脑胶质瘤干细胞衍生的外泌体lncRNA HOXA-AS2促进脑胶质瘤的增殖、迁移、侵袭和干细胞特性

Glioma stem cell-derived exosomal lncRNA HOXA-AS2 promoted proliferation,migration,invasion and stemness in glioma

背景与目的:外泌体是介导肿瘤微环境中肿瘤细胞与受体细胞间相互作用的重要信使。然而,细胞外泌体长链非编码RNA(long non-coding RNA,lncRNA)在脑胶质瘤干细胞(glioma stem cell,GSC)和脑胶质瘤细胞的细胞间通信中的作用尚不清楚。本研究探究外泌体衍生的lncRNA对脑胶质瘤增殖、迁移、侵袭和干细胞特性的影响。方法:从中国脑胶质瘤基因组图谱(the Chinese Glioma Genome Atlas,CGGA)和癌症基因组图谱(the Cancer Genome Atlas,TCGA)数据库下载包含低级别脑胶质瘤(low-grade glioma,LGG)和高级别脑胶质瘤(high-grade glioma,HGG)lncRNA表达数据的数据集,识别LGG和HGG组织之间的差异表达lncRNA(differentially expressed lncRNA,DelncRNA),并分析HOXA-AS2水平与胶质瘤患者总生存期(overall survival,OS)之间的关系。从人胶质瘤细胞系SHG44中分离GSC,用流式细胞术检测CD133^(+)富集的细胞,再用蛋白质印迹法(Westernblot)检测干细胞相关蛋白(CD133、SOX2和OCT4)的表达水平。提取和识别SHG44-GSC衍生的外泌体,并用PKH26细胞膜染料进行荧光标记;再将转染了Cy3标记HOXA-AS2的SHG44-GSC与SHG44细胞进行间接共培养;后用实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)检测SHG44-GSC和SHG44-GSC衍生外泌体中HOXA-AS2的水平。使用pLVX-IRES-PUROHOXAAS2慢病毒质粒和含靶向HOXA-AS2质粒的慢病毒shRNA进行慢病毒转染。采用细胞计数试剂盒-8(cellcountingkit-8,CCK-8)和transwell实验检测SHG44-GSC衍生的外泌体HOXA-AS2对SHG44细胞增殖和侵袭能力的影响。结果:HOXAAS2在胶质瘤中呈现高表达,且与患者较差的OS相关(P<0.01)。SHG44-GSC中CD133^(+)细胞比例明显高于SHG44细胞(P<0.0001),SHG44-GSC中干细胞相关蛋白(CD133、SOX2和OCT4)的表达水平明显高于亲代SHG44细胞(P<0.0001),并且SHG44-GSC中HOXA-AS2水平显著升高(P<0.0001)。PKH26标记的外泌体被SHG44细胞吸收,且SHG44细胞中可观察到Cy3标记的HOXA-AS2;HOXA-AS2OE转染的SHG44-GSC细胞(SHG44-GSC/HOXA-AS2OE)和SHG44-GSC/HOXA-AS2OE衍生的外泌体(SHG44-GSC/HOXA-AS2OE-Exo)中HOXA-AS2水平显著升高(P<0.01),在与SHG44-GSC/HOXA-AS2OE细胞共培养的SHG44细胞中HOXA-AS2水平显著升高(P<0.01)。SHG44-GSC/HOXAAS2 OE-Exo可显著促进SHG44细胞增殖、迁移和侵袭。结论:来自SHG44-GSC的外泌体HOXA-AS2能显著促进胶质瘤细胞增殖、迁移、侵袭和干细胞特性,提示HOXA-AS2可能是脑胶质瘤潜在的治疗靶点。

Background and purpose:Exosomes are important messengers that mediate the crosstalk between cancer cells and recipient cells in the tumor microenvironment;however,the role of extracellular exosomal long non-coding RNA(lncRNA)in the cell-cell communications of glioma stem cells(GSCs)and glioma cells remains unclear.This study investigated the effects of exosome-derived lncRNAs on proliferation,migration,invasion and stemness in glioma.Methods:The datasets containing lowgrade glioma(LGG)and high-grade glioma(HGG)lncRNA expression data were downloaded from the Chinese Glioma Genome Atlas(CGGA)and the Cancer Genome Atlas(TCGA)databases.Differentially expressed lncRNA(DelncRNA)between LGG and HGG tissues was identified,and the relationship between HOXA-AS2 levels and overall survival(OS)of glioma patients was analyzed.GSCs were isolated from human glioma cell line SHG44,and CD133^(+)enriched cells were detected by flow cytometry.The expression levels of stem cell-related proteins(CD133,SOX2 and OCT4)were detected by Western blot.Exosomes derived from SHG44-GSCs were extracted and identified,and labeled with PKH26 cell membrane dye.Then SHG44-GSCs transfected with Cy3-labeled HOXA-AS2 were indirectly co-cultured with SHG44 cells.The levels of HOXA-AS2 in SHG44-GSCs and SHG44-GSCderived exosomes were detected by real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR).The plVX-IRESPURO HOXA-AS2 lentivirus plasmid and lentivirus shRNA containing targeting HOXA-AS2 plasmid were used for lentivirus transfection.The effect of HOXA-AS2 derived from SHG44-GSC on the proliferation and invasion of SHG44 cells was detected by cell counting kit-8(CCK-8)and transwell assays.Results:HOXA-AS2 was highly expressed in gliomas,and was associated with worse OS in patients(P<0.01).The proportion of CD133^(+)cells were significantly higher in SHG44-GSC than in SHG44 cells(P<0.0001).The expression levels of stem cell related proteins(CD133,SOX2 and OCT4)in SHG44-GSC cells were significantly higher compared with parental SHG44 cells(P<0.0001).HOXA-AS2 level was significantly increased in SHG44-GSC cells(P<0.0001).PKH26-labeled exosomes were absorbed by SHG44 cells,and Cy3-labeled HOXA-AS2 could be observed in SHG44cells.HOXA-AS2 levels were significantly increased in HOXA-AS2 OE transfected SHG44-GSCs cells(SHG44-GSC/HOXA-AS2OE)and SHG44-GSC/HOXA-AS2 OE-derived exosomes(SHG44-GSC/HOXA-AS2 OE-Exo)(P<0.01).In addition,HOXA-AS2levels were significantly increased in SHG44 cells co-cultured with SHG44-GSC/HOXA-AS2 OE cells(P<0.01).HOXA-AS2 could be transferred from SHG44-GSC to SHG44 cells through exosomes.Functionally,SHG44-GSC/HOXA-AS2 OE-Exo significantly promoted the proliferation,migration and invasion of SHG44 cells.Conclusion:Exosomal HOXA-AS2 derived from SHG44-GSCs significantly promoted glioma cell proliferation,migration,invasion and stemness,suggesting that HOXA-AS2 may be a potential therapeutic target for glioma.