Downregulation of microRNA-144 inhibits proliferation and promotes the apoptosis of myelodysplastic syndrome cells through the activation of the AKAP12-dependent ERK1/2 signaling pathway

Highlights

• This is a report that miR-144 regulates AKAP12-mediated ERK1/2 signaling pathway.

• MiR-144 is highly expressed in MDS cells while AKAP12 is lowly expressed.

• MiR-144 promotes MDS by inhibiting expression of AKAP12, a target gene of miR-144.

• Down-regulation of miR-144 can activate the ERK1/2 axis and treat MDS.

• This study provides a basis for proliferation and apoptosis in MDS cells.

Abstract

Background

Myelodysplastic syndromes (MDS) represent a family of hematopoietic stem cell disorders characterized by ineffective hematopoiesis. While the functions of many microRNAs have been identified in MDS, microRNA-144 (miR-144) remains poorly understood. Thus, the aim of the present study was to determine the effects of miR-144 on cell proliferation and apoptosis in MDS cells and mechanism thereof.

Methods

MDS-related microarrays were used for screening differentially expressed genes in MDS. The relationship between miR-144 and A-kinase anchoring protein 12 (AKAP12) was determined by a dual luciferase reporter gene assay. Subsequently, gain- and loss-function approaches were used to assess the effects of miR-144 and AKAP12 on cell proliferation, cell cycle and cell apoptosis by MTT assay and flow cytometry. Following the induction of a mouse model with MDS, the tumor tissues were extract for evaluation of apoptosis and the expression of miR-144, AKAP12, and the relevant genes associated with extracellular-regulated protein kinases 1/2 (ERK1/2) signaling pathway and apoptosis.

Results

We observed significantly diminished expression of AKAP12 in MDS samples. miR-144 directly bound to AKAP12 3′UTR and reduced its expression in hematopoietic cells. Downregulation of miR-144 or upregulation of AKAP12 was observed to prolong cell cycle, inhibit cell proliferation, and induce apoptosis, accompanied by increased expression of AKAP12, p-ERK1/2, caspase-3, caspase-9, Bax, and p53, as well as decreased expression of Bcl-2. The transplanted tumors in mice with down-regulated miR-144 exhibited a lower mean tumor diameter and weight, and increased apoptosis index and expression of AKAP12 and ERK1/2.

Conclusion

Taken together, these studies demonstrate the stimulative role of miR-144 in MDS progression by regulating AKAP12-dependent ERK1/2 signaling pathway.

Introduction

Myelodysplastic syndrome (MDS) belongs to a highly heterogenous family of hematopoietic tumors featuring hemopoietic insufficiency, peripheral cytopenia as well as increased susceptibility to acute myeloid leukemias [1]. Patients with MDS usually exhibit several clinical phenotypes including bone marrow cellularity and blast count, cytopenia, and a higher risk of acute myeloid leukemias [2]. The therapeutic regimen for patients with MDS is largely influenced by the multifaceted pathogenetic factors, different heterogeneous stages, as well as the age of the patient, all contributing to the complexity involved in determining an appropriate treatment approach [3]. MicroRNAs (miRNAs) have been shown to play a pivotal role in regulating the physiological functions and progression of hematopoietic stem cells, with reports suggesting a correlation between them and hematologic diseases including MDS [4].

miRNAs are known to occupy an important place in normal hematopoiesis and hematological malignancies [5]. Previous studies have suggested a strong relationship between microRNA-144 (miR-144) and the progression of colorectal and bladder cancer [6,7]. In murine erythroleukemia cells, particularly high expression of miR-144 in blood has been previously demonstrated [8]. More specifically, the overexpression of certain miRNAs can act to suppress erythroid growth, while the down-regulated expression of certain miRNAs such as miR-144/451 cluster may be effective in disease-related ineffective erythropoiesis [9]. The ERK1/2 signaling pathway is a major cellular signal transduction pathway that converts a mass of extracellular signals into cellular responses by phosphorylating and inducing a plethora of downstream targets [10]. Furthermore, the ERK1/2 signaling pathway has been reported to promote cell survival in a series of specific manners [11]. A-Kinase anchoring protein 12 (AKAP12) is a scaffold protein known to regulate cell cycle [12]. Interestingly, a pervious study found that 5’ CpG island of the AKAP12 failed in the methylation process of normal hematopoietic progenitors and granulocytes, but its methylation occurs in leukemic myeloblasts [13]. Additionally, another report found that a total of 139 genes including Gravin/AKAP12 were downregulated in at least half of its experimental subject patients with MDS [14]. Taken together, the reviewed literature highlights the links between miR-144, ineffective erythropoiesis and AKAP12 which is downregulated in patients with MDS. Hence, we hypothesized that miR-144 is closely related to AKAP12 based on the possibly targeted relationship that was predicated on-line using the bioinformatics website. Thus an investigation into how miR-144 exerts its effects on patients suffering from MDS in vivo and in vitro was conducted.