Regulation of autophagy by aspartate β-hydroxylase

Objective To investigate the regulation of autophagy by aspartate β-hydroxylase (ASPH). Methods ASPH plasmid was transfected into OUMS-29 and HEK-293T cells. Then the expression of p62 and microtubule-associated protein 1 light chain 3 (LC3)-Ⅱ was verified by Western blotting. OUMS-29 cells were co-transfected with ASPH and pEGFP-LC3 plasmid. The ratio of green fluorescent protein (GFP)-LC3-Ⅱ punctate positive to GFP positive cells was calculated under fluorescence microscope. ASPH was knocked down in HuCCT1 and SSP25 cells. Western blotting was performed to examine the expression of p62 and LC3-Ⅱ. Then, the effect of autophagy inducer rapamycin (Rapa) or metformin (Met) on cell growth rate was detected by methyl thiazol tetrazolium (MTT) assay. Results ASPH overexpression could inhibit LC3-Ⅱ expression in OUMS-29 and HEK-293T cells (44% lower than control) (P=0.002, P=0.008), but did not alter the p62 expression level (P=0.060, P=0.798). ASPH overexpression could lower the ratio of GFP-LC3-Ⅱ punctate positive cells to GFP positive cells in OUMS-29 cells from 50.84% to 28.31% (P=0.002). LC3-Ⅱ expression level increased in HuCCT1 and SSP25 cells with ASPH knocked down (87% and 133% higher than control, respectively) (P=0.001, P=0.000). However, p62 expression level did not change in these cells (P=0.677, P=0.168). In HuCCT1 and SSP25 cells, Rapa or Met could mask the effect of ASPH knock-down on cell growth. Conclusion ASPH suppresses autophagy via inhibiting the generation of LC3-Ⅱ. ASPH might regulate cell growth by modulating autophagy. Key words: Aspartate β-hydroxylase; Autophagy; Cell growth