2034 ONCOLOGY LETTERS 18: 2034-2042, 2019 MicroRNA‑144 targets APP to regulate AML1/ETO+ leukemia cell migration via the p‑ERK/c‑Myc/MMP‑2 pathway LING JIANG1*, WEI MENG2*, GUOPAN YU1, CHANGXIN YIN1, ZHIXIANG WANG1, LIBIN LIAO1 and FANYI MENG3 1Department of Hematology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510500; 2Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong 510515; 3Department of Hematology, Kang Hua Hospital, Dongguan, Guangdong 523080, P.R. China Received February 20, 2018; Accepted December 4, 2018 DOI: 10.3892/ol.2019.10477 Abstract. Extramedullary infiltration (EMI) is common in targets the APP gene and regulates cell migration via the patients with acute myeloid leukemia (AML) and is closely APP/p‑ERK/c‑Myc/MMP‑2 pathway. associated with the prognosis of disease. We previously reported that patients carrying the AML1/ETO (A/E) fusion Introduction gene and expressing the amyloid precursor protein (APP) tended to develop EMI, and had a poor prognosis. In the Due to the continued improvement of chemotherapeutic agents, present study, the relapse‑free survival (RFS) time and overall the remission rate of leukemia has significantly improved (1-3). survival (OS) time were significantly lower in patients with However, the recurrence rate remains as high as 30‑40% (4). EMI. The results demonstrated that the EMI incidence was Extramedullary infiltration (EMI) of acute leukemia includes significantly higher (P<0.05), while the RFS and OS rates were a wide variety of clinically significant phenomena that often significantly lower (P<0.05), in patients with high APP expres- pose therapeutic dilemmas. The acute myeloid leukemia 1 sion. Kasumi‑1 cells, which are A/E+, and the APP gene were protein/protein ETO (AML1/ETO; A/E) fusion gene, caused used as the in vitro cell model to detect the mechanism of action by a t(8;21) translocation, accounts for 15% of AML cases and in detail. Following the knockdown of APP expression, cell is identified in 15‑26.7% of young patients with EMI (5), with migration was significantly reduced (P<0.05). Furthermore, a 5‑year overall survival (OS) rate of 61% (6). It is hypoth- western blotting demonstrated that the protein expression of esized that being A/E+ is indicative of a good prognosis and phosphorylated extracellular‑signal‑regulated kinase (p‑ERK), it has been reported that ≤40% of A/E+ leukemia cases have matrix metalloproteinase‑2 (MMP‑2) and c‑Myc was markedly EMI (7-9). Our previous study demonstrated that patients with reduced following interference of APP, while the expression high expression of amyloid precursor protein (APP) were more of CXCR4 and MMP‑9 was not altered. Kasumi‑1 cells were likely to develop EMI (10). The aim of the present study was co‑cultured with p‑ERK or c‑Myc inhibitors and demonstrated to investigate the molecular mechanisms of EMI in detail that the APP/p‑ERK/c‑Myc/MMP‑2 pathway was involved in vitro. in signal transduction and regulation of cell migration. C‑X‑C motif chemokine receptor 4 (CXCR4) serves an MicroRNA‑144 (miR‑144) mimics and transfected Kasumi‑1 important role in cell migration, with certain studies hypoth- cells were generated. Reverse transcription‑quantitative poly- esizing that cell migration is predominantly dependent on the merase chain reaction and western blotting demonstrated that CXCR4/stromal‑derived factor‑1 (SDF‑1) axis (11). CXCR4 miR‑144 was a negative regulator of APP. Taken together, the and its ligand SDF‑1 are primarily studied for their crucial findings of the present study suggest that miR‑144 negatively roles in the homing of stem and progenitor cells in the bone marrow, chemotaxis, cell arrest, angiogenesis, metastasis and cell survival (12). Tavor et al (13) reported that AML cells constitutively secrete and express SDF‑1‑dependent cell surface elastase, which regulates their migration and prolif- Correspondence to: Professor Fanyi Meng, Department of eration. Our previous study revealed that A/E+ patients highly Hematology, Kang Hua Hospital, 1000 Dongguan Road, Dongguan, expressed CXCR4. Furthermore, it was found that APP regu- Guangdong 523080, P.R. China E‑mail: [email protected] lated cell migration via matrix metalloproteinase (MMP)‑2. MMP‑2 and MMP‑9 serve important roles in metastasis due *Contributed equally to their capacity to degrade the extracellular matrix (14,15); they are hypothesized to be particularly important for cell Key words: microRNA‑144, amyloid precursor protein, migration, as these proteinases act on type IV collagen (11). AML1/ETO+, migration, matrix metalloproteinase‑2 Experimental evidence has demonstrated that MMP‑2 and MMP‑9 are not only involved in the invasion and metastasis of solid tumors, but are also overexpressed in a variety of JIANG et al: miR‑144 TARGETS APP TO REGULATE LEUKEMIA CELL MIGRATION 2035 acute and chronic leukemia (13,16), suggesting that they may reverse transcription‑quantitative polymerase chain reac- serve an