Inhibition of MicroRNA-149-5p Induces Apoptosis of Acute Myeloid Leukemia Cell Line THP-1 by Targeting Fas Ligand (FASLG)

Abstract

BACKGROUND This study was aimed to reveal the role of miR-149-5p in acute myeloid leukemia (AML) cells apoptosis and the possible mechanism involved. MATERIAL AND METHODS The expression of miR-149-5p in leukemia cell lines, as well as the blood and bone marrow (BM) samples from leukemia patients, were monitored by reverse-transcription polymerase chain reaction (RT-PCR). AML cell line THP-1 was transfected with miR-149-5p mimic or inhibitor, and then cell apoptosis was determined using the APO Percentage assay kit. The target of miR-149-5p was predicted by using the microRNA.org database, and verified by RT-PCR, Western blot, and Dual-Luciferase reporter assays. Further, small interfering RNA (siRNA) against the target gene was co-transfected with miR-149-5p inhibitor, and then the cell apoptosis and the expression of apoptosis-related proteins were assessed. RESULTS MiR-149-5p was significantly up-regulated in leukemia cell lines and samples from leukemia patients (P<0.01 or P<0.001), especially in THP-1 cells and samples from AML patients. Cell apoptosis was significantly decreased by miR-149-5p overexpression (P<0.01) and increased by miR-149-5p suppression (P<0.05). Fas Ligand (FASLG) was a direct target of miR-149-5p, and was negatively regulated by miR-149-5p. More importantly, the inductive effects of miR-149-5p suppression on cell apoptosis were abrogated by si-FASLG (P<0.01). Furthermore, the up-regulative effects of miR-149-5p suppression on the phosphorylated form of Fas-associated via death domain (p-FADD), caspase-8, caspase-2, caspase-3, and the cleaved forms of these caspases were abrogated by si-FASLG. CONCLUSIONS Inhibition of miR-149-5p can induce apoptosis in THP-1 cells. These inductive effects might be via targeting FASLG and activating FADD and caspases.

Figure 1

Relative miR-149-5p expression at mRNA level in leukemia cell lines and the BM and blood samples from leukemia patients. The miR-149-5p expression at mRNA level in 3 leukemia cell lines, THP-1, Molt-4, and K562 was measured by RT-PCR analysis (A). The mRNA level of miR-149-5p expression was detected in the BM samples (B) and fresh blood (C) from 3 types of leukemia: AML, T-ALL, and CML. BM – bone marrow; RT-PCR – real-time reverse transcription polymerase chain reaction; AML – acute myeloid leukemia; T-ALL – T cell acute lymphoblastic leukemia; CML – chronic myeloid leukemia; ** P<0.01; *** P<0.001.

Figure 2

Inhibition of miR-149-5p induced THP-1 cells apoptosis. MiR-149-5p mimic or inhibitor was transfected into THP-1 cells, and the efficiency of transfection was detected by RT-PCR analysis (A). Afterward, cell apoptosis was measured by using APO Percentage assay kit (B). RT-PCR – real-time reverse transcription polymerase chain reaction; * P<0.05; ** P<0.01; *** P<0.001.

Figure 3

FASLG was predicted and verified as a direct target of miR-149-5p. The microRNA.org database was used to predict the target gene of miR-149-5p (A). The mRNA and protein level of FASLG expression in miR-149-5p overexpressed cells or miR-149-5p suppressed cells were detected by RT-PCR (B) and Western blot (C). Dual-Luciferase reporter assay was performed to confirm miR-149-5p targeted the 3′-UTR of FASLG (D). FASLG – Fas ligand; RT-PCR – real-time reverse transcription polymerase chain reaction; 3′-UTR – 3′-untranslated region; ** P<0.01; *** P<0.001.

Figure 4

Inhibition of miR-149-5p induced cell apoptosis via targeting FASLG. si-FASLG or its negative control was transfected into THP-1 cells, and then the mRNA level of FASLG expression was measured by RT-PCR (A). si-FASLG or/and miR-149-5p inhibitor were transfected into THP-1 cells, and the expression of FASLG protein was detected by Western blot (B). si-FASLG or/and miR-149-5p inhibitor were transfected into THP-1 cells, and the cell apoptosis was determined using the APO Percentage assay kit (C). FASLG – Fas ligand; RT-PCR – real-time reverse transcription polymerase chain reaction; ** P<0.01; *** P<0.001.

Figure 5

MiR-149-5p targeted FASLG and activated FADD and caspases. si-FASLG or/and miR-149-5p inhibitor were transfected into THP-1 cells, and then the protein expressions of p-FADD, FADD, caspase-8, cleaved caspase-8, caspase-2, cleaved caspase-2, caspase-3, and cleaved caspase-3 were determined by Western blot. FASLG – Fas ligand; FADD – Fas-associated via death domain.