Study Of Tumor Suppressor Gene TERE1(UBIAD1) In Ras-MAPK Signal Transduction Pathway And Cell Apoptosis

TERE1(Transitional epithelial response gene1), also known as UBIAD1(UbiAprenyltransferase domain containing1), The TERE1(UBIAD1) gene was mapped tochromosome1p36between the micro-satellite markers D1S2667and D1S434throughFISH (Fluorescence in situ Hybridization), encodes a protein with338amino acids (36.8KD). The TERE1(UBIAD1) gene is ubiquitously expressed in human tissues. Aprotein-protein interaction between TERE1(UBIAD1) and ApoE was identified. TERE1(UBIAD1) plays an important role in lipid metabolism and regulates cholesterol transportand storage. The mutation of TERE1(UBIAD1) could cause SCCD (Schnyder CrystallineCorneal Dystrophy) disease in hunman eyes. TERE1(UBIAD1) protein, a potentialprenyltransferase has10a-helical transmembrane domains with its C and N terminallocalized inside of the plasma membrane. TERE1(UBIAD1) was also identified as a novelhuman menaquinone-4biosynthetic enzyme, processing side-chain cleavage andprenylation ability. TERE1(UBIAD1) modulates lipid metabolism in human andsuppresses bladder tumor progression. We speculate TERE1(UBIAD1) involved in lipidmetabolism, cholesterol regulation, tumorigenesis and SCCD formation by protein-proteininteractions with ApoE and with the help of substrate MK-4.To study the interaction between TERE1(UBIAD1) and Ras, siRNA was used.Activiation of ERK in Ras-MAPK signal pathway was detected by siRNA to knock downthe expression of TERE1(UBIAD1) in HEK293cells in this thesis. Phosphorylated ERKwas decreased when Sulindac Sulfide and GW5074were added to block the Ras-MAPKsignal pathway, demonstrating that TERE1(UBIAD1) may exact its effect on Ras proteinor proteins upstream of the Ras-MAPK signal pathway. TERE1(UBIAD1) regulates theactiviation of Ras-MAPK signal pathway. Combined with other people’s FRET and IPexperiments, we found that TERE1(UBIAD1) interacts with H-Ras protein and TERE1(UBIAD1) was a negative regulator of Ras protein. In further study we also found that theRas accumulation in plasma membrane was significantly increased when using SiRNA toknock down TERE1(UBIAD1) expression in HEK293cells transfected with BFP-H-Rasand RBD (Ras binding domain) plasmid. We transfected bladder cell lines T24with exogenous TERE1(UBIAD1) throughLipofectamineTM2000. We counted T24cell number transfected with TERE1(UBIAD1)after48hours. The cells number declined sharply. Also we used MTT method to test cellsvitality of T24cells transfected with TERE1(UBIAD1). It is observed that vitality wassignificantly decreased in experimental group compared to control group. FCM (flowcytometry) was used to detect cell cycle of T24cells transfected with TERE1(UBIAD1),we detected apoptosis peak in TERE1(UBIAD1) treated T24cells. FITC and PI dye wereused to detect cell apoptosis, following FCM experiment.93.7%cells went to apoptosis.We found that TERE1(UBIAD1) induced apoptosis in bladder cancer cell lines T24.Through experiments with HEK293and T24in this thesis, we proved TERE1(UBIAD1) had interactions with Ras protein. TERE1(UBIAD1) was negative regulator ofRas, involving in Ras-MAPK signal transduction pathway. We found that TERE1(UBIAD1) induced apoptosis in bladder cancer cell lines T24. Our research worksupplements the Ras-MAPK signal pathway. Since TERE1(UBIAD1) induces cancercells apoptosis, TERE1(UBIAD1) could be used to design anti-cancer drugs and offerreference to cancer research.