微囊泡介导胚胎细胞miRNA转运调控黑色素瘤细胞侵袭性的研究

miRNA plays an important role in determining tumor fate as important signal molecules of microvesiclesin cell communication. We found that mouse embryonic could invade malignant cells in vitro and that malignant invasive properties of B16 cells were inhibited during B16 melanoma cells and mouse embryonic cellswere co-cultured. Further, in addition to its mechanism associated with the regulation of the extracellular matrix molecules signal ,there could exist other pathways that may lead tumor cells to change their own way of invasion. Thus, further study of the mechanism is what we need. It is newly discoveredthat microvesicles communication can pass from one cell to another cellhorizontally by miRNA signal, "reprogramming" their gene expression and influence function and behavior of cell.We have recently discovered the existence of microvesiclesof mouse blastocyst, thus we guess: early embryonic cells secrete functional miRNA of microvesicles to tumor cells miRNA so that inhibit tumor cell invasiveness by post-transcriptional level of miRNAmolecules regulating gene expression in tumor cells.We intend to use highly invasive metastatic mouse melanoma B16-F10 cells, co-culture system in vitro and the C57BL / 6 mouse melanoma animal model.To investigate the inhibition of embryonic cells microvesiclesto tumor cell invasion, we usemiRNA expression profiling microarray combining high-throughput miRNA tumor database analysis so as to predict key miRNAand their targets. Further, we identifythe signaling pathway of loyal transport functionmiRNA regulation in tumor cell.Thus, It demonstrates microvesocles communication form in level of microvesicles and miRNA molecular and clarifies the mechanism thatmiRNA-mediated embryonic cell can regulate invasiveness of malignant cells signal transduction. It should provide new ideas and new research targets for prevention and treatment of malignant melanoma.

miRNA在决定肿瘤运命中发挥重要作用。微囊泡通讯能将miRNA信号从一个细胞传递至另一个细胞而调控细胞的基因表达，影响细胞的功能和行为。我们发现了小鼠胚胎体外侵袭恶性肿瘤细胞的现象，其机制除与细胞外基质分子信号的调控有关外可能还存在其它信号途径。近期又发现小鼠囊胚细胞胞外微囊泡的存在，因此推测在胚胎细胞与恶性肿瘤细胞之间存在由胚胎细胞释放的微囊泡介导的信号途径。项目拟以C57BL/6小鼠囊胚与小鼠恶性黑色素瘤B16-F10细胞共培养模型及囊胚胞外微囊泡与肿瘤细胞共培养模型，研究小鼠囊胚胞外微囊泡控制肿瘤细胞侵袭性的作用；miRNA微阵列技术分析小鼠囊胚胞外微囊泡miRNA表达谱特征，并结合miRNA高通量数据库的生物信息学分析预测调控肿瘤细胞侵袭性的微囊泡功能性miRNA并实验验证功能miRNA的作用。从微囊泡层次证明胚胎细胞与肿瘤细胞间的微囊泡通讯方式，分子水平阐明胚胎细胞微囊泡传递胚胎细胞miRNA信号调控肿瘤细胞侵袭性的机制。

微囊泡通讯能将miRNA信号从一个细胞传递至另一个细胞，以调控纳入它们的细胞的基因表达，影响细胞的功能和行为。项目以植入前小鼠囊胚为材料，采用超高速密度梯度离心富集胚胎细胞胞外微囊泡。建立胚胎细胞胞外微囊泡-B16F10肿瘤细胞共培养模型，采用RTCA实时监测在与微囊泡共培养下的肿瘤细胞侵袭性的变化。采用miRNA表达谱微阵列分析小鼠胚胎微囊泡miRNA表达谱，结合miRbase database miRNA高通量数据库的生物信息分析，预测控制肿瘤细胞侵袭性的功能miRNA及其调控通路。采用LC-MS/MS技术进行胚胎胞外微囊泡蛋白质组学分析，结合UniProtKB和STRING的数据库挖掘进行蛋白功能预测。发现早期胚胎细胞胞外微囊泡存在，揭示了小鼠胚胎细胞的胞外微囊泡抑制恶性黑色素瘤细胞侵袭性的现象可能与其转运miRNA信息有关，验证了小鼠胚胎细胞胞外微囊泡转运胚胎细胞功能micRNA调控肿瘤细胞侵袭性的微囊泡通讯的科学假设，筛选出miR-34c、miR-410、mir-214、miR-154、miR-409-3p、mir-320等与控制恶性黑色素瘤细胞侵袭性相关的微囊泡功能性miRNA。小鼠胚胎胞外微囊泡表达181种蛋白，与代谢、生长发育、细胞侵袭与迁移、细胞分化的调控相关，其中65种膜蛋白中Krt17、Thbs1、A2m、Pzp、Col1a1、Lifr等与细胞识别微囊泡及触发靶细胞胞吞作用相关，提示靶细胞摄入胚胎细胞胞外微囊泡可能与脂伐介导的胞吞及网格蛋白介导的胞吞作用相关。在微囊泡层次证明胚胎细胞与肿瘤细胞间的微囊泡通讯方式，分子水平阐明胚胎细胞微囊泡传递胚胎细胞miRNA信号调控肿瘤细胞侵袭性的机制。项目成果对扩充细胞通讯理论具有重要的意义，对抑制肿瘤细胞侵袭性的新靶标的发现，降低恶性肿瘤的发生控制癌症死亡率具有重要的社会意义。