| BackgroundProstate cancer is the first most common male malignant cancer in America and Europe. However, with the length of average life span and the continuous change of life style as well as the structure of diet, both the morbidity and mortality of prostate cancer are gradually increased in China, especially in developed districts. Take Shanghai for example, the morbidity of prostate cancer is higher than that of bladder tumor and renal carcinoma, prostate cancer becomes the fifth most common male malignant cancer and the first most common urinary tumor. Prostate cancer can develop from focal lesions into locally advanced prostate cancer and advanced prostate cancer. At present, the therapeutic regimens of prostate cancer depend on the its clinical stage, patients in early stage and locally advanced stage can be cured by following radical prostatectomy or radiotherapy; and patients in advanced stage and suffered from metastasis can receive androgen deprivation therapies such as surgical castration, chemical castration and so on. Nevertheless, most patients present good reaction to androgen deprivation therapies in the early18-24months, then some patients inevitably occur many characteristics, such as the increase in prostate specific antigen (PSA), regrowth of tumor and multiple bone metastasis, then prostate cancer develops into hormone refractory prostate cancer (HRPC). No ideal therapy for HRPC is developed now. Therefore, the oncogenesis and development of prostate cancer, especially the development of process, becomes the international hotspot.Multiple studies showed that the disturbed cell generation cycle played a key role in oncogenesis and development, and the disturbance of promotion in Gl stage was closely associated with oncogenesis and development. P27protein is one of the important members of cyclin-dependent kinase inhibitor (CDKI), it can negatively regulate cell cycle. p27plays its inhibitory effect on cyclin-CDK complexes mainly by binding to cyclin. p27plays its inhibitory effects on CDK in two aspects:firstly, it can inhibit the activity of activated CDK binding to cyclin; secondly, it can also inhibit the progression from G1stage to S stage by inhibiting the activation of CDK. P27is considered as a tumor-inhibiting factor at present, and plays a key role in oncogenesis and development. P27primarily plays its inhibitory effects by the negative regulation to cell cycle, inducing cell apoptosis and inducing cell differentiation. However, multiple studies observed the decrease in p27protein in many tumor tissues, suggesting the decrease in p27protein was closely related to oncogenesis and development, but its exact mechanism remained uncertain.P27gene mutation rarely occurs in malignant tumors, and it has no correlation with any hereditary tumors. However, Cave et al. observed a single nucleotide transition at the109th codon in the polymorphism of p27gene, which resulted in the amino acid change from Val to Gly. This polymorphism accounts for11%-26%in patients with tumors, but it accounts for about39%in normal people and tumor adjacent tissues. Keibel et al. studies the correlation between Val109Gly polymorphism of p27gene and the onset risk of prostate cancer, among96patients with advanced prostate cancer,67(70%) were VV homozygotes,24(25%) were VG heterozygotes,5(5%) were GG homozygotes, statistical analysis showed that the polymorphism of p27gene at109th codon was correlated with the susceptibility of advanced prostate cancer. Patients with VV genotype were more susceptible to HRPC (OR=2.11,95%CI=1.05-4.22). But rare studies on the correlation between the polymorphism of p27gene and prostate cancer are reported so far.The oncogenesis and development of tumors attribute to multiple factors in vivo and in vitro, more and more studies show that the polymorphism of human genome is closely associated with the oncogenesis and development of tumors. Different genotypes of the same gene may have absolutely opposite effect in different tumors. As an important tumor-inhibiting factor, p27plays key roles in regulating cell cycle and inducing cell apoptosis and cell differentiation, therefore, the polymorphism of p27gene probably affects the progression and prognosis of prostate cancer. However, the correlation between the polymorphism of p27gene and the progression as well as prognosis of prostate cancer is rarely studied at present. This study aims to investigate the relationship between the val109Gly polymorphism of p27gene and the regulation of cell cycle as well as cell apoptosis of prostate cancer in detail by using in vitro experiment, and clarify whether the Val109Gly polymorphism of p27gene affects the progression and prognosis of prostate cancer combined with clinical data.Part â… Eukaryotic expression vector construction of human p27gene mutantObjectiveTo study the Val109Gly polymorphism of p27gene by constructing the eukaryotic expression vector of p27gene mutant using point mutation.Materials and MethodspcDNA3.1-p27plasmid (kindly provided by Dr. Wang ping from Department of Urinary Surgery, the First Affiliated Hospital of Zhejiang University) was used as the template to design the point mutation primers, all procedures were performed according to the protocol of D0206gene site-directed mutagenesis kit (Beyotime). Then the products were sequenced, cloned and amplified.ResultsThe eukaryotic expression vector of p27gene mutant p27(G/G) was successfully constructed, this vector could effectively express in prostate cancer cells. This