| Objective: Gliomas occupy 80% of the malignant primary tumors of the human central nervous system,and their prognosis is extremely poor.Glioma is an angiogenic solid tumor,its malignant progression relies on the formation of blood vessels in tumor tissue.Although some advances have been made in anti-angiogenic therapy applied to glioma,the effect is still insufficient to be relied upon as a primary therapy.One of the limiting factors of anti-angiogenesis is related to the generation of vasculogenic mimicry(VM)in glioma cells.At the translational level,the regulation of gene expression depends primarily on the specific structure and function of the 5' and 3' untranslated regions(UTR)of mRNA.Upstream open reading frames(uORFs),commonly found in the 5'UTR of eukaryotic genes,are of importance in the initiation of translation.Studies have reported that uORFs are present in 44%-49% of human genes,and uORFs in a gene activate nonsense-mediated RNA Decay(NMD),which promotes the degradation of the gene.ZNRD1-AS1(zinc ribbon domain-containing 1 antisense RNA 1)is located on chromosome 6p22.1.Emerging evidence has confirmed that ZNRD1-AS1 expression is great in lung cancer tissues,and that ZNRD1-AS1 and its functional Cis-eQTL promote lung cancer.Presently,there is no report on the expression of ZNRD1-AS1 in glioma and its involvement in functional regulation.miR-449a-5p is located on the chromosome5q11.2.Recent studies have reported that miR-499a-5p acted a tumor-suppressive role in gliomas,and inhibited migration and invasion of glioma cells and promoted apoptosis.ELF1(E74 like ETS transcription factor 1),which belongs to the ETS family,is located in the 13q13.3-13q14.11 region.ELF1 is highly expressed in endometrial and ovarian cancers.EMI1(early mitotic inhibitor-1),also known as FBXO5(F-box only protein 5),is a key cell cycle regulator.EMI1 is highly expressed in hepatocellular carcinoma and breast and ovarian clear cell carcinoma tissues.At present,there are no reports about the expression and function of ELF1 and EMI1 in gliomas.The purpose of this study is to reveal the molecular mechanisms by which ZNRD1-AS1-144aa-uORF regulates biological behaviors such as VM of glioma cells through the ZNRD1-AS1/miR-499a-5p/ELF1/EMI1 pathway.It provides a new basis for the occurrence and development of glioma,and also provides a new target for moleculartargeted therapy of glioma.Methods: Real-time PCR was performed to detect the expression of ZNRD1-AS1,miR-499a-5p,ELF1 and EMI1,and Western blot was used to elucidate the expression levels of 144aa-uORF,ELF1 and EMI1 in glioma tissue and cells.CCK-8,Transwell,and tube formation experiments were conducted to reveal cell proliferation,migration invasion,and VM formation ability changes,respectively.The plasmids of overexpression 144aa-uORF,inhibiting ZNRD1-AS1,EMI1,and overexpression and expression inhibiting ELF1,miR-499a-5p were transfected into cells to construct corresponding cell lines.ZNRD1-AS1,ELF1 mRNA and miR-499a-5p expression changes were described by Real-time PCR.144aa-uORF,ELF1,and EMI1 protein expression changes were detected by Western Blot.RIP and RNA pull down experiments verified the binding of ZNRD1-AS1 and UPF1.Actinomycin D method confirmed the half-life change of ZNRD1-AS1.The dual luciferase gene reporter system was initiated to investigate whether ZNRD1-AS1 and miR-499a-5p,and miR-499a-5p and ELF1 have targeted binding effects.ChIP experiments analyzed the direct binding of ELF1 and EMI1 promoter regions.Nude mouse xenograft experiments were performed to test the effects of 144aa-uORF,ZNRD1-AS1 and ELF1 on the growth of subcutaneous xenografts and survival time of orthotopic xenografts in nude mice.Results: This study found that ZNRD1-AS1 is highly expressed in glioma tissues and cells,and down-regulation of ZNRD1-AS1 inhibits VM formation in glioma cells.144aa-uORF is down-regulated in glioma,promoting the degradation of ZNRD1-AS1 through the NMD pathway and inhibiting VM formation of glioma cells.miR-499a-5p is reduced in glioma tissues and cells,and ZNRD1-AS1 binds to miR-499a-5p to regulate VM formation.ELF1 is up-regulated in glioma tissues and cells,and miR-499a-5p binds to ELF1 to regulate VM formation.As a role of ceRNA,ZNRD1-AS1 competitively binds to miR-499a-5p,and negatively regulates the inhibitory effect of miR-499a-5p on ELF1.The expression of EMI1 is increased in glioma tissues and cells,and ELF1 directly binds to the promoter of EMI1 to regulate VM formation.Notably,In in vivo experiments,the overexpression of 144aa-uORF and underexpression of ZNRD1-AS1,ELF1 in combination allowed the nude mice to achieve the minimal xenograft volume,the longest survival time,and the most sparse tumor VM density.Conclusions:1.ZNRD1-AS1-144aa-uORF and miR-499a-5p are under-expressed in glioma tissuesand cells,and ZNRD1-AS1,ELF1,and EMI1 are over-expressed.Knockdown ofZNRD1-AS1,ELF1,EMI1 and overexpression of 144aa-uORF and miR-499a-5p caninhibit the proliferation,migration,invasion and VM of glioma cells.2.144aa-uORF reduces the stability of ZNRD1-AS1 through the NMD pathway.ZNRD1-AS1 targets miR-499a-5p.Overexpression of 144aa-uORF inhibits theexpression of ZNRD1-AS1 by reducing its stability.3.Knocking down ZNRD1-AS1 weakened its binding effect with miR-499a-5p andinhibited the proliferation,migration,invasion and vasculogenic mimicry of gliomacells.4.miR-499a-5p targets ELF1 mRNA 3'UTR.ZNRD1-AS1,inhibiting the negativeregulatory effect of miR-499a-5p on ELF1 by binding miR-499a-5p,competitivelycombines miR-499a-5p to reduce its negative regulatory effect on ELF1,therebypromoting glioma VM formation.ELF1 directly binds to the promoter of EMI1 andenhances its activity.Overexpression of miR-499a-5p inhibits the transcription ofEMI1 by reducing the expression of ELF1,and inhibits the formation of VM in gliomacells.5.ZNRD1-AS1-144aa-uORF/ZNRD1-AS1/miR-499a-5p/ELF1/EMI1 pathway plays animportant role in regulating VM of gliomas. |
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