Mechanism Of LINC00665 Stabilized By TAF15 Regulating The Biological Behaviors Of Glioma Cells Via Stau1-mediated MRNA Degradation

Objective:Glioma is the most common,and most lethal primary malignant tumor in brain.Based on the grading criteria of World Health Organization(WHO),glioma are classified into four categories: low-grade glioma(including category I and II)and high-grade glioma(including category III and IV).High-grade glioma cells could easily infiltrate into extracellular matrix of human brain cells,a characteristic that leads to multiple problems: difficulties in radical cure,especially in eradicating glioma through traditional methods,such as surgery or radio-chemical therapy,poor prognosis,high recurrence rate and low survival rate.Finding tumor biomarkers and developing molecular targeted therapy for glioma,therefore,have become hot topics in current studies.Long non-coding RNAs(lnc RNAs)are a kind of endogenous RNA molecule which represents a class of transcripts longer than 200 nucleotides with limited protein-coding potential.lnc RNAs have various biological features and functions,such as regulating the occurrence and development of tumor tissues.For instance,lnc RNAs perform the role of carcinogenic factor or tumor suppressor involved in the biological process of tumor cell cycle change,proliferation,transportation,invasion and apoptosis.RNA-binding proteins(RBPs)is a group of RNAs that participate in the modulation of RNA metabolism via binding to proteins.The dynamic complex of RNA and RBPs,mediates virtually every stage of RNA lifecycle.Research indicates that alterations in RNA metabolism due to RBPs dysfunction may lead to entire changes in the transcriptome and proteome of the cell which affects growth,proliferation,invasion and apoptosis of tumor cells.In our study,we verified the intrinsic expression of RBP-TAF15,lnc RNA-LINC00665,transcription factor MTF1,YY2 and target protein GTSE1,and their effect on glioma cells biological behaviors.We further investigated the possible interaction among the factors and the molecular mechanisms modulating the biological behaviors of glioma cells.Our study aims to clarify novel molecular mechanisms of glioma magnificant progression,to provide new insights and targets in glioma therapy.Methods:Cell culture of U87 and U251 glioblastoma cell line,normal human astrocytes(HA)and human embryonic kidney cell HEK293 T.Normal brain tissues(NBTs)and glioma tissues obtained from patients at Shengjing Hospital of China Medical University.Expression of LINC00665,MTF1 and YY2 were detected by quantitative real-time PCR(q RT-PCR).The expression and location of LINC00665,MTF1 and YY2 in HA,U87 and U251 glioma cells,were detected by RNA Fluorescence In Situ Hybridization(RNA FISH)assay.Protein expression of TAF15,MTF1,YY2 and GTSE1 in NBTs,glioma tissues,HA and glioma cells,was detected by Western blot.Cell proliferation was determined by Cell Counting Kit-8(CCK-8).Cell apoptosis was tested by flow cytometry.Cell migration and cell invasion was tested by Transwell assay.Cell lines with stable knockdown or overexpression of TAF15,LINC00665,MTF1,YY2 and GTSE1 were established by cell transfection.The expression of TAF15,LINC00665,MTF1,YY2 and GTSE1 were detected by q RT-PCR.The nascent LINC00665 expression was detected by Nascent RNA Capture Assay.RNA half-life was measured with Actinomycin D treated.RIP assay and RNA pull-down assay were used to test the interactions between TAF15 and LINC00665.The interaction and the binding sites between LINC00665 and MTF1,LINC00665 and YY2 were clarified by Dual-Luciferase reporter assay.Chromatin immunoprecipitation assay was used to verify the interacrion between MTF1 and GTSE1,YY2 and GTSE1 promoter.Nude mice glioma xenograft models were established to detect the effects of TAF15,LINC00665,MTF1,and YY2 on tumor growth and survival.Results:The study demonstrated that TAF15 and LINC00665 were downregulated in glioma tissues and cells.MTF1,YY2 and GTSE1 were upregulated in glioma tissues and cells.TAF15 and LINC00665 overexpression,as well as MTF1,YY2 and GTSE1 knockdown,markedly repressed the proliferation of U87 and U251 glioma cells,promoted cell migration and invasion,and attenuated cell apoptosis.TAF15 directly interacted with LINC00665.Overexpressing TAF15 significantly stabilized LINC00665 and upregulated expression of LINC00665.LINC00665 bound with MTF1 and YY2 m RNA,thus promoting degradation of MTF1 and YY2 m RNA via SMD pathway.MTF1 and YY2 directly bound with GTSE1 promoter.Downregulation of MTF1 and YY2 inhibited the GTSE1 expression transcriptionally,and repressed the malignant progression of glioma cells.Upregulation of TAF15 and LINC00665,as well as MTF1 and YY2 downregulation resulted in smaller nude mice xenografts and longer survival.Conclusion:1.TAF15 repressed the malignant progression of glioma cells via binding to LINC00665 and enhacing the stability of LINC00665.2.LINC00665 specifically bound to the Alu elements of MTF1 and YY2 m RNA 3'UTR,promoting the degradation of MTF1 and YY2 m RNA,to inhibite the malignant progression of glioma cells.3.MTF1 directly bound to GTSE1 promoter,and transcriptionally activated GTSE1 expression,to facilitate the malignant progression of glioma cells.4.YY2 directly bound to GTSE1 promoter,and transcriptionally activated GTSE1 expression,to facilitate the malignant progression of glioma cells.5.TAF15/LINC00665/MTF1(YY2)/GTSE1 axis played a crucial role in the regulation of the biological behaviors of glioma cells.