OBJECTIVE: To study the effect of FAM172A protein related to diabetic macroangiopathy on apoptosis and proliferation in HEK293 cells. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Li LX, Zhou WB, Tao Z, Deng WJ, Liang WC, Yang ZH, Ye WW, Bao YQ, Jia WP, Hu RM (2010) [Effect of FAM172A protein on apoptosis and proliferation in HEK293 cells] Zhonghua Yi Xue Za Zhi90: 2424-7
Li LX, Zhou WB, Tao Z, Deng WJ, Liang WC, Yang ZH, Ye WW, Bao YQ, Jia WP, Hu RM (2010) Zhonghua Yi Xue Za Zhi90: 2424-7
