore the relationship among has_circ_0001922, exosomal miRNAs and PCa metastasis and prognosis through a case-control study.

外泌体miRNAs在PCa转移中发挥着重要作用，然而PCa转移相关miRNAs如何进入外泌体内却不清楚。在前期研究中我们通过RNA测序发现hsa_circ_0001922与PCa转移相关，并在PC-3M-1E8(高转移)中证实其表达显著高于低转移PC-3M-2B4，进而发现前者外泌体显著促进后者迁移，它们外泌体miRNAs表达差异明显，且PC-3M-1E8中hnRNPA1高表达。这些提示has_circ_0001922可能参与PCa外泌体miRNAs分选影响PCa转移，但其作用机制却不清楚。在本研究中，我们将通过PCa转移细胞模型分析hsa_circ_0001922调控外泌体miRNAs分选的分子机制，确认外泌体PCa转移相关miRNAs表达谱及其在PCa转移中的作用；进而通过裸鼠实验验证；最终通过病例-对照分析has_circ_0001922、外泌体miRNAs与PCa转移及预后的关系。

肿瘤微环境在肿瘤的发生发展中扮演着极其重要的作用。通过不断分泌外泌体，肿瘤细胞将特异性的物质运送至其他受体细胞，培育出适合其生存及进展的微环境。由于miRNA靶标众多，外泌体miRNAs常常涉及受体细胞中多个生理和病理进程，然而，肿瘤细胞如何将特异性miRNAs分选至外泌体进而调控微环境，目前机制还不清楚。本项目以前列腺癌miRNAs的外泌体分选为出发点，从非编码RNA调控和巨噬细胞极化两方面探索肿瘤细胞通过miRNAs的外泌体分选培育微环境的作用机制。首先，我们确认环状RNA hsa_circ_0001922在前列腺癌进展中作用，并探明其与miR-100-5p和miR-146a外泌体分选的关系；其次，明确前列腺癌外泌体特异miRNA参与巨噬细胞功能调控的途径，并阐明调控该外泌体miRNA分选的分子机制。在本项目中，我们发现，hsa_circ_0001922在前列腺癌细胞PC-3中表达显著上升，其通过上调Vimentin、降低E-cadherin表达促进前列腺癌细胞EMT进程，靶向抑制hsa_circ_0001922后，前列腺癌细胞增殖、迁移和侵袭能力显著减弱，其外泌体中miR-146a和miR-100-5p也表达下降；同时，我们也证实，PCa外泌体可以通过AKT和STAT3信号通路诱导巨噬细胞向M2表型分化，而外泌体抑制剂GW4869阻碍PCa细胞对巨噬细胞的培育作用，并在体内外抑制前列腺癌的进展，提示GW4869可以作为肿瘤外泌体分泌的抑制剂在治疗前列腺癌转移中发挥重要作用；更关键的是，我们首次发现，PC-3中EIF3B能够上调这些外泌体中miR-100-5p丰度进而导致巨噬细胞的吞噬作用显著抑制，其机制是通过miR-100-5p/NOX4途径减少巨噬细胞ROS产生，敲低PC-3中EIF3B导致其外泌体miR-100-5p显著下降，同时恢复巨噬细胞的吞噬功能。这些结果不但探讨了环状RNA参与前列腺癌发生发展的表观机制，而且确定了外泌体内容物对肿瘤微环境的调控角色。重要的是，我们首次报告了EIF3B在miR-100-5p外泌体分选中的关键作用，阐明了外泌体运送miR-100-5p调控巨噬细胞功能的分子机制，进一步确认了通过外泌体干预进行前列腺癌免疫治疗的可能。