LINC00680 And TTN-AS1 Stabilized By EIF4A3 Promoted Malignant Biological Behaviors Of Glioblastoma Cells

Objective: Gliomas are the most common malignant intracranial tumor.Almost half of them are the most lethal form,glioblastomas.Even after surgery and chemoradiotherapy glioblastomas have a tendency of recurrence with a low five-year survival rate.Therefore,new therapy needs to apply to the treatment of glioblastoma,such as molecular targeted therapy.RNA binding proteins(RBPs)are involved in metabolism of RNA in many aspects,including RNA splicing,location and transportation.Dysregulation of RBPs may cause different diseases,cancers,for example.RBP EIF4A3 is a core component of exon junction complex(EJC),which plays important role in RNA metabolism.EJC could regulate the development of brain,growth and activity of neuron.However,how EIF4A3 acts in glioblastoma has not been studied.Long non-coding RNAs(lncRNAs)are a group of RNAs with molecular weight of >200 bases in length that generally do not code for proteins and that is vital for tumor formation and development.It might be a new therapy for glioblastoma targeting lncRNAs.LINC00680 might be a protective biomarker and an independent prognostic indicator of soft-tissue sarcoma.TTN-AS1 promotes esophageal squamous cell carcinoma cell proliferation and invasion.The clinical significance of lncRNA LINC00680 and TTN-AS1 in glioblastomas remains unclear.MicroRNAs(miRNAs)are endogenous small non-coding RNAs of 18-24 nucleotides,acting as modulators in post-transcriptional and translational gene expression.Up-regulation of miR-320 b could promote apoptosis and inhibit proliferation,migration and invasion of glioma cells.However,its mechanism is indistinct.EGR3(early growth response 3)is a member of zinc finger transcription factor family.EGR3 is highly expressed in glioblastoma.But the function of EGR3 on biological behavior and its mechanism in glioblastoma has not been stated.The plakophilin-2(PKP2)is a member of armadillo-like protein subfamily that mainly localizes to cell desmosomes.PKP2 could directly activate epidermal growth factor receptor(EGFR)signaling.It has been reported that PKP2 is up-regulated and related to the progression of gliomas.But how it affects the biological behavior and its mechanism in glioblastoma is still unclear.Methods: 1.The expression of EIF4A3,LINC00680,TTN-AS1 and EGR3 were detected in glioma tissues and glioblastoma cells.PCR was used to detect the expression ofLINC00680,TTN-AS1 and mRNA of EGR3.Western blot was used to detect the expression of EIF4A3,EGR3 and PKP2.2.U87 and U251 cells were stably transfected with plasmid to knockdown EIF4A3,LINC00680,TTN-AS1 and EGR3 and transiently transfected with plasmid of agomir-320 b and antagomir-320 b.3.The expression of EIF4A3,LINC00680,TTN-AS1,miR-320 b,EGR3 and PKP2 were detected by PCR or Western blot.4.Proliferation ability of cells were detected by CCK-8.5.Transwell was used to determine the migration and invasion of cells.6.Flow cytometer was used to detect the apoptosis of cells.7.RNA pulldown and RIP was performed to detect the binding between EIF4A3,LINC00680 and TTN-AS1.8.Stability of LINC00680 and TTN-AS1 was detected.9.Nascent RNA of LINC00680 and TTN-AS1 was detected.10.Dual luciferase reporter assay was carried out to detect the binding between miR-320 b and LINC00680 or TTN-AS1,miR-320 b and EGR3.11.RIP was used to detect the binding between miR-320 b and LINC00680 or TTN-AS1 in RISC.12.ChIP was used to detect the binding of EGR3 and promotor of PKP2.13.Tumor xenografts in nude mice was used to assess the tumorigenesis ability of cells.Results: 1.EIF4A3,LINC00680,TTN-AS1 and EGR3 were highly expressed in glioma tissues and glioblastoma cells;2.EIF4A3 targeted LINC00680 and TTN-AS1.Knockdown of EIF4A3 could down-regulate the expression of LINC00680 and TTN-AS1.Knockdown of EIF4A3,LINC00680 and TTN-AS1 could inhibit the proliferation,migration and invasion and promote apoptosis of glioblastoma cells;3.Knockdown of EIF4A3 didn’t change the synthesis of LINC00680 or TTN-AS1 but inhibit the stability of them;4.miR-320 b formed RISC by targeting LINC00680 and TTN-AS1.Knockdown of LINC00680 and TTN-AS1 could down-regulate the expression of miR-320b;5.Knockdown of miR-320 b could promote the proliferation,migration,invasion and inhibit apoptosis of glioblastoma cells.Overexpression of miR-320 b could inhibit the proliferation,migration,invasion and promote apoptosis of glioblastoma cells;6.MiR-320 b targeted mRNA of EGR3.Knockdown of miR-320 b up-regulated the expression of EGR3 while overexpression of miR-320 b down-regulated the expression of EGR3;7.Knockdown of EGR3 inhibited the proliferation,migration,invasion and promote apoptosis of glioblastoma cells;8.EGR3 could bind to the promotor of PKP2;9.Knockdown of EIF4A3,LINC00680,TTN-AS1 and EGR3 whileoverexpression of miR-320 b could down-regulate the expression of PKP2.Knockdown of miR-320 b could up-regulate the expression of PKP2;10.The binding of miR-320 b with mRNA of EGR3 could down-regulate the expression of PI3K/Akt;11.Knockdown of EIF4A3,LINC00680 and TTN-AS1 alone or together could inhibit the growth of xenograft and prolong the survival of nude mice.Conclusion:1.EIF4A3,LINC00680,TTN-AS1 and EGR3 were highly expressed in glioma tissues and glioblastoma cells.Knockdown of EIF4A3,LINC00680,TTN-AS1 and EGR3 could inhibit the proliferation,migration,invasion and promote apoptosis of glioblastoma cells.2.EIF4A3 exerts its function in glioblastoma via promoting the stability of LINC00680 and TTN-AS1.LINC00680 and TTN-AS1 could bind to miR-320 b while miR-320 b could bind to 3’-UTR of EGR.EGR3 could bind to the promotor of PKP2 to facilitate its transcription.3.Knockdown EIF4A3 reduces stability of LINC00680 and TTN-AS1,thus to reduce the binding of LINC00680/TTN-AS1 and miR-320 b,upregulating the negative regulation of miR-320 b on EGR3 and inhibiting the transcription ability of PKP2 promoted by EGR3,which resulted in the inhibiting of proliferation,migration and invasion and promoting apoptosis of glioblastoma cells.4.Knockdown EIF4A3,LINC00680 and TTN-AS1 alone or together could inhibit the growth of xenograft and prolong the survival of nude mice.5.EIF4A3/LINC00680(TTN-AS1)/miR-320b/EGR3/PKP2 axis could regulate malignant behavior of glioblastoma cells.