Circ-0047641 Serves As A Sponge For MiR-338-5p To Promote Prostate Cancer Proliferation,Metastasis And Enzalutamide Resistance Via Upregulating RRM2 Expression

ObjectiveProstate cancer(PCa)is the predominant solid malignancy in men and its morbidity is on the upswing.Because of the absence of typical clinical symptoms in the early stages,many patients have progressed to high-risk PCa when diagnosed.Besides radical resection,advanced PCa required adjuvant combination therapy,especially endocrine therapy.However,majority of high-risk PCa continued to proceed to castration-resistant PCa(CRPC)and even metastatic PCa,which has become one of the leading causes of prostate tumor-related deaths。Recent studies have found that enzalutamide delayed CRPC progression,but only for 4-6months.Circular RNAs(circ RNAs)were a class of non-coding RNAs widely found in eukaryotes,with covalently closed,circular,single-stranded RNA molecules that are highly stable in structure.It could combine with various non-coding RNAs to form regulatory networks that could regulate the epigenetics of tumors,and play an active role in the development of many tumors.We aimed to search for enzalutamide resistance-related circ RNAs in PCa,construct enzalutamide resistance-related circ RNA/mi RNA/m RNA ce RNA regulatory network and risk score prognostic model,and investigate the expression of key circ RNAs and their effects for proliferation,migration and invasion,enzalutamide resistance and other biological roles in PCa.We would also explore the specific molecular mechanisms and signaling pathways of the circ RNAs,screen and identify specific and sensitive circ RNAs,which could provide theoretical guidance for the search of molecular targets for PCa diagnosis,prognosis and treatment,and for the precise treatment of PCa.Methods1.In part 1,to search enzalutamide resistance-related circ RNA,we downloaded the transcriptomic dataset from the GEO database and performed differential analysis using limma package in R language.Simultaneously,we downloaded mi RNAs and m RNAs expression data and performed differential expression analysis,then obtained candidate mi RNA and m RNA by overlapping with mi RNA and m RNA predicted by bioinformatics prediction website.Spearman analysis was applied to identify the correlation between candidate circ RNA and mi RNA as well as mi RNA and m RNA.Then the Cytoscape software and STRING database were used to construct the circ RNA-mi RNA-m RNA ce RNA regulatory network and PPI network,respectively.In addition,univariate and LASSO COX regression analyses were applied to construct risk score prognostic models.Subsequently,ROC curves and Kaplan-Meier survival analysis were employed to assess the reliability and sensitivity of the models,and Spearman analysis was used to evaluate the correlation with clinicopathological characteristics.Finally,the prognosis-related ce RNA regulatory sub-networks were constructed by combining the ce RNA regulatory network,risk score prognostic model and Hub gene.2.In Part II,we performed Sanger sequencing,agarose gel assay and verified the stable closed loop structure of circ-0047641 by RNase R and Act D treatment.Then cytoplasmic nucleoplasm separation assay and FISH assay were used to verify its intracellular sublocalization.Subsequently,we examined the expression of circ-0047641 in PCa cells and tissues by q RT-PCR and in situ hybridization(ISH)assay,respectively.We examined the effect of circ-0047641 on the proliferation and migration and invasion ability of PCa cells by CCK-8and Ed U cell proliferation assays and Transwell assay.We constructed enzalutamide-resistant LNcap cells(LNcap-Enz R),and examined the IC50 values of LNcap cells and LNcap-Enz R cells by CCK-8 cytotoxicity assay to detect the effect of circ-0047641 on enzalutamide-resistant PCa.Finally,to verify the effect of circ-0047641 on the proliferation ability of PCa,we grew LNcap cells with stable low expression of circ-0047641,and inoculated them into nude mice and observed their ability of subcutaneous tumorigenic ability compared with PCa cells with normal expression of circ-0047641.3.In Part III,FISH assays were carried out to determine the intracellular sublocalization of circ-0047641 and mi R-338-5p to confirm whether they had direct interactions.Then,RIP experiments were performed to verify whether circ-0047641 could enrich mi R-338-5p and verify whether circ-0047641 could bind with mi R-338-5p.Next,we designed circ-0047641wild-type and mutant reporter gene plasmids,and followed with a dual luciferase reporter gene assay to further validate the direct binding of circ-0047641 with mi R-338-5p.We designed the