| Prostate cancer(PCa)is one of the most common cancer diseases of male reproductive system in China.The morbidity of PCa has rapidly increased in recent years in China.PCa is high heterogeneous and hormone sensitive.Although the mechanism of PCa has not been fully elucidated yet,mostly researchers have generally accepted that high grade intraepithelial neoplasia(HGPIN)is a precancerous lesion of PCa.HGPIN?latent PCa?clinical PCa--metastasis may be the main mode of the PCa progression.The progression of clinical PCa was that tumors confined to the prostate?invasion and breakthrough of prostate capsule?invasion of seminal vesicle gland?metastasis to adjacent lymph nodes?metastasis to bone(most commonly seen)and other organs.Androgen deprivation treatment(ADT)remains the standard treatment for patients with advanced PCa.However,patients inevitably recur with a more aggressive castration-resistant prostate cancer(CRPC).With the use of abiraterone or enzalutamide,a subset of patients with late-stage CRPC eventually develops neuroendocrine prostate cancer(NEPC),which is associated with extremely poor overall prognosis.The mechanisms of PCa progression,particularly pathways involved in the development of CRPC and NEPC,need to be better understood in order to develop more effective treatments.SOX4(sex-determining region Y-box 4)gene is a critical developmental transcription factor.Previous studies found that SOX4 was up-regulated in a variety of tumor tissues,such as bladder cancer,liver cancer,acute myeloid leukemia,prostate cancer,endothelial cell cancer,etc.We and others have found that overexpression of SOX4 has been associated with high Gleason score and poor clinical outcome of PCa patients.Most recently,we defined an important role for SOX4 promoting PCa cell proliferation and invasion by orchestrating an epithelial-mesenchymal transition(EMT).Additionally,we demonstrated that SOX4 is a dihydrotestosterone(DHT)-repressed androgen receptor(AR)target gene and is overexpressed in CRPC tumors as compared to the hormone-dependent PCa.These results suggest that SOX4 may be involved in the progression of PCa.MicroRNAs(miRNAs)are a class of small noncoding RNA species.They are usually combined with messenger RNA(mRNA)through base pairing principle,regulating the expression and stability of mRNA at the post transcription level.Aberrant expression of many miRNAs has been linked to multiple human diseases including PCa cancers.Similar to classical oncogenes and tumor suppressors,miRNAs play important roles in PCa development and progression.In our previous study,we identified miRNAs that play an important role in the development of PCa,such as miR-30a,miR-33b,miR-200b/c,miR-204,miR-573,etc.As a transcription factor,SOX4 can bind to the promoters to regulate many genes that play significant roles in PCa progression including EZH2,EGFR,HSP70,CUL4B,Tenascin C.However,whether SOX4 regulates miRNAs expression in PCa has not been reported.Therefore,we propose scientific questions:whether SOX4,as a transcription factor,can regulate the expression of miRNAs and their target genes;we aim to clarify the mechanism of SOX4 promoting PCa progression.In this study,PCa cell lines were used to study the function and mechanism of SOX4.The results were as follows:Part ? SOX4/miR-17-92/RB1 Axis Promotes Prostate CancerProgressionPreviously,we and others found that SOX4 promotes PCa cell proliferation,migration and invasion in vitro.SOX4 promotes PCa progression by orchestrating an epithelial-mesenchymal transition(EMT).MiRNAs are involved in the occurrence and development of PCa.We then propose the scientific questions:whether SOX4,as a transcription factor,can regulate the expression of miRNAs and their target genes to promote PCa progression?The mechanism of SOX4 promoting PCa progression was further elucidated.In this study,PCa cell lines were used to study the function and mechanism of SOX4.The results were as follows:1.SOX4 knockdown inhibits PCa tumor growth and metastasis in vivoTo extend the role of SOX4 on PCa cells in vivo,we used VCaP subcutaneously xenograft mouse model to evaluate the function of SOX4 inhibition.The results showed that the tumor of control(VCaP-shNC)group grew rapidly.However,there was no tumor formation and grew until the end of the experiment in the SOX4 knockdown(VCaP-shSOX4)group.In addition,three mice of the control group(VCaP-shNC)had lung metastasis,while the SOX4 knockdown(VCaP-shSOX4)group had no lung metastasis.These results suggested that SOX4 knockdown significantly inhibited the PCa tumor formation and metastasis.2.Bioinformatics analysis of downstream miRNAs of SOX4We preformed high-throughput miRNA expression profiling in SOX4-knockdown VCaP cells.A total of 92 miRNAs