| Background:Glioma originates from glial cells or precursor cells and is divided into astrocytoma,ependymoma and oligodendroglioma,It is the most common malignant primary tumor of the central nervous system(CNS).In addition,the World Health Organization(who)divides gliomas into four categories according to the degree of malignancy.WHO grade 1-2 gliomas are called low-grade gliomas(LGG),including angiocentric gliomas and diffuse astrocytomas,while WHO grade 3-4 gliomas are regarded as high-grade gliomas(HGG),including stromal astrocytoma and glioblastoma multiforme(GBM).GBM has a high mortality and recurrence rate and is the most malignant disease of the central nervous system.At present,the standard for the treatment of GBM is still to remove the tumor with temozolomide through neurosurgery(TMZ)chemotherapy and radiotherapy.Unfortunately,even with this combination therapy,the median survival time of GBM patients is only 15 months.In the past few years,bevacizumab has achieved relatively good efficacy in a variety of tumors,including glioma,but more than 30% of patients do not respond to anti angiogenesis therapy at first,and eventually the relevant patients will relapse and die soon In this disease.There is evidence that noncoding RNA,especially lncRNA and micro RNA,plays an important role in the regulation of angiogenesis.Long noncoding RNAs(Lnc RNA)represent more than 200 nucleotides and have no obvious protein coding potential.Long noncoding RNA(lncRNAs play a wide range of functions in physiological and pathological processes including tumor progression.It is reported that dysregulated lncRNAs can affect angiogenesis.Angiogenesis is a complex multi-step process that drives the formation of new blood vessels and accelerates cancer progression by providing nutrition and energy.However,lncrnas regulate glioma cell proliferation,invasion and migration The underlying mechanism of multiple biological behaviors is unclear.We compared the differential genes in normal brain tissues and gliomas with different malignant degrees by high-throughput sequencing,and finally selected lncRNA WARS2-IT1.The expression level of lncRNA WARS2-IT1 in gliomas was significantly higher than that in normal brain tissues,and was positively correlated with the malignant degree of gliomas.According to the website of lncRNA subcellular localization,lncRNA WARS2-IT1 is located in the cytoplasm,but no relevant research on the tumor of this gene has been retrieved,and its functional mechanism is not clear.Therefore,based on previous studies,this study further summarizes the progress in the field of lncRNA regulation in glioma in recent years,and explores the specific mechanism of lncRNA WARS2-IT1 in the occurrence,development and angiogenesis of glioma,in order to study accurate biomarkers and reliable therapeutic targets.Objective:Based on the previous work,this study first verified the role of lncRNA WARS2-IT1 and miR-299-3p in glioma proliferation and invasion at the molecular and cellular levels.The role of lncRNA WARS2-IT1 in glioma angiogenesis through miR-299-3p/VEGFA signal axis was verified by tubule formation experiment;The molecular mechanism of the regulatory relationship between lncRNA WARS2-IT1/miR-299-3p/VEGFA signal axis and downstream signal pathway related proteins was verified at the overall level;After glioma surgery,the specimens were collected and transplanted tumors were extracted through animal experiments.The RNA expression level of lncRNA WARS2-IT1/miR-299-3p was verified at the tissue level,so as to explain the molecular mechanism of lncRNA WARS2-IT1 promoting glioma angiogenesis by activating PI3K/AKT pathway through miR-299-3p/VEGFA axis,so as to provide a practical and theoretical basis for exploring the targeted treatment of glioma.Methods:Part Ⅰ expression and clinical significance of lncRNA WARS2-IT1 in Glioma1.Tissue sequencing was used to detect the expression of lncRNA in 4 cases of glioma and 2 cases of normal brain tissue,and the lncRNA with obvious difference was screened out;2.The expression level of lncRNA WARS2-IT1 was detected by real time PCR in glioma and normal brain tissues;3.Detect the expression of lncRNA WARS2-IT1 in glioma cell lines and normal brain tissue cell lines;4.Analyze the correlation between the expression level of lncRNA WARS2-IT1 and the clinical characteristics of glioma patients,and analyze the correlation between the expression level of lncRNA WARS2-IT1 and the prognosis of glioma patients in the biological information database.Results:1.Comprehensive analysis in up-regulated differential lncRNAs,the expression level of lncRNA WARS2-IT1 in glioma tissue samples was significantly increased(P< 0.05).2.RT-PCR was used to detect lncRNA WARS2-IT1 in glioma and normal brain tissues.The results showed that the expression of lncRNA WARS2-IT1 was significantly higher than that in normal brain tissues,of which 55.8%(29/52)was significantly higher in glioma samples(P < 0.05).Compared with human normal glioma HEB cell line,the expression of lncRNA WARS2-IT1 in glioma U87,U118,U251 and T98 G cell lines was significantly increased(P < 0.05).The expression abundance in T98 G and U251 cells was higher,and the expression abundance in the other two cells was medium.3.The expression level of lncRNA WARS2-IT1 and Glioma patients was correlated with tumor size,WHO grade and KPS score(P < 0.05),but no significant correlation with gender,age,tumor location and cumulative brain lobe.4.According to the results of biological information database,taking the median expression of lncRNA WARS2-IT1 as the cut-off