The Research On The Function And Mechanism Of LncRNA RP11-252E In Glioma Angiogenesis

Glioma originates from the central nervous system(CNS)and is a highly vascularized intracranial malignant tumor.The abundant blood vessels can meet the needs of tumors for oxygen and nutrients,which is a necessary condition for tumor growth and metastasis.Tumor angiogenesis refers to the process of forming new capillaries from peritumoral or intratumoral blood vessels.It is a complex and highly ordered process that depends on a wide network of signal transduction between endothelial cells,parietal cells and other cell types.The process of angiogenesis is regulated by a variety of angiogenesis-related factors and signaling pathways.It involves many biological processes such as vascular endothelial cell activation,basement membrane degradation,endothelial cell proliferation and migration,luminal structure formation and fusion as well as extracellular matrix reconstruction.Long noncoding RNAs(LncRNAs)are types of RNA transcripts that are generally greater than 200 nucleotides and could not encode proteins.LncRNAs,as an important "medium" in cells,can interact with one or more of DNA,RNA and proteins,through mediated chromatin remodeling and histone modification in epigenetic level,transcription level and post-transcriptional level regulate the expression of genes and proteins.And at the same time it participates in multiple biological processes such as cell proliferation,differentiation and apoptosis.In tumor diseases,lncRNAs mainly involved in tumor growth,diffusion and metastasis by acting as oncogenes or tumor suppressor genes.According to literature reports,lncRNA RP11-252E was significantly differentially expressed between glioma tumor tissue and normal brain tissue,suggesting that it may play a crucial part in the occurrence and development of glioma.The previous study of our group found that lncRNA RP11-252E could affect the proliferation,apoptosis and migration and alter their cell cycle in glioma cells U251 and U87.However,the biological function of lncRNA RP11-252E in glioma angiogenesis remains unknown.Therefore,in this study,we explored the function of lncRNA RP11-252E in the process of glioma angiogenesis.It is hoped to further reveal the mechanism of glioma angiogenesis,and provide reference for clinical diagnosis and treatment of glioma as well as the research and development of anti-angiogenic drugs.ObjectionsExplore the biological function and mechanism of lncRNA RP11-252E in the process of glioma angiogenesis.Meanwhile,provide new research ideas for some specifically expressed lncRNAs as clinical diagnostic markers and potential therapeutic targets for glioma.Methods1.RT-PCR and QPCR to detect the expression of lncRNA RP11-252E in several different tumor cells.2.Combining bioinformatics technology with RNA fluorescence in situ hybridization technology(RNA FISH)to clarify the basic characteristics of lncRNA RP11-252E.3.Constructing lncRNA RP11-252E knockdown and overexpressed plasmids and prepare lentivirus,lentivirus infection to obtain lncRNA RP11-252E low-expression and over-expression U251、U87 cell lines.4.Exploring the effect of lncRNA RP11-252E on glioma angiogenesis:Collecting lncRNA RP11-252E knockdown and overexpressed U251、U87 cell medium,then stimulate HUVEC cells,CCK-8 assay to analyze the effect of lncRNA RP11-252E on HUVEC cell proliferation;Co-culture U251、U87 with HUVEC cells or stimulate HUVEC cells with conditioned medium,Trans well assay to analyse the effect of lncRNA RP11-252E on the migration ability;tube formation assay to detect the effect of lncRNA RP11-252E on the angiogenesis ability of HUVEC cells.5.Exploring the molecular mechanism of lncRNA RP11-252E in glioma angiogenesis:RT-PCR to detect the expression of VEGFA and bFGF in U251、U87 cells,and ELISA to detect the content of VEGFA in cell culture supernatant,analysis of the effect of lncRNA RP11-252E on the expression of angiogenesis-related factors;Combining bioinformatics with luciferase reporter assay to screen and validate miRNAs bound by lncRNA RP11-252E;Combining bioinformatics with luciferase reporter assay to find miRNA-binding proteins.Results1.The expression of lncRNA RP11-252E is different in different tumor cells,it is significantly higher in glioma cells U251、U87 than that in lung cancer cells H1299、A549 and liver cancer cells SMCC-7721.2.LncRNA RP11-252E shows very low coding potential,mainly localized in U251、U87 cytoplasm.3.Down-regulating the expression of lncRNA RP11-252E in U251、U87 cells significantly inhibite the proliferation,migration and tube-forming abilities of HUVEC;Overexpression of lncRNA RP11-252E in U251、U87 cells significantly promote the proliferation,migration and tube-forming abilities of HUVEC.4.Down-regulating the expression of lncRNA RP11-252E in U251、U87 cells significantly reduce the expression of angiogenesis-related factors VEGFA and bFGF;Overexpression of lncRNA RP11-252E in U251、U87 cells significantly up-regulate the expressions of angiogenesis-related factors VEGFA and bFGF.5.In U251、U87 cells,lncRNA RP11-252E could directly bind to miR-185-3p and indirectly affect the expression of VEGFA.ConclusionsLncRNA RP11-252E is a newly discovered long non-coding RNA which mainly located in the cytoplasm of U251、U87 cell.It is highly expressed in glioma cells and affects the process of glioma angiogenesis.In glioma U251 and U87 cells,lncRNA RP11-252E promoted the expression of angiogenesis-related factors VEGFA and bFGF,and then enhanced the proliferation,migration and tube-forming ability of vascular endothelial cells.Preliminary mechanistic studies have shown that lncRNA RP11-252E plays an important role in glioma angiogenesis mainly by directly binding to miR-185-3p to regulate the expression of VEGFA.