| FAM3A is one of the members of cytokine family with sequence similarity 3, which is regulated by PPARG and participate in the metabolism of lipid in liver. This experiment was to study the transcriptional regulatory mechanism of FAM3A in preadipocytes and its role in the process of adipogenesis. The main results are as follows:1. Overexpress C/EBPbeta in the 3T3L1 cells, and detecte FAM3A gene expression changes. We found that C/EBPbeta can promote FAM3A expression (P<0.01).2. Establish double fluorescent report vectors of FAM3A promoter delections, which were Dl(-1478/+46), D2(-1187/+46), D3(-951/+46), D4(-442/+46), D5(-285/+46) and D6(-32/+46), and then transfect 3T3L1, C2C12 and CHO cells to detect fluorescence activity. The results showed that all of the FAM3A promoter delections were active in three kinds of cells and have similar trends of activity. Among them, D2(-1187/+46) got the highest activity, while the activity of D6(-32/+46) was the lowest and just posess the basic activity. We deduced that the core promoter was located around the region of-32bp/+46bp relative to the transcriptional start site. Moreover, the fragment from-285bp to-32bp in relation to the transcriptional start site may enriched with positive regulatory elements, while more suppressant element must distributed in the region between-1478 bp to-285bp.3.Cotransfect 3T3L1 cells with C/EBPbeta eukaryotic expression vector and FAM3A promoter delections to detect fluorescence activity. The result showed that the activity of each FAM3A promoter delections was significantly enhanced (P<0.01), including the delection D(-1187/+46) which was effected most by C/EBPbeta that the activity was elevated by 3.55 times. Continue to site-directed mutate C/EBPbeta binding sites on the promoter and cotransfect 3T3L1 cells with C/EBPbeta eukaryotic expression vector shows that the activity of mutation promoter was almost not influenced by C/EBPbeta. Then a further evidence of EMSA experiment indicated that C/EBPbeta protein can combine with the C/EBPbeta binding sites on the promoter.4. Induce 3T3L1 cells differentiation with MDI after transfect 3T3L1 cells with builded FAM3A eukaryotic expression vector, it turned out that the efficiency of preadipocytes to differentiate into adipocytes was inhibited by FAM3A, and the triglycerides content decreased significantly after 8 days’differentiation. Detection of adipose differentiation marker gene expression indicated that the expression level of PPARG2 and FABP4 genes were significantly degraded (P<0.05) in FAM3A overexpression cells differentiate for 2 days. After FAM3A overexpression cells differentiate for 8 days, C/EBPα, SCD1, DGAT1, LPL and FABP4 genes down expressed and reached significant level (P<0.05), while the expression of genes HSL, ACC2, AdipoRl, FAS, ACOX1 and UCP2 were not effected by FAM3A.6.Transfect 3T3L1 cells with FAM3A eukaryotic expression vector to measure cell cycle after 72h. It showed that G1 phase cells of experimental group increased from 63.5% to 72.7%, while S phase of cell proportion fell from 24.8% to 18.3%, two groups of cells did not differ between the G2 phase of cell number, demonstrated that FAM3A can inhibit the cell cycle of 3T3L1 cells. |
