【作者】 朱光辉；

【导师】 肖涛；

【作者基本信息】 中南大学 ， 外科学， 2022， 博士

【摘要】 背景:长链非编码RNA X非活性特异性转录本（Long non-coding RNA X-inactive specific transcript,Lnc-RNA XIST）调控多种肿瘤的进展,包括骨肉瘤（Osteosarcoma,OS）。骨髓间充质干细胞（Bone marrow mesenchymal stem cells,BMSCs）可被招募至骨肉瘤组织,并可通过分泌外泌体影响骨肉瘤进展。然而BMSCs来源外泌体是否传递LncRNA-XIST调控骨肉瘤生长和转移以及其中的机制如何,目前尚不明确。方法:本研究中,我们采用BMSCs来源外泌体处理人骨肉瘤细胞系MG63和143B,基于si RNA干预BMSCs中LncRNA-XIST水平,通过CCK-8,EdU标记以及transwell小室分析细胞增殖、迁移和侵袭能力变化。采用生物信息学、RNA pulldown和双荧光素酶报告基因实验研究LncRNA-XIST与miR-655结合,以及miR-655与ACLY的互作。用143B/LUC细胞系建立原位骨肉瘤动物模型,验证BMSCs来源外泌体LncRNA-XIST通过miR-655对骨肉瘤生长和转移的影响。用油红O染色、western blot和商业化试剂盒检测脂质沉积和蛋白表达变化。结果:首先,通过表征生物学特征,我们证实BMSCs状态、纯度良好,具有成骨和成脂分化潜能;发现BMSCs^exo可以被骨肉瘤细胞系MG63和143B吸收,并有助于骨肉瘤细胞系MG63和143B中LncRNA-XIST的上调;进一步构建si XIST,证实BMSCs来源外泌体通过传递LncRNA-XIST促进骨肉瘤细胞系MG63和143B增殖、迁移和侵袭能力。其次,基于starbase 3.0预测LncRNA-XIST与miR-655存在相互作用关系,miR-655介导了BMSCs来源外泌体LncRNA-XIST促进骨肉瘤进展的作用;进一步发现BMSCs来源外泌体通过LncRNA-XIST结合miR-655促进了骨肉瘤细胞增殖、迁移和侵袭能力,并通过在体实验结果证明BMSCs来源外泌体可通过LncRNA-XIST结合骨肉瘤miR-655从而促进肿瘤生长和转移过程。最后,采用starbase 3.0在线数据库预测miR-655下游靶基因分别为ARPP19、TOB1和ACLY;BMSCs来源外泌体可促进骨肉瘤细胞中ACLY表达,而抑制LncRNA-XIST,则ACLY表达水平下降。在此基础上,下调miR-655的表达,ACLY的表达得到回复,而ARPP19和TOB1的蛋白水平在各处理组间均没有变化,在体水平与体外结果相一致;BMSCs来源外泌体LncRNA-XIST可通过结合miR-655进而促进骨肉瘤细胞中ACLY的表达,进而导致骨肉瘤中脂质沉积。进一步检测细胞活力、增殖、迁移和侵袭能力,发现BMSCs来源外泌体LncRNA-XIST通过miR-655/ACLY信号升高骨肉瘤细胞增殖、迁移和侵袭能力。结论:1.BMSCs来源外泌体处理骨肉瘤细胞,可升高细胞中LncRNA-XIST水平,促进骨肉瘤细胞增殖、迁移和侵袭。2.BMSCs来源外泌体LncRNA-XIST进入骨肉瘤细胞后,可通过ce RNA机制结合并下调骨肉瘤细胞中miR-655水平,并且通过该机制促进体外骨肉瘤细胞增殖、迁移和侵袭,以及在体骨肉瘤生长和肺转移过程;3.BMSCs来源外泌体LncRNA-XIST结合并下调miR-655水平后,促进了骨肉瘤细胞中ACLY的表达,并进一步增加了细胞中脂质水平以及β-catenin信号活性,并通过该效应促进骨肉瘤细胞增殖、迁移和侵袭过程。图41幅,表12个,参考文献101篇更多还原