overexpression sequence and interference sequence of mi R-338-5p,and verified the expression of mi R-338-5p and its regulation by upstream circ-0047641 via q RT-PCR in PCa.Subsequently,the effects of mi R-338-5p on the proliferation and migration and invasion ability of PCa cells were examined by CCK-8 cell proliferation assay and Transwell migration and invasion assay.Afterwards,RRM2 wild-type and mutant reporter gene plasmids were designed,and then the dual luciferase reporter gene assays were performed to validate that RRM2 was a target gene for mi R-338-5p.Furthermore,the expression of RRM2 and its regulation by upstream mi R-338-5p in PCa were verified by q RT-PCR and western blot assays,and the expression of RRM2 and its correlation with clinicopathological features(T-stage,lymph node metastasis,Gleason score)in PCa tissues were verified by IHC assays,and the public database data to validate and explore the impact of RRM2 on the prognosis of PCa.We explored the effect of RRM2 on the prognosis of PCa in public database.In addition,we verified the regulation of RRM2 expression at transcriptional and protein levels by q RT-PCR and western blot assays.Moreover,we investigated that circ-0047641 regulated the proliferation,migration and invasion of PCa cells via sponged mi R-338-5p by CCK-8 cell proliferation assays and Transwell assasy.Finally,we explored the downstream activated signaling pathways by bioinformatics analysis and combined with literature reports and verified them by western blot experiments.Results1.In part 1,We screened and obtained 13 enzalutamide resistance-associated differentially expressed candidate circ RNAs,and 210 candidate mi RNAs and 467 candidate m RNAs.The Cytoscape software was applied to construct a ce RNA regulatory network containing 2 upregulated circ RNAs(hsa-circ-0047641 and hsa-circ-0068697)and 2 down-regulated circ RNAs(hsa-circ-00000919 and hsa-circ-0000036),as well as 6 mi RNAs and 167 downstream target genes,and the PPI network was used to predict 10 Hub genes,including RRM2.Next,a riskscore prognostic model containing 8 target genes was constructed,and the ROC curve suggested that its AUC value was 0.816,and the AUC values at the 3-,5-,and 10-year survival outcomes were 0.848,0.772,and 0.950,respectively,which suggested that the riskscore prognostic model had good predictive potential for PCa.The multivariate ROC curve revealed that the AUC value of the signature was 0.985,indicating that it had more desirable stability and sensitivity.Besides,based on the univariate and multivariate COX regression analysis,riskscore was confirmed as an independent prognostic factor for PCa.Finally,a prognosisrelated ce RNA regulatory network was constructed,containing one down-regulated expressed circ RNAs(hsa-circ-0000036)and two up-regulated circ RNAs(hsa-circ-0047641 and hsa-circ-0068697),and five mi RNAs(hsa-mi R-326 hsa-mi R-338-3p,hsa-mi R-338-5p,hsa-mi R-671-5p,hsa-mi R-885-5p)and 8 m RNAs,and we selected circ-0047641/ mi R338-5p/RRM2 ce RNA regulatory network for in-depth validation to explore its role in PCa development,enzalutamide resistance and possible regulatory mechanisms.2.In part II,we confirmed that circ-0047641 was derived from the DYM gene,resulting from the back-splicing of exons 11,12,and 13.And we constructed Sanger sequence to confirm that circ-0047641 was a covalently closed continuous loop.it could be amplified by divergent primers,which excluded the possibility of gene recombination and rearrangement interference,and could be resistant to RNase R and Act D.Then the cytoplasmic nucleoplasm separation assay and FISH assay demonstrated that the majority of circ-0047641 was located in the cytoplasm of PCa cells.Secondly,q RT-PCR experiments confirmed that circ-0047641 was highly expressed in PCa cells relative to normal prostate cells,while ISH experiments revealed that circ-0047641 was highly expressed in PCa tissues compared with normal prostate tissues,and its expression was linked to the clinicopathological parameters of PCa(AJCC T,N stage and Gleason score),with a statistically significant difference.Next,we found that the proliferation ability of PCa cells transfected with circ-0047641 interference sequence was significantly decreased compared with the control group by CCK-8 cell proliferation assay(p<0.05).Similarly,the migration and invasion ability of PCa cells transfected with circ-0047641 interference sequence was also significantly decreased.Besides,we successfully constructed enzalutamide-resistant LNcap cells(LNcap-Enz R)and confirmed that the expression of circ-0047641 in LNcap-Enz R cells was significantly higher than that in parental LNcap