Molecular Mechanism Of The Role Of TERE1/UBIAD1 In Bladder Cancer Suppression And Structural And Factional Analysis Of TERE1/UBIAD1

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.