【作者】 夏炎枝；

【导师】 王西明；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 目的检测分析临床膀胱癌组织和正常膀胱组织TERE1/UBIAD1表达及Ras-MAPK信号通路的变化。方法采用免疫组化检测分析人正常膀胱组织及膀胱癌组织中TERE1和端粒逆转录酶(hTERT)的蛋白表达水平：用RT-PCR及western blotting检测膀胱癌组织中TERE1和hTERT的表达水平及Ras-MAPK信号通路关键分子的变化。结果1.免疫组化结果表明相对于正常膀胱组织,膀胱癌组织中TERE1染色明显减低。按组织学分级G3-4期TERE1染色阳性率为16.13%(5／31例数),与G2期阳性率TERE1为51.85%(14／27)相比具有显著性差异,与G1期阳性率为76.00%(19／25)相比差异具有极显著性；膀胱癌按临床分期T2期TERE1染色阳性率为27.27%(6／22),T3-4期TERE1染色阳性率为16.00%(4／25),与Ta期94.12%(16／17)和T1期阳性率为68.42%(13／19)相比,具有显著性差异。2.膀胱癌组织中TERE1的mRNA和蛋白表达水平与正常膀胱组织相比明显下降(P<0.05)。3.癌组织中hTERT的mRNA和蛋白表达与正常膀胱组织相比明显上升(P<0.05)。4.膀胱癌组织中磷酸化ERK、磷酸化MEK(1／2)、磷酸化B-Raf(Ser455)及磷酸化C-Raf(Ser338)蛋白表达水平与正常组织相比明显增加(P<0.05),而膀胱癌组织与正常膀胱组织中总的ERK、MEK(1/2)、A-Raf、B-Raf, C-Raf及磷酸化A-Raf (Ser299)蛋白表达没有明显差异。结论膀胱癌中TERE1/UBIAD1表达较正常组织明显下调,且与膀胱癌分级分期密切相关,高恶性度肿瘤未能检测到TERE1/UBIAD1的表达。膀胱癌中Ras-MAPK信号通路关键分子较正常组织相比明显被激活；hTERT表达增加。TERE1/UBIAD1可能通过Ras-MAPK信号通路激活影响膀胱癌的形成。第二部分TERE1／UBIAD1抑制膀胱癌的分子机制研究目的研究TERE1/UBIAD1抑制膀胱癌的作用机制是否通过Ras-MAPK信号通路及作用的分子靶点。方法1.采用RT-PCR及western blotting检测膀胱癌细胞系T24中TERE1和hTERT的mRNA和蛋白表达水平及Ras-MAPK信号通路中关键分子的变化。2. siRNA干扰技术实验：分阴性对照组、转染试剂对照组、TERE1-291-siRNA组、TERE1-322-siRNA组、TERE1-396-siRNA组,将化学修饰的TERE1-siRNA oligos转染L02正常细胞系48小时后,用RT-PCR及western blotting检测TERE1和hTERT的mRNA及蛋白表达水平,western blotting分析Ras-MAPK信号通路关键分子的变化,用细胞计数和MTT法检测细胞增殖情况。3.MEK抑制剂U0126实验：分阴性对照组、TERE1-291-siRNA组、TERE1-322-siRNA组,将化学修饰的TERE1-siRNA oligos转染L02正常细胞系48小时后再加入MEK抑制剂U0126作用30分钟,western blotting检测Ras-MAPK信号通路关键分子的变化。结果1.膀胱癌细胞系T24与正常细胞系L02比较,TERE1的mRNA及蛋白表达水平明显下调,Ras-MAPK信号通路分子磷酸化的ERK表达明显上升；hTERT的mRNA及蛋白表达增加。2.与阴性对照组或转染试剂组相对比,化学修饰的TERE1-291-siRNA oligos和TERE1-322-siRNA oligos转染正常细胞48小时后能明显将细胞TERE1基因沉默,使hTERT的mRNA和蛋白表达水平上升(P<0.05),磷酸化的ERK蛋白表达水平显著增加(P<0.01),同时细胞数目增多、细胞增殖加快。3.TERE1-291-siRNA和TERE1-322-siRNA作用组,加抑制剂U0126与不加抑制剂U0126相比,Ras-MAPK信号途径中关键分子ERK的蛋白磷酸化被抑制,hTERT的蛋白表达被部分抑制。结论TERE1基因沉默能激活细胞Ras-MAPK信号通路及导致细胞增殖明显加快,MEK抑制剂能阻滞TERE1下调引起的Ras-MAPK信号通路的激活,部分阻滞hTERT蛋白表达。TERE1可能作为Ras-MAPK信号通路的负向调节因子,在膀胱癌的发生中有重要作用。更多还原

【Abstract】 Part 1 Analysis of TERE1/UBIAD1 expression and Ras-MAPK signalling pathway in bladder cancerObjective Detection of TERE1 (transitional epithelial response gene)/UBIAD1(UbiA prenyltransferase domain containing 1) expression and Ras-MAPK signal pathway molecules changes in clinical bladder cancer tissues and normal bladder tissues.Methods Analysis of TERE1 and human telomerase reverse transcriptase (hTERT) protein expression in normal bladder tissue and bladder cancer tissues using immunohistochemistry; the expression of TERE1、hTERT in bladder cancer tissues and normal bladder tissues and the Ras-MAPK signaling pathway molecules changes were determined by RT-PCR and western blotting.Results 1. Immunohistochemistry results showed that compared with normal bladder tissue, TERE1 staining in bladder cancer tissues were reduced. According to histological grade, the positive rate 16.13%(5/31 the number of cases) on G3-4 TERE1 staining was different compared with 51.85%(14/27) on grade G2, the positive rate 16.13%on G3-4 TERE1 staining was significant difference compared to the positive rate 76.00%(19/25) on grade G1; according to clinical bladder cancer stage, the positive rate of TERE1 was 27.27%(6/22) in T2 stage, TERE1 positive staining rate was 16.00%(4/25) in T3-4 stage,68.42%(13/19) in T1 stage and 94.12%(16/17) in Ta stage.2. The mRNA and protein expression levels of TERE1 in bladder cancer tissues were decreased significantly compared with normal bladder tissues (P<0.05).3. The mRNA and protein expressions of hTERT in bladder carcinoma significantly increased.4. Compared with normal tissue, phosphorylated