| BackgroundProstate cancer is the most common cancer in man and the second leading cause of death in European counties. Prostate cancer (CaP) is becoming the increasing malignant tumor in China when the quality of Chinese people lives and the number of the people increase greatly. Since androgen ablation is administrated to treat advanced prostate cancer (Pca) and gains significant therapeutic efficacy by Huggins in 1941, hormone therapy has been an important therapy for PCa. In this stage called androgen-dependent prostate cancer (ADPC) the treatment is effective, and the valid rate is up to 70%. After androgen ablation therapy for 12-16 months, androgen- dependent cancer cells inevitably progress to an androgen-independent status (AIPC).And the cancer cells spread into other organs rapidly, and lead to death finally. But no effective treatment has been developed. The treatments of CaP are decided after punatura examation of PSA, which greatly raise the patients'pains. Thus specific, sensitive marker focused on diagnosis and treatments of CaP are expected. So it is become a main task to find an effective medicine to prevent CaP from becoming AIPC.Many studies have been done on the roles of AIPC by microarray and proteomics.Some molecules such as OC, PSAM, PAP, PSMA are the targets in order to treating prostate cancer. But the lower specificity and sensitivity compared with PSA confine them to a small scope, and it is difficult to improve their function.The reason for the occurrence and progression of CaP remain largely unknown. Recent studies suggest that microRNAs (miRNAs) are involved in human tumori genesis. Some aberrantly- expressed miRNAs have been discovered in CaP cell lines and clinical tissues and these CaP-related miRNAs may play critical roles in the pathogenesisCurrently, the studies of microRNA focus on the tumour development, progression and drug resistance. Some microRNAs studies have applied for patients, and the importance of microRNA attract many researchers attentions. Up to 30% of all gene coding proteins are negatively regulated by miRNAs in human cells. It is now evident that 52% of miRNAs located in the fragile site of genes associated with cancers.The regulations of miRNAs expressions can lead to the cancers development and progression.MicroRNAs are coming to be the new clinical assistant methods.1,MicroRNAs are diagnosis tools. The diagnosis of metastatic carcinoma is the main purpose.2,MicroRNAs are prognosis tools. It attracted more attention that poorly differentiated tumours are suitable for the application of microRNAs.And prostate cancers belong to poorly differentiated cancers. And more reports on miRNA differential expression in the CaP and BPH. Thus the study aimed on the transition of PIN into CaP is in the early stage.And the miRNA that works in the progression of ADPC into AIPC need to be deeply studied.In this study we collected 5 ADPC specimens and 5 AIPC specimens,and in gene chip company the differential expression of microRNAs were got by ISH. The results were confirmed through Northern blotting, and the expression of miR-221was the most different one. In our study we conclud that miR-221 plays an important role in the progression of androgen-independence.The function and expression of miR-221 is closely related with the fate of prostate cancer cells.In order to identifying the function of miR-221,we detect the contribution of miR-221 in prostate cancer cell lines. We can find some significant objectives in the treatment of the advanced prostate cancer.ObjectiveGene screening was operated through miRNA chip on the basis of 5 ADPC specimen and 5 AIPC specimen.And miRNA expression database was constructed. The regulation mechanism of miR-221 was identified through prostate cancer cell lines. Seeking the target proteins that were regulated by miR-221, and elucidating the relationship with the AIPC progression.Methods1.MiRNAs screening were operated on the basis of 5 ADPC specimen and 5 AIPC specimen,and the different expression of miRNAs were confirmed through Northern andRT-PCR.2.Constructing AIPC cell lines and verifying the differential expression of miRNA in LNCaP cell lines and LNCaP-AI cell lines. Confirming the prostate cancer cell lines used in study of the biological function. And selecting miR-221 to be studied.3. Constructing the prostate cancer cells invasive model and confirming the effect of androgen on miR-221 and the biological function of miR-221.Studying the roles of miR-221 in the AIPC progression