Study On RNA Interference Targeting SPK1 In Prostate Cancer DU145 Cell Lines

BackgroundProstate cancer is one of the most common malignant tumor of the male urogenital system. In China, it' s mobidity has been rising yearly in the recent decade . Endocrine therapy of androgen blockade is the mainly treatment for prostate cancer at present. But most patients after endocrine therapy usually change into androgen-independent and become hormone refractory prostate cancer ,recurrence or metastasis .Traditional surgery, radiotherapy, chemotherapy and endocrine therapy are ineffective for hormone refractory prostate cancer. Many scholars explore at the level of gene regulation and control to the treatment of prostate cancer,and look for a new target for treatment to make a breakthrough . In recent years, studies have proved that membrane sphingomyelin derivatives include ceramide (Cer), sphingosine (Sp), sphingosine-1- phosphate (S1P) and other metabolites .They plays a key role in the dynamic equilibrium regulation of cell proliferation and apoptosis . The sphingosine kinase (SPK) is important rate-limiting enzyme to maintain the intracellular balance of these substances. It was important regulatory factors of cell proliferation and survival. The cell levels of these sphingolipid metabolites are regulated by sphingosine kinase (SPK) which phosphorylates Sp to form S1P and regulates its cellular levels. Sphingosine kinase signaling pathway are closely related with cell apoptosis, growth and proliferation.RNA interference (RNAi) is recently developed a new gene therapy and diagnostic technology. RNA interference happen in a variety of biological cells .It is special phenomenon that double-stranded RNA mediated homologous sequence specific mRNA degradation and led to gene silencing. We envisage that RNA interference cause SPK1 gene silencing, thereby inhibiting the expression of SPK1, regulating ceramide, sphingosine-l-phosphate,sphingosine intracellular level, Induced apoptosis of hormone-independent prostate cancer cell. It was explored for hormone-independent prostate cancer gene therapy.OBJICTIVER: SPK1 signal molecule expression were examined in the hormone-independent prostate cancer cells line Du145. SPK1 and Myeloid cell leukemia-1 (Mcl-1) expression were examined after recombinant adenovirus Ad-H1-SPK1 mediated SPK1 interference on Dul45 cell .It was discussed that SPK1 interference play role on Du145 cell apoptosis and possible mechanism. We want to look for a new target for hormone-independent prostate cancer gene therapy.Method: The hormone-independent prostate cancer Du145 cell was selected as experimental subject. Recombinant adenovirus Ad-H1-SPK1 which carry SPK1 specific interference sequence was built and prepared, as an research tool to mediat RNA interference for SPK1 gene. RT-PCR method was used to measure SPK1 signal molecule expression on Du145. Infected efficiency of Du145 was tasted with adenovirus Ad-GFP as a reference. Adenovirus Ad-H1 which not to carry SPK1 specific interference sequence was as control. SPK1 and Mcl-1 expression was measured by Western blotting. Influence of SPK1 and Mcl-1 protein expression were evaluated after SPK1 interference. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide( MTT) dye reduction assay. Cell viability was assessed by MTT after the indicated times in Dul45 cells treated with doxorubicin 1.0μg/ml and infected by adenovirus Ad-H1 or Ad-H1-SPK1. Doxorubicin-induced proliferation inhibition on Du145 was evaluate after SPK1 interference by MTT. The percentages of apoptotic cell were determined by flow cytometry analysis. Camptothecin-induced apoptosis of Du145 was evaluated after SPK1 interference.Result: The results showed that Du145 cell expressed SPK1 signal molecule. Infected efficiency of Dul45 cells by the adenovirus is 99.92% percent(MOI=100) and meet the need of the follow-up experiment. SPK1 and Mcl-1 protein expression were significantly inhibited. Cell viability of (Ad-H1-SPK1 and doxorubicin 1μg/ml )group was lower control group (Ad-H1 + doxorubicin 1μg/ml), 48 hours for 71.2% of control and 72 hours 61.3% of control. Average cell apoptosis rate of (Ad-H1-SPK1 and camptothecin 1μM) group (58.3%) was higher than (Ad-H1 and camptothecin 1μM )group(29.1%).The difference between grops was significant (P<0.01).Conclusion: Taken together ,these results show that SPK signaling pathway can regulate prostate cancer cell apoptosis, SPK1 interference can enhance the hormone-independent prostate cancer cells Du145 docrobicin-induced proliferation and camptothecin-induced apoptosis. These results suggest that SPK1 may be a potential new therapeutic target in hormone-independent prostate cancer cell.