Effects of Promyelocytic Leukemia (PML) Gene Knockdown on Apoptosis and Proliferation of Human Myeloid Leukemia THP-1 Cells and Its Potential Mechanism

Abstract: The fusion of promyelocytic leukemia (PML) gene and the retinoic acid receptor α (RARα) gene resulting to oncogenic PML-RARα was the main molecular mechanism in the pathogenesis of acute promyelocytic leukemia (APL)， whereas few investigation are focused on the role of PML in acute myeloid leukemia subtypes other than APL. This study mainly aimed at investigating the role of PML in the proliferation and apoptosis activity of myeloid leukemia cells and its potential mechanism. Firstly， the levels of PML mRNA and protein in five myeloid leukemia cell lines were determined by qPCR and Western blot， respectively. Next， THP-1 cells with high PML expression were infected with shRNA lentivirus targeting PML (shPML group) and its negative control (Scramble group)， followed by puromycin selection. THP-1 cells untreated were named as Mock group. shRNA efficiency was confirmed by qPCR and Western blot. Cell proliferation activity was assessed by CCK-8 in vitro; Colony formation ability was analysed by colony-forming assay. The apoptosis rate was determined by flow cytometry. The protein levels of apoptosis related proteins Bcl-2 and Bax were measured by Western blot. In addition， the expression of phosphorylated-AKT (pAKT) and phosphorylated-Foxo3a (pFoxo3a) were detected by Western blot. The results showed different levels of PML mRNA and protein in myeloid leukemia cell lines， and THP-1 cells exhibited higher PML expression. We transduced THP-1 cells with shRNA lentivirus targeting PML and confirmed knockdown of PML at mRNA and protein levels. Compared with Mock and Scramble groups， silencing PML significantly promoted cell proliferation activity and colony formation ability in vitro (P<0.05)， and inhibited cell apoptosis (P<0.05).In addition， knockdown of PML up-regulated Bcl-2 protein levels and down-regulated Bax protein levels. Furthermore，suppressing PML significantly enhanced the levels of AKT phosphorylation at residues Ser473 and its downstream Foxo3a phosphorylation at residues Ser253 (P<0.05)， but had no remarkable change of total protein levels of AKT and Foxo3a. These data provide evidence that PML as a tumor suppressor plays an important role in leukemogenesis involving AKT/Foxo3a signaling pathway， indicating the multiple role of PML in leukemia.

CSTR: 32200.14.cjcb.2016.07.0006