【Abstract】 Background:Long non-coding RNA X-inactive specific transcript（LncRNA-XIST）regulates the progression of various tumors,including osteosarcoma.Bone marrow mesenchymal stem cells（BMSCs）can be recruited to osteosarcoma tissues and can affect osteosarcoma progression by secreting exosomes.However,whether BMSCs-derived exosomes transmit LncRNA-XIST to regulate the growth and metastasis of osteosarcoma and the mechanism thereof are still unclear.Methods:In this study,we used BMSCs-derived exosomes to treat human osteosarcoma cell lines MG63 and 143B.Based on si RNA intervention on the level of LncRNA-XIST in BMSCs,we analyzed the changes in cell proliferation,migration and invasion capabilities by CCK-8,EdU labeling and transwell chambers.Bioinformatics,RNA pulldown and dual luciferase reporter gene experiments were used to study the binding of LncRNA-XIST to miR-655,and the interaction between miR-655 and ACLY.The 143B/LUC cell line was used to establish an orthotopic osteosarcoma animal model to verify the effect on growth and metastasis of osteosarcoma by the LncRNA-XIST from BMSCs exosomes through miR-655.Lipid deposition and protein expression changes were detected by Oil Red O staining,western blot and commercial kits.Results:First,through characterization of biological characteristics,it was confirmed that BMSCs were in good state,good purity,and had osteogenic and adipogenic differentiation potential;it was found that BMSCs^exo could be absorbed by osteosarcoma cell lines MG63 and 143B,and contributed to the upregulation of LncRNA-XIST in osteosarcoma cell lines MG63 and 143B;further construction of si XIST confirmed that BMSCs-derived exosomes promoted the proliferation,migration and invasion of osteosarcoma cell lines MG63 and 143B by delivering LncRNA-XIST.Secondly,based on starbase 3.0,it was predicted that LncRNA-XIST had an interaction relationship with miR-655,and miR-655 mediated the effect of BMSCs-derived exosome promoted the proliferation,migration and invasion of osteosarcoma cells through LncRNA-XIST binding to miR-655.The results of in vivo experiments confirm that BMSCs-derived exosomes can bind osteosarcoma miR-655through LncRNA-XIST to promote tumor growth and metastasis.Finally,the starbase 3.0 online database was used to predict that the downstream target genes of miR-655,which were ARPP19,TOB1 and ACLY;BMSCs-derived exosomes could promote the expression of ACLY in osteosarcoma cells,inhibit LncRNA-XIST,and decrease the expression level of ACLY.On this basis,the expression of miR-655 was down-regulated,and the expression of ACLY was restored,while the protein levels of ARPP19 and TOB1 did not change among the treatment groups,and the in vivo levels were consistent with the in vitro results;BMSCs-derived exosomal LncRNA-XIST can promote the expression of ACLY in osteosarcoma cells by binding to miR-655,which leads to lipid deposition in osteosarcoma.Further detection of cell viability,proliferation,migration and invasion ability showed that BMSCs-derived exosomal LncRNA-XIST increased the proliferation,migration and invasion ability of osteosarcoma cells through miR-655/ACLY signaling.Conclusions:1.Treatment of osteosarcoma cells with BMSCs-derived exosomes can increase the level of LncRNA-XIST in cells,and promote cell proliferation,migration and invasion.2.After BMSCs-derived exosome LncRNA-XIST enters osteosarcoma cells,it can bind and down-regulate the level of miR-655 in osteosarcoma cells through the ce RNA mechanism so as to promotes osteosarcoma cells proliferation,migration and invasion in vitro,as well as in vivo osteosarcoma growth and lung metastasis.3.After BMSCs-derived exosome LncRNA-XIST binds and down-regulates the level of miR-655,it promotes the expression of ACLY in osteosarcoma cells,and further increases the lipid level andβ-catenin signaling activity in the cells.And through this effect,it promotes the process of osteosarcoma cell proliferation,migration and invasion.更多还原