Mechanism Of Piwil3/Oip5-AS1/miR-367-3p/CEBPA And TDP43/ SNHG12/miR-195/SOX5 Feedback Loops Regulating The Biological Behavior Of Glioma Cells

Objective: Glioblastoma(GBM)is a brain tumor of neuroepithelial origin.GBM is the most malignant and invasive glioma according to WHO Classification of Brain Tumors,causing poor prognosis for patients.Patients with GBM have a 5-year survival rate of less than 5% despite having been treated with aggressive surgery and postsurgical chemotherapy and radiation.GBM’s resistance to chemotherapy and radiation is the key factor affecting therapeutic effect.Currently,scientists study the pathogenesis of GBM,by utilizing various approaches,such as genomics,genetics,epigenetics,etc.Studies involving the pathogenesis of GBM has become crucial to diagnosis and targeted therapy.Non-coding RNAs(nc RNAs),consisting of short and long non-coding RNAs(lnc RNAs),are among the transcription products of human genome.Short non-coding RNAs comprise micro RNAs(mi RNAs),short-interfering RNAs(si RNAs)and PIWI-interacting RNAs(pi RNAs).Pi RNAs usually bind to Piwi subfamily proteins of Argonaute proteins,exerting functions of gene silencing,regulation and maintenance of germline DNA stability.Lnc RNAs’ major biological functions are transcriptional and post-transcriptional regulations.Mi RNAs function crucially in the regulation of gene expression and cell function.RNA-binding proteins(RBPs)are a group of RNAs that bind to proteins,participating the regulation of RNA metabolism.RBPs major functions are mediating RNA’s maturation,transfer,localization and translation.RBPs are universally expressed in various tumor cells,participating in the regulation of tumor cells’ biological behaviors.A regulatory network consisting of RBPs,various nc RNAs,transcription factors and target genes,with nc RNAs at the center,facilitates the tumorigenesis and progression.In the first part of our study,we validated the intrinsic expression of PIWIL3,pi R-30188,OIP5-AS1,mi R-367-3p,CEBPA and TRAF4 and their effect on the biological behaviors of GBM.We further investigated the possible interaction between these factors and their molecular mechanism regulating the biological behaviors of GBM.In the second part of our study,we first validated the expression of TDP43,SNHG12,mi R-195 and SOX5 in GBM cells and tissues.We further investigated the regulatory relationship among TDP43,SNHG12,mi R-195,SOX5 and Gelsolin and their regulatory effect on the biological behaviors of GBM.This study aims to reveal novel molecular mechanisms of tumorigenesis and progression of GBM and provide new insights and targets into treatment for GBM.Methods: 1.Cell cultures of human GBM cell lines U87 and U251,normal human astrocytes and human embryonic kidney cell line HEK293 T.2.Glioma tissues acquired from patients at Shengjing Hospital of China Medical University.3.The expression of pi R-30188,mi R-367-3p,OIP5-AS1,SNHG12 and mi R-195 were detected by Real-time PCR and Fluorescence In Situ Hybridization(FISH).4.The protein expression levels of PIWIL3,TRAF4,CEBPA,TDP43,SOX5 and Gelsolin in normal brain tissues,glioma tissues,normal human astrocytes,glioma cells and glioma stem cells were determined by Western blot.5.Cell proliferation were assessed by Cell Counting Kit-8(CCK-8).6.Cell migration and invasion were detected by Transwell migration assay.7.Cell apoptosis was detected by flow cytometry.8.Cell lines with stable overexpression or silencing of PIWIL3,pi R-30188,OIP5-AS1,mi R-367-3p,TRAF4,CEBPA,TDP43,SNHG12,mi R-195 and SOX5 were established by cell transfection.9.The expression levels of PIWIL3,pi R-30188,OIP5-AS1,mi R-367-3p,TRAF4,CEBPA,TDP43,SNHG12,mi R-195 and SOX5 were detected by Real-time PCR.10.Half-life of the RNAs was measured by Actinomycin D.11.The interactions between pi R-30188 and PIWIL3,SNHG12 and TDP43 were examined by RIP assay and RNA pull-down assay.12.The binding and binding sites between pi R-30188 and OIP5-AS1,OIP5-AS1 and mi R-367-3p,SNHG12 and mi R-195 were verified by Dual-Luciferase reporter assay.13.The direct binding between CEBPA and TRAF4,CEBPA and PIWIL3 promoter,SOX5 and Gelsolin,SOX5 and SNHG12 promoter were verified by chromatin immunoprecipitation assay.14.Tumor xenograft models were established in nude mice to detect the effects of PIWIL3,OIP5-AS1,mi R-367-3p,TDP43,SNHG12 and mi R-195 on tumor growth and survival.Results: 1.PIWIL3 and pi R-30188 were down-regulated in U87 and U251 glioma cell lines and glioma tissues.Overexpression of PIWIL3 and pi R-30188 significantly attenuated the proliferation,migration and invasion of U87 and U251 glioma cell lines while promoting apoptosis.2.OIP5-AS1 expression was elevated in U87 and U251 glioma cell lines and glioma tissues.Knockdown of OIP5-AS1 significantly attenuated the proliferation,migration and invasion of U87 and U251 glioma cell lines while promoting apoptosis.3.Over-expression of