miR-149-3p suppressed epithelial–mesenchymal transition and tumor development in acute myeloid leukemia

ABSTRACT Objective Acute myeloid leukemia (AML) is a form of primary acute leukemia with high mortality. Our previous study demonstrated that miR-149-3p was down-regulated in chemoresistant acute leukemia cells. However, the biological function of miR-149-3p in AML needs to be further explored. Methods Herein, the expression of miR-149-3p was overexpressed/silenced in U-937 human AML cells via transfection with miR-149-3p agomir/antagomir. The effect of miR-149-3p on U-937-induced tumor growth was investigated using a xenograft nude mouse model. Results The results showed that miR-149-3p overexpression inhibited the proliferation and increased the apoptosis of U-937 cells. In addition, miR-149-3p suppressed epithelial–mesenchymal transition in U-937 cells, as demonstrated by the miR-149-3p agomir-induced increase in E-cadherin expression and decrease in vimentin expression. The in vivo experiments demonstrated that miR-149-3p suppressed tumor progression. Conclusion In conclusion, the findings revealed the association of miR-149-3p with the development of AML and suggest that miR-149-3p is a potential therapeutic candidate for AML.


Introduction
Acute leukemia is a heterogeneous type of malignant disorder defined by changes in mechanisms of differentiation, self-renewal, and proliferation in hematopoietic progenitor cells committed to the lymphoid or myeloid [1]. Acute myeloid leukemia (AML) is a form of primary acute leukemia in adults characterized by the cumulation of malignant precursors of the myeloid lineage in the peripheral blood and bone marrow due to the loss of hematopoietic progenitor cells' ability to differentiate into normal hematopoietic cells [2][3][4]. In 2015 alone, an determined 27080 new case of acute leukemia were diagnosed in the United States, with over 76% of the cases being AML [5]. The morbidity of AML increases with age, from ∼1.3 in every 10000 patients less than 65 years old to 12.2 in every 10000 patients over 65 years old [6]. Despite decades of effort in clinical studies, the mainstream AML treatment is limited to chemotherapy, such as anthracyclines like daunorubicin, and the prognosis remains poor due to relapse and drug resistance [7][8][9][10]. Targeted treatments development provides promise for more effective therapies with reduced side effects [11,12].
MicroRNAs (miRNAs) are small endogenous RNAs with 19-25 nucleotides that modulate gene expression post-transcriptionally [13]. They are involved in the regulation of cellular processes such as apoptosis, proliferation, and epithelial-mesenchymal transition (EMT) [14,15]. The emergence of miRNAs has been one of the most decisive developments in tumor biology over the past decade and has provided new therapeutic and diagnostic opportunities for cancer [16]. Accumulating studies have shown that miRNAs are related to the initiation and development of leukemia and act as promise therapeutic candidates in AML therapy [17,18]. For example, Vandewalle et al. found that miR-21-5p and miR-15a-5p were up-regulated in chemoresistant AML patients and contributed to chemoresistance in AML by targeting ARL2, BTG2, and PDCD4 [19]. We previously demonstrated that miR-149-3p was down-regulated in chemoresistant acute leukemia cells, indicating that it may play an important function in chemotherapy failure [20]. However, the biological function of miR-149-3p in AML needs further exploration.
To investigate the effect of miR-149-3p in AML, the expression of miR-149-3p was overexpressed or silenced in U-937 human AML cells via transfection with miR-149-3p agomirs or antagomirs, respectively. Cell proliferation and apoptosis were evaluated, and the effect of miR-149-3p on U-937-induced tumor growth was investigated in vivo.

Cell counting kit-8
Cell counting kit-8 (CCK-8) was performed to evaluate cell proliferation after transfection. Harvested transfected U-937 cells and HL-60 were grown in 96-well plates (3 × 10 3 cells in 100 μl of medium per well) and maintained at 37°C with 5% CO2 for 24, 48, and 72 h. After 10 μl of CCK-8 solution (Solarbio) was added, the cells were further cultured for 4 h and the optical density was measured at 450 nm using an AMR-100 apparatus (Leica).
Hoechst 33258 staining 5 × 10 5 transfected cells were fixed in 0.5 ml of fixative for 30 min and washed twice with phosphate-buffered saline (PBS). The cells were resuspended in 50 μl of PBS, dropped onto a glass slide, and stained with 0.5 ml of Hoechst 33258 staining solution (Solarbio) for 5 min. After coating with antifade solution, the glass slide was viewed using an inverted fluorescence microscope (Leica) at 200× amplification.
Flow cytometry 1 × 10 6 transfected cells were resuspended in medium and centrifuged at 400×g, 4°C for 5 min. The cells were then resuspended in 200 μl of PBS and stained with 10 μl of Annexin V-fluorescein isothiocyanate (BD) and 10 μl of propidium iodide (BD) in the dark for 30 min. After adding 300 μl of PBS, flow cytometry was performed using a NovoCyte apparatus (ACEA Biosciences).

Tumor xenograft in nude mