| BackgroundProstate cancer (PCa) remains the second leading cause of cancerrelated death among American men. Traditional therapy for PCa has not changed substantially over the last few decades in that surgicalremoval of the prostate (prostatectomy) remains the basis for primaryPCa treatment. Ablative radiation therapies and cytotoxic chemotherapeutic agents in numerous clinical trials and in practice have an overall limited treatment efficacy. after prostatectomy of primary PCa, although generally efficacious in the short term, fails when a subset of the carcinoma cells becomescastrate-resistant (insensitive to withdrawal of androgen and/or androgensignaling blockade by administration of androgen antagonists,such as bicalutamide). The generation of new molecularly targeted therapies is required to improve PCa patient care, treatment, and prognosis. Evasion of apoptosis (programmed cell death to prevent uncontrolled growth) is a known hallmark of cancer progression and a means by which cancers develop resistance to chemotherapeutic treatments. Castrate-resistant PCa (CRPC) may be overcome by either sensitizing cancer cells to apoptotic stimuli or circumventing upstream blockages in apoptotic signaling. Such restoration of apoptosis in PCa cells would counterbalance the proliferative and invasive burdens imposed on the host by localized primary and disseminated metastatic tumor cells.The growth and survival of prostate cancer cells in androgen depleted conditions has been attributed to a variety of mechanisms. Advanced-stage breast cancers frequently metastasize to the bones and cause bone destruction, butthe underlying mechanism is not fully understood. This study presents evidence that TGF-β-activated protein kinase1(TAK1) signaling in tumor cells promotes bone destruction bymetastatic breast carcinoma cells, controlling expression of pro-metastatic factors, including MMP-9and COX2. Suppression of TAK1signaling by dominant-negative (dn) TAK1in breast carcinoma MDA-MB-231cells impairs bone colonization by carcinoma cells and bone osteolysis in the intra-cardiac injection model. Mechanistic studies showed that inhibition of TAK1by dn-TAK1or siRNA blocked expression of factors implicated in bone metastasis, such as MMP-9,COX2/PTGS2, PTHrP, and IL8.So this study silence TAK1gene expression by siRNA of people prostate cancer DU145cell line, gene knockout TAK1preliminary research on prostate cancer DU145cell proliferation, apoptosis, invasion, migration and drug sensitivity of change and mechanism of action. In prostate cancer development, transfer, resistance, etc. to lay a good foundation for further study on the mechanism of action of TAK1gene.Objective Research the interference effect of TAK1siRNA target, and detecting the effect of silence TAK1expression on cell proliferation, apoptosis, invasion, migration and the influence of the drug sensitivity and related protein expression of prostate cancer DU145, and then to discuss its mechanism of action.Method1. Using RNAi silencing to influence the TAK1gene in high expressing cell lines of DU145. Using SqRT-PCR to detect the expression and silencing efficiency of TAK1mRNA, and calculate the TAKlgene silencing rate.2. Application of western blot to assay the expression changes of TAK1protein before and after the transfection.3. After TAK1gene expression was successfully silenced, drawing the growth curve to detecting the change of cell proliferation of prostate cancer.4. Using Transwell Chambers and scratch test to analyze the efffect of TAK1siRNA on the invasion and migration ability on prostate cancer cells.5. Using extracellular matrix induced bone marrow (BM-ECM) and transwell Chambers (8.0μm and8.0μm) to build bone metastasis model in vitro, and observe the BM-ECM effect on the promote growth and chemotaxis on prostate cancer cells.6. Using flow cytometry instrument to test the apoptosis changes before and after the prostate cancer cell was transfected TAK1siRNA.7. Using MTT method to detect the effect of TAK1siRNA on three kinds of chemotherapy drugs sensitivity docetaxel, L-OHP and5-fluorouracil (5FU)) on prostate cancer DU145cells.8. Before and after TAK1siRNA was transfected,The transcription and expression change of JNK、Bcl-2、COX-2、(3-catenin、MMP-2、9、EGFR were detected by qRT-PCR and Western blot.Results1. Using Lipofectamine2000transfect TAK1siRNA can effective knockout TAK1gene expression, and RT-PCR and western blot analysis suggests that TAK1siRNA can significantly inhibit TAK1gene transcription and translation, the difference was statistically significance (P<0.05).2. Cell growth curve suggests that when TAK1of prostate cancer DU145was silenced using TAK1siRNA, cell proliferation ability was weakened. The difference was statistically significance Starting from the fourth day (P<0.05). 3. Transwell and scratch test suggests that the invasion and migration ability of prostate cancer DU145cell were declined when transfected by TAK1siRNA, the difference was statistically significance (P<0.05).4. The apoptosis rate of prostate cancer cells before and after transfected were4.13±0.11%, and13.21±1.41%, knockout TAK1gene expression can significantly increase the apoptosis rate of prostate cancer DU145cells. Comparison between the two groups, the difference was statistically significance (P<0.05).5. Extracellular matrix induced by bone marrow (BM-ECM) have promote growth, invasion and migration effect on prostate cancer DU145cells have to promote growth, invasion and migration effect, the difference was statistically significance (P<0.05).6. When transfected by TAK1siRNA, the IC50of docetaxel, L-OHP and5-fluorouracil on prostate cancer DU145cells were significantly lower than the negative control group. TAKl gene silence can significantly increase sensitivity to chemotherapy drugs of prostate cancer DU145cells, the difference was statistically significance (P<0.05); Between the control group, the difference of IC50of three drugs have no statistically significance (P>0.05).7. When the prostate cancer wasTransfected after48h, the transcription and translation of COX-2, the Bcl-2, JNK,β-Catenin, MMP2and MMP9in TAK1siRNA group were lower than that of in the negative control group and the control group, the difference was statistically significance (P<0.05); while between the control group and the negative control group, the expression of these proteins in prostate cancer cells has no statistical significance (P>0.05).ConclusionDownregulate the expression of TAK1gene can effectively inhibit the expression of Bcl-2, JNK, COX-2, EGFR, then the apoptosis was enhanced, the cell proliferation ability was suppressed and the drug sensitivity were augmented. Silencing the TAK1gene can also reduce the expression of MMP-2,MMP-9and COX-2gene expression, and these gene were related with the migration and invasion of prostate cancer DU145cells. So we guss the TAK1gene may be a key target in prostate cancer cell signal transduction network. |
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