Inhibitory Effect of Calcium/Calmodulin-dependent Protein Kinase Ⅱ Inhibitor1(CAMK2N1) on Prostate Cancer Cell Growth
|School||Huazhong University of Science and Technology|
|Keywords||Calcium/calmodulin-dependent protein kinase Ⅱ inhibitor1 Expression plasmid Genetransfer Gene expressionProstate cancer In vitro Cell proliferation Cell cycleProstate cancer Protein expression Immunohistochemistry Clinicopathology|
Part1Amplifying CAMK2N1plasmid vector and up-regulating CAMK2N1expression in vitroObjectiveTo amplify CAMK2N1plasmid vector (pCMV6-AC-GFP-CAMK2N1) and test if it could up-regulate CAMK2N1expression in prostate cancer cells PC3.Methods(1) pCMV6-AC-GFP-CAMK2N1was transferred into E. colli competent cells DH5a and amplified. CAMK2N1plasmid vector sequence was evaluated by DNA sequencing.(2) pCMV6-AC-GFP-CAMK2N1was transferred into prostate cancer cells PC3. After24hours, the expression level of CAMK2N1mRNA and protein was detected by PCR and Western blotting respectively. Results(1) Enough CAMK2N1plasmid vector was harvested and its sequence was confirmed.(2) After transferring pCMV6-AC-GFP-CAMK2N1plasmid into PC3cells, the mRNA and protein expression level of CAMK2N1was up-regulated.ConclusionpCMV6-AC-GFP-CAMK2N1plasmid could up-regulate CAMK2N1expression successfully, which established the essential provision for our further study. Part2CAMK2N1inhibits the growth of prostate cancer cells in vitroObjectiveTo study the impact of up-regulating CAMK2N1on human prostate cancer cells by transfering pCMV6-CAMK2N1plasmid into PC3cells, including cell growth, cell proliferation, cell cycle, cell apoptosis and cell invasion.MethodsPC3cells were grown in RPMI1640medium containing10%(v/v) FBS,0.37%(w/v) NaHCO3, penicillin (100U/ml) and streptomycin (100U/ml) in a humidified incubator under5%CO2and95%air at37℃, and divided into three groups:blank group, control group, CAMK2N1group.24hours after control group transferred with pCMV6-AC-GFP and CAMK2N1group transferred with pCMV6-AC-GFP-CAMK2N1, cell growth was determined by Cell Counting Kit-8, cell proliferation was detected by5-ethynyl-2’-deoxyuridine assay. Cell cycle and apoptosis were determined by flow cytometric assay. Cell invasion was detected by matrigel transwell invasion test.ResultsCompared to blank group and control group, the cell growth, cell proliferation and cell invasion of CAMK2N1group was suppressed, and the cell cycle was arrested at G0/G1phases. There were no differences in cell apoptosis test.ConclusionpCMV6-CAMK2N1plasmid could effectively up-regulate CAMK2N1expression in vitro. Overexpression of CAMK2N1could inhibit the growth, cell proliferation and cell invasion of PC3cells, lead to cell cycle arrest at G0/G1phases. This study shows CAMK2N1probably has the nature of tumor suppressor genes in prostate cancer. Part3Expression of CAMK2N1in human prostate cancer and its clinical significanceObjectiveIn last part we have showed that overexpression of CAMK2N1could inhibit the prostate cancer cells growth in vitro. The aim of this part is to explore the expression of CAMK2N1in human prostate cancer and its clinical significance.MethodsThe prostate tissue samples were obtained from78patients undergoing prostatectomy or prostate biopsy in Tongji hospital from2007.10～2011.4, and were divided into3groups:(1) prostate cancer group (n=34);(2) benign prostatic hyperplasia (BPH) group (n=28);(3) normal prostate tissue group (n=16). The localization and expression of CAMK2N1in prostate tissues were determined by immunohistochemical and immunofluorescence. The relationships between CAMK2N1expression level and clinicopathologic variances were analyzed.Results(1) CAMK2N1protein expressed in all three goups, which localized in cytoplasm.(2) The expression level of CAMK2N1protein in prostate cancer group was significantly lower than that in BPH group and normal group. In prostate cancer group, the expression level of CAMK2N1was negatively correlated with extraprostatic extension, tumor stage, histological grade and Gleason score.(3) The expression level of CAMK2N1was negatively correlated with that of Ki-67and positively with that of p27Kip1.ConclusionThe expression of CAMK2N1is downregulated in prostate cancer, and the abnormal degree was correlated with clinicopathologic variances. CAMK2N1may participate in the pathogenesis of prostate cancer through effecting some proteins involved in cell proliferation and cell cycle.