PIWIL3,pi R-30188,and knockdown of OIP5-AS1 resulted in the inhibition of malignant growth of U87 and U251 glioma cell lines.PIWIL3 and pi R-30188 over-expression as well as OIP5-AS1 knockdown attenuated the proliferation,migration and invasion of U87 and U251 glioma cell lines while promoting apoptosis,with the effect of the combined application being the most significant.4.Over-expression of PIWIL3 and pi R-30188 significantly inhibited the expression of OIP5-AS1 in U87 and U251 glioma cell lines,with the effect of the combined application being the most significant.5.There was targeted combination between PIWIL3 and pi R-30188.6.There was targeted combination between OIP5-AS1 and pi R-30188.7.Mi R-367-3p was down-regulated in U87 and U251 glioma cell lines and glioma tissues.Over-expression of mi R-367-3p significantly attenuated the proliferation,migration and invasion of U87 and U251 glioma cell lines while promoting apoptosis.Knockdown of mi R-367-3p significantly promoted the proliferation,migration and invasion of U87 and U251 glioma cell lines while attenuating apoptosis.8.Over-expression of PIWIL3 and pi R-30188,and silencing of OIP5-AS1 significantly increased mi R-367-3p expression in U87 and U251 glioma cell lines.PIWIL3 and pi R-30188 over-expression as well as OIP5-AS1 knockdown significantly elevated mi R-367-3p expression in U87 and U251 glioma cell lines,with the effect of the combined application being the most significant.9.Up-regulation of mi R-367-3p significantly lowered OIP5-AS1 expression in U87 and U251 glioma cell lines.Knockdown of mi R-367-3p significantly increased OIP5-AS1 expression in U87 and U251 glioma cell lines.10.There was targeted combination between OIP5-AS1 and mi R-367-3p in a RISC-dependent manner.11.Over-expression of PIWIL3 and pi R-30188,and silencing of OIP5-AS1 significantly suppressed CEBPA and TRAF4 expression in U87 and U251 glioma cell lines,with the effect of the combined application being the most significant.12.Up-regulation of mi R-367-3p significantly attenuated CEBPA m RNA and protein expression in U87 and U251 glioma cell lines.Knockdown of mi R-367-3p significantly elevated CEBPA m RNA and protein expression in glioma cells.13.Up-regulation of mi R-367-3p significantly lowered TRAF4 protein expression in U87 and U251 glioma cell lines.Knockdown of mi R-367-3p significantly increased TRAF4 protein expression.14.There was specific binding between mi R-367-3p and 3’UTR of CEBPA.Mi R-367-3p mediated the proliferation,migration,invasion and apoptosis of U87 and U251 glioma cell lines via binding to 3’UTR of CEBPA.15.CEBPA was up-regulated in U87 and U251 glioma cell lines and glioma tissues.Knockdown of CEBPA significantly inhibited the proliferation,migration and invasion of U87 and U251 glioma cell lines while promoting apoptosis.16.Knockdown of OIP5-AS1 with over-expression of mi R-367-3p significantly decreased CEBPA expression in U87 and U251 glioma cell lines,inhibiting the proliferation,migration and invasion of glioma cells while promoting apoptosis.Whereas knockdown of OIP5-AS1 and mi R-367-3p simultaneously showed no significant effect on CEBPA expression in U87 and U251 glioma cell lines,as well as the proliferation,migration and invasion of glioma cells.17.TRAF4 was up-regulated in glioma cells and tissues.Knockdown of TRAF4 significantly suppressed the proliferation,migration and invasion of U87 and U251 glioma cell lines while promoting apoptosis.18.Knockdown of CEBPA significantly attenuated TRAF4 m RNA and protein expression in U87 and U251 glioma cell lines.19.CEBPA directly bound with TRAF4 promoter.20.Knockdown of CEBPA significantly decreased PIWIL3 m RNA and protein expression in U87 and U251 glioma cell lines.CEBPA directly bound with PIWIL3 promoter.21.Up-regulation of PIWIL3 and mi R-367-3p,as well as silencing of OIP5-AS1 resulted in significantly smaller xenografts and longer survival of nude mice.Significantly,simultaneous over-expression of PIWIL3 and mi R-367-3p with OIP5-AS1 knockdown resulted in the smallest xenografts and longest survival.22.TDP43 and SNHG12 were over-expressed in U87 and U251 glioma cell lines and glioma tissues.SNHG12 was localized in both nucleus and cytoplasm of glioma cells.Silencing of TDP43 and SNHG12 significantly inhibited the proliferation,migration and invasion of glioma cells while promoting apoptosis.23.There was targeted combination between TDP43 and SNHG12.24.Silencing of TDP43 significantly reduced half-life of SNHG12.25.Mi R-195 was down-regulated in U87 and U251.Mi R-195 was localized in both nucleus and cytoplasm of glioma cells.Over-expression of mi R-195 significantly inhibited the proliferation,migration and invasion of U87 and U251 glioma cell lines while promoting apoptosis.Silencing of mi R-195 significantly promoted the proliferation,migration and invasion of U87 and U251 glioma cell lines while attenuating