| Prostate cancer is the most commonly malignant tumor and the second leading cause of death cancer in North American and European men. Its morbidity is much more lower in Chinese men than in North American and European. However, the morbidity has increased in recent years, and prostate cancer has been becoming the third genitourinary system tumor. Prostate cancer is becoming a severe threat to people'health. Pathogenesy of prostate cancer are drawing more and more attention. The traditional methods are hormone therapy, radiotherapy, chemotherapy and operation. Researchers are searching for new therapies.There was new wish for oncotherapy since gene therapy emerged in last century. Now researchers are searching for new gene therapies. A20 was first studied in human umbilical vein endothelial cells and identified as a tumor necrosis alpha (TNF) inducible primary response gene. Isolation of the full length cDNA followed by sequence analysis has revealed that A20 coded a zinc finger protein. The ability of A20 to inhibit opoptosis induced by TNF has already been observed. High and stable expression of A20 can inhibit opoptosis induced by TNF in many cell lines: MCF-7 breast cancer cells, beta cell of islet, human umbilical vein endothelial, WEHI 164 and NIH 3T3 fibroblast cell lines. However, A20 does not increase in human hepatoma HepG2 cells, lung epithelial A549 cell, or human cervix carcinoma Hela cells. The reason why some cell lines are protected by A20 and others are still unclear.RNAi is a process of mRNA cleavage and degradation that is induced by double-stranded RNA in a sequence-specific manner. Since the process is triggered after transcription, it is also named post transcriptional gene silencing ( PTGS ). Now, because of its high efficiency and specificity, RNAi are generally applied in researches of gene function and treatment.To study the expression and significance of A20 in prostate cancer cell lines PC-3,PC-3M,DU145,LNcap and 22RV1, RT-PCR and Western blot were used. The results showed that level of protein and mRNA are higher in PC-3 and PC-3M cell lines than in DU145,LNcap and 22RV1 cell lines (p<0.01). The results demonstrates that expression of A20 in prostate cancer cell lines which may be related with genesis mechanism of prostate cancer.According to the A20 gene sequence and principle of siRNA design, three pairs complementary oligonucleotides were designed and synthesized, Then three small hairpin RNA ( shRNA ) expression vector were constructed. We get 3 recombinate siRNA expression vectors: pSilencer3.1-A1 ,pSilencer3.1-A2 and pSilencer3.1-A3; and then, DNA sequence analysis to recombinate siRNA expression vectors were correct; last, the recombinated expression vectors were transfected, and cell clones were screened with optimizing hygromycin concentration. Then RT-PCR and Western blot were used to detect expression of A20; anti-apoptosis index and cell cycle were detected by FCM; survival curve of PC-3 cell was demonstrated by MTT assay. The results of RT-PCR and Western blot indicated that recombinated pSilencer3.1-A1 and pSilencer3.1-A3 vector inhibited expression of A20 at mRNA and protein level (p<0.01); the apoptosis index increased by 11.9% and 7.7%; however, there is no obvious change with the survival curve and cell cycle in PC-3 cell.In conclusion, A20 is highly expressed and plays anti-apoptosis role in PC-3 cell, that is to say, A20 can protect PC-3 cell to some extent. These results suggest that A20 probably become a novel target of gene therapy in prostate cancer. |
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