Study On The Role Of CIRBP-HIF1?-PTGIS Axis In Bladder Cancer

Background Bladder cancer(BCa)is the ninth most common malignancy worldwide,Cold-inducible RNA binding protein(CIRBP)has been reported to be associated with distinct tumorigenesis,the involvement of CIRBP in BCa has not yet been investigated.CIRBP is an RNA-binding protein and binds specifically to the 3?-untranslated regions(3?-UTR)of many target mRNAs,UniGene data base showed that HIF-1? is one of CIRBP-targeted transcripts.As a master regulator in hypoxic cancer progression,HIF-1?(hypoxia inducible factor-1?)activates transcription of many genes,two publications of microarray hybridization analysis have reported that PTGIS is an important target gene of HIF-1?,and our microarray analysis indicate that PTGIS is a significantly downregulated gene in BCa tissues compared with normal bladder tissues.PTGIS(Prostaglandin I2 Synthase)encodes prostaglandin synthase,catalyzes the conversion of prostglandin H2 to prostaglandin I2,and have been reported as a tumor suppressor in many cancers.Objective To investigate the role of CIRBP-HIF1?-PTGIS axis in BCa.Methods To evaluate the expression level of target genes,RT-PCR,Western-blot,immunofluroscence staining,Confocal,were conducted.To study the effects of CIRBP and PTGIS on the biological behaviors of BCa,MTT assay,clonogenic survival assay and transwell migration assay were used to evaluate the effect of CIRBP and PTGIS on cell proliferation and migration abilities in vitro,while xenograft model and pulmonary metastasis model were used to evaluate the effect of CIRBP in vivo.To explore whether CIRBP upregulate HIF-1? expression through binding to the 3'-UTR and increases its mRNA stability,mRNA stability assay,cycloheximide assay,RNA-binding protein immunoprecipitation assay and biotin pull-down assay were performed.Chromatin immunoprecipitation assay and luciferase reporter assay were used to verify the regulation way of HIF-1? on PTGIS.Results In this study,we investigated the role of CIRBP in human bladder cancer(BCa),indicating that CIRBP is overexpressed in BCa tissues and cell lines to promote proliferation and migration.Moreover,CIRBP could induce expression of HIF-1? via binding to the 3'-UTR of its mRNA to increase the mRNA stability in BCa cells.Furthermore,we demonstrated that PTGIS is a HIF-1? targeted gene,our results suggested that overexpression of HIF-1? may suppress the expression of PTGIS in BCa cells,by binding to HRE sequence at the promoter region of PTGIS.In addition,we found a strongly downregulation of PTGIS in BCa tissue and transcriptionally inhibited by HIF-1? in BCa cells,which could be triggered by its DNA methylation.Further result suggested that knockdown of CIRBP could promote the expression of PTGIS,meanwhile knockdown of PTGIS could partially rescue CIRBP-deficiency induced inhibition of migration and proliferation in BCa cells.Conclusions Taken together,our study indicated that CIRBP could be a novel oncogene in human bladder cancer inducing transcription of HIF-1?,which could inhibit expression of methylated PTGIS.