protein levels of ERK、MEK(1/2)、B-Raf (Ser455) and C-Raf (Ser338) in bladder cancer significantly increased (P<0.05), total protein levels of ERK、MEK (1/ 2)、A-Raf, B-Raf, C-Raf and phosphorylated protein levels of A-Raf (Ser299) in bladder cancer tissues and normal bladder tissues were no significant difference.Conclusion TERE1/UBIAD1 expression in bladder cancer tissues was significantly lower than normal tissues, TERE1/UBIAD1 expression was closely related with the grade and stage of bladder cancer. The expression of TERE1/UBIAD1 was no detected in high grade tumors. Ras-MAPK signaling pathway molecules in bladder cancer were significantly activated compared to normal bladder tissues; The hTERT expression in bladder cancer was significantly increased. TERE1/UBIAD1 possibly affected the formation of bladder cancer through activated Ras-MAPK signaling pathway. Part 2 Molecular mechanism of the role of TERE1/UBIAD1 in bladder cancer suppressionObjective To study the molecular mechanism of the inhibition role of TERE1/UBI ADI in bladder cancer through the Ras-MAPK signaling pathway and the molecular targets in Ras-MAPK signaling pathway.Methods 1. The mRNA and protein expression levels of TERE1 and hTERT in bladder cancer cell line T24 were determined by RT-PCR and western blotting, the Ras-MAPK signaling pathway was also detected using western blotting.2. SiRNA interference experiments:experiments were divided into negative control group、the transfection reagent control group、TERE1-291-siRNA group、TERE1-322-siRNA group、TERE1-396-siRNA group, the chemical modification of TERE1-siRNA oligos transfected L02 normal cells for 48 hours, the mRNA and protein levels of TERE1 and hTERT were measured by RT-PCR and western blotting, the Ras-MAPK signaling pathway molecules were analyzed by western blotting, cell proliferation assay were determined using cell technology and MTT assay.3. MEK inhibitor U0126 tests:experiments were divided into negative control group、TERE 1-291-siRNA group、TERE1-322-siRNA group, the chemical modification of TERE1-siRNA oligos normal L02 cells transfected for 48 hours, then pretreated adding MEK inhibitor U0126 for 30 minutes, the Ras-MAPK signaling pathway molecules changes were detected using western blotting.Results 1. The mRNA and protein levels of TERE1 in T24 bladder cancer cell line were significantly reduced compared with the normal cell line L02, the phosphorylation protein levels of ERK in Ras-MAPK signaling pathway was significantly increased; mRNA and protein expression of hTERT were also increased.2. Compared to the negative group or the transfection reagent group, TERE1 gene in normal cells was significantly silenced by chemical modification of TERE1-291-siRNA oligos and TERE1-322-siRNA oligos, the mRNA and protein levels of hTERT were increased (P<0.05), phosphorylated ERK protein levels was increased significantly (P<0.01), while cell number were increased andcell proliferation was accelerated.3. Adding inhibitor U0126, the ERK protein phosphorylation levels was inhibited in TERE1-291-siRNA group and TERE1-322-siRNA group compared to without inhibitor U0126, the protein expression of hTERT was partially inhibited.Conclusions TERE1 gene silencing can activate Ras-MAPK signaling pathway and lead to cell proliferation, MEK inhibitor can suppress the activation of Ras-MAPK signaling pathway due to down-regulation of TERE1, partially block the expression of hTERT protein. TERE1 might serve as a negative regulator Ras-MAPK signaling pathway and might have an important role in bladder carcinogenesis.更多还原