Mechanism Of MCM3AP-AS1/miR-211/KLF5/AGGF1 Pathway And ANKHD1/LINC00346/ZNF655 Feedback Loops Regulating Glioma Angiogenesis

Objective:Glioma is the most common tumor of the human central nervous system,which is characterized by its high degree of malignancy,high invasiveness,and poor prognosis.The median survival time of patients with glioma is still only 12 to 18 months,despite improvements in surgery,radiotherapy,and chemotherapy.Numerous studies have shown that the formation of new blood vessels is involved in the process of tumor growth and metabolism.Thus,angiogenesis is considered to be a marker of the development and progression of malignant tumors.Glioma is a solid tumor of which the growth and metastasis are dependent on angiogenesis.Therefore,anti-angiogenesis therapy has become an effective method for the treatment of gliomaNon-coding RNAs(ncRNAs)include long non-coding RNAs(lncRNAs)and micro RNAs(miRNAs),which are not translated into protein.Long non-coding RNAs(lncRNAs)are non-coding RNAs longer than 200 nucleotides.More and more studies have shown that lncRNAs are abnormally expressed in many malignant tumors and participate in the regulation of tumor occurrence and development.Mi RNAs are small non-coding RNAs with a size of about 20 nucleotides and highly conserved sequences.They can directly target and bind messenger RNAs(mRNAs),thereby negatively regulating the expression of target genes.RNA binding proteins(RNA binding proteins,RBPs)can play an important regulatory role in multiple stages of regulating gene expression such as transcription and post-transcription,and participate in the processes of mRNA splicing,RNA stability maintenance,intracellular localization and translation regulation.Recent studies have shown that RBPs are involved in regulating the occurrence and development of many tumors.Therefore,research on the mechanism of regulating the occurrence and development of glioma by molecular regulatory networks composed of ncRNAs,including RBPs,transcription factors and target genes,can provide new ideas for comprehensive treatment of anti-glioma.The first part of this study first clarified the expression levels of MCM3AP-AS1,miR-211,KLF5 and AGGF1 in glioma vascular endothelial cells and its effect on glioma angiogenesis,and further studied the regulatory relationship between the above factors.And the influence of these regulatory effects on glioma angiogenesis;the second part first clarifies the expression levels of ANKHD1,LINC00346,ZNF655 in GECs and the regulatory effects on glioma angiogenesis.Further study the molecular regulatory network formed by these factors in the role and mechanism of glioma angiogenesis.This study can not only reveal the new mechanism of the molecular regulatory network with ncRNAs as the core in regulating glioma angiogenesis,but also provide new targets and new strategies for anti-angiogenesis therapy for glioma.Methods:Glioma U87 cells,human microvascular endothelial cells(ECs),and human embryonic kidney cells(HEK-293T)were cultured,and glioma vascular endothelial cells(GECs)were obtained using U87 cell conditioned medium.Quantitative real-time reverse transcription-PCR(qRT-PCR)experiment was used to detect the endogenous expression levels of MCM3AP-AS1,miR-211,KLF5 mRNA,AGGF1 mRNA,ANKHD1 mRNA,LINC00346 and ZNF655 mRNA;in situ hybridization experiment was used to detect The nucleoplasmic distribution and expression level of LINC00346;Western blot method was used to detect the endogenous expression level of KLF5,AGGF1,ANKHD1 and ZNF655.Construction of MCM3AP-AS1 and KLF5 stably expressing silent GECs cell lines and ANKHD1,LINC00346 and ZNF655 stably expressing silent/overexpressing GECs cell lines by cell transfection method,constructing miR-211 silent/overexpressing GECs by transient transfection Cell line to construct a dual-intervention GECs cell line.The qRT-PCR experiment detected the changes in the RNA expression levels of MCM3AP-AS1,miR-211,KLF5 mRNA,AGGF1 mRNA,ANKHD1 mRNA,LINC00346 and ZNF655 mRNA.Western blot method was used to detect the protein expression level of KLF5,AGGF1,PI3 K,p-PI3 