of prostate cancer.4. Indentifying accurate binding site of miR-221 target protein in the gene through bioinformatics and miRNA datebase.Verifying the relationship of the disease with the target gene by Western blot and RT-PCR.Results1. It was found in our study that most AIPC LNCaP-AI lines increased miRNA expression compared with ADPC line, and that five miRNAs (miR-221, miR-222 miR-21, miR-205 and miR-125b) were up-regulated, while two miRNAs (miR-15a and miR-101) were down-regulated in AI LNCaP-AI cell lines compared with their parental AD LNCaP line, by Northern and RT-PCR.And miR-221 was upregulated significantly.2. qRT-PCR was performed to evaluate miR-221 expression in LNCaP, LNCaP-AI and PC3 cell lines. It was found that miR-221 expression was up-regulated in LNCaP-AI independently derived AIPC cell lines and down-regulated in LNCaP dependently derived ADPC cell lines, suggesting that these differentially expressed miRNAs might contribute to the progression of CaP cells.3. Cell growth was assessed by using the CCK-8 cell proliferation assay. Treatment with miR-221 significantly stimulated the growth of LNCaP cells and LNCaP-AI cells, while the miRNA negative control (miR-NC) without treatment failed to affect cell growth. The influence of miR-221m on the S-phase fraction was also upregulated by flow cytometry.4. NE differentiation was studied in LNCaP cells stimulated with increased miR-221 levels.NSE mRNA levels increased rapidly on day.We examined the level of NSE protein in miR-221-and miR-NC-treated LNCaP cells by Western blot. Transfection with miR-221 led to remarkable up-regulation of NSE in LNCaP cell lines on day 3,5. LNCaP cells transiently transfected with miR-221 or miR-NC were grown until confluence in a wound healing assay. Migration of LNCaP-AI cells transfected with anti-miR-221 migrated into the open area was significantly increased.and the ability of migration was upregulated.6. A Matrigel invasion assay was also performed to obtain a measure of the migration of these transfected cells. The results clearly revealed a significant increase in migration and invasiveness in anti-miR-221-treated LNCaP-AI cells. It was concluded that miR-221 was an important determinant of motility and invasiveness of CaP.7. Analyses of the migration relative genes were performed using the Sanger miRNA database target search program.RT-PCR analysis of RNA extracted from anti-miR-221-treated LNCaP-AI cells showed that there was a significant increase in the expression of DVL2. Treatment of LNCaP-AI cells with anti-miR-221 caused a great increase in DVL2 protein in LNCaP-AI cells by Western blot. And DVL2 may play a role in the invasive ability of LNCaP-AI cells.8. We next analyzed the level of miR-221 in the plasma samples from ADPC and AIPC patients by TaqMan qRT-PCR.MiR-221 expression was upregulated in ADPC patients compared with AIPC patients.MiR-221 could be decided as a new tumor-derived marker. Conclusions1. In our study miR-221 promotes the growth of LNCaP and LNCaP-AI cells. Consistently, ectopic introduction of miR-221 in low expressers of LNCaP cells strongly increased their growth potential by inducing a G1-S shift in the cell cycle. In this study, we showed that miR-221 could promote neuroendocrine differentiation of LNCaP cells in an androgen deprived environment. Thus miR-221 plays an important role in the progression of androgen independence.2. Down-regulation of miR-221 in LNCaP-AI cells greatly increased the number of cells migrating through the transwell lambers. We therefore conclude that miR-221 might not affect the ability of migration of LNCaP cells significantly; Rather, a poor invasive CaP cell line and miR-221 level might inversely correlate with the invasiveness in androgen independence CaP. We have demonstrated the importance of miR-221 in the migration on LNCaP-AI cells that are strongly invasive and androgen-independent in nature. In the present study, we chose one target mRNA DVL2 that was correlated with the migration. As expected, the expression of DVL2 was up-regulated in LNCaP-AI cells transfected with anti-miR-221.And the reason for it need to be investigated deeply.3. We analyzed the level of miR-221 in the plasma samples from ADPC and AIPC patients by TaqMan qRT-PCR.MiR-221 expression was upregulated in ADPC patients compared with AIPC patients. These results suggest that tumor-derived miR-221 in plasma would be a blood-based miRNA biomarker candidates for CaP. |
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