| Backgroud and Objective: Prostate cancer(PCa) is one of the most common diseases threat to men’s health. The incidence presents an exponential growth for the men older than50 years old especially. Compared with the developed areal such as Europe and the United States etc, china has a lower incidence. In 2002, the incidence of PCa standardized by age in china was 1.6 per 100,000 lower than 124.8 per 10,000 in USA. However, the incidence rate has been increasing year by year with the change of life span and living condition. To date, the pathogenesis of PCa is unclear, and clinical symptom of PCa is atypical,accompanied with the lack of the screening for prostate specific antigen(PSA).About 1/3 of patients can not be operated with the existence of local advanced and/or distant metastasis at the time of diagnosis. Retrospective studies show that the median age of PCa is 72 years old, consideration of the life expectancy and health condition, androgen deprivation therapy(ADT) plays an important role for delaying progression of PCa and improving quality of life.Since 1940, the Huggins and Hodges has proved the positive effect of ADT for PCa,which became a main treatment strategy for PCa, although the most of patients will irreversibly develop into castration resistant prostate cancer after 12-30 months for ADT.However, the signal pathway of androgen has always being actived, which makes sense for the progression of PCa.Our previous experiment has proved that androgens upregulate the aquaporin9(AQP9)expression in the prostate, the relationship between androgen and AQP9 is linkage, We therefore speculate that prostatic epithelial cells may survive while they both express AQP9 and maintain cellular osmotic pressure by androgen. Whether prostate cancer cells have the similar nature and will undergo apoptosis after interfering the expression the AQP9 are in question. The present experiment is on the basis of this hypothesis, and try to find a way to explain the relationship between AQP9 and apoptosis, so we can make a new opinion for PCa gene therapy.Objective:(1) To culture prostate cancer cell line of pc-3, to investigate the expression of AQP9 in the cells and to explain the differences.(2) To synthetic small interference RNA(si RNA)for AQP9(human, h), to interfere the expression AQP9 in pc-3 cell line and to examine the AQP9 m RNA and protein content after interfering. To detect the effects of AQP9 silenced on pc-3 cell line proliferation and apoptosis, to explore a possible gene delivery strategy for PCa gene therapyMethods:(1) Pc-3 cell line were cultured in Ham’s F12. Lncap cell line were cultured in RPMI 1640. Cell immunofluorescence method and western blot were used to analyze the expression of AQP9 in prtostate cancer cell lines. Set hepatic tissue as a positive control.(2) The experiment were divided into three groups: si RNA transfection,lipofectamine 2000 control, and blank control. Pc-3 cells were transfected with designed si RNA using the liposomes method when the cells’ confluence meets 60%-80%, the expressions of AQP9 were determined by Q-PCR and Western blot(3) the proliferation and apoptosis of the pc-3 cell line in the three groups were detected by MTT and flow cytometry.Results:(1) The positivity immunoreactivity were mainly observed in the cytoplasm of pc-3 and lncap cell lines, western blot analysis revealed that the bands at 26-34 k Da.The AQP9 protein expression level of pc-3 cell lines was 0.90 土 0.01, compared with the lncap cell lines 0.57 土 0.01, hepatic tissue was 8.1±0.01, There was significant difference of AQP9 protein level between in the pc-3 cell line and the lncap cell line,but the exprssion of AQP9 protein in hepatic tissue was the highest(P <0.01).(2) The expression level of AQP9 m RNA 0.12±0.02 in the transfected pc-3 cell line was decreased, with statistically significant difference from the blank control group6.77±0.39 and lipo2000 control group 6.67±0.3(P<0.01) by real-time q PCR detected.The AQP9 protein expression level of the transfected group was 0.0363±0.02 at 48 hours, markedly decreased as compared with the blank control group 0.109±0.001 and lipo2000 control group 0.12±0.011(P<0.05) with western blot analyzed. There were not significant difference between blank control group and lipo2000 control group(P>0.05).(3) The pc-3 cell line growth was inhibited by RNAi with MTT test. There was significant differece between transfection group and blank control group(P < 0.01). The A450 of transfected group was 0.32±0.03, blank control group 0.58±0.08,lipo2000 control group 0.61±0.11; The pc-3 cell lines growth inhibition rate was 43.6% at 48 hours after transfection, There were not significant difference between blank control group and lipo2000 control group(P>0.05).(4) The rate of apoptosis pc-3 cell line was 4.63% ± 0.09% in the experimental group significant difference higher(P<0.01) compared with the blank group and the lipo2000 control group 1.6% ± 0.15% and 1.61% ± 0.05%,There were not significant difference between blank control group and lipo2000 control group(P>0.05).Conclusion:(1) AQP9 is expressed in pc-3 and lncap cells. The positive immunoreactivity were mainly observed in the cytoplasm. High expression level of AQP in pc-3 cell is associated with the high metabolic state.(2) The expression of AQP9 gene in pc-3 cell lines can be inhibited obviously both at m RNA and protein levels by the RNAi that targeting AQP9. Target-silencing AQP9 gene can inhibit cell proliferation and induced apoptosis in pc-3 cell lines,the apoptotic cells were mainly at the early apoptotic stage. There may be a possible gene delivery strategy for PCa gene therapy... |
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