important role in breaking through the bone marrow tion (RT‑qPCR). The prognosis was analyzed and compared barrier. In the present study, the different molecular expres- between these two groups (Table II). sion levels of MMP‑2 and MMP‑9 were measured following interference with APP expression. Cell culture and detection. Kasumi‑1 cells (American Type MicroRNA (miRNA/miR)‑144 is an important tran- Culture Collection, Manassas, VA, USA), which are a human scriptional regulator in the process of hematopoiesis, and its A/E+ cell line derived from AML, were plated at 3x105 cells/ml abnormal expression is closely associated with the patho- in RPMI‑1640 medium (Gibco; Thermo Fisher Scientific, Inc., genesis of hematological malignancies (17,18). At present, an Waltham, MA, USA) supplemented with 20% fetal bovine increasing amount of research is focusing on miR‑144 and its serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 4 mM role in erythroid formation; however, there are relatively few glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin studies pertaining to its role in leukemia (19,20). Liu et al (21) at 37˚C in a humidified atmosphere containing 5% CO2. FISH, reported that miR‑144 increased the sensitivity of leukemia morphological detection using Wright's dye, and karyotype cells to imatinib and was closely associated with the c‑Myc analysis were performed as described previously (10). gene. Liang et al (22) reported that miR‑144 could inhibit human embryonic trophoblast cell invasion. Based on these Cell migration assay. The procedure was performed as results, we hypothesize that miR‑144 may serve an important described previously (10). To evaluate the migration ability role in the migration of Kasumi‑1 cells. the method was adjusted as follows: Non‑invading cells were The present study investigated the association between removed from the upper surface of the Transwell membrane miR‑144 and the APP gene, and demonstrated that APP with a cotton swab and invading cells on the lower membrane regulates cell migration through the APP/phosphorylated surface were fixed in methanol at the room temperature for extracellular‑signal‑regulated kinase (p‑ERK)/c‑Myc/MMP‑2 15 min. The membrane was then stained with 0.1% crystal pathway. This finding may provide novel insights into the violet for 15 min, washed with water three times and dried development of therapeutic strategies for the treatment of for 3 h at the room temperature. Images were captured using patients with AML, with particular focus on A/E+ patients a light microscope (magnification, x200) and cells in five with EMI. randomly selected fields were counted. Each invasion experi- ment was performed in triplicate. Materials and methods Target in vitro luciferase reporter assay. pMIR‑REPORT Patient characteristics. A/E+ AML patients (n=123), diagnosed plasmids for the miR‑144 target APP 3'‑untranslated region according to the World Health Organization 2008 criteria (23), (UTR) were constructed as wild‑type (WT) pmiR‑APP were enrolled in the present study between February 2002 containing two tandem repeats of miR‑144 response element and June 2013 at the Nanfang Hospital, Southern Medical from the APP 3'‑UTR and a mutant (MUT) pmiR‑APP University, (Guangzhou, China). All patients enrolled in the by replacing two nucleotides within the ‘seed sequence’ present study provided written informed consent, and the study (psi‑CHECK‑APP‑mut‑UTR). The oligonucleotides were was approved by the Ethics Committee of Nanfang Hospital annealed and inserted into the pMIR‑REPORT vector (Guangzhou, China). Patients were examined using marrow (Promega Corporation, Madison, WI, USA). The site‑directed cytology analysis, karyotype analysis and fluorescence in situ mutagenesis was performed using the Quick Change kit hybridization (FISH). All patients received follow‑up until (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, December 2013, with a median time of 46 months (6 ‑141 months). USA). The empty vector (pMIR‑REPORT) was used as a nega- As this was a retrospective study, certain data were not obtained tive control. Cells were transfected using Lipofectamine 2000 for all individuals included in the present study. (Invitrogen; Thermo Fisher Scientific, Inc.), according to All patients completed 1‑2 cycles of induction chemo- the manufacturer's protocol, with 0.2 µg Target reporter therapy, the majority of whom received the ‘3+7’ regimen plasmids (catalog no. 64158; Addgene, Inc., Cambridge, MA, consisting of anthracyclines and cytarabine. Following remis- USA) and 0.01 µg pMIR‑REPORT Control Plasmid (catalog sion, 109 patients received a median of 3 (range, 1‑8) intrathecal no. 26280; Addgene, Inc.), and 200 nM miR‑144‑MIMIC injections of 10 mg methotrexate or 50 mg cytarabine plus (miR‑34c precursor; Addgene, Inc.) and NC control (Shanghai 5 mg dexamethasone, which were used alternately. A total of GenePharma Co., Ltd.) per well on 96‑well plates.