间充质干细胞源性外泌体介导miRNA-21—Hedgehog通路参与骨肉瘤细胞增殖的机制研究

Mesenchymal stem cells (MSCs) play an important role in the proliferation of osteosarcoma cells, but the mechanism is unknown. MSCs affect osteosarcoma cells proliferation via exosomes. In previous experiments, we have isolated MSCs and purified MSCs exosomes. The ultra-structure of exosomes body was observed under electron microscope. MSCs-exosomes were internalized by cancer cells imaging with confocal microscopy. It was confirmed that the proliferation ability of osteosarcoma cells increased after MSCs-exosomes uptake and the Hedgehog pathway was upregulated in osteosarcoma cells. However, it is not clear that what exact molecule was mediated by MSCs-exosomes on osteosarcoma cells. In our pre-experiment ,We have isolated MSCs from the microenvironment of osteosarcoma and found its high expression of miRNA-21. MiRNA-21 is known as a oncogene and can up-regulate the Hedgehog pathway of tumor cells. Therefore, we speculate that "MSCs-exosomes shuttle miRNA-21 involvement in osteosarcoma cell proliferation" is a new mechanism for the proliferation of osteosarcoma cells. We plan to use RNA-Seq technique to detect the MSCs-exosomes miRNA-21 in the microenvironment of osteosarcoma. The miRNA-21—Hedgehog feedback loop mediated by MSCs exosomes is involved in the proliferation mechanism of osteosarcoma cells in vivo and in vitro. By in vivo and in vitro experiments, we confirm the mechanism that MSCs-exosomes mediated miRNA-21—Hedgehog signaling pathway involved in proliferation of osteosarcoma cells.

间充质干细胞（MSCs）在骨肉瘤细胞增殖中起重要作用，但机制不详。MSCs通过外泌体作用于骨肉瘤细胞而影响肿瘤增殖。前期实验中我们已分离MSCs并纯化其外泌体，电镜观察到外泌体超微结构，共聚焦显微镜观察到肿瘤细胞摄取外泌体，实验证实外泌体摄取后的骨肉瘤细胞增殖能力增强、Hedgehog通路上调。但MSCs外泌体介导何种分子作用于骨肉瘤细胞，目前尚不明确。我们预实验从骨肉瘤微环境中分离MSCs并发现其高表达miRNA-21。miRNA-21是促癌基因，能上调肿瘤细胞Hedgehog通路。因此我们推测“MSCs外泌体介导miRNA-21参与骨肉瘤细胞增殖”是MSCs促骨肉瘤细胞增殖的新机制。我们拟采用RNA-Seq技术检测骨肉瘤微环境中MSCs外泌体miRNA-21。通过体内、外实验证实MSCs外泌体介导miRNA-21—Hedgehog信号通路参与骨肉瘤细胞的增殖机制假说。

研究背景：人源性骨髓间充质干细胞在骨肉瘤发病机制和进展中的作用至今尚未完全阐明。.研究内容：在这项研究中，建立了人骨髓间充质干细胞与骨肉瘤细胞共培养模型，并研究了人骨髓间充质干细胞介导的外泌体在骨肉瘤增殖和侵袭中的作用。为了找到骨髓间充质干细胞源性外泌体活化的miRNA和激活miRNA的靶基因，我们挖掘了GEO数据集并通过实时定量PCR检测表达差异miRNA。使用miranda、targetscan、encori和pictar对miRNA靶基因预测，并通过双荧光素酶报告基因检测验证预测基因靶点是否正确。然后我们进一步研究间充质干细胞是否通过外泌体介导目的miRNA促进骨肉瘤细胞的增殖和侵袭，调控骨肉瘤细胞可能的信号通路。.研究结果：人骨髓间充质干细胞外泌体得到成功分离，透射电镜观察到外泌体超微结构，Western Blot检测到外泌体表面标记物CD81、CD63。共聚焦显微镜观察到骨肉瘤细胞MG63摄取外泌体。骨髓间充质干细胞源性外泌体被骨肉瘤细胞MG63摄取后，骨肉瘤细胞增殖、侵袭能力明显增强、凋亡能力明显减弱。我们还发现miR-21-5p在间充质干细胞和间充质干细胞来源的外泌体中显著过度表达。转染miR-21-5p的间充质干细胞源性外泌体可显著增强骨肉瘤细胞的增殖和侵袭。生物信息学分析和双荧光素酶报告基因分析验证了外泌体miR-21-5p和PIK3R1之间的靶向关系。此外，我们还证明了来源于人骨髓间充质干细胞、富含miR-21-5p的外泌体可以通过抑制骨肉瘤细胞中PIK3R1的表达激活PI3K/Akt/mTOR通路而促进骨肉瘤细胞增殖与侵袭。.本研究发现：1,人骨髓间充质干细胞源性外泌体具有促进骨肉瘤细胞增殖与侵袭作用；2，骨髓间充质干细胞源性外泌体介导miR-21-5p靶向PIK3R1从而激活下游PI3K/Akt/mTOR通路而促进骨肉瘤细胞增殖与侵袭。.研究意义：我们的发现为人骨髓间充质干细胞和骨肉瘤细胞在肿瘤相关微环境中的相互作用提供了新的见解，这为骨肉瘤发生发展机制研究提供新的思路，为骨肉瘤化疗治疗研究领域提供新的研究基础。