plasmid and wild type pcDNA3.1-p27(V/V) were respectively transfected into prostate cancer cells to systematically investigate the effect of the polymorphism of p27gene on the functions of p27in prostate cancer cells.Conclusion1. In this part of experiment, the eukaryotic expression vector of p27gene mutant p27(G/G) was successfully constructed.Part â…¡ The effect of the polymorphism of p27gene on LNCap cell proliferationObjectiveTo analyze the effect of gene polymorphism on the antiproliferative action of p27by determining the indexes of cell proliferation in groups transfected with plasmid [blank pcDNA3.1, wild type pcDNA3.1-p27(V/V), mutant type pcDNA3.1-p27(G/G)].Materials and MethodsEmpty plasmid, wild type pcDNA3.1-p27(V/V) and mutant type pcDNA3.1-p27(G/G) were respectively transfected into PC3cells, and the empty plasmid group was served as the control group. Then the cell proliferation was determined at0h,24h and48h by using MTT.ResultsMTT assay found that wild type pcDNA3.1-p27(V/V) showed no statistical difference compared with control group at24h (p<0.05); ConclusionWild type pcDNA3.1-p27(V/V) showed different inhibitory effect on cell proliferation compared with control group at48h.Part â…¢ The effect of the polymorphism of p27gene on LNCap cell cycleObjectiveTo analyze the effect of the polymorphism of p27gene on PC3cell cycle by determining the respective cell cycle of PC3transfected with different plasmids [empty plasmid, wild type pcDNA3.1-p27(V/V), mutant type pcDNA3.1-p27(G/G)].Materials and MethodsEmpty plasmid, wild type pcDNA3.1-p27(V/V), mutant type pcDNA3.1-p27(G/G) were respectively transfected into PC3cells, and cells were collected at48h to determine the distribution of cell cycle in each group by using flow cytometry (FCM)(PI staining).ResultsCompared with control group, the proportion of G0/G1stage cells was gradually increased in wild type pcDNA3.1-p27(V/V) and mutant type pcDNA3.1-p27(G/G) transfection group, and the proportions of S stage and G2/M stage cells were relatively decreased, but no statistical difference was observed between wild type pcDNA3.1-p27(V/V) transfection group and mutant type pcDNA3.1-p27(G/G) transfection group. At48h, wild type pcDNA3.1-p27(V/V) and mutant type pcDNA3.1-p27(G/G) transfection group showed difference in G0/G1stage compared with that of control group (p<0.05), this difference was also observed between wild type pcDNA3.1-p27(V/V) and mutant type pcDNA3.1-p27(G/G).Conclusion1. p27could block PC3cells in G0/G1stage, but mutant type pcDNA3.1-p27(G/G) showed a stronger block effect than wild type pcDNA3.1-p27(V/V).Part â…£ The effect of the polymorphism of p27gene on the cell apoptosis and apoptosis-related proteins of PC3cellsObjectiveTo analyze the effect of the polymorphism of p27gene on PC3cells and preliminarily explore its apoptotic mechanism by determining the apoptotic rate and apoptosis-related proteins of PC3cells transfected with different plasmids [empty plasmid, wild type pcDNA3.1-p27(V/V), mutant type pcDNA3.1-p27(G/G)] at different time points.Materials and MethodsEmpty plasmid, wild type pcDNA3.1-p27(V/V), mutant type pcDNA3.1-p27(G/G) were respectively transfected into PC3cells, and cells were collected at24h and48h to determine the apoptotic rate by using FCM (AV+PI staining). The expressions of apoptosis-related proteins (bcl-2, bax, caspase-3, PARP) at24h and48h were determined by using Western blot.ResultsCompared with control group, the apoptotic rate of PC3cells was increased in wild type pcDNA3.1-p27(V/V) and mutant type pcDNA3.1-p27(G/G) transfection group. At48h, mutant type pcDNA3.1-p27(G/G) showed a stronger promotion of cell apoptosis compared with wild type pcDNA3.1-p27(V/V). Mutant type pcDNA3.1-p27(G/G) showed an increase in bcl-2and bax expression and caspase-3activation compared with wild type pcDNA3.1-p27(V/V), which was well coincidence with the apoptotic rate results determined by using FCM.Conclusion1. Both wild type pcDNA3.1-p27(V/V) and mutant type pcDNA3.1-p27(G/G) could increase the apoptotic rate of PC3cells.2. Mutant type pcDNA3.1-p27(G/G) showed a stronger effect on inducing cell apoptosis than wild type pcDNA3.1-p27(V/V).Part V The effect of the polymorphism of p27gene on the prognosis of patients with prostate cancerObjectiveTo understand the distribution of the polymorphism of p27gene in our hospital, and explore the relationship between the polymorphism of p27gene and the prognosis of patients with prostate cancer.Materials and MethodsA total of67patients with prostate cancer and received androgen deprivation therapy in our hospital from2006to2008were admitted in our study to analyze the p27genotypes and follow up their prognosis, for patients followed up for more than two years or died, the time of hormonal resistance and death time of were recorded. The relationship between the polymorphism of p27Kip and death was analyzed by Oneway ANOVA, and Fisher's Exact testwere used to analyze the risk factors affecting the prognosis of patients.ResultsAmong these67patients,57were V/V homozygotes(androgen-dependent/androgen-independent22/35),5were V/G heterozygotes(androgen-dependent/androgen-independent3/2),5were G/G homozygotes((androgen-dependent/androgen-independent4/1).ConclusionThe result showed,the The distribution of p27genotypes were not different between the patients with prostate cancer and contral team. But, the polymorphism of p27Kip have relationship with prognosis of patients.
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