The Study On The Mechanism Of TRIM29Retrieve The Sensitivity To Oxaliplatin In P53Mutant And Drug-Resistant Colon Cancer

Part1Construction of the pIRES2-ZsGreenl-TRIM29plasmidObjective:To construct the pIRES2-ZsGreenl-TRIM29plasmidMethods:Use the pDONR223-TRIM29as the template for CDS of TRIM29PCR amplification.The products were electrophoresed and the specific band of TRIM29were excised and extracted by Gel Extraction Kit. The TRIM29PCR products and the vector pIRES2-ZsGreenl were digistived by two restriction enzyme-BglII and EcoRI. Then the TRIM29PCR products and the vector pIRES2-ZsGreenl were linked together. The recombinant plasmids were transfected into E. coli DH5a for amplification, and the positive clones were selected in kan+medium. After then, small-scale plasmid extraction from the postive clones were implimented. The ricombinant plasmids were verified by two restriction enzyme digestion and DNA sequence alignment.Result:1. The TRIM29PCR products were electrophoresed and the specific band was about1.8K, as the same location as TRIM29’s.2. The ricombinant plasmids were digestived by two restriction enzyme-Bglll and EcoRI, and two fragments were demonstrate-one fragment of1.5k TRIM29and one fragment of5.3k plasmid.3. DNA sequence alignment displayed the seqence of recombinant plasmid and TRIM29gene were entierly identical.Conclusion:Successfully constructed pIRES2-ZsGreenl-TRIM29expression plasmid Part2TRIM29greatly increase the sensitivity to oxaliplatin in mutp53HT29colon cancer cellsObjiective:To explore the impact of TRIM29on sensitivity to oxaliplatin in colon cancer cells in different P53status.Method:Use of wild-type p53colon cancer cell HCT116and mutant p53colon cell HT29as control study subjects. Analyse the growth rate and IC50of oxaliplatin change by MTT method in these two colon cancer cells after TRIM29transfection.Result:1. After transfected by TRIM29plasmid, the mRNA and protein of TRIM29expression were both increased indicated the transfection succeeded.2. After transfected by TRIM29plasmid, the growth rate of HCT116increased at day4, and the number of cells was1.4folds of the parent cells’at day7. Whereas the growth rate of HT29decreased at day3, and the number of cells was73%campare to the parent cells at day7.3. After transfected by TRIM29plasmid, the IC50of oxaliplatin in HCT116 increased lightly from34.89umol/L to47.26umol/L, the durg resistant index was1.35. In sharp contrast, the IC50of oxaliplatin in HT29decreased drasticly from11.54umol/L to0.98umol/L, the drug resistant index was0.08.Conclusion:1. In CRC, when cells express the wild-type p53, TRIM29promoted the tumor growth; howerver when cells express mutant p53, TRIM29inhibited the tumor growth.2. In wild-type p53HCT116, TRIM29increased the drug resistance of oxaliplatin lightly indicated wild-type p53mild-related with the sensitivity of oxaliplatin.3. In sharply contrast, TRIM29greatly increased the sensitivity of oxaliplatin in mutant p53HT29indicated mutant p53highly correlated with drug resistance of oxaliplatin. Part3The establishment of oxaliplatin resistant cell model in HT29Objective:To establish the oxaliplatin resistant cell model in HT29Method:Use of sustained-low concentration culture method to establish the oxaliplatin resistant cell model in HT29. Firstly, The HT29cultured with oxaliplatin at the concentration of4umol/L(about1/3of IC50) for 2-4weeks untill the cell multiplication appeared. Then continued to culture at this concentration for another2weeks. Elevated the oxaliplatin concentration to6umol/L and this cycle was repeated until drug concentration was reached at which cell multiplicationno longer occurred.The final concentration is12umol/L.Result:1. Successfully obtain three oxaliplatin-resistant HT29cells, named as HT29-OX-4umol/L, HT29-OX-8umol/L, HT29-OX-12umol/L.2.The IC50of oxaliplatin in three oxaliplatin-resistant HT29cells were74.42umol/L,136.50umol/L,188.90umol/L separately.3. Consistant with the increase drug-resistant ablilty in three cells, MDR1expression gradually increased both in mRNA and protein.Coclusion:successfully established three oxaliplatin-resistant HT29cells which laid the foundation for further study on the effect of TRIM29invloved in oxaliplatin resistance in p53mutant colon cancer. Part4TRIM29successfully reverse the sensitivtiy to oxaliplatin in oxaliplatin-resistant HT29cell model via downregulation of MDR1through binding mutant P53Objective:To verify whether TRIM29could reverse the sensitivtiy to oxaliplatin in oxaliplatin-resistant HT29cell model and explore the mechanism involved in it.Method:Analyse the IC50of oxaliplatin change by MTT method in HT29-OX-12umol/L after TRIM29transfection. Use of co-ip to detect whether TRIM29binding to mutant P53. Use of Q-PCR and Westernblot to mesure the expression of TRIM29and mutant P53in three oxaliplatin-resistant cells. Use of Q-PCR and Westernblot to mesure the expression of MDR1and mutant p53before and after TRIM29transfection.Result:1. After TRIM29transfection, the IC50of oxaliplatin in HT29-OX-12umol/L decreased from188.90umol/L to22.59umol/L, the Drug-resistant Index decreased from16.37to1.96.2.TRIM29could bind to mutant P53in HT29commutatively.3. With the increase drug-resistant ablilty in three cells, the TRIM29expression gradually decreased and the P53expression gradually increased both in mRNA and protein.4.After TRIM29transfection, MDR1expression drop drasticly, however P53expression was not effected.Conclusion:1. TRIM29inhibit the mutant P53function by physically bind antagonism, not by downregulating the mutant P53expression.2. TRIM29successfully reverse the sensitivtiy to oxaliplatin in oxaliplatin-resistant HT29cell model via downregulation of MDR1through binding mutant P53.