miR-199a靶向AXL信号通路调控骨肉瘤细胞凋亡和侵袭及其分子机制的研究

miRNAs are short single stranded RNA molecules, which serve as master regulators of gene expression in normal and tumor cells. miR-199a is a microRNA discovered recently, which may be one of the important regulators during the malignant progression of many tumors. Nevertheless,the molecular mechanism of miR-199a in osteosarcoma is still unknown. In our pre-experiment, we have shown that miR-199a expression was obviously lower in the aggressive osteosarcoma cell line MG63 than in the normal fibroblastic cell line NIH3T3. Through the software to predict microRNA targets in mammals scanning,we found that the recptor tyrosine kinase AXL was one of the target genes of miR-199a. We also proved that the expression of AXL protein decreased obviously in MG63 after the transfection of miR-199a mimic. It indicated that AXL maybe one of the important target genes of miR-199a in osteosarcoma cells. More pre-experiment have also proved that phosphor-AXL was highly expressed in osteosarcoma cells, which was significantly related to lung metastasis and poor prognosis of the patients. In osteosarcoma cell lines, it was shown that AXL activated by its ligand Gas6 also protected the osteosarcoma cells from apoptosis caused by serum free, and promoted the cell lines migration and invasion in vitro. Then, our question is whether miR-199a directly target AXL gene in osteosarcoma cells? Is there the miR-199a-AXL regulative pathway in osteosarcoma cell? Do this pathway regulate the apoptosis and invasion of osteosarcoma cell? And what is the molecular mechanisms of this pathway? We designed this project to ensure that there is AXL signal pathway downregulated by miR-199a in osteosarcoma,at the samt time, to confirm that miR-199a is related with the tumor cells apoptotic rate and invasive ability.The research methods including luciferase-reporter assays,qRT-PCR, pre-miRNA stable transfection, apoptosis antibody array will be used. We also want to reveal the molecular mechanisms for the miR-199a-AXL pathway to regulate the apoptosis and invasion of osteosarcoma cells, and try to show the potential therapy role of miR-199a to osteosarcoma. The purpose of this project is to know deeply the mechanism of the malignant progression of osteosarcoma, and build the theory basis to find the prognostic impactors and the potential therapy target for osteosarcoma patients.

国内外研究提示miR-199a是多种肿瘤恶性进展中重要的调控性miRNA。我们前期研究证实高侵袭性骨肉瘤细胞中 miR-199a 低表达,初步筛选发现骨肉瘤细胞内受体酪氨酸激酶AXL是miR-199a重要的靶向基因；继而证明磷酸化-AXL在骨肉瘤细胞中高表达,与患者肺转移率和不良预后显著正相关,AXL活化后细胞凋亡率降低,侵袭力增强。那么,骨肉瘤中miR-199a对AXL是否直接靶向调控？该通路是否调控肿瘤细胞的凋亡和侵袭？其分子机制如何？本项目拟应用荧光素酶报告基因系统、qRT-PCR、pre-miRNA稳定转染、凋亡抗体Array等技术明确骨肉瘤细胞中miR-199a对AXL直接靶向调控,进而研究该通路对骨肉瘤凋亡和侵袭的调控作用及其相关分子机制，最后验证相关调控蛋白在骨肉瘤的临床肿瘤学意义。旨在深入认识miR-199a-AXL对骨肉瘤的调控机理，寻找相应肿瘤预后标记物和潜在治疗靶点。

本研究利用常规培养骨肉瘤细胞系MG63,143B,U2OS,HOS,SOS-2 和纤维母细胞系NIH3T3，应用real-time qRT-PCR法研究发现miR-199a-3p在骨肉瘤细胞系中的表达水平显著低于正常对照组织和细胞系；且 miR-199a-3p不影响几株骨肉瘤细胞系的增殖速率；瞬时转染 miR-199a-3p mimic能够抑制骨肉瘤细胞的体外迁移和侵袭，miR-199a-3p的作用可能是通过下调磷酸化AKT的作用来实现，其影响的详细信号通路有待进一步深入研究。骨肉瘤细胞系MG63,U2OS,143B,HOS中miR-199a -3p能够负性调控AXL mRNA和蛋白的表达；且双荧光素酶报告检测系统发现AXL 3´-UTR区域存在miR-199a-3p的直接结合位点，是miR-199a-3p的直接靶基因。这一结果提示，骨肉瘤细胞中miR-199a-3p通过对AXL的负性调控而抑制骨肉瘤细胞迁移和侵袭。石蜡包埋和新鲜冻存骨肉瘤组织real-time PCR检测结果发现骨肉瘤中miR-199a-3p的水平显著低于瘤旁正常对照组织，且miR-199a-3p的表达水平与患者的复发和肺转移率以及生存时间呈显著性负相关。应用骨肉瘤细胞系143B 接种入裸鼠体内，建立原位移植瘤模型和肺转移模型，体内注入miR-199a-3p激动剂模拟裸鼠体内miR-199a高表达的状况，研究发现，与对照组相比较，应用miR-199a-3p 激动剂的实验组原位移植瘤的增殖速率并无显著性变化，但是实验组裸鼠肿瘤肺转移灶的数目、转移灶的大小明显减少，坏死率明见减低，证明miR-199a-3p 具有体内抑制骨肉瘤侵袭转移的作用。应用Gas6刺激骨肉瘤细胞系MG63,143B和U2OS, Hoechst检测细胞凋亡速率，转染AXL siRNA以及细胞毒性和细胞周期检测，并应用化疗药物DDP和MTX，发现Gas6能够特异性与其受体AXL结合，并抑制细胞凋亡，却没有影响细胞周期和细胞增殖速率。应用41例骨肉瘤组织进行TUNEL凋亡信号检测以及免疫组化染色，发现骨肉瘤中凋亡相关蛋白BCL-2和BAX均高表达，且P-AXL的表达与肿瘤细胞的凋亡速率呈反比。本研究结果有助于深入认识骨肉瘤发生发展的调控机理，并为临床寻找相应的肿瘤预后标记物和治疗靶点提供了理论基础。