CircRPPH1-ATF3 Signal Promotes The Malignant Phenotype Of Glioma Stem Cells Via TGF-β1/Smad Pathway

Objective: Glioblastoma(GBM)is the most lethal intracranial malignant brain tumor in adults.It has a poor prognosis due to diffuse infiltrative growth and the complex molecular mechanisms behind it.Non-coding circular RNAs(circRNAs)are more stable in vivo due to their circular structural properties which promote tumor development through protein binding and protein stabilization.In this study,we focus on the expression of circRPPH1 in glioma stem cells in malignant glioblastoma and investigate the intrinsic regulatory mechanism.This study involves both glioma stem cells and animal models,focusing on the effects of circRPPH1 on the proliferation,invasion,and self-renewal ability of glioma stem cells,and examining the transcriptional regulation of circRPPH1-ATF3 and the activation of TGF-β1/Smad signaling pathway downstream of circRPPH1,in order to identify new therapeutic targets and provide a basis for targeted therapy.Methods: Glioblastoma stem cell lines GSC35 and GSC38 were extracted from fresh tumor tissues of glioblastoma patients for in vitro knockdown and overexpression of circRPPH1 and ATF3.After transfection of circRPPH1 knockdown and overexpression,the proliferation,invasion and self-renewal ability of GSCs were tested by MTS proliferation assays,Transwell invasion assays,and limited dilution assays after circRPPH1 knockdown and overexpression.The interaction of circRPPH1 with ATF3 was verified by RNA pull down.RT-qPCR and Western Blot were used to detect the expression of stemness markers in GSCs after transfection.GSEA enrichment,dual luciferase reporter gene,RT-qPCR,ELISA,and Western Blot were used to detect the transcriptional regulatory role of ATF3 and its downstream TGF-β1/Smad signaling pathway.Finally,we found the alteration of proliferative properties of glioblastoma by knockdown as well as overexpression of circRPPH1 in vivo by intracranial tumorigenesis assay in BALB/c nude mice.Results: Tissue RT-qPCR results showed that circRPPH1 was highly expressed in glioblastoma.MTS assays,Transwell assays,and limited dilution assays showed that knockdown of circRPPH1 and ATF3 could inhibit the proliferation,invasion,and selfrenewal of GSC,while gene overexpression could promote this effect.RNA pulldown assays,Western Blot assays demonstrated that circRPPH1 could bind ATF3 protein and enhance ATF3 stability.Western Blot assays demonstrated that circRPPH1 could bind ATF3 protein to enhance the stability of ATF3.Dual luciferase reporter assays demonstrated that ATF3 transcriptionally regulated the expression of nestin.Western Blot assays demonstrated that ATF3 promotes downstream TGF-β1/Smad signaling and that it promotes the expression of stemness markers,thereby allowing tumors to receive growth signals,continue to proliferate and infiltrate,and increase its malignancy.In vivo experiments demonstrated that knockdown of circRPPH1 expression inhibited glioma proliferation,while overexpression of circRPPH1 promoted tumor growth.Conclusion: Circ RPPH1 shows high expression in glioma stem cells and leads to poor patient prognosis.circRPPH1 can promote proliferation,invasion,and self-renewal of glioblastoma by binding ATF3 for maintaining its stability and prompting ATF3 transcriptional regulation of nestin expression.It also activated the expression of downstream TGF-β1/Smad signaling.This suggests that circRPPH1-ATF3 and TGF-β1/Smad signaling plays an important role in regulating the proliferation of glioma stem cells.Targeting circRPPH1 may be a new target for glioblastoma treatment.