ABSTRACT: To study the effect of FAM172A protein related to diabetic macroangiopathy on apoptosis and proliferation in HEK293 cells.
The pDrive-FAM172A plasmid constructed in our previous study was used as a template to amplify human FAM172A open reading frame by a polymerase chain reaction. The resulting PCR products were subcloned into the eukaryotic expression vector PDC315 to construct recombinant PDC315-FAM172A plasmid. PDC315-FAM172A plasmid was identified by enzyme cleavage and sequencing analysis. HEK293 cells were transiently transfected respectively with appropriate PDC315 or PDC315-FAM172A plasmid by Lipofectamine 2000 according to the manufacturer's instruction. XTT assay and growth curve were used to observe the effect of over-expression of FAM172A gene on HEK293 cell proliferation. PI and Annexin V/PI staining method were used to assess the effect of FAM172A gene on apoptosis and cell cycle of HEK293 cell.
Eukaryotic expression vector PDC315-FAM172A was successfully constructed and identified by enzyme cleavage and sequencing analysis. Compared with PDC315 plasmid transfection, the XTT assay showed that optical density (A) value increased by 52% when transfected with PDC315-FAM172A plasmid (0.21±0.07 vs 0.32±0.06, P<0.01). Growth curve revealed that HEK293 cells transfected with PDC315-FAM172A plasmid proliferated faster than those transfected with PDC315 plasmid. PI staining showed that, as compared with PDC315 plasmid transfection, the apoptotic rate of HEK293 cells transfected with PDC315-FAM172A plasmid decreased by 38.5% (23.79±1.36 vs 14.64±0.95, P<0.01), cell percentage of G0-G1 phases significantly decreased (66.79±1.73 vs 58.16±0.75, P<0.01) and cell percentage of S phases significantly increased (22.62±1.16 vs 33.56±0.94, P<0.01). Annexin V/PI staining revealed that, as compared with PDC315 plasmid transfection, the percentage of early and advanced apoptotic cells decreased by 28% (13.63±0.56 vs 9.79±0.39, P<0.01) and 29% (7.70±0.29 vs 5.43±0.29, P<0.01) respectively.
FAM172A protein promotes cell proliferation, inhibits cell apoptosis and facilitates S-phases entry. It indicates that FAM172A protein is involved in cell growth regulation. Our findings provide a clue for further study on its physiological functions and roles in diabetic macroangiopathy.
Zhonghua yi xue za zhi 09/2010; 90(34):2424-7.