Regulation Mechanism Of PDLIM5 On Proliferation And Metastasis In Prostate Cancer

[Background]Prostate cancer(PCa)is a common malignancy of urinary system in middle-aged and elderly men.Currently,the incidence and mortality of prostate cancer in the world ranks second and sixth in all male malignancies.In developed countries,prostate cancer has become the most prevalent tumor in men,and according to the latest cancer statistics in 2018,there are an estimated 164,690 new cases of prostate cancer in the United States,resulting in an estimated 29,430 deaths each year.Compared with Western countries,the incidence and mortality of prostate cancer in Asia is relatively low.Although the incidence and mortality of prostate cancer in China are below the world average,with the popularity of early screening techniques for prostate cancer represented by screening of prostate specific antigen(PSA)in recent years,the incidence rate of prostate cancer in China is rising rapidly.According to the latest data released in 2015,the incidence of prostate cancer in China reached 60.3 / 100,000,ranking sixth in the incidence of malignant tumors in men and increasing rapidly,and the PCa mortality rate also reached at 26.6 / 100000.Early screening led to early detection of prostate cancer in most patients,providing a relatively favorable prognosis and long-term survival through radical treatment.However,many patients are diagnosed with advanced prostate cancer for the first time,or eventually progression to castrated resistant prostate cancer(CRPC)because of tumor recurrence.Although standardizing androgen deprivation therapy(ADT)can delay the progression of locally advanced or metastatic PCa progression.However,the treatment of these patients is limited,the prognosis is usually poor.Tumor metastasis is responsible for 90% of cancer-related deaths.PCa is one of the most susceptible human cancers for bone metastases.Patients with CRPC,particularly those with metastatic CRPC(mCRPC),have had limited benefit from ADT.Therefore,it is a hot topic in recent years to explore the potential pathways of PCa metastasis and to search for new and effective biomolecular targets for prostate cancer metastasis in order to make better CRPC treatment strategies and prevention strategies for bone metastasis.PDZ and LIM domain 5(PDLIM5)is a 63 kDa cytoplasmic protein composed of a PDZdomain in the N-terminus and three consecutive LIM domains in the C-terminus.PDLIM5 was first discovered in 1996 by Kuroda using the yeast two-hybrid technique with PKC as bait protein.PDLIM5 is anchored to the actin cytoskeleton through its PDZ domain and recruits the actin filament-associated protein.It is thought to be involved in cytoskeletal organization,cell lineage specification,organ development and oncogenesis.The functions of PDLIM5 have been studied mainly in relation to its functions in modulation of synaptic strength.Therefore,large numbers of studies have focused on the association between PDLIM5 and psychiatric diseases such as schizophrenia and depression.Besides,PDLIM5 was considered necessary in cell migration by forming lamellipodia,and phosphorylation inactivation of PDLIM5 was reported to weaken the cell metastasis capacity by inactivating RAC1.According to recent studies,PDLIM5 may participate in the progression of many types of cancer including thyroid cancer,lung cancer,breast cancer and gastric cancer.Koutros et al reported that PDLIM5 mRNA was overexpressed in PCa tissues by using gene chip detection.Ma et al reported that PDLIM5 may prove to be a diagnostic molecular target for serum and urine assays for adjuvant diagnosis and have a potential value in predicting the risk of advanced PCa progression.Cell migration and invasion are known to be closely related to dynamic changes of the cytoskeleton,especially the actin-containing microfilament skeleton.The arrangement of cytoskeleton is a key factor in determining cell morphology and motility.Knowing that PDLIM5 is a cytoskeleton related protein and may play an important role in the invasion and metastasis of PCa cells,an understanding about the molecular mechanisms of PDLIM5 in PCa initiation,development and metastasis would help improve the treatment of PCa patients,even those in the CRPC stage.AMP-activated protein kinase(AMPK)is an energy receptor that is activated during hypoxia,ischemia,glucose loss,and stress.Studies have demonstrated that activation of AMPK is closely associated with tumor growth and proliferation,cell cycle,apoptosis,neovascularization and metastasis.In this study,we found that PDLIM5 was over-expressed in PCa tissues as compared with normal prostate tissues both by database-mining and experimental investigation.In addition,PDLIM5 may exert a physiological function by reacting with AMPK.So we silenced PDLIM5 gene in PC-3 and DU145 cells by using lentivirus expressing short hairpin RNA to observe the phenotype changes of cancer cells and uncover the underlying regulatory mechanism in the tumorigenesis of PCa of PDLIM5.[Objective]Explore the expression of PDLIM5 in prostate cancer tissues and cell lines in order to clarify the high expression of PDLIM5 in prostate cancer.Further investigate the biological function of PDLIM5 in tumorigenesis and metastasis and its regulatory mechanism in prostate cancer cell lines with high expression of PDLIM5.