[Knockdown of ALAS2 Affects Erythroid Differentiation by Down-regulating Mitophagy Receptor BNIP3L]

AbstractObjective: To investigate the effect of ALAS2 downregulation on the expression of BNIP3L and erythroid differentiation in K562 cells.

The expression of ALAS2 was down-regulated by transfection of lentivirus, then quantitative real-time PCR was performed to detect the transfection efficiency. Flow cytometry analysis was applied to evaluate apoptosis of cells, erythroid differentiation, mitochondrial membrane potential and reactive oxygen species (ROS) level. Western blot was used to detect the BNIP3L expression, Co-immunoprecipitation was performed to analyze the relationship between ALAS2 and BNIP3L.

Compared with sh-NC group, knockdown of ALAS2 induced downregulation of BNIP3L mRNA and protein expression(P<0.01) and erythroid related transcription factors GATA1, Nrf2 expression, as well as reduction of ROS level(P<0.05). Mitochondrial membrane potential of control (sh-NC) group was lower than that of shALAS2 group(P<0.05), but there was no significant change of cell apoptotic rate in two groups. CD71highCD235ahigh + CD71lowCD235ahigh cells of sh-NC and shALAS2 groups were 53.5%, 92.9% at 96 h after hemin induction, respectively. No direct action between ALAS2 and BNIP3L was observed.

The intracellular heme level can affect the expression of BNIP3L which may be related with the regulation of ROS and transcription factors GATA1 and Nrf2. Higher BNIP3L facilitates cell differentiation but lower BNIP3L is favorable for cells survival.