Establishment Of Inducible Cell Line With Stable Expression Of TRIM28 And Its Potential Functional Analysis

Objectives:This study was aimed (1) to clone TRIM28 into an inducible expression vector to establish stable cell lines expressing TRIM28, (2) to investigate the regulation of expression level of TRIM28 protein and its effect on the stability of target genes, revealing new biological functions and (3) to explore the ubiquitination role of TRIM28 in regulating TWIST expression. Methods:First, the TRIM28 was cloned into inducible expression vectors by using TRIM28 gene cDNA as a template, polymerase fidelity and PCR amplification of the full length gene. The expression vector pcDNA5/FRT/TO, digested with EcoRV and NheI, was ligated to the same restriction enzyme digestion TRIM28. Then, restriction enzyme digestion, PCR and sequencing the positive clones were performed. We obtained a recombinant plasmid pcDNA5/FRT/TO/TRIM28. Secondly, we established a stably expressed TRIM28 cell line and analyzed the expression and possible function. For this, the pcDNA5/FRT/TO/TRIM28 recombinant plasmid was transfected into a host cell line Flp-InTM-RExTM-293, positive clones were screened by using hygromycin B. Then, the obtained pcDNA5/FRT/TO/TRIM28 was transfected into endogenous expression TWIST 4T1 cells, and the possible regulation of TWIST by TRIM28 was studied by Real-Time PCR and WB analysis. In this thesis the following experiments has been performed:(1) restriction enzyme digestion, PCR and sequencing test of the positive clones; (2) by using different doses of DOX and different time for pcDNA5/FRT/TO-TRIM28 HEK293 (293-TRIM28) cell lines induced expression, the expression of TRIM28 was detected by Western blotting; (3) the recombinant plasmid pcDNA5/FRT/TO/TRIM28 was transfected into cells, and its expression in cells was analyzed by western blotting; (4) its endogenous expression TWIST in 4T1 cells, with transfected TRIM28 were studied by WB for the investigation of the regulation of TWIST; (5) The protein ubiquitination inhibitor MG-132 was used to treat MDA-MB-435 cells, for different times,WB analysis the stability of TWIST; (6) Using DOX induced pcDNA5/FRT/TO-TRIM28 HEK293 (293-TRIM28) cell line, while overexpression of TWIST and UB, with WB detect the ubiquitination of TWIST. Results:The recombinant plasmid pcDNA5/FRT/TO/TRIM28 was constructed and it was induced by DOX in induced cell lines with stably expressing TRIM28 after the empty vector and the recombinant plasmid was transfected into cells. We found that hosted Flp-InTM-RExTM-293 cells transfected with pcDNA5/FRT/TO empty vector (as control) did not show any TRIM28 expression, no matter whether the DOX was added or not, but transfection with pcDNA5/FRT/TO-TRIM28 into the Flp-InTM-RExTM-293, DOX treatment showed high level TRIM28 expression. DOX at 0.01 μg/ml significantly enhanced the expression of pcDNA5/TRIM28 than DOX treatment of 0.001 μg/ml in inducible cell line. We also found that Effect of DOX is time dependent. As treatment of 2 hours (hs) significantly enhanced the expression than that of 1 h. Western blot analysis and PCR reactions have identified. The expression of TWIST mRNA was lower in TRIM28 transfected cells than in empty vector transfected in 4T1 cells, but the difference was not significant (P>0.05), we also found the expression of TWIST protein was lower in TRIM28 transfected cells than in empty vector transfected, and the difference was significant (P<0.05). The protein ubiquitination inhibitor MG-132 was used to treat MDA-MB-435 cells, for different times, and TWIST expression was studied by WB analysis. Result shows that using MG-132 increases the TWIST protein level and also improve its stability, This also indicate TWIST presence ubiquitination regulation. Using DOX induced pcDNA5/FRT/TO-TRIM28 HEK293 (293-TRIM28) cell line, while overexpression of TWIST and UB, with WB detect the ubiquitination of TWIST, Results indicate TRIM28 can promoted the ubiquitination of TWIST. Conclusion:This study established an inducible cell line with stable TRIM28 expression. Also, this study shows TRIM28 do not significantly reduce Twist mRNA expression, but it significantly reduce the Twist protein expression. TWIST presence ubiquitination regulation.This shows that TRIM28 regulation for TWIST expression might be at transcriptional level rather than translational level, specific regulatory mechanism needs to be studied in the future.