| Glioma is the most common malignant tumor with strong invasion,heterogeneity and susceptibility.Although it can be treated by surgical resection,radiotherapy and chemotherapy,the median survival time is only 13-16 months.The main reasons for poor prognosis and high recurrence rate of glioma are abundant blood flow,rapid growth,easy invasion and resistance to radiotherapy and chemotherapy.We therefore believe that the further study on the surrounding cells and molecular biology of glioma will bring broad research prospects for the study of the pathogenesis of Glioma,and find new treatment options to explore its important clinical significance.As "dark matter",non-coding RNAs(ncRNAs)account for more than 60%of human transcripts,which not only have multiple functions under physiological and pathological conditions,but also play an important regulatory role in the pathogenesis of glioma.According to the length of the RNA strand,ncRNA can be divided into two categories:one is short non-coding RNA,which is generally less than 200nt in length;another one is long non-coding RNA(LncRNA),which is longer than 200nt.The most typical short non-coding RNA is microRNA(miRNA),which has been deeply and comprehensively studied in glioma.However,in the past decade,Long non-coding RNA has attracted great attention from scientists.Studies have shown that LncRNA has an important impact on the occurrence,development and metastasis of glioma,and may become an important target for gene therapy of glioma in the future.In order to explore the role of lncRNA in glioma,we screened and analyzed by gene chip technology,and found that an important lncRNA-PVT1 was significantly up-regulated in glioma tissue.PVT1 gene is also called Plasmcytoma Variant Tralocation Gene 1(PVT1),and its transcript belongs to intergenic lncRNA.The PVT1 gene is located on chromosome 8q24 and on the sense strand of the chromosome.It spans a genomic interval of more than 300 kb.However,the influence of PVT1 on glioma cell proliferation,migration,invasion and angiogenesis and their regulatory mechanisms remain to be further studied.The purpose of the present study was to investigate and clarify the important mechanism of PVT 1 in the occurrence and development of glioma,and to provide obvious relevant diagnostic markers for clinical treatment and potential therapeutic targets for future clinical treatment.This study will make a systematic exposition from four aspects:Part Ⅰ:Screening and expression of PVT1 in gliomaObjective:To screen the significantly differentially expressed lncRNAs in gliomas and to investigate the expression and clinical significance of PVT1 in gliomas.Methods:Three pairs of glioma and adjacent normal tissues were selected to screen the differentially expressed lncRNAs in glioma by cDNA microarray.PVT1,the most significantly up-regulated gene with conserved homology,was selected to analyze the possible effects of PVT1 on biological functions and signal transduction pathways by bioinformatics of gene chip.Using TCGA data to deeply explore the differential expression of PVT1 in various types of tumors,the expression changes in normal(non-tumor)brain tissues and glioma tissues,low-grade(grade Ⅰ and Ⅱ)and high-grade(grade Ⅲ and Ⅳ)glioma tissues,and the correlation with the staging and prognosis of glioma;The expression of PVT1 in normal(non-tumor)brain tissue and glioma tissue,low-grade Ⅰ-Ⅱglioma and high-grade Ⅲ-Ⅳ glioma tissue was proved by RT-qPCR.Results:1.there were 327 high expression levels and 559 low expression levels in the multiple change≥ 1.5 and p<0.05 in the differentially expressed lncRNA of gene chip.Among the dysregulated lncRNA transcripts,lncRNA-PVT1 and LINC004831 showed the most significant changes,with lncRNA-PVT1 having a log2FC of 4.32.2.The high expression of lncRNA-PVT1 was correlated with WNT pathway,cell adhesion,DNA damage,T cell receptor and oxidative stress in glioma.3.Compared with other types of tumors,the expression of PVT1 was higher in glioma.The expression of PVT1 was meaningfully higher in high grade glioblastomas(p<0.01),but not in low grade gliomas(p>0.05).In gliomas of different grades,the expression of PVT1 increased with the higher ground of tumor stage(p<0.01).Overall Survival(OS)and disease-free survival(DFS)were significantly lower in tumors with high expression of PVT 1 than in tumors with relatively low expression of PVT1(p<0.01).4.Compared with normal tissues,PVT1 was highly expressed in glioma tissues(p<0.001).The expression of PVT1 in Ⅲ-Ⅳgliomas was meaningfully higher than that in low-grade Ⅰ-Ⅱ tumors(p<0.01).Conclusion:Among the differentially expressed lncRNAs screened by cDNA microarray,we found a highly expressed lncRNA-PVT1 in glioma,which may be related to the development of glioma by bioinformatics analysis.PVT1 was not only significantly associated with the pathological grade of gliomas,but also was an independent prognostic factor for high-grade gliomas.Part Ⅱ:The effects of PVT1 on the proliferation,invasion and angiogenesis of glioma cellsObjective:To knock down PVT1 expression in glioma cells by RNA interference(RNAi),and to investigate the effects of lncRNA-PVT1 on proliferation,invasion and migration.Methods:1.The expression of PVT1 was detected by RT-qPCR in normal human astrocytes(HEB)and glioma cell lines(U87,U251,SHG-44 and H4).2.Small hairpin