The Role Of LASP-1 In Prostate Cancer Invasion And Metastasia And Its Mechanism

Part one:Expression of LASP-1 in prostate cancer and correlation with clinicopathologic dataBackground and objective:prostate cancer is the most common malignant tumor in male reproductive system,incidence rate of prostate cancer in continuously increased in china,prostate cancer is also the third cause of death among cancers,which seriously threaten the healthy life of men.No specific symptoms can be observed in early stage of prostate cancer,the use of PSA to screen prostate cancer is not yet completed,more than one third of patients are locally invasive and distantly metastatic when diagnosed,then high-risk prostate cancer,5-year recurrence rate of high-risk prostate cancer beyond 50%,control of local invasion and distant metastasis is tough,but can largely improve prognosis of patients.Therefore,the present research aims to explore the biomarkers or targets which closely relate with the development of prostate cancer to provide guidance for precisely personal treatment.Methods:① tissue samples from patients with prostate cancer were collected From January 2015 to December 2016 in XXX hospital,32 samples were diagnosed as prostate cancer,15 samples were prostatic hyperplasia as control.② 10 samples were randomly chosen,then differentially expressed genes between carcinoma and para-carcinoma were performed by gene chip technology.③highly expressed LASP-1 in carcinoma were considered as our interested gene by the result of gene chip and previous citations.④ the expression of p65 and IκBα in tissues were detected by immunohistochemistry(IHC),RT-qPCR and western blot.⑤ clinical information,such as TNM staging,Gleason score,level of tPSA and fPSA,ECT,were prepared to investigate their correlation with LASP-1 expression.Results:① IHC,RT-qPCR and western blot displayed compared with control group,LSAP-1 was highly expressed in carcinoma,both in cytoplasm and nuclei;IκBα and p65 in NF-κB signaling pathway were also highly expressed.② correlation analysis showed there are no obvious correlation between LASP-1 and tPSA,fPSA,TNM stage and Gleason score.However,expression of LASP-1 was higher in high-risk patients(PSA>20ng/ml or Gleason score≥8)compared with medium/low-risk patients.③ compared with patients without metastasis,expression of LASP-1 were much higher in patients with bone and lymph nodes metastasis.④5 of all samples were from recurrent patients,compared with patients without recurrence,LASP-1 was highly expressed in recurrent patients.Conclusions:① LASP-1 was highly expressed in prostate cancer.②Compared with medium/low-risk patients patients,expression of LASP-1 was higher in high-risk patients.The increased expression of LASP-1 at cytoplasm and the positive expression of the nucleus in prostate cancer tussue may indicate the high invasive ability and poor prognosis.Part two:LSAP-1 regulates the development of prostate cancer via NF-κB pathwayObjective:LASP-1 and NF-κB pathway were found to relate with prostate cancer in part one,in this part,we will further explore how LSAP-1 regulate the development of prostate cancer via NF-κB pathway on PC3 and Du145 cell lines.Methods:① interference of LASP-1 expression by ShRNA-LASP1.② mRNA and protein level of p65 and IKBa after ShRNA interference were detected by RT-qPCR and western blot.③ cell viability after ShRNA interference was investigated by CCK-8 assay.④ cell apoptosis and cell cycle arrest after ShRNA interference were performed by flow cytometry.⑤ colony formation ability after ShRNA interference was observed by colony forming assay.⑥ invasion and migration ability after ShRNA interference were explored by Transwell experiment.⑦ location and expression of LASP-1,p65,IκBα and p-IκBαafter ShRNA interference in cells were visualized by immunofluorescence staining(IF).Results:① cell viability,both PC3 and Du145,were decreased after ShRNA-LASP1 interference.② cell apoptosis was increased after ShRNA-LASP1 interference.③knockdown of LASP-1 induced G1/S phase cell arrest.④invasion and migration ability were decreased after knockdown of LASP-1.⑤mRNA and protein level of p65 and IκBαwere down-regulated after knockdown of LASP-1,protein level of p-IκBα was down-regulated,both at cytoplasm and nuclei.Conclusions:① LASP-1 closely related with the development of prostate cancer,knockdown of LASP-1 in prostate cell lines decreased cell viability,invasion and migration ability and colony formation ability.② LASP-1 could regulate the development of prostate cancer via NF-κB pathway.③ LASP-1 may become one of targets for prostate cancer treatment.Part three:The role of LASP-1 in the development of prostate cancer by in vivo animal experimentObjective:relationship between LASP-1 and prostate cancer were observed both in cell lines and tissues,we further verify the role of LASP-1 in prostate cancer by in vivo animal experiment.Methods:① C57BL/6J mice were randomly separated into four groups,10 mice for each group,two of four group were intraventricularly injected with PC3 and Du145 transfected with ShRNA-nc respectively,another two group were intraventricularly injected with PC3 and Du145 transfected with ShRNA-LASP1 respectively.② 1-2 weeks later,analysis of tumor metastasis in mice by whole-body optical imaging system.Results:compared with mice injected with cells transfected with ShRNA-nc,fluorescent intensity in ShRNA-LASP1 group was obviously decreased,which suggested that knockdown LASP-1 in prostate cell could decrease prostate cancer metastasis.Conclusion:targeted inhibition of LASP-1 could decrease prostate cancer metastasis.