K,AKT,p-AKT,ERK1/2,p-ERK1/2,ANKHD1,ZNF655,EGFL7 and Robo4.The half-life experiment was used to detect the half-life levels of LINC00346 and ZNF655 mRNA;qRT-PCR was used to detect the expression level of newborn LINC00346.The RIP experiment was used to detect the binding effect and binding sites of MCM3AP-AS1 and miR-211,ANKHD1 and LINC00346,and LINC00346 and ZNF655 mRNA.The dual luciferase gene reporter experiment was used to detect the binding effect and binding sites of MCM3AP-AS1 and miR-211,LINC00346 and ZNF655 mRNA.RNA pull-down experiment detects the binding effect and binding site of ANKHD1 and LINC00346.Co-immunoprecipitation experiment(Ch IP)was used to clarify the promoter regions of KLF5 and AGGF1,and the binding effects and binding sites of ZNF655 with the promoter regions of ANKHD1,EGFL7 and Robo4.The CCK-8 experiment was used to detect the change of cell proliferation ability,and the Transwell experiment was used to detect the change of cell migration ability.The tube formation experiment was used to detect changes in cell tube formation ability.The matrigel plug experiment in nude mice was used to detect the effects of MCM3AP-AS1 and miR-211,ANKHD1,LINC00346 and ZNF655 on the angiogenesis ability of gliomas in vivo.Results:1.Knockdown of MCM3AP-AS1 Inhibits the Angiogenesis of GECs.2.Overexpression of miR-211 Inhibits the Angiogenesis of GECs.3.miR-211 Targets MCM3AP-AS1 in GECs.4.Knockdown of KLF5 Inhibits the Biological Behaviors of GECs by Inhibiting the Expression of AGGF1 and the Activity of PI3K/AKT/ERK1/2 Signaling Pathways.5.KLF5 Is a Target of miR-211.6.Knockdown of AGGF1 Inhibits the Angiogenesis of GECs.7.Knockdown of MCM3AP-AS1,Overexpression of miR-211,and Their Combined Application Inhibits the Angiogenesis of GECs In Vivo.8.ANKHD1 Is Upregulated in GECs,and Knockdown of ANKHD1 Inhibits the Angiogenesis of GECs.9.LINC00346 Is Upregulated in GECs,and Knockdown of LINC00346 Inhibits the Angiogenesis of GECs.10.ANKHD1 Targets LINC00346 and Regulates the Angiogenesis of GECs viaStabilizing LINC00346.11.ZNF655 Is Downregulated in GECs,and Overexpression of ZNF655 Inhibits the Angiogenesis of GECs.12.LINC00346 Binds to STAU1 and Involves SMD to Promote the Degradation of ZNF655 mRNA by Targeting ZNF655 mRNA and Forming the SBS.13.LINC00346 Interacts with ZNF655 to Regulate the Angiogenesis of GECs.14.ZNF655 Inhibits ANKHD1 Expression via Targeting Its Promoter Region and Forms the ANKHD1/LINC00346/ZNF655 Feedback Loop.15.Knockdown of ANKHD1 and LINC00346 Combined with Overexpression of ZNF655 Restrains Glioma Angiogenesis In Vivo.Conclusion:1.MCM3AP-AS1 is highly expressed in GECs,and miR-211 is low expressed in GECs.MCM3AP-AS1 targets miR-211 and negatively regulates its expression,playing a regulatory role in promoting the proliferation,migration and tube formation of GECs.2.KLF5 is highly expressed in GECs,miR-211 targets the 3,UTR region of KLF5,andplays a regulatory role in inhibiting GECs proliferation,migration and tube formation.3.AGGF1 is highly expressed in GECs,KLF5 directly binds to the promoter region ofAGGF1 to promote its transcription and play a regulatory role in promoting theproliferation,migration and tube formation of GECs.4.MCM3AP-AS1/miR-211/KLF5/AGGF1 pathway plays an important regulatory role in glioma angiogenesis.5.ANKHD1 targets LINC00346 and increases its stability,up-regulates its expression,and plays a role in promoting GECs angiogenesis.6.LINC00346 binds to the Alu element of ZNF655 mRNA through the Alu element to form an SBS site,recruit STAU1 and UPF1,and then participate in the mRNA degradation pathway mediated by STAU1,reduce the half-life of ZNF655,and negatively regulate its expression to promote GECs The role of angiogenesis.7.ZNF655 directly binds to the promoter regions of EGFL7 and Robo4,negatively regulates their transcription,and plays a role in inhibiting the angiogenesis of GECs.8.ZNF655 directly binds to the promoter region of ANKHD1 to form a positive feedback loop and regulate GECs angiogenesis.9.ANKHD1/LINC00346/ZNF655 feedback loop plays an important role in the regulation of GECs angiogenesis.