Study Of CaMK?? Promotes Prostate Cancer Invasion And Metastasis Based On Bioinformatics

1.Apply methods of the bioinformatics to screen potential genes associated with metastatic prostate cancer,in order to improve the understanding of the mechanisms underlying PCa metastasis.2.Examine the samples of clinical prostate cancer to verify the expressions of differentially expressed genes(CaMK ??)and analyze its clinical influence.3.Investigate the effects of CaMK ?? on the proliferation,migration and invasion of human prostate cancer(PC-3)cells and explore the possible molecular mechanisms that promote the invasion and metastasis of prostate cancer.Methods:1.The GSE3325 microarray dataset,which was downloaded from the Gene Expression Omnibus database,consist of seven clinically localized PCa samples,six hormone-refractory metastatic PCa samples and six benign prostate tissue samples.The Linear Models for Microarray Data package was used to identify differentially expressed genes(DEGs)and a hierarchical cluster analysis for DEGs was performed with the pheatmap package.Furthermore,potential functions for the DEGs were predicted by a functional enrichment analysis.Subsequently,microRNAs(miRNAs)potentially involved in the regulation of PCa metastasis were identified by WebGestalt software,and the DEGs-miRNA regulatory network was visualized using Cytoscape.In addition,the KEGG-based pathway enrichment of the DEGs was analyzed in the regulatory network.2.Thirteen fresh frozen specimens of radical prostatectomy and 60 samples of fixed specimens of paraffin were obtained from Renmin hospital of Wuhan University.The expression of CaMK ?? mRNA was detected in prostate cancer tissues and adjacent tissues with RT-PCR.Immunohistochemistry was used to detect the expressions of CaMK II 8 protein and ?-catenin and analyze the relationship between CaMK ?? and prostate cancer PSA,Gleason score,T stage,pathological stage,and lymph node metastasis and the relationship between CaMK ?? and?-catenin expressions.3.We used in vitro approaches to determine whether silencing of CaMK ?? affects cell proliferation,migration,and invasiveness.Human CaMK ?? siRNA was transfected into PC-3 cells by Lipofectamine RNAiMAX.CaMK ?? mRNA and protein were detected by real-time PCR and Western blot.MTT assay was performed to measure cell proliferation,wound migration assay and transwell assays were used to measure the cell migration and invasion.4.CaMK ?? downexpression effect on the Wnt signaling pathway was investigated through TOP/FOP flash assay.The effect of CaMK ?? downexpression on the Wnt signaling pathway was further investigated through WB analysis against downstream genes of the pathway.Expression level of total ?-catenin,nuclear?-catenin protein and downstream target protein such as LEF-1,Axin2,C-myc,Cyclin-D1 was detected after CaMK ?? siRNA was transfected into PC-3 cells.Results:1.A total of 306 and 2,073 genes were differentially expressed in the clinically localized PCa and the metastatic PCa groups,respectively,as compared with the benign prostate group,of which 174 were differentially expressed in both groups.A number of the DEGs,including CaMK ?? and SH3BP4,were significantly enriched in the cell cycle,and others,such as MAF,were associated with the regulation of cell proliferation.Furthermore,some DEGs(CaMK ?? and PCDH17)were observed to be regulated by miR-30,whereas others(ADCY2,MAF,SH3BP4 and PCDH17)were modulated by miR-182.Additionally,ADCY2 and CaMK ?? were distinctly enriched in the calcium signaling pathway.2.CaMK ?? mRNA and protein are overexpressed in human prostate cancer tissues,and the expression is positively correlated with pathological stage,lymph node metastasis,and T stage of prostate cancer,but is not related to PSA and Gleason score.These findings indicate that CaMK ?? protein expression is closely related to the invasion and metastasis of prostate cancer.The expression of CaMK ?? protein is significantly correlated with that of ?-catenin protein in prostate cancer tissues.3.CaMK ?? siRNA was stably transfected into PC-3 cells,compared with untreated cells,the protein and mRNA expression levels of CaMK ?? in PC-3 cells transfected with CaMK ?? siRNA were decreased significantly,MTT assay revealed the proliferation of PC-3 cells was significantly inhibited when transfected with CaM ?? siRNA,and the abilities of migration and invasion of PC-3 were decreased(P<0.01).4.The TOP/FOP flash luciferase reporter plasmid showed that Wnt/p-catenin signaling pathway was inhibited.Silent CaMK ?? gene expression inhibits the intranuclear ?-catenin(nuclear ?-catenin),but without affecting the expression of total ?-catenin in the cell,and the downstream expression of LEF-1,Axin2,C-myc and Cyclin D1 were inhibited in the Wnt/?-catenin signaling pathway(P<0.01).Conclusions:1.Our bioinformatics study identified novel DEGs,including CaMK ??,ADCY2,MAF,SH3BP4 and PCDH17,that may be involved in the metastasis of PCa.2.CaMK ?? is overexpressed in human prostate cancer tissues,and the expression is related to the invasion of prostate cancer and the metastasis of lymph node.3.RNAi silencing of CaMK ?? inhibits the proliferation,migration,and invasion of prostate cancer PC-3 cells in vitro,which may be related to its inhibition of Wnt/?-catenin signaling pathway.