apoptosis.26.Silencing of TDP43 and SNHG12 significantly elevated mi R-195 expression in U87 and U251 glioma cell lines,with the effect of the combined application being the most significant.27.Up-regulation of mi R-195 significantly attenuated SNHG12 expression in U87 and U251 glioma cell lines.Silencing of mi R-195 significantly increased SNHG12 expression in U87 and U251 glioma cell lines.28.There was targeted combination between SNHG12 and mi R-195 in a RISC-dependent manner.29.Knockdown of SNHG12 with over-expression of mi R-195 significantly suppressed the proliferation,migration and invasion of U87 and U251 glioma cell lines while promoting apoptosis.Whereas knockdown of SNHG12 and mi R-195 simultaneously exhibited no significant effect on the proliferation,migration and invasion of U87 and U251 glioma cell lines.30.SOX5 was up-regulated in U87 and U251 glioma cell lines and glioma tissues.Silencing of SOX5 significantly inhibited the proliferation,migration and invasion of U87 and U251 glioma cell lines while promoting apoptosis.31.Knockdown of TDP43 and SNHG12 significantly decreased SOX5 protein expression in U87 and U251 glioma cell lines,with the effect of the combined application being the most significant.32.Up-regulation of mi R-195 significantly lowered SOX5 expression in U87 and U251 glioma cell lines.Silencing of mi R-195 significantly increased SOX5 protein expression.Knockdown of SNHG12 with over-expression of mi R-195 significantly decreased SOX5 protein expression in U87 and U251 glioma cell lines.Whereas knockdown of SNHG12 and mi R-195 simultaneously exhibited no significant effect on SOX5 protein expression in U87 and U251 glioma cell lines.33.There was specific binding between mi R-195 and 3’UTR of SOX5.Mi R-195 mediated the proliferation,migration,invasion and apoptosis of U87 and U251 glioma cell lines via binding to 3’UTR of SOX5.34.Silencing of SNHG12 and over-expression of mi R-195 significantly decreased Gelsolin protein expression in U87 and U251 glioma cell lines.Knockdown of mi R-195 significantly increased Gelsolin protein expression in U87 and U251 glioma cell lines.Knockdown of SNHG12 with over-expression of mi R-195 significantly lowered Gelsolin protein expression in U87 and U251 glioma cell lines.Whereas knockdown of SNHG12 and mi R-195 simultaneously exhibited no significant effect on Gelsolin protein expression in U87 and U251 glioma cell lines.35.Silencing of SOX5 significantly attenuated Gelsolin protein expression in U87 and U251 glioma cell lines.Silencing of mi R-195 significantly elevated Gelsolin protein expression in U87 and U251 glioma cell lines.Whereas knockdown of SOX5 and mi R-195 simultaneously exhibited no significant effect on Gelsolin protein expression in U87 and U251 glioma cell lines.36.SOX5 bound to Gelsolin and SNHG12 promoters.37.Up-regulation of SOX5 significantly increased SNHG12 expression,whereas knockdown of SOX5 significantly decreased SNHG12 expression in U87 and U251 glioma cell lines.38.Silencing of TDP43 and SNHG12,as well as over-expression of mi R-195 resulted in significantly smaller xenografts and longer survival of nude mice.Significantly,simultaneous silencing of TDP43 and SNHG12 with mi R-195 over-expression resulted in the smallest xenografts and longest survival.Conclusion: 1.Pi R-30188 inhibited the malignant biological behaviors of glioma cells via binding to PIWIL3.Pi R-30188 specifically bound to OIP5-AS1 and negatively regulated its expression,modulating the biological behaviors of glioma cells.2.OIP5-AS1 specifically bound to mi R-367-3p and negatively regulated its expression,promoting the malignant biological behaviors of glioma cells.3.Mi R-367-3p specifically bound to 3’UTR of CEBPA,suppressing the malignant biological behaviors of glioma cells.4.CEBPA bound to TRAF4 promoter and positively regulated its transcription,facilitating the malignant biological behaviors of glioma cells.5.CEBPA bound to PIWIL3 promoter.6.PIWIL3/pi R-30188/OIP5-AS1/mi R-367-3p/CEBPA feedback loop played an important role in the regulation of the biological behaviors of glioma cells.7.TDP43 specifically bound to SNHG12 and promoted its stability,facilitating the malignant biological behaviors of glioma cells by upregulating SNHG12.8.SNHG12 specifically bound to mi R-195,facilitating the malignant biological behaviors of glioma cells by downregulating mi R-195.9.Mi R-195 specifically bound to 3’UTR of SOX5,suppressing the malignant biological behaviors of glioma cells.10.SOX5 bound to Gelsolin promoter and positively regulated its transcription,facilitating the malignant biological behaviors of glioma cells by downregulating mi R-195.11.SOX5 bound to SNHG12 promoter,forming a positive feedback loop to regulate the biological behaviors of glioma cells.12.TDP43/SNHG12/mi R-195/SOX5 feedback loop played an important role in the regulation of the biological behaviors of glioma cells.