| Objective:Glioma is the most common primary tumor in adults,accounting for about 60% of intracranial tumors.The tumor’s invasive growth characteristics make it difficult to cure and easy to relapse.In recent years,despite the continuous improvement of treatment methods such as surgery,radiotherapy,chemotherapy,and molecular targeted therapy,the prognosis of patients with glioma is still very poor,and the mortality rate is extremely high.Due to the existence of VM in gliomas,it is difficult for clinical anti-angiogenesis drugs to achieve the expected curative effect.In recent years,the research on the molecular regulatory network of glioma VM and the corresponding targeted drugs has become a frontier hot spot in the field of glioma treatment.Therefore,this study has conducted an in-depth exploration of the mechanism of glioma VM and the related malignant biological behaviors of glioma cell growth,migration and invasion.In the first part of this study,we clarified the expression levels of factors such as USF1,SNHG16 and Linc00667 in glioma tissues and cells,and further studied the regulatory relationship between the factors and their influence on the formation of VM in glioma cells.We used bioinformatics software to predict that long non-coding RNAs(Long non-coding RNAs,lnc RNAs)SNHG16 and Linc00667 had potential binding sites for USF1 in the promoter regions.Next,we applied the database to predict that SNHG16 had a binding site with miR-212-3p,Linc00667 had a binding site with miR-429,ALDH1A1 was a potential target gene for miR-212-3p and miR-429.By constructing a glioma cell line that knocked down the expression of USF1,we studied the transcriptional regulation of SNHG16 and Linc00667 by USF1,and further clarified that SNHG16 and Linc00667 interact with miRNAs through ce RNA,thereby regulating the expression of ALDH1A1,and ultimately affect the molecular mechanism of VM in glioma.In the second part,we first clarified the expression of UBE2 I,PUM2,CEBPD and DSG2 in glioma tissues and cells.Through bioinformation analysis and experimental detection,it was found that there was a SUMOylation site on PUM2,and through the regulation of its site mutation and UBE2 I,it was found that PUM2 was involved in regulating the formation of glioma VM.Using the data in the low-grade glioma group and glioblastoma multiforme group in the TCGA database,the genes related to PUM2 and UBE2 I were analyzed,and the downstream gene CBD of PUM2 was screened out through experiments.Next,through experimental studies,we clarified the molecular mechanism by which CEPBD regulates the VM-related gene DSG2 through transcription,thereby mediating the formation of glioma VM.This study aims to clarify the key role of USF1 and PUM2 related molecular regulatory network in the formation of glioma VM,and to explore the molecular mechanism of glioma VM formation in the regulatory network and the corresponding therapeutic targets,so as to provide new strategies for the treatment of gliomas.Methods:1.Real-time fluorescent quantitative PCR(qRT-PCR)was used to detect the expression levels of USF1,SNHG16,Linc00667,miR-212-3p,miR-429,ALDH1A1,UBE2 I,PUM2,CEBPD and DSG2.2.Western blot was used to detect the expression levels of USF1,ALDH1A1,UBE2 I,PUM2,CEBPD,DSG2,VE-cadherin,Eph A2,MMP-2 and MMP-14.3.Chromatin immunoprecipitation(ChIP)experiment was used to detect the binding of USF1 to the promoter region of SNHG16 and Linc00667;the binding of CEBPD to the promoter region of DSG2.4.Double luciferase reporter gene detection was used to detect SNHG16 and miR-212-3p,Linc00667 and miR-429,miR-212-3p and miR-429 and ALDH1A1 3’UTR binding effect and binding site.5.RNA binding protein immunoprecipitation(RIP)experiment was used to detect the binding effect of SNHG16/miR-212-3p and Linc00667/miR-429 with Ago2;the binding effect of PUM2 with CEBPD.6.Co-immunoprecipitation(Co-IP)test was used to detect the binding effect of PUM2 and SUMO2/3 protein.7.Observe the nuclear and cytoplasmic distribution of PUM2 and SUMO2/3 by laser confocal microscope,and observe the co-localization of PUM2 and SUMO2/3 in the cytoplasm.8.CCK-8 experiment was used to detect cell proliferation ability,Transwell experiment was used to detect cell migration and invasion ability,Hstudio M4 system was used to observe cell migration ability,three-dimensional tube generation experiment was used to detect VM formation ability,nude mouse xenograft experiment was used to detect tumor formation ability,immune group