value,the Overall Survival of patients with high expression(n = 254)and low expression(n = 256)of lncRNA WARS2-IT1 was compared between the two groups.There was a significant difference in Disease-Free Survival(P < 0.05),and the survival rate of patients with high expression of lncRNA WARS2-IT1 was significantly lower(P < 0.05).Part Ⅱ Silencing lncRNA WARS2-IT1 inhibits proliferation,invasion and angiogenesis of glioma Methods:1.Construct the recombinant lentivirus vector that silences the expression of lncRNA WARS2-IT1.Construction of lentivirus sh WARS2-IT1 stable transformation cell lines of T98 G,U251 and HBMVEC.The expression of lncRNA WARS2-IT1 was detected by RT-PCR.2.The effects of silencing lncRNA WARS2-IT1 on the proliferation,invasion and migration of T98 G and U251 cells were detected by scratch test,cell counting kit-8(CCK-8),Transwell test and EDU analysis.3.The effect of silencing lncRNA WARS2-IT1 on the apoptosis of T98 G and U251 cells was detected by flow cytometry.4.Tube formation assay detected angiogenesis after silencing lncRNA WARS2-IT1.Results:1.The results showed that the silencing transfection efficiency of lentivirus sh WARS2-IT1 stable cell lines T98 G and U251 was high,and T98 G and U251 cell lines silenced lncRNA WARS2-IT1 were successfully obtained.2.The results of scratch test,CCK-8,Transwell and EDU showed that the proliferation,invasion and migration of cells were inhibited after silencing lncRNA WARS2-IT1.3.The results of flow cytometry showed that after silencing lncRNA WARS2-IT1,the number of apoptosis of T98 G and U251 cells increased significantly.4.Silencing lncRNA WARS2-IT1 can significantly inhibit glioma angiogenesis.Part Ⅲ Silencing lncRNA WARS2-IT1 inhibits glioma proliferation,invasion and angiogenesis by regulating miR-299-3P /VEGFA axis Methods:1.Firstly,the subcellular localization of lncRNA WARS2-IT1 was detected.2.Use bioinformatics analysis websites or tools to analyze the miRNAs with common response elements with lncRNA WARS2-IT1 and VEGFA,and verify their expression levels in T98 G and U251 cells.3.Mimics and inhibitor of miR-299-3p were transfected into T98 G and U251 cells,respectively.The targeting relationship between lncRNA WARS2-IT and miR-299-3p,miR-299-3p and VEGFA was identified by Dual-Lucifase Reporting System.4.T98 G and U251 cells were transfected with mimics and inhibitor of miR-299-3p.The effects on cell proliferation,invasion,migration and apoptosis were detected by scratch test,CCK-8,EDU analysis,Transwell and flow cytometry.The effect of HBMVEC transfection on angiogenesis was observed.5.The rescue experiment further verified that lncRNA WARS2-IT1 could affect the phenotypic function of glioma cells by regulating miR-299-3p/VEGFA axis.Results:1.lncRNA WARS2-IT1 is mainly expressed in the cytoplasm and serves as the ce RNA of miRNA to regulate the occurrence and development of tumors.2.Bioinformatics analysis showed that miR-299-3p shared a common response with lncRNA WARS2-IT1 and VEGFA,and was underexpressed in glioma cells.3.Mimics and inhibitor of miR-299-3p were successfully transfected into T98 G and U251 cells,respectively.Dual-Lucifase reports showed that lncRNA WARS2-IT and miR-299-3p had targeted regulatory effects on VEGFA.4.The results of scratch test,CCK-8,EDU analysis,Transwell and flow cytometry showed that after transfecting mimics of miR-299-3p,the proliferation,invasion and migration of T98 G and U251 cells were significantly inhibited,and the apoptosis was promoted,the ability of HBMVEC to form tubules was inhibited in glioma.5.The rescue experiment further confirmed that lncRNA WARS2-IT1 could affect the phenotypic function of glioma cells by regulating miR-299-3p/VEGFA axis.Part IV Silencing lncRNA WARS2-IT1 inhibits glioma proliferation,invasion and angiogenesis through PI3K/AKT signaling pathway Methods:1.Construct the stable cell line of recombinant lentivirus vector silencing the expression of lncRNA WARS2-IT1,and detect the expression of VEGFA,CD34,PI3 K,p-PI3 K,AKT,p-AKT,Caspase-3,Caspase-7,Bax,Bcl-2,MMP2 and MMP9 by Western blot.2.The mimics and inhibitor of miR-299-3p were transfected respectively,and then the expressions of VEGFA,CD34,p-PI3 K,AKT,p-AKT,Caspase-3,Caspase-7,Bax,Bcl-2,MMP2 and MMP9 in T98 G and U251 cells were detected by Western blot.3.The recombinant lentivirus vector stable cell line silencing the expression of lncRNA WARS2-IT1 was constructed to carry out the subcutaneous tumorigenesis experiment in nude mice,and its effects on the tumorigenicity,VEGFA,CD34 protein and the number of blood vessels in the tumor were observed.Results:1.The expression levels of VEGFA,CD34,P-PI3 K,P-AKT,Bcl-2,MMP2 and MMP9 in lncRNA WARS2-IT1 was silenced and miR-299-3p was overexpressed were significantly decreased by Western-blot analysis.There was no significant effect on the expression levels of PI3 K and AKT proteins,the expression levels of caspase-3,Caspase-7 and BAX were significantly increased.2.The tumor formation experiment in nude mice confirmed that silencing lncRNA WARS2-IT1 could significantly reduce the tumor volume,VEGFA,CD34 protein and the number of tumor blood vessels in nude mice(P < 0.05).Conclusions:1.Lnc RNA WARS2-IT1 was highly expressed in glioma;2.Lnc RNA WARS2-IT1 promotes the proliferation,invasion,angiogenesis and tumorigenesis of glioma;3.Lnc RNA WARS2-IT1 can inhibit miR-299-3p molecular level and promote VEGFA protein expression,thereby regulating PI3K/AKT signaling pathway and promoting glioma proliferation,migration and HBMVEC angiogenesis. |
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