Effects And Mechanism Of CIT Knockdown On The Proliferation,Migration And Invasion Of The Prostate Cancer PC-3 Cell Line

Background and objectiveProstate cancer(PCa)is the second common malignancies cancer in men worldwide,which caused over 300,000 cancer-related death one year.Radical prostatectomy and androgen deprivation therapy are the major treatment for the localized stage of PCa patients.Unfortunately,approximately 90%of PCa patients would finally develop metastasis.For this stage,rare treatments were effective and the overall survival is only 12-15 month.The technology of next-generation sequencing has accelerated our understanding of the cancer related genes underlying the development and progression of PCa and identify many gene mutation and copy number variants that associated with PCa progression.However,these massive amounts of sequencing data on PCa also bring new challenges.Besides next-generation sequencing,we need to establish functional relevance and gaining mechanistic insight in cell culture and animal model system to comprehend the new findings.Citron Rho-Interacting Serine/Threonine kinase(CIT)is a downstream substrate of Rho protein.During cytokinesis,CIT plays an important role in the formation of midbody through the regulation of multiple molecular networks.Recent studies have shed light on the pathological role of CIT.Loss of CIT leads to cytokinesis failure and apoptosis in mammals’neuronal progenitors and germ cells,causing microcephaly and testicular hypoplasia.Moreover,CIT is also associated with the development of human cancer.The expression of CIT is found to be up-regulation in colon cancer and knockdown of CIT reduces cell proliferation.Furthermore,in PCa,Whitworth et al.found that CIT deficiency would decrease cell growth in both androgen-dependent and castration-resistant PCa cells.However,the role of CIT in metastasis,the most fatal cancer stage,remains unknown.1.Identify the cancer related gene based on TCGA and MSKCC databasesTo screen the new prostate cancer related gene,we access the TCGA and MSKCC prostate cancer sequencing databases.The results showed that based on TCGA database,the CIT mRNA level positively correlated with T stage.In addition,in 51 paired prostate cancer samples,the mRNA level of CIT is higher in the cancer samples than the adjacent normal samples.The similar results also identified in MSKCC database.The patients with high CIT mRNA level also showed high N stage,M stage and Gleason score.Overall,these results revealed that CIT might promote the metastasis and progression of prostate cancer.2.The biological functional analysis in prostate cancer cell line with CIT knockdownTo investigate the mechanism of CIT in prostate cancer,we used siRNA to build CIT-silenced cell model for analyzing the function changes in prostate cancer cells when CIT deficiency.The results of MTT cell proliferation assay showed cell proliferation was reduced after KMT2D silenced.The wound healing assay and Transwell assay without revealed the deficiency of CIT inhibited the migration ability of PC-3 cells.In addition,the Transwell assay with Matrigel also indicated that the knockdown of CIT decreased the invasion ability of PC-3 cells.Overall,these data supported that CIT plays important role in PCa progression.The deficiency of CIT would suppress the PCa metastasis.3.The molecular mechanism involved in the CIT associated PCa metastasisAs the knockdown of CIT inlhibited the migration and invasion ability in PC-3 cells,we further detect the changes of EMT associated protein after CIT knockdown.The results showed that the E-cadherin was increased while the N-cadherin and Vimentin decreased in CIT silenced cells.Meantime,the EMT regulated factor,Snail and Slug was decreased consistently.In addition,the Hippo-YAP pathway was also tested in CIT knockdown and negative control cells.We found the CIT deficiency would increase the level Mskl/2 and Lats2,promoting the phosphorylation of YAP and reduce the YAP level.Thus,in prostate cells,CIT might inhibit the phosphorylation of YAP via block the activity of Mstl/2 and Latsl,which contribute to maintain the intracellular level of YAP.The YAP would further increase the expression of Snail and Slug.These two proteins mediate the"cadherin switch"and promote the EMT in prostate cancer cells.This mechanism would finally lead the PCa metastasis.