Molecular Mechanism Of ZFP36 Negatively Regulating CDK6 In Prostate Cancer Cell Cycle

Obective: Globally,prostate cancer(PCa)is the most common tumor in men,especially in western countries.In about 90% of cases,prostate cancer is still limited to organs or local progression only at the time of diagnosis,which makes prostatectomy or local radiotherapy effective.However,30%-40% of patients usually experience disease progression.At this stage,tumor growth depends on androgen,so the most effective treatment is androgen deprivation therapy,which aims to block androgen secretion and / or activity.This treatment is based on pharmacological castration and is obtained by administering gonadotropin releasing hormone(Gn RH)agonists,alone or in combination with antiandrogens,however,despite the good initial response,most patients relapsed within 2-3 years and the tumor progressed to the state of castration resistance.Treatment options need to be improved for castration resistant patients,taxane based treatment and immunotherapy,as well as new therapies using enzalutamide and abiraterone,usually provide a progression free survival of several months,so this treatment is often used as a treatment after the progression of prostate cancer.In parallel,80% of advanced PCa frequently develop bone metastases,usually treated with radiotherapy and chemotherapy,which is related to a considerable morbidity and has an adverse effect on the quality of life and some bone-related events.Therefore,more accurate markers should be found to predict specific diagnosis and prognosis.The determination of cell fate is guided by a complex regulatory mechanism to interpret genomic information.The regulatory mechanisms include epigenetic modification,RNA post-transcriptional modification and proteomic post-translational modification.This disorder of precise mechanisms leads to abnormal cell behavior,which can lead to malignant transformation: such as cancer.RNA binding protein tristetraprolin(TTP),also known as zinc finger protein36(ZFP36),plays an important role in various physiological and pathological processes,including differentiation,reprogramming,metabolism,proliferation,tumorigenesis and immunity.ZFP36 can bind and target the m RNA containing ARE,and then rapidly degrade the m RNA level,resulting in the down-regulation of the expression of a large number of key genes,thereby acting as a tumor suppressor gene.It is reported that the loss of ZFP36 is associated with poor prognosis in a variety of human tumors.Our previous study also found that the expression of ZFP36 in prostate cancer tissues was significantly lower than that in benign prostate tissues,indicating the potential of ZFP36 as an auxiliary marker for prostate cancer.Therefore,we would like to further evaluate the biological function of ZFP36 dysregulation involved in the malignant phenotype of PCa in clinical sample evaluation and in vitro and in vivo experiments,to determine its potential mechanism for tumor regulation,and to provide a new theoretical basis for gene therapy of prostate cancer.Methods:1.To explore the expression of ZFP36 in prostate tissue and prostate cancer tissue,and to analyze the relationship between ZFP36 and clinical features of prostate cancer patients:(1)TCGA and Taylor public database were used to evaluate the m RNA expression level of ZFP36 in prostate cancer and normal prostate tissues.(2)Use the TCGA and Taylor prostate cancer database to analyze the correlation between ZFP36 m RNA expression and clinical patient age,preoperative PSA,Gleason score,clinical stage,pathological stage,metastasis,overall survival,biochemical recurrence,and surgical margins.(3)Based on the follow-up data of TCGA and Taylor database,the survival curve and overall survival curve of prostate cancer patients with m RNA expression levels of ZFP36 were plotted,and the cox risk regression model was used to evaluate the prognostic value of ZFP36 in patients with prostate cancer.2.To explore the effect of ZFP36 on the biology of prostate cancer cells:(1)Construct the prostate cancer cell lines 22RV1 and DU145 with overexpression and knockdown of ZFP36,perform Q-PCR and Western blot verification,and use cell function experiments to verify the effect of ZFP36 on the biological behavior of prostate cancer cells.(2)Construct a prostate cancer cell line overexpressing ZFP36,establish a nude mouse subcutaneous tumor model,observe the growth of the tumor,extract the tumor-bearing protein,and perform Western blot to verify the relevant markers.3.To explore the downstream pathways of ZFP36’s biological impact on prostate cancer:(1)Use immunohistochemistry to detect changes in markers related to invasion in ZFP36 overexpression prostate cancer cell lines,such as Vimentin,MMP9,E-Cadherin,Ki67,etc.(2)Transcriptome sequencing and biosynthesis analysis to screen downstream target genes;constructed ZFP36 overexpression and knockdown cell lines,real-time PCR and Western blot methods are used to verify the regulation of ZFP36 on downstream pathways.(3)Dual-luciferase and RNA co-immunoprecipitation experiments were used to verify the targeted regulation of ZFP36 on downstream target genes.Results: 1.The expression of ZFP36 protein was high in human normal prostate tissues,but low in prostate tumors.2.The ZFP36 m RNA expression correlation and the clinical characteristics of patients with prostate cancer was analyzed by TCGA and Taylor public database.In TCGA database,the m RNA expression ZFP36 was negatively correlated with Gleason(p = 0.002),pathological stage(p = 0.002)and biochemical recurrence(p = 0.003).In Taylor database,the m RNA expression level of ZFP36 was negatively correlated with Gleason score(p <0.001),pathological stage(p = 0.016),distant metastasis(p < 0.001),overall survival rate(p < 0.001)and biochemical recurrence(p < 0.001).Kaplan-meier method showed that the expression of ZFP36 was high expressed,and the survival rate of non-biochemical recurrence and metastasis non-biochemical recurrence significantly increased.Cox risk regression model also suggests that ZFP36 can be used as an independent prognostic factor to predict the survival rate of prostate cancer patients without biochemical recurrence.3.In vitro cellular function experiments show that overexpression of ZFP36 could inhibit the proliferation,invasion and migration of prostate cancer DU145 and 22RV1 cells.4.The results of subcutaneous xenograft tumors in nude mice showed that overexpression of ZFP36 can inhibit the growth of prostate tumor cells.5.The results of immunohistochemistry showed that the overexpression of ZFP36 could reduce the expression of MMP9,Vimentin,Ki-67 and promote the expression of E-cadherin,thereby inhibit the proliferation,invasion and metastasis of tumor cells.6.RNA sequencing screened out CDK6 as the downstream target gene of ZFP36.Western Blot/Q-PCR demonstrated that overexpression of ZFP36 can reduce the expression of CDK6 at the cell level and animal tissues,and the dual luciferase experiment and RIP experiment proved that CDK6 is the downstream target of ZFP36.It was further proved that the levels of CDK2,CDK6,cyclin D1,cyclin D2,E2F1 and c-myc in DU145 cells with ZFP36 overexpression decreased,despite the p27 upgrade,while the knockout DU145 cell line showed the opposite results.It proves that CDK6 is the downstream target of ZFP36,and ZFP36 could inhibit the growth of tumor cells by blocking the cell cycle at G1 stage.Conclusions:1.The expression of ZFP36 in prostate cancer tissue is lower than that in normal prostate tissue.The lower the expression of ZFP36 protein in the tissues of patients with prostate cancer,the worse the clinical prognosis and the lower the survival rate without biochemical recurrence.ZFP36 can be used as an independent prognostic factor for patients with prostate cancer without biochemical recurrence after operation.2.In prostate cancer,ZFP36 may be used as a tumor suppressor gene to inhibit the growth,invasion and migration of prostate cancer.3.ZFP36 inhibits the progression of prostate cancer by targeting CDK6 signaling pathway,thereby affecting the cyclin in the downstream pathway of CDK6,and preventing the prostate cancer cell cycle in the G1 phase to inhibit tumor progression,thus providing a new theoretical basis and a possible new target for the clinical treatment of PCa.