Characterization Of SOX4-miR-106b～25-PTEN Negative Feedback Loop In The Progression Of Prostate Cancer

Prostate cancer is one of the most common malignant tumors of male reproductive system.According to the most recent data released in 2022,prostate cancer is the leading cause of male malignancies and the second leading cause of death.The biological behavior of prostate cancer is highly heterogeneous,most of which are relatively indolent and progress slowly.Only 10-15%of prostate cancer is invasive,and many patients with invasive prostate cancer have extensive distant organ metastasis at the first diagnosis,and the prognosis is very poor.In terms of treatment,for Localized prostate cancer,Radical prostatectomy(RP)is usually selected clinically.The main treatment for Advanced prostate cancer is Androgen deprivation therapy(ADT),but most patients develop biochemical relapse after 1-3 years of treatment.Castration-resistant prostate cancer(CRPC)develops gradually.The European Urology Association recommended that localized prostate cancer patients with PSA levels of>2ng/mL following initial treatment with RP be defined as Biochemical recurrence.Patients with elevated PSA after RP have about a 60%chance of developing distant metastasis within 10 years and a 20%chance of dying from prostate cancer.Therefore,how to accurately determine the "Indolent" and "Aggressive" of prostate cancer,predict which prostate cancer patients are more prone to biochemical recurrence and tend to progress to CRPC,and how to provide better diagnosis and treatment for patients after RP surgery to delay the progression of the disease.Both are important issues of clinical concern.The key molecular mechanisms underlying the progression of prostate cancer need to be further elucidated.SOX4(Sex-determining-region Y-box 4)is a member of the C subgroup of SOX transcription factor family.SOX4 gene has been proved to be highly expressed in breast cancer,bladder cancer,lung cancer etc,and plays an important role in the invasion and metastasis of tumors.Previously,our research group and others found that SOX4 overexpression is an independent poor prognostic factor for prostate cancer patients in China.SOX4 has been proved to promote the proliferation,migration and invasion of prostate cancer cells in vivo and in vitro.SOX4 and fusion gene TMPRSS2-ERG synergistically promote epithelial-mesenchymal transition(EMT)in tumor cells.In addition,our latest study found that SOX4 may promote neuroendocrine transformation of prostate cancer cells by regulating microRNA(miRNAs)and its downstream target genes.PTEN is an important tumor suppressor gene.The occurrence and development of prostate cancer and the process of androgen independent transformation are related to the loss of PTEN gene expression.It has been reported that the expression of PTEN protein is significantly negatively correlated with Gleason score,pathological grade and clinical stage of prostate cancer,suggesting that the expression level of PTEN protein may decrease with the increase of the malignant degree of prostate cancer.Loss of PTEN or inactivation of protein expression can activate the PI3K-AKT signaling pathway,leading to the occurrence and development of prostate cancer and promoting its transition to CRPC.In addition,studies have shown that SOX4 is necessary to maintain the activity of PTEN-PI3K-AKT signaling pathway.To further study the role and molecular mechanism of SOX4 in the malignant progression of prostate cancer,we first used GEO(Gene Expression Omnibu)and TCGA(The Cancer Genome Atlas)to analyze prostate Cancer public databases and found that The SOX4 expression level gradually increased with the progression of prostate cancer.Prostate cancer patients with high SOX4 mRNA expression levels have a higher risk of biochemical recurrence.Importantly,Gene Set Enrichment Analysis(GSEA)analyzed the public prostate cancer database and showed that activation of PI3K-AKT signaling pathway may play an important role in biochemical recurrence of prostate cancer,and that PTEN may be a downstream target gene of SOX4.In addition,by analyzing GEO and TCGA prostate cancer public databases,we found a significant negative correlation between SOX4 and PTEN expression at the mRNA level.Our research group has preliminarily constructed three Tissue microarray(TMA),including 206 patients with prostate cancer after RP.Immunohistochemistry(IHC)results show that SOX4 is negatively correlated with PTEN protein expression in prostate cancer patients.The proportion of patients with both high SOX4 expression and low PTEN expression(SOX4+/PTEN-)increased significantly in the biochemical recurrence of prostate cancer,while the proportion of patients with both low SOX4 expression and high PTEN expression(SOX4-/PTEN+)was higher in the nonbiochemical recurrence of prostate cancer.These results suggest that the interaction between the two may play an important role in the biochemical recurrence and malignant progression of prostate cancer.To verify this hypothesis,through the cluster analysis of the public database of prostate cancer,we found that SOX4+/PTEN-prostate cancer patients were significantly enriched in the aggressive prostate cancer group and had poor prognosis.The above results suggest that SOX4+/PTEN-prostate cancer may be an aggressive prostate cancer subtype with poor prognosis.To further study the