不同遗传背景下miRNA在外泌体介导的肺癌与外环境间的信息传递及预后调控中的作用研究

miRNAs have an impact on the assembling, transfer and secretion of exosomes. miRNAs in exosomes could affect target cells by inducing the development and metastasis of tumors. Our previous studies discovered that mutations in miR-149, miR-608, miR-196a2, miR-30c1, miR-27a and miR-26a1 were associated with prognosis of lung cancer. Based on these mutations, the clinical information and prognosis information of patients, the present study will screen the differential expressed miRNAs in exosomes by next-generation sequencing technology and explore the association between these mutations and lung cancer in cohort study. On the basis of differential expression of transcripts between intracellular matrix and extracellular matrix, we will investigate the role of exosomes in miRNAs selection and transfer, explore how the dysregulation of miRNAs associated with prognosis influence the transfer of miRNAs between cells, and verify the hypothesis that miRNAs could influence survival of lung cancer mediated by exosomes. We will alter the expression of some miRNAs through various ways to observe the miRNA sponge effect of exosomes, changes of cell functions and tumor suppression effect in vivo, then illuminate the mechanisms of miRNAs in exosomes to influence prognosis of lung cancer with different genetic environments.

miRNAs影响细胞外泌体的组装、转运和分泌，外泌体分泌miRNAs对靶细胞作用，诱导肿瘤进展、转移等生物学行为。本研究拟以前期发现的与肺癌预后相关miR-149、miR-608、miR-196a2、miR-30c1、miR-27a、miR-26a1等突变多态位点为遗传背景，结合肺癌患者临床及预后等信息，利用二代测序技术筛选外泌体差异表达miRNAs，并进行肺癌专病队列验证；基于细胞内外转录组的表达差异分析外泌体在miRNAs选择和转运中的作用，探讨预后相关miRNAs失调控关系对细胞间miRNAs转运的影响，并验证外泌体介导的miRNAs影响肺癌预后的假说；进一步进行细胞功能学和动物实验验证，通过不同方式的外界刺激和不同途径改变细胞内、外的特定miRNAs表达，观察外泌体miRNAs的海绵效应、细胞功能变化及实验动物的抑瘤效应，阐明不同遗传背景下外泌体miRNAs对肺癌预后影响的机制。

在肺癌专病队列中，研究已发现的miR-338-3p、miR-149-5p、miR-141-3p、miR-130b-3p、miR-146a-5p、miR-27a-3p、miR-21-5p等多态位点的突变状态作为参比，结合肺癌患者临床特征及生存期等信息，比较各组中外泌体差异表达。肺癌患者不同遗传背景影响外泌体对miRNAs的包裹和转运，外泌体miR-338-3p在非小细胞肺癌患者中表达下调，并与患者M分期、淋巴结远端转移正相关。外泌体miR-338-3p表达上调癌细胞内CHL1基因表达下调，且癌细胞增殖能力减弱、凋亡细胞占比增加，同时MAPK通路后活性下调。外泌体进入肿瘤外微环境释放miR-149-5p，通过与AMOTL2基因靶向结合抑制其表达后刺激癌细胞增殖并干扰癌细胞凋亡；miR-141-3p表达上调组细胞增殖能力较强，并通过与CHRDL1基因靶向结合抑制其表达。CHRDL1基因高表达患者生存期长。外泌体miR-130b-3p表达上调细胞内DEPDC1基因表达下调，DEPDC1基因低表达患者生存期长。外泌体miR-146a-5p表达上调，癌细胞增殖能力降低，且癌细胞凋亡占比增加，PSMC6基因在非小细胞肺癌组织中表达上调，且PSMC6基因低表达患者生存期长。miR-27a-3p在肺腺癌组织中表达上调，通路预测分析发现miR-27a-3p参与ErbB通路，且miR-27a-3p与ING5基因表达负相关。外泌体miR-21-5p下游调控靶BMPR2基因、ZNF367基因、ADNP基因、TNRC6B基因，其中TNRC6B、BMPR2基因下调促进抑制癌细胞耐药性，且在癌细胞中TNRC6B、BMPR2基因突变率较高。TNRC6B、BMPR2、ADNP基因高表患者预后时间长于TNRC6B、BMPR2、ADNP基因低表达患者。ZNF367基因高表达患者预后时间短于ZNF367基因低表达患者。在不同遗传背景的肺癌患者中，由于高异质性和个体遗传差异，研究发现外泌体的miR-338-3p、miR-149-5p、miR-141-3p、miR-130b-3p、miR-146a-5p、miR-27a-3p、miR-21-5p等对肺癌生物学行为和功能有作用，阐明其生物学机制时，采取有针对性的靶向干预和治疗措施对于改善肺癌预后、延长患者生存期具有重要意义。