Immunohistochemistry experiment was used to detect the ability of VM formation in glioma tissue.Results:1.USF1 was up-regulated in glioma tissues and cells;knocking down USF1 significantly inhibited the proliferation,migration,invasion and VM formation of glioma cells;USF1 promoted expression of SNHG16 and Linc00667 by combining respectively the promoter regions of SNHG16 and Linc00667.2.SNHG16 and Linc00667 were up-regulated in glioma tissues and cells;knocking down SNHG16 or knocking down Linc00667 significantly inhibited the proliferation,migration,invasion and VM formation of glioma cells.3.SNHG16 down-regulated the expression of miR-212-3p;and Linc00667 downregulated the expression of miR-429.4.The expression of miR-212-3p and miR-429 was down-regulated in glioma tissues and cells.Overexpression of miR-212-3p or miR-429 significantly inhibited the proliferation,migration,invasion and VM formation of glioma cells.5.The rescue experiment verified that SNHG16(Linc00667)regulated the proliferation,migration,invasion and VM formation of glioma cells by effecting miR-429(miR-429).6.ALDH1A1 was up-regulated in glioma tissues and cells,and promoted the proliferation,migration,invasion and VM formation ability of glioma cells;knocking down ALDH1A1 significantly inhibited the proliferation,migration,invasion and VM formation ability of glioma cells;miR-212-3p and miR-429 regulated the expression of ALDH1A1 by binding to ALDH1A1 3’UTR.7.Inhibitors of multiple targets of USF1,SNHG16 and Linc00667,alone or in combination,significantly inhibited tumor formation in nude mice transplanted tumor models,increased the survival time of nude mice,and reduced VM formation in transplanted tumor tissues.8.UBE2 I modified PUM2 by regulating its combination with SUMO2/3 to cause sumoylation which degraded the PUM2 protein.9.UBE2 I was up-regulated in glioma tissues and cells;knocking down UBE2 I significantly inhibited the migration,invasion and VM formation ability of glioma cells.10.PUM2 expression was down-regulated in glioma tissues and cells;overexpression of PUM2 significantly inhibited the migration,invasion and VM formation ability of glioma cells.11.PUM2 inhibited CEBPD protein expression by binding CEBPD m RNA,thereby inhibiting the migration,invasion and VM formation ability of glioma cells.12.CEBPD was up-regulated in glioma tissues and cells;knocking down CEBPD significantly inhibited the migration,invasion and VM formation ability of glioma cells.13.DSG2 was up-regulated in glioma tissues and cells,and promotes the migration,invasion and VM formation ability of glioma cells;knocking down DSG2 significantly inhibited the migration,invasion and VM formation ability of glioma cells.14.CEBPD regulated the expression level of DSG2 by transcription to promote the migration,invasion and VM formation ability of glioma cells.15.Inhibitors and agonists of multiple targets of UBE2 I,PUM2 and CEBPD,alone or in combination,significantly inhibited tumor formation in nude mice xenograft models,increased the survival time of nude mice,and reduced VM formation in xenograft tissues.Conclusion:1.USF1 was up-regulated in glioma tissues and cells,and promoted VM formation in glioma cells.2.The up-regulated USF1 expression in glioma cells was expressed in glioma tissues and cells through transcriptional activation of SNHG16 and Linc00667.3.The up-regulated expression of SNHG16 and Linc00667 in glioma cells combined respectively with miR-212-3p and miR-429,through the ce RNA mechanism to increase the expression of ALDH1A1 in glioma tissues and cells,and promote the VM formation ability of glioma cells.4.USF1 regulated ALDH1A1 through SNHG16/miR-212-3p and Linc00667/miR-429,thereby regulating biological behaviors such as VM formation in glioma cells.5.UBE2 I was up-regulated in glioma tissues and cells,and promoted VM formation in glioma cells;UBE2I,which was up-regulated,modified PUM2 by regulating its combination with SUMO2/3 to cause sumoylation which degraded the PUM2 protein.6.The down-regulated PUM2 expression in glioma cells attenuated its inhibitory effect on CEBPD m RNA translation,thereby promoting the VM formation ability of glioma cells.7.CEBPD,which was up-regulated in glioma cells,promoted the expression of DSG2 through transcription,thereby promoting the VM formation ability of glioma cells.8.PUM2 regulated CEBPD through SUMO modification to further regulate the VM formation ability of glioma cells. |
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