specific regulatory mechanism between SOX4 and PTEN,we first used Western blot to detect the protein expression level of SOX4 in prostate cancer cell lines,and combined with the expression of PTEN in prostate cancer cell lines(PTEN wild-type prostate cancer cell VCaP and DU 145).The VCaP cells with the highest SOX4 expression were selected for siRNA knockdown.DU 145 cells with the lowest SOX4 expression were selected for overexpression.Western blot and qRT-PCR showed that SOX4 down-regulated PTEN protein expression,but PTEN mRNA level had no significant effect.These results suggested that SOX4 may regulate PTEN protein expression at the post-transcriptional level.Further experiments showed that SOX4 had no significant regulation on PTEN protein stability.MiRNAs are a class of endogenous non-coding RNAs that regulate the translation and stability of mRNA at the post-transcription level by interacting with the 3 ’-untranslated region(UTR)of mRNA.In our previous work,we found that SOX4,as a transcription factor,can regulate the expression of a variety of miRNAs.Therefore,we hypothesized that SOX4 may down-regulate PTEN protein by regulating miRNAs expression.Public database data,miRNAs-Seq chip data of siSOX4 and TargetScan bioinformatics analysis and prediction software were used to screen out miRNAs related to SOX4 and targeting PTEN,including miR-106b-25 clusters(members include:miR-106b-5p,miR-93-5p,miR-25-3p).Other studies have found that the high expression of miR-106b-25 clusters can promote the proliferation and migration of prostate cancer cells,and is associated with poor prognosis of prostate cancer patients,and all three members of miR-106b-25 clusters can target PTEN.Next,by analyzing GEO database,we found that members of miR-106b～25 clusters were significantly positively correlated with SOX4 expression levels in prostate cancer patients.qRT-PCR results showed that SOX4 up-regulated the expression of miR-106b～25 cluster members in prostate cancer cells VCaP and DU 145.Considering that SOX4 acts as a transcription factor,we hypothesized that SOX4 may transcriptionally regulate the expression of miR-106b-25 clusters.qRT-PCR results showed that SOX4 up-regulated the expression of pre-miR-106b-25 cluster members in VCaP and DU 145 of prostate cancer cells.It has been reported that clusters of miR-106b～25 are located in the 13th intron of MCM7 gene,suggesting that clusters of miR-106b～25 share the same promoter region with host gene MCM7.In addition,we found a positive correlation between SOX4 and MCM7 mRNA expression levels in prostate cancer patients,and qRT-PCR results showed that SOX4 upregulated MCM7 and miR-106b～25 clusters.Based on the above results,we hypothesized that SOX4 may regulate miR-106b～25 clusters by binding to the promoter region of MCM7.ChIP assay and dual luciferase assay showed that SOX4 could bind to the promoter region of MCM7,thereby up-regulating the expression of host gene MCM7 in clusters miR-106b～25 and then up-regulating the expression of members of clusters miR-106b～25.In addition,Western blot and dual luciferase assay demonstrated that miR-106b～25 cluster members directly bind to the PTEN 3’-UTR region and negatively regulate the expression of PTEN protein.Rescue experiments demonstrated that miR-106b～25 cluster members mediate the regulation of SOX4 on PTEN protein.Finally,we discuss whether there is mutual regulation between SOX4 and PTEN.Western Blot results showed that PTEN down-regulated the expression of SOX4 protein in prostate cancer cells.Through cell rescue experiments,MTS,EdU and Transwell experiments further confirmed that overexpression of PTEN could partially reverse the enhancement of proliferation,migration and invasion of prostate cancer cells caused by overexpression of SOX4.SOX4 and PTEN form a negative feedback loop to promote the progression of prostate cancer.At present,PTEN-PI3K-AKT signaling pathway inhibitors have made progress in the treatment of triple negative breast cancer,chronic myeloid leukemia and ovarian cancer.LY294002 is a known inhibitor of PI3K-AKT signaling pathway.Several studies have shown that LY294002 can inhibit the proliferation,migration and invasion of prostate cancer cells.Results of MTS,EdU and Transwell experiments showed that overexpression of SOX4 could increase the resistance of DU145 cells to LY294002,and LY294002 could partially reverse the proliferation,migration and invasion of prostate cancer cells caused by overexpression of SOX4.In conclusion,this study is the first to confirm that SOX4 is associated with biochemical recurrence of prostate cancer.In prostate cancer patients,SOX4 is negatively correlated with PTEN expression level,and SOX4+/PTEN-may be a highly invasive subtype with poor prognosis.In vitro experiments,we found and clarified that SOX4 can down-regulate the expression of PTEN protein by up-regulating miR-106b～25 clusters in transcription,and a negative feedback loop can be formed between SOX4 and PTEN,promoting the malignant progression of prostate cancer.Overexpression of SOX4 can increase resistance of prostate cancer cells to PI3K-AKT pathway inhibitors.This study provides us with a further understanding of the molecular mechanisms related to the aggressive progression of prostate cancer,and provides a potential target and theoretical basis for the diagnosis and targeted therapy of prostate cancer.