cells.The CCK-8 cytotoxicity assay was applied to construct a dose-response curve for LNcap cells and LNcap-Enz R cells.The results showed that the IC50 value of the drug response to enzalutamide was 18.7μM/L in parental LNcap cells and 60.29μM/L in LNcapEnz R cells.The IC50 value of parental LNcap cells transfected with si-circ-0047641 was7.749μM/L,which indicated that LNcap cells with knocking down circ-0047641 significantly reduced the enzalutamide resistance of parental LNcap cells.Similarly,we transfected si-circ-0047641 into LNcap-Enz R cells to knock down the expression of circ-0047641 and its IC50 value was reduced to 16.82μM/L,which also suggested that circ-0047641 significantly altered the enzalutamide resistance of LNcap-Enz R cells.Finally,we found that knockdown of circ-0047641 significantly inhibited tumor growth in vivo by subcutaneous tumorigenesis assay in nude mice.3.In Part III,it was first confirmed by FISH experiments that circ-0047641 co-localized with mi R-338-5p in the cytoplasm,which provided a theoretical basis for the next confirmation that circ-0047641 could act as a sponge for mi RNA.Then,we confirmed by RIP experiments that AGO2 antibody could enrich more circ-0047641 and mi R-338-5p compared with Ig G antibody,and the dual luciferase reporter gene assay showed that mi R-338-5p mimics could directly bind to wild-type circ-0047641 to inhibit the fluorescence activity of the reporter gene,but could not reduce the activity of the mutant circ-0047641 reporter gene.It suggested that circ-0047641 could directly bind with mi R-338-5p to exert biological effects.Secondly,we demonstrated that mi R-338-5p was lowly expressed in PCa by q RT-PCR assay.Moreover,based on CCK-8 cell proliferation assay and Transwell assay,we realized that PCa cells transfected with mi R-338-5p mimics had enhanced proliferation ability and improved migration and invasion ability in comparison with the controls.Additionally,q RT-PCR experiments showed that mi R-338-5p expression was regulated by upstream circ-0047641.Secondly,the dual luciferase reporter gene assay confirmed that mi R-338-5p could directly bind to RRM2,which indicated that RRM2 was a target gene for mi R-338-5p.Furthermore,q RTPCR and western blot assays showed that the expression of RRM2 was regulated by mi R-338-5p.Subsequently,we selected pathological sections of PCa samples for IHC,and the results showed that RRM2 was lowly expressed in normal prostate tissues compared with prostate tumor tissues,and RRM2 expression was significantly linked to the clinicopathological features for PCa.Moreover,we downloaded transcriptomic and clinical data regardinig RRM2 from TCGA database to verify the expression of RRM2 and its relevance with clinicopathological parameters.The findings were in accordance with the previous results.Also,by Kaplan-Meier survival analysis,we found that patients with high RRM2 expression had significantly lower overall survival,disease-specific survival and progression-free interval than those with low RRM2 expression(log-rank p<0.05).In addition,q RT-PCR and western blot experiments confirmed that circ-0047641 could regulate RRM2 expression at transcriptional level and protein level.Thirdly,we confirmed that circ-0047641 could regulate the expression of RRM2 via sponged mi R-338-5p and further mediate the proliferation,migration and invasion of PCa.Finally,by western blot assay,we confirmed that circ-0047641/ mi R338-5p/RRM2 ce RNA regulatory network affected the biological function of PCa through activation of PI3K/AKT/m TOR signaling pathway.ConclusionsWe screened 13 enzalutamide resistance-associated circ RNAs by bioinformatics analysis,constructed circ RNA-mi RNA-m RNA ce RNA regulatory network and PPI network,and then established a reliable and robust riskscore prognostic model for PCa.Subsequently,we selected enzalutamide resistance-related circ-0047641/mi R-338-5p/RRM2 prognosis-related ce RNA network for further study.We demonstrated that circ-0047641 had a stable covalently closedloop structure,which mainly localized in the cytoplasm and was highly expressed in PCa cells and tissues and correlated with the clinicopathological parameters of PCa.Functionally,we found that circ-0047641 promoted the proliferation,migration and invasion of PCa,and could promote the enzalutamide resistance of PCa.Mechanistically,circ-0047641 could serve as a ce RNA to regulate RRM2 expression through sponge mi R-338-5p,which activated PI3K/AKT/m TOR signaling pathway further and finally had an effect on the biological function of PCa,and was expected to be a novel biomarker and therapeutic target for PCa diagnosis and prognosis.