RORA Regulates The TGF-β1/Smad Signaling Pathway Involved In The Aggressive Phenotype And Epithelial-mesenchymal Transition In Multifocal Glioblastomas

Objective:Multifocal glioblastoma is a very rare and refractory subtype of high-grade glioma with a very poor prognosis.Retinoic acid receptor-related orphan receptor A(RORA)is a transcription factor and specifically binds to the promoter of target genes to regulate its expression and function changes.Our previous study showed that the expression of RORA was downregulated in glioma,and RORA inhibited the tumorigenesis and proliferation of glioma.The epithelial-mesenchymal transition(EMT)is an important cause of enhanced glioblastoma invasiveness and tumor recurrence.This study was to explore the regulatory effect and mechanism of RORA on migration,invasion,and EMT in multifocal glioblastoma.Methods: The glioblastoma cell line T98 G and the patient-derived glioma stem cell GSC4 D were selected to study the overexpression of RORA in vitro.The expression level of RORA in clinical glioblastoma multiform(GBM)was detected by q PCR,Western blotting,and immunohistochemistry.RORA-overexpressed T98 G cells and GSC4 D were constructed by lentivirus transfection.To verify the regulatory effect of RORA on migration,invasion,and epithelial-mesenchymal transition of glioblastoma cell line T98 G and the patient-derived glioma stem cell GSC4 D,we performed migration assay,transwell assay,immunofluorescence,and western blotting.Gene Set Enrichment Analysis(GSEA),q PCR,ELISA,western blotting,and luciferase reporting assay were used to detect the transcriptional regulation of RORA on TGF-β1/Smad signaling pathway.Results: q PCR,Western blotting,and immunohistochemistry confirmed that the expression level of RORA was decreased in glioblastoma,and the RORA expression in multifocal glioblastoma was significantly lower than that in unifocal glioblastoma,moreover,lower RORA expression was significantly associated with poor prognosis in patients with glioblastoma.Migration assay,Transwell assay,immunofluorescence,and Western blotting confirmed that RORA overexpression inhibited the migration,invasion,and EMT of T98 G or GSC4 D.Gene Set Enrichment Analysis(GSEA),q PCR,ELISA,Western blotting,and luciferase reporting assay confirmed that RORA inhibited the expression of TGF-β1 and its mediated Smad signaling pathway through transcriptional regulation.Conclusion: The expression of RORA is downregulated in multifocal glioblastoma,and RORA transcriptionally regulates the TGF-β1/Smad signaling pathway to inhibit the migration,invasion,and epithelial-mesenchymal transition of multifocal glioblastoma.RORA may become a new target for molecular targeted therapy of glioblastoma.