[Methods]Part ?:Expression Analysis of PDLIM5 in Prostate Cancer Related Database and Verification in Tissue SpecimensCollect information of the expression,metastasis and prognosis of PDLIM5 in prostate cancer samples from Oncomine and PROGGENE databases to verify the differential expression of PDLIM5 in cancerous and normal glands and further explore the correlation between PDLIM5 overexpression and prostate cancer metastasis and prognosis.44 cases of prostate cancer tissue specimens were collected after radical prostatectomy in Changzheng Hospital to detect the expression of PDLIM5 in tissues of cancer and adjacent tissues by immunohistochemistry.Part ?:Select and establish silencing PDLIM5 prostate cancer cell lines and verify the silencing efficiencyPDLIM5 expression was analyzed by Western Blot and qPCR in the common prostate cancer cell lines,and the metastatic prostate cancer cell lines DU145 and PC-3 highly expressing PDLIM5 were selected as the follow-up experimental subjects.A lentivirus containing a plasmid targeting the silencing PDLIM5 was constructed for cell infection.The fluorescence intensity of the reporter gene on the plasmid was observed,and the knockdown efficiency of PDLIM5 was verified respectively at the mRNA and protein levels to confirm the successful construction of the PDLIM5-silenced cell lines.Part ?:Detection of cancer cell proliferation,cell cycle and apoptosis levels changes,as well as the impact on invasion and metastasis after PDLIM5 silencingAfter silencing PDLIM5 in the DU145 and PC-3 cells,the proliferation ability of tumor cells was evaluated by MTT and clone formation experiments.Cell cycle and apoptosis were detected by flow cytometry to determine the effect on cell cycle arrest and apoptosis induce when PDLIM5 knockout.The effects of PDLIM5 on tumor migration and invasion abilitywere detected by cell scratch healing experiment and TRANSWELL experiment.Part ? : A preliminary study of the regulatory mechanism of PDLIM5 on the proliferation and metastasis of prostate cancerAccording to the results of cell phenotype experiment and the analysis of database information,PDLIM5 has a significant influence on the metastasis and invasion ability of tumor cells.The Epithelial-mesenchymal transition(EMT)-related molecules were detected by Western Blot and qPCR in the PDLIM5 silencing state.Then we constructed a plasmid to overexpresse PDLIM5 in the same cell line,the up-regulation of EMT-related molecule expression was detected and the correlation analysis factors of bone metastasis in prostate were detected under PDLIM5 knockdown and overexpression respectively.Finally,the interaction between PDLIM5 and AMPK was determined by co-immunoprecipitation.The phosphorylation of AMPK was detected after PDLIM5 knockdown,and the regulatory mechanism of PDLIM5-AMPK-EMT was explored.Part ?:The function of PDLIM5 in vivo was studied through animal experimentsDU145 cells with silencing PDLIM5 were used for subcutaneous tumor formation in nude mice to observe the effect of PDLIM5 knockdown on tumorigenesis and proliferation of cells.Subsequently,the tumor tissue was lysed and the expression of PDLIM5 protein was detected by Western Blot.The silencing efficiency of PDLIM5 was determined and the expression of AMPK and EMT related signaling pathways were detected to clarify the regulation of PDLIM5 on cell proliferation and metastasis mechanism in vivo.[Results]1.PDLIM5 was found to be abnormally high expression in prostate cancer tissues and was associated with biochemical recurrence and survival of prostate cancer.Through immunohistochemistry and bioinformatics analysis,it was confirmed that PDLIM5 expression in prostate cancer was higher than that in normal prostate tissue,and PDLIM5,which was abnormally high expressed in database chips,was associated with poor prognostic factors such as metastasis,high Gleason score,advanced TMN staging.At the same time,multivariate analysis and survival analysis found that high expression of PDLIM5 was an independent risk factor for biochemical recurrence of prostate cancer and led to a decrease in overall survival.2.We successfully established DU145 and PC-3 cell lines with stable silencing PDLIM5.Through related cytological experiments,we found that silencing PDLIM5 inhibited the proliferation,colony formation,invasion and metastasis of prostate cancer cells,and induced G2 / M arrest and apoptosis.3.Detection of epithelial-mesenchymal transition(EMT)related molecules expression after PDLIM5 knockdown,Western Blot and qPCR showed that the expression of ZO1,Vimentin and Snail were down-regulated in different degree,while expression of the EMT key molecule E-cadherin increased,confirming that the EMT process was inhibited.4.Overexpression of PDLIM5 in DU145 and PC-3 cells could up-regulate the expression of SNAIL and decrease the expression of the key EMT factor E-cadherin in DU145 and PC-3cells.It was confirmed that the effect of PDLIM5 on cell invasion and metastasis was closely related to EMT.In addition,CTGF,PTHrP and uPA are known to be involved in the bone metastasis of prostate cancer.qPCR results showed that the expression of these pro-tumor metastasis factors was significantly down-regulated in PC-3 cells after silencing PDLIM5.While expression of these molecules was up-regulated when PDLIM5 was overexpressed and similar results were also observed in DU145 cells,further confirming that PDLIM5 plays a role in promoting tumor metastasis5.Co-immunoprecipitation assay showed that PDLIM5 could bind to AMPK,and knockdown of PDLIM5 decreased AMPK protein and phosphorylated AMPK protein.Subsequently,the upstream protein synthesis was blocked by actinomycin,and AMPK protein levels were detected at different time points after administration.As a result,AMPK protein degradation was accelerated in PDLIM5 silencing group.PDLIM5 can promote the proliferation and metastasis of prostate cancer by maintaining the stability of AMPK and activating the AMPK pathway.6.Nude mice tumorigenicity experiments show that in vivo conditions,silencing PDLIM5 can inhibit tumor cell growth.PDLIM5 also regulates the proliferation and metastasis of prostate cancer cells through AMPK-EMT-related pathways in vivo by animal experiments.[Conclusion]This study confirmed that the expression level of PDLIM5 was up-regulated in PCa tissues,indicating that PDLIM5 may be an oncogene in PCa formation.Through bioinformatics analysis to explore the relationship between PDLIM5 and PCa recurrence and metastasis,indicating that high expression of PDLIM5 prostate cancer prognosis is related topoor prognosis.In addition,both cellular and animal experiments show that PDLIM5 can promote the proliferation of tumor cells and enhance the EMT mediated invasion and metastasis through the activation of AMPK pathway.Exploring the relevant pathways and targets of PDLIM5 may help to enrich the mechanisms of PCa progression and metastasis and bring new therapeutic strategies to mCRPC patients.