were upregulated and 154 miRNAs were downregulated in the SOX4-knockdown VCaP cells as compared to the negative control(fold change>2.0).The most obvious expression changes were miR-30a-5p,miR-19a-3p,miR-19b-3p,miR-20a-5p,miR-93-5p,etc.Furthermore,the top miRNAs correlated to SOX4 expression in The Cancer Genome Atlas(TCGA)were chosen by Pearson correlation.The result showed that the expression of miR-20a,miR-19a,miR-200b,miR-183 were correlated with the expression of SOX4 in PCa patients.Next,we analyzed the top miRNAs that were increased in castration-resistant xenograft tissues as compared to androgen-dependent xenograft tissues from GSE55829 dataset.The result showed that miR-19a,miR-20a,miR-26b and miR-30b were up-regulated in CRPC xenograft tissues.MiR-19a and miR-20a belonged to the intersections of high-throughput miRNA expression profiling and bioinformatics analysis of miRNAs.Importantly,these two miRNAs belong to miR-17-92 cluster(include miR-17-5p,miR-18a-5p,miR-19a-3p,miR-19b-3p,miR-20a-5p and miR-92a-3p).All above results suggested that SOX4 might regulate miR-17-92 cluster expression in PCa.3.SOX4 promotes miR-17-92 cluster expression by binding to its promoterSOX4 promotes the expression of miR-17-92 cluster in PCa cells by RT-qPCR assay.Considering that SOX4 is a transcription factor,we hypothesized that SOX4 might regulate the expression of miR-17-92 at the transcription level.To test this hylpothesis,we confirmed that SOX4 can promote the expression of pri-,pre-miR-17-92 in PCa cells.The chromatin immunoprecipitation(ChIP)assay showed that SOX4 protein can bind to miR-17-92 promoter 313-322 position in PCa cells.Moreover,SOX4 was able to induce miR-17-92 promoter activity but failed to do so in mutant miR-17-92 promoter luciferase activities in VCaP and HEK293T cells.These results confirmed that SOX4 transcriptionally regulated the expression of miR-17-92 cluster in PCa cells.4.SOX4-induced PCa cell proliferation,migration and invasion are mediated by miR-17-92 cluster membersEDU and Transwell assays showed that SOX4-knockdown inhibited PCa cell proliferation,migration and invasion,whereas reconstitution of miR-17-92 cluster members partially blocked the repressed effects due to SOX4-knockdown.Additionally,reconstitution of miR-17-92 cluster members' inhibitors partially blocked SOX4-overexpression-induced cell proliferation,migration and invasion.Collectively,these findings suggest that SOX4-induced PCa cell proliferation,migration and invasion are mediated by miR-17-92 cluster members.5.RB1 is a downstream gene of SOX4/miR-17-92 axisUsing Gene Set Enrichment Analysis(GSEA),we analyzed the genes that are potentially regulated by SOX4 from GSE11914 dataset which is the whole genome expression microarray of knockdown and overexpression SOX4 in LNCaP cells.Bioinformatics analysis was carried out to analyze the target genes of miR-17-92 cluster.The result showed that RB1 gene might be a downstream target gene of SOX4/miR-17-92 axis.Western Blot and RT-qPCR results showed that SOX4 inhibited RBI protein expression but had no significant effect on mRNA expression level of RB1 in PCa cells,suggesting that SOX4 might regulate RB1 protein expression at the post-transcriptional level.We assumed that miR-17-92 cluster may participate in this regulation process.Furthermore,Western Blot and Dual luciferase assay results confirmed that RBI was the target gene of miR-17-5p and miR-20a-5p in PCa cells.In addition,the rescue experiment showed that miR-17-5p and miR-20a-5p partially reversed the RB1 protein increase caused by SOX4-knockdown;whereas inhibitors of miR-17-5p and miR-20a-5p partially reversed the RB1 protein decrease caused by SOX4 overexpression.All above result showed that RBI is the target gene of SOX4/miR-17-92 axis.SOX4 repressed RBI protein expression via upregulation of miR-17-92 cluster expression in PCa cells.6.Co-expression of SOX4,miR-17-92 and RB1 in vivoOur result showed that the expression of miR-17-92 cluster members was positively correlated with SOX4 expression in PCa patients from the GSE26367 dataset.Furthermore,we examined whether SOX4 regulation of miR-17-92 cluster is physiologically relevant in PCa clinical specimens collected in this study.We found that the expression of miR-17-5p and miR-19a-3p was higher in SOX4-high patients as compared to SOX4-low patients.Analysis of GSE35988 showed that RBI expression was significantly negatively correlated with SOX4 expression in PCa patients(R=-0.7882,P<0.0001).Using TMA and IHC,we found that SOX4 was overexpressed in 23.0%(32/140)of Chinese PCa patients.IHC showed that approximately 8%of PCa patients in our cohort demonstrated RB1 protein loss.Of the seven PCa cases with small cell carcinoma,IHC also