RNA(shRNA)interference plasmid was used to construct and package high titer lentivirus.PVT1 expression was knocked out by RNA interference in glioma cell lines with high PVT1 expression.3.unde that condition of knocking down PVT1,to confirm the effect of PVT1 silencing on cell proliferation and to observed the cell invasion and to detect the change of angiogenesis in vitro,we detected CCK-8 assay,Transwell assay and angiogenesis assay,which were important characteristics of tumor invasion and metastasis and angiogenesis in tumor tissues.Results:1.Compared with normal astrocytes,the expression of PVT1 in glioma cell lines(U87,SHG-44,U251)was significantly increased(P<0.05),and U87 and U251 were relatively higher,so these two cell lines could be used for RNA interference experiments;2.Compared with the negative control group,after RNA interference of PVT1 gene expression in U87 and U251 cells,both shRNA-1 and shRNA-2 targets could significantly knock down PVT1 expression in U87 cells.The difference was statistically significant(P<0.05),and the three targets of shRNA-1,shRNA-2 and shRNA-3 could significantly reduce the expression of PVT 1 in U251 cells.The proliferation,invasion and angiogenesis of U87 and U251 glioma cells were effectively inhibited by ShRNA-1 and shRNA-2(P<0.05).Conclusion:PVT1 is highly expressed in glioma cell lines(U87,U251 and SHG-44)compared with normal human glioma cells(HEB),and decreased PVT1 expression significantly inhibited proliferation,invasion and angiogenesis of glioma cell lines U87 and U251.Part Ⅲ:PVT1 regulates miR-1207-3p targeting HNF1B/EMT axis to promote the mechanism of glioma progressionObjective:To explore the mechanism of PVT1 regulating miR-1207-3p targeting HNF1B/EMT axis in glioma,and to clarify the relationship between PVT1 regulating miR-1207 and miR-1207 targeting HNF1B to affect EMT regulation.Methods:1.The distribution of PVT1 in glioma cells and cytoplasm was determined by nucleocytoplasmic segregation.2.Bioinformatics analysis combined with miR-1207 of PVT1;3.The expression of miR-1207-3p in different tumors and its relationship with the overall survival and disease-free survival of glioma;4.Dual-luciferase reporter experiment verifies that bin relationship between miR-1207-3p and PVT1 and the binding relationship between miR-1207-3p and HNF1B;Western Blot analysis confirmed that miR-1207-3p regulates HNF1B expression at the translational level.Results:1.PVT1 was mainly distributed in the cytoplasm and a small amount in the nucleus by RT-qPCR with nucleocytoplasmic separation.2.Bioinformatics revealed that PVT1 was the target gene of Mir-1207-3p.3.The expression level of miR-1207-3p in high grade gliomas was obviously lower than that in low grade gliomas(P<0.05).The expression of Mir-1207-3p was highly correlated with the prognosis of glioma.4.Luciferase activity assay showed that miR-1207-3p mimetic could significantly reduce the expression of wild-type PVT1 and wild-type HNF1B 3’-UTR compared with the control group,but there was no significant change in mutant type.(P<0.01);The HNF1B protein showed significant downregulated expression by miR-1207-3p mimics compared with the control group(P<0.05).Conclusion:PVT1 distributed in the cytoplasm of glioma cells can reduce miR-1207-3p through endogenous adsorption sponge effect,miR-1207-3p expression is positively correlated with glioma prognosis,miR-1207-3p can target the expression of HNF1B,and the reduction of miR-1207-3p leads to the deinhibition of HNF1B.Thereby up-regulating the expression of HNF1B and affecting the function of the glioma.Part Ⅳ:The effect of PVT 1 knockdown on tumorigenicity and EMT of glioma cells in vivoObjective:To study the effect of PVT1 gene knockout on tumogenesis of xenograft in nude mice,and to verify that the silencing of PVT1 is regulated by HNFB1/EMT pathway in vivo.Methods:1.Ten nude mice were divided into 2 groups with 5 mice in each group,and the tumor was inoculated subcutaneously.Tumor volume was measured and growth curves were plotted at 5-day intervals.Mice,whose tumor tissue was collected,were sacrificed 6 weeks after inoculation.2.Western Blot assay was performed to detect the expression of HNFB1/EMT pathway in tumor tissues.Results:1.The tumor growth in the PVT1 knockout group,compared with the control group,was significantly inhibited(P<0.01).2.Western blot analysis showed that the expression of genes related to EMT(Vimentin and N-cadherin)and MAPK pathway(Foxm1,VEGFA and PKP4)were significantly decreased by the inhibition of lncRNA-PVT1 expression(P<0.05).Conclusion:Silencing PVT1 can inhibit the growth of glioma in nude mice,and PVT1 can regulate tumor proliferation and invasion through HNF1B/EMT pathway.Full text conclusion1.LncRNA-PVT1 was significantly overexpressed in gliomas,and the expression level of PVT 1 was correlated with the pathological grade of gliomas.The up-regulation of PVT1 expression was an independent factor for poor prognosis of high-grade gliomas.2.Decreased expression of PVT 1 inhibits proliferation,invasion,and angiogenesis in glioma cell lines U87 and U251.3.PVT1 regulates miR-1207-3p-targeted HNF1B expression via a ceRNA mechanism.4.In nude mice,silencing PVT1 can inhibit the growth of glioma in nude mice,and PVT1 can regulate tumor proliferation and invasion through HNF1B/EMT pathway.. |
PDF Full Text Request