The Role Of CircRNA In TR4-induced Invasion And Metastasis In Prostate Cancer

Background and objective:Prostate cancer(PCa)is the second leading cause of cancer related deaths among men in the United States,and its progression depends on androgen/androgen receptor(AR)signaling.Targeting AR using the androgen deprivation therapy(ADT)via either chemical or surgical castration has been an effective therapy to suppress the PCa progression.However PCa in most patients eventually turn into the castration-resistant prostate cancer(CRPC)after ADT treatment.Testicular nuclear receptor4(TR4)is a transcriptional regulator that belongs to the nuclear receptor superfamily.Early in vivo mice studies indicated that TR4 might play important physiological roles in normal spermatogenesis,cerebellum development,oxidative stress,glucose metabolism and insulin resistance.Importantly,the clinical survey from PCa patients found the TR4 expression in PCa might be linked to the pathological Gleason scores,and recent studies also suggested thatTR4 might play positive roles to promote the PCa cell invasion.Circular RNAs(circRNAs)are competing endogenous RNAs(ceRNAs)which play an important role in the initiation and progression.The aim of this study is to explore the role of circRNA in TR4-induced invasion and metastasis in prostate cancer.Methods:1、 Prostate cancer cells were treated with siRNA and cDNA of TR4,and observe the influence on the invasion and metastasis through the Transwell experiment.Using the database of TCGA to analyze the expression of TR4 in clinical samples and identify the correlation between expression of TR4 and clinical stage through IHC staining.2、 We predict some circular RNAs base on the proteins related tumor invasion and metastasis which are regulated by TR4.Circular RNA was screened and is circular RNA indeed through RNase R test.Then we construct the overexpression circPLCL2 and knock down plasmid,which are transfect into prostate cancer cells.And we observe the influence on the invasion and metastasis by TR4.3、 To test TR4 can regulate the expression of circPLCL2 through the regulation of host gene PLCL2.And we observe the influence of PLCL2 on the invasion and metastasis by TR4.4、 To test whether circPLCL2 can bind to miR-425-5p like miRNA sponge.Using Western blot to examine the expression of downstream gene and whether TR4-circPLCL2-miR-425-5p enhanced PCa cell invasion via altering FGF9/FGFR/P-AKT signals pathway.To analyze the correlation between expression of TR4 and FGF9 via the database of TCGA and observe the influence of miR-425-5p and FGF9 on the invasion and metastasis by TR4.5、 In vivo mouse model to confirm TR4-circPLCL2-miR-425-5p enhanced PCa cell invasion via alteringFGF9/FGFR/P-AKT signalsResults:1、 TR4 can enhance cell invasion in three PCa cell lines.In the clinical samples of prostate cancer,the expression of TR4 is higher in metastasis patients than in localized patients.The expression of TR4 has positive correlation with Gleason Scores.2、 We screened 21 circular RNAs and test circPLCL2 is circular RNA indeed.TR4 can regulate the expression of circPLCL2 in three PCa cell lines.Circ PLCL2 can reverse partially the TR4 influence on cell invasion in 3 PCa cells.3、 CircPLCL2 is generated by host gene PLCL2 and TR4 can regulate the expression of circPLCL2 in three PCa cell lines via regulation of the expression of PLCL2.TR4 can modulate the expression of PLCL2 in transcriptional level.Transwell test show PLCL2 could not reverse the TR4 influence on cell invasion in C4-2,CWR22Rv1 and PC-3 cells.4、 Overexpresstion of TR4 could not influence the expression of mi R-425-5p and circPLCL2 could interact with miR-425-5p like miRNA sponge effect.TR4-circPLCL2 may function via sponging/altering the miR-425-5p to enhance the PCa cell invasion.Using western blot assay,we confirmed that adding TR4 in PCa cells significantly increased the expression of FGF9 and p-AKT at the protein level.Significantly expression of sh-circPLCL2,miR425-5p could partially reverse the TR4-induced up-regulation of FGF9 as well as downstream genes p-FGFR/p-AKT.Importantly,using interruption approaches,we found that adding mi R-425-5p or FGF9 neutralizing antibody could significantly reverse the TR4-enhanced cell invasion in all 3 PCa cells.Importantly,using a database in TCGA to analyze the PCa samples which included gene expression data from 550 patients,we also found the positive correlation between TR4 and FGF9 in total PCa samples and 309 metastatic PCa samples.In addition,we also found that metastatic PCas have a higher FGF9 expression than those localized PCa.5、 Results from in vivo mouse model studies are in agreement with the in vitro studies and demonstrated that TR4 could enhance PCa cell invasion/metastasis via circPLCL2/miRNA-425-5p/FGF9/FGFR/p-AKT signals.Conclusion:1、 TR4 enhances PCa cell invasion2、 TR4 modulates the circPLCL2 expression3、 TR4 modulates circPLCL2 host gene expression at transcriptional level in PCa cells4、 TR4 enhances PCa cell invasion via altering the circPLCL2 expression5、 TR4-circPLCL2 enhanced PCa cell invasion via sponging/altering the miR-425-5p expression.6、 TR4-circPLCL2-miR-425-5p enhanced PCa cell invasion via altering FGF9/FGFR/P-AKT signals7、 In vivo mouse model to confirm TR4-circPLCL2-miR-425-5p enhanced PCa cell invasion via alteringFGF9/FGFR/P-AKT signals.