携带miR-155的exosome在缺氧调控MDSC/Treg营造口腔鳞癌转移前微环境中的机制研究

Myeloid derived suppressor cells (MDSCs) and regulatory T cells (Treg) play important roles in the formation of pre-metastatic niche. Hypoxia micro-environment can affect the function of MDSC/Treg in the pre-metastatic organ, but how hypoxia regulates MDSC/Treg distantly remains unknown. Recently, the discovery of exosome as a cargo of protein, mRNA, and microRNA provides a possibility that cancer cell may function on MDSC/Treg distantly by releasing exosomes. In this study, we are going to study if miR-155 in the exosome can be regulated by hypoxia, and if exosomes that carrying miR-155 can function on the MDSC/Treg and contribute to the formation of pre-metastatic niche of oral squamous cell carcinoma. Using in vitro cell culture, animal model, and clinical sample, we will study what's the function of hypoxia on the miR-155 expression in the exosomes; which signaling pathway in MDSC/Treg can be regulated by miR-155 carried by exosomes; and what function of MDSC/Treg could be affected by miR-155? The results of this study will add new knowledge in the mechanism of how hypoxia regulates the pre-metastatic niche formation in distant organ, and provide novel insight in the preventing metastasis of oral squamous cell carcinoma for clinicians.

髓源抑制细胞（MDSC）和调节性T细胞（Treg）在营造转移前微环境、促进肿瘤转移中发挥重要作用。肿瘤缺氧微环境可以影响转移靶器官内MDSC和Treg功能，但如何影响尚不清楚。最近发现exosome可携带蛋白，mRNA和microRNA至远处器官，为阐述肿瘤细胞远程作用于MDSC和Treg从而营造转移前微环境的机制提供了新的思路。本课题拟在体外细胞、动物模型和临床样本中，从exosome携带miRNA影响MDSC和Treg功能的角度出发，探讨缺氧对肿瘤细胞exosome及其携带的miR-155有何影响？exosome携带的miR-155能够作用于MDSC和Treg中哪些关键信号通路？调控MDSC和Treg哪些功能？研究结果将从exosome携带miRNA作用于MDSC和Treg，从而营造转移前微环境的新视野，诠释缺氧促进口腔鳞癌转移的机制，从而为临床防治口腔鳞癌侵袭转移提供新的思路和策略。

最近发现exosome能够携带细胞特异的信息，并将相应信息传递给其它细胞和远处组织。作为细胞-细胞间通讯的媒介，exosome在生理、病理状态下可能发挥着重要的调控作用。本课题提出了科学假设：缺氧肿瘤细胞可能通过释放exosome携带更多的miR-21，并将缺氧细胞miR-21传递至常氧细胞中，促使常氧细胞具有缺氧细胞的特征。拟探讨①缺氧肿瘤细胞来源exosome中miRNA表达谱变化；②缺氧调控exosomal miR-21表达的机制；③ exosomal miR-21对OSCC侵袭、迁移和增殖活性的调控作用；④ OSCC患者血清exosomal miR-21表达水平的临床病理意义。. 我们采用RNA-seq技术检测常氧和缺氧培养OSCC细胞来源的exosome中miRNA表达谱的差异，发现108个miRNA在常氧和缺氧来源的exosome中表达存在显著差异。我们发现miR-21的表达水平明显高于其他exosomal miRNAs，而且其缺氧条件下表达显著升高，因此我们进一步探讨了exosomal miR-21在缺氧调控OSCC侵袭转移中所扮演的角色。通过ChIP技术，我们发现，缺氧条件下HIF-1α和HIF-2α均可与miR-21的启动序列中缺氧调控原件（HRE）相结合，证实HIF-1α和HIF-2α对miR-21的直接调控。缺氧条件下，miR-21沉默后的exosome显著抑制了常氧培养细胞的EMT、迁移和侵袭，结果表明，缺氧exosome促进细胞迁移、侵袭有赖于其所携带的miR-21。再者，exosomal miR-21促进了OSCC细胞中Vimentin的表达同时抑制了E-cadherin的表达。最后我们从健康志愿者和OSCC患者血清样本中分离了exosome，并通过定量PCR证实OSCC患者血清exosomal miR-21表达水平显著高于健康志愿者血清中exosomal miR-21水平，且exosomal miR-21表达水平与肿瘤的缺氧状态密切相关。. 综上所述，我们得出以下结论，缺氧肿瘤细胞通过释放富含miR-21的exosome，这些exosome可以作用于临近的非缺氧细胞，调节这些细胞的EMT，从而进一步影响细胞的迁移、侵袭能力，最终促进肿瘤的生长和转移。