沉默lncRNA-MALAT1对胶质瘤血管生成拟态的影响

Effect of silencing lncRNA MALAT1 on formation of vasculogenic mimicry of glioma

目的 探讨沉默长链非编码RNA转移相关肺腺癌转录本1(Metastasis-associated lung adenocarcinoma transcript 1,MALAT1)对胶质瘤血管生成拟态的影响。方法 实时荧光定量(qPCR)法检测人胶质瘤细胞U251、U87和正常人星形胶质细胞NHA中lncRNA-MALAT1的表达量。京都基因与基因组百科全书(KEGG)和基因本体论(GO)分析lncRNA-MALAT1的信号通路及生物学功能。qPCR法检测沉默效率,将沉默效率最高的片段序列作为实验组(LV-MALAT1组),携带无意义片段的重组慢病毒感染人胶质瘤细胞U251、U87作为阴性对照组(LV-NC组),未干预的人胶质瘤细胞U251、U87作为空白对照组(BLANK组),并检测分析3组中lncRNA-MALAT1的表达量。细胞三维培养法观察分析U251、U87细胞的体外血管生成拟态(Vasculogenic mimicry, VM)形成能力。CCK-8实验和划痕实验评估细胞的增殖和迁移能力。Transwell实验观察细胞的迁移和侵袭能力。Western-blot检测VM相关蛋白VE-cadherin、MMP2、VEGF以及PI3K/AKT/FOXO1信号转导通路蛋白及其磷酸化蛋白表达量。结果 与NHA细胞比较,lncRNA-MALAT1在胶质瘤细胞U251、U87中高表达(P<0.000 1);KEGG和GO富集分析结果显示,lncRNA-MALAT1参与肿瘤细胞信号通路PI3K/AKT途径、细胞黏附、迁移及细胞外基质生成等细胞分子生物学过程;体外VM形成能力实验结果显示,与U251及U87细胞LV-NC组比较,LV-MALAT1组VM形成数量和长度明显减少,差异有统计学意义(P<0.01);Western-blot结果显示,与U251及U87细胞LV-NC组比较,LV-MALAT1组VM相关蛋白VE-cadherin、MMP2、VEGF表达显著降低,差异有统计学意义(P<0.05);CCK-8实验结果显示,在细胞培养48 h后,与U251及U87细胞LV-NC组比较,LV-MALAT1组的细胞增长速率明显降低(P<0.000 1);划痕实验结果显示,与U251及U87细胞LV-NC组比较,LV-MALAT1组在24 h、48 h后划痕愈合面积明显减小(P<0.000 1);Transwell实验结果显示,与U251及U87细胞LV-NC组比较,LV-MALAT1组的细胞迁移和侵袭数量显著减少(P<0.001);与U251及U87细胞LV-NC组比较,LV-MALAT1组的PI3K、Phospho-PI3K、AKT、Phospho-AKT表达量显著下降,FOXO1、Phospho-FOXO1表达量显著升高(P<0.05)。结论 沉默lncRNA-MALAT1既能抑制胶质瘤细胞U251、U87血管生成拟态的能力,又可使其增殖、迁移及侵袭能力明显降低,其机制可能与PI3K/AKT/FOXO1通路表达水平变化相关。

Objective To investigate the effect of downregulation of lncRNA metastasis-associated lung adenocarcinoma transcript 1(MALAT1)on glioma vasculogenic mimicry.Methods lncRNA-MALAT1 ex-pression levels in human glioma cells U251,U87 and normal astrocytes NHA were detected by qPCR.The Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene Ontology(GO)analyzed the signaling pathway and biological function of lncRNA-MALAT1.The silencing efficiency was detected by qPCR.The fragment sequence with the highest silencing efficiency was selected as the experimental group(LV-MALAT1 group),recombinant lentivirus infected human glioma cells U251 and U87 carrying meaningless fragments were selected as negative control group(LV-NC group),and the untreated human glioma cells U251 and U87 were selected as BLANK control group.The expression level of lncRNA-MALAT1 in the 3 groups was detected and analyzed.The VM formation ability of U251 and U87 cells in vitro was observed and analyzed by 3-dimensional cell culture.Cell proliferation and migration were evaluated by CCK-8 assay and scratch assay.Transwell assay was used to observe the migration and invasion ability of cells.The ex-pression of vasculogenic mimicry(VM)related proteins VE-cadherin,MMP2,VEGF and PI3K/AKT/FOXO1 signal transduction pathway protein and its phosphorylated protein were detected by Western blot.Results lncRNA-MALAT1 was highly expressed in glioma cells U251 and U87 compared with NHA cells(P<0.0001).KEGG and GO enrichment analysis showed that lncRNA-MALAT1 was involved in the cellular molecular biological processes of tumor cell signaling pathway,such as PI3K/AKT pathway,cell adhesion,migration and extracellular matrix generation.The results of in vitro VM formation ability test showed that compared with U251 and U87 cells LV-NC group,the number and length of VM formation in LV-MALAT1 group were significantly reduced,with statistical significance(P<0.01).Western-blot re-sults showed that compared with U251 and U87 cells LV-NC group,the expressions of VM-related pro-teins VE-cadherin,MMP2 and VEGF in LV-MALAT1 group were significantly decreased,with statistical significance(P<0.05).The results of CCK-8 experiment showed that after 48 h of cell culture,compared with U251 and U87 cells in LV-NC group,the cell growth rate of LV-MALAT1 group was significantly decreased(P<0.01).The scratch test results showed that compared with U251 and U87 cells LV-NC group,the scratch healing area of LV-MALAT1 group was significantly reduced after 24 h and 48 h(P<0.0001).The results of Transwell experiment showed that compared with U251 and U87 cells in LV-NC group,the number of cell migration and invasion in LV-MALAT1 group was significantly reduced(P<0.001).Compared with U251 and U87 cells in LV-NC group,the expressions of PI3K,Phospho-PI3K,AKT and Phospho-AKT in LV-MALAT1 group were significantly decreased,and the expressions of FOXO1 and Phospho-FOXO1 were significantly increased(P<0.05).Conclusion Down-regulation of lncRNA-MALAT1 inhibited the ability of glioma cells U251,U87 vasculogenic mimicry and significantly reduced their proliferation,migration and invasion,the mechanism of which may be related to the altera-tion of PI3K/AKT/FOXO1 pathway.