手术诱导单核样髓系抑制细胞来源外泌体促肿瘤转移及其机制研究

Surgical excision of primary tumor is a mainstay of therapy in many tumor types, but surgical stress may promote the metastasis of malignant cells. It has been reported recently that exosomes derived from tumor cells and myeloid-derived suppressor cells (MDSC) play a key role in promoting tumor metastasis. Our preliminary studies demonstrated that monocytic MDSC (M-MDSC) significantly increased after lung cancer surgery and surgery-induced M-MDSC were more efficient in promoting tumor metastasis. Furthermore, the exosome released from surgery-induced M-MDSC promote tumor cell proliferation in vitro. Based on these results, we hypothesize that surgery-induced M-MDSC may regulate tumor cells and promote tumor metastasis through releasing exosomes. In the present study, we will focus on the exosome released from surgery-induced M-MDSC and investigate its pro-metastatic effect firstly. To clarify the molecular mechanism underlying this phenomenon, we will screen the differential expressed microRNA released from pre- and post-operative M-MDSC and identify the target gene regulated by this microRNA. Finally, we will further verify whether regulating this microRNA could inhibit tumor metastasis after tumor surgery. Our research will provide a foundation for verifying the mechanisms involved in post-operative tumor metastasis through a new point of surgery-induced M-MDSC, which will provide a new strategy for preventing post-operative tumor metastasis.

手术是肿瘤的重要治疗手段，但手术应激会增加术后肿瘤转移的机会。最新研究显示肿瘤细胞和髓系抑制细胞(MDSC)来源的外泌体具有促肿瘤转移作用。我们的研究表明肺癌术后单核样髓系抑制细胞(M-MDSC)较术前显著升高且手术诱导的M-MDSC促肿瘤转移，手术诱导M-MDSC来源外泌体体外促肿瘤细胞增殖。为此，我们提出假说：手术诱导的M-MDSC可能通过外泌体调控肿瘤细胞并促进肿瘤术后转移。本研究以手术诱导M-MDSC来源外泌体为研究对象，首先探讨M-MDSC来源外泌体促肿瘤转移的作用，进而从手术前、后M-MDSC来源外泌体差异表达microRNA分子及其调控的靶基因筛选和鉴定等方面揭示手术诱导M-MDSC来源外泌体促肿瘤转移的分子机制，并通过动物实验验证调控microRNA表达是否抑制手术后肿瘤转移。本研究将从手术诱导M-MDSC揭示手术后肿瘤转移的机制，从而为预防肿瘤手术后转移提供新的思路。

手术是肿瘤的重要治疗手段，但手术应激会增加术后肿瘤转移的机会。研究显示肿瘤细胞和髓系抑制细胞来源的外泌体具有促肿瘤转移作用。本研究从手术诱导的未成熟髓系细胞（IMC）揭示手术后肿瘤转移的机制。研究结果表明：1）荷瘤小鼠切除肿瘤后（TR）骨髓和外周血中CD11b+Ly6G+细胞（IMC）显著升高。体外实验和小鼠成瘤实验证实TR-IMC促肿瘤细胞增殖。2）将TR-IMC与肿瘤细胞混合后接种至B6小鼠侧腹部皮下，并给予外泌体抑制剂GW-4869，结果显示抑制外泌体分泌后TR-IMC促肿瘤生长作用减弱，即TR-IMC通过分泌外泌体促肿瘤细胞增殖。对TR-IMC来源的外泌体（TR-IMC-Exo）鉴定结果表明TR-IMC-Exo为直径100nm的囊泡，表达CD9和CD63。体外实验和小鼠成瘤实验均表明TR-IMC-Exo促肿瘤细胞增殖。3）芯片法筛选TR-IMC-Exo中长链非编码RNA（LncRNA）表达谱，结果显示TR-IMC-Exo高表达LncSNHG14。RT-qPCR检测结果表明TR-IMC和TR-IMC-Exo中均高表达LncSNHG14。4）通过StarBase数据库分析与LncSNHG14结合的miRNA，发现LncSNHG14具有miR-340的结合位点。双荧光素酶报告实验结果表明LncSNHG14可特异性结合miR-340。在LLC细胞中干扰或过表达LncSNHG14并不改变miR-340表达量，提示LncSNHG14 通过碱基互补吸附miR-340从而阻断其对mRNA的调控作用。5）将肺癌细胞与TR-IMC-Exo共孵育后发现肺癌细胞吞噬Exo。体外实验及小鼠模型均证实，TR-IMC-Exo可显著上调肺癌细胞中LncSNHG14和CDK4表达水平，干扰LncSNHG14可抑制肺癌细胞中CDK4表达。上述实验结果表明TR-IMC-Exo中LncSNHG14通过调控miR-340/CDK4促进肺癌细胞增殖。6）肺癌细胞中Hsa_circ_0046264表达下调，其通过靶向调控hsa-miR-1245/BRCA2影响肺癌细胞凋亡、增殖和侵袭。本研究揭示了手术诱导IMC来源外泌体促肿瘤细胞增殖的作用及其相关机制，为肿瘤术后转移、复发的防治的临床应用奠定了坚实的工作基础。发表SCI收录论著2篇，中文科技核心期刊发表论著2篇。培养硕士研究生两名。