revealed that 71%(5/7)showed overexpression of SOX4 and 57.1%(4/7)demonstrated RBI loss.Notably,IHC revealed that RB1 protein expression was negatively correlated with SOX4 expression in PCa patients with small cell carcinoma.Collectively,our data suggest that there is a significant relationship between the co-expression of SOX4,miR-17-92,and RB1,which indicates that the SOX4/miR-17-92/RB1 axis may promote PCa progression.7.SOX4 overexpression is associated with PCa progression and NE phenotypePrevious studies reported that RB1 loss is one characteristic of NEPC and that RB1 dysfunction promotes PCa metastasis and CRPC progression.Therefore,we hypothesized that the SOX4/miR-17-92/RB1 axis might be involved in the neuroendocrine-like(NE-like)phenotype of PCa.Using GEO datasets and cbioportal database,we analyzed the expression level of SOX4 in normal prostate tissue,PCa,CRPC and NEPC patients.The results showed that the expression level of SOX4 gradually increased with the development of PCa,and SOX4 overexpression was associated with NE-like phenotype.8.SOX4 knockdown restrains NE phenotype and PCa cell proliferationAccording to previous studies,LNCaP-NE cells with NE-like phenotype change were established.SOX4 knockdown was able to convert LNCaP-NE cells with the NE phenotype to the rounded shape that is typical of epithelial cell clusters.Furthermore,SOX4 knockdown was shown to reduce LNCaP-NE cells' proliferation and the expression of NEPC markers(NCAM1,CHGA,CHGB,SYP).In conclusion,SOX4 downregulates RB1 protein expression via transcriptionally upregulating miR-17-92 cluster expression in PCa.Clinically,the expression of miR-17-92 cluster members was positively correlated with SOX4 expression,while RB1 was negatively correlated with SOX4 expression.The SOX4/miR-17-92/RB 1 axis may promote PCa progression and development of the NE phenotype.Part ? SOX4 Promotes Castration-Resistant Prostate CancerProgression by Upregulation of BMI1 via miR-224-452 ClusterIn our previous work,we demonstrated that SOX4 is a dihydrotestosterone(DHT)-repressed androgen receptor(AR)target gene and is overexpressed in CRPC tumors as compared to the hormone-dependent PCa.However,the role and mechanism of SOX4 in castrated resistant prostate cancer have not been elucidated.Therefore,bioinformatics analysis and molecular biological experiments were carried out to study the role and mechanism of SOX4 in CRPC cells.The results were as follows:1.SOX4 is overexpressed and associated with poor prognosis in CRPCUsing publicly available PCa and CRPC datasets as well as using TMA and IHC,we demonstrated that SOX4 expression is significantly higher in CRPC patients compared to primary PCa patients.GSEA analysis showed that SOX4-knockdown downregulated gene signatures were significantly enriched for metastatic CRPC patients as well as CRPC xenograft tumors.These results suggested that SOX4 overexpression might participate in CRPC progression.2.SOX4 suppression inhibits the CRPC cell proliferation and invasionEDU and cell colony formation assays showed that the SOX4 knockdown inhibited CRPC cell proliferation.Importantly,SOX4-knockdown contributed to reduced cell proliferation to a greater extent under CSS culture vs.FBS culture.To extend the in vitro results,we used a castration-resistant C4-2B xenograft mouse model to evaluate the therapeutic potential of SOX4 inhibition in CRPC.SOX4 knockdown significantly reduced tumor growth compared to control group.At the endpoint,the average tumor volume and weight were significantly reduced in C4-2B-shSOX4 group.Moreover,the Ki67 percentage score of tumor cells in C4-2B-shSOX4 group significantly decreased compared to that in shNC group.Taken together,above results indicated that SOX4 suppression inhibits CRPC cell proliferation under androgen deprivation.Transwell assays demonstrated that SOX4 knockdown displayed less migration and invasion capability of CRPC cells under CSS culture vs.FBS culture.Next,castration-resistant C4-2B xenograft mouse showed that the C4-2B-shNC group displayed signs of outside-invasion of the fibrotic capsule and lung metastases,whereas the majority of xenograft tumors of the C4-2B-shSOX4 group were well encapsulated without muscle invasion and no lung metastases.Taken together,all above results indicated an important role of SOX4 in proliferation,invasion and metastasis of CRPC.3.Bioinformatics analysis of downstream genes and miRNAs of SOX4To further elucidate the mechanism of SOX4 regulating CRPC cell growth and invasion,we preformed gene expression profiling of siSOX4 vs control(NC)in C4-2B cells under CSS culture.GSEA analysis indicated that SOX4 was related to Wnt signaling pathway,SHH signaling pathway,ESC signaling pathway;PRC2-EZH2,PRC1-BMI1,HOXA9,MYC and RB1 protein etc.Using publicly available datasets TCGA and GSE26367,top miRNAs negatively correlated with SOX4 expression were chosen by Pearson correlation analysis.Cross-comparison of clustered miRNAs allowed us to generate a "consensus" of five miRNAs:miR-221,miR-222,miR-224,miR-31 and miR-205.Notably,BMI1 is the target gene of miR-221,miR-222 and miR-224.Previous studies showed that BMI1 is involved in ESC signaling pathway.4.BMI1 is the downstream gene of SOX4Western Blot and immunofluorescence results showed that SOX4 knockdown inhibited the expression of BMI1 protein in CRPC cells,whereas SOX4 overexpression promoted the expression of BMI1 protein.RT-qPCR showed that SOX4 had no significant regulation on the mRNA expression of BMI1.Additionally,BMI1 expression was positively correlated with SOX4 expression in CRPC patients and tumor xenografts.Further,the rescue experiments confirmed that SOX4 knockdown/overexpression induced CPRC cell proliferation and invasion could be partially reversed by overexpression/knockdown of BMI1.We found that BMI1 was the downstream gene of SOX4 and mediated SOX4-induced CRPC cell proliferation and migration.5.SOX4 inhibits miR-224-452 clusters expression in CRPC cellsOur results suggested that SOX4 induced BMI1 protein expression at the post transcriptional level.We hypothesized that miRNAs may participate in this regulation.Using publicly available datasets TCGA and GSE26367,top miRNAs negatively correlated with SOX4 expression were chosen by Pearson correlation analysis.Next,miRNAs targeting BMI1-3'UTR were predicted by TargetScan.The result showed that miR-224-452 cluster might be regulated by SOX4 and participate in the regulation BMI1 by SOX4.RT-qPCR showed that SOX4 inhibited the expression of miR-224-452 cluster members in CRPC cells,and EZH2 mediated the regulation of miR-224-452 cluster by SOX4.Western Blot detected that SOX4 induced EZH2 expression in CRPC cells.The CoIP assays confirmed that SOX4 protein interacted EZH2 protein in CRPC cells.ChIP assay showed that EZH2,H3K27 protein bind to the promoter region of GABRE which is the host gene of miR-224-452 cluster.In addition,the rescue experiments showed that miR-224-452 inhibitors mediated SOX4-induced CRPC cell migration.Our results showed that miR-224-452 cluster were target miRNAs of SOX4 in CRPC cells.6.SOX4 upregulates BMI1 protein expression through repressing miR-224-452 cluster expressionWestern Blot showed that miR-224-5p and miR-452-5p mimics inhibited the expression level of BMI protein,while miR-224-5p and miR-452-5p inhibitors promoted the expression level of BMI1 protein in CRPC cells.The relative luciferase activity of the wild-type BMI1-3'UTR was inhibited by both miR-224-5p and miR-452-5p mimics but increased by their respective inhibitors in C4-2B cells.However,such effects were not observed with the mutant BMI1-3'UTR.Furthermore,the rescue experiment showed that miR-224-452 cluster could partially reverse the increase of BMI 1 protein caused by SOX4 overexpression,whereas miR-224-452 inhibitors partially reverse the decrease of BMI 1 protein caused by SOX4 knockdown.7.BMI1 mediates SOX4-induced stem-like phenotype of CRPCAccumulated evidences have indicated that cancer stem cells(CSCs)are an important cause of tumor initiation,metastasis,recurrence and intrinsic resistance of standard treatment in PCa.Previous studies have found that BMI1 promotes PCa progression by regulating PCa stem-like phenotype.Moreover,SOX4 mediates cell development and differentiation.We hypothesized that SOX4 might promote CRPC stem-like phenotype and BMI1 might mediates this regulation.GSEA analysis showed that stem-cell-related genes were enriched in CRPC patients and SOX4 overexpression groups.The expression of tumor stem cell markers(BMI1,SOX2,CD133,CD44)were positively correlated with SOX4 expression in CRPC patients from the Trento/Cornell/Broad 2016 cohort.In addition,the sphere formation assay showed that SOX4 overexpression increased the sphere formation ability of C4-2B cells,while the BMI1-knockdown decreased the sphere formation ability caused by SOX4.Furthermore,RT-qPCR and Western Blot showed that SOX4 overexpression significantly increased the expression of stem markers(CD44,CD 133,NKX3.1 and NANOG),whereas BMI1 knockdown reduced the expression of these stem markers.In conclusion,SOX4 expression was increased in CRPC and SOX4 knockdown played a stronger role in inhibiting CPRC cell proliferation and metastasis under androgen deprivation.SOX4 induced BMI1 protein expression via inhibiting the expression of miR-224-452 cluster.There may be a SOX4/miR-224-452/BMI1 axis promoting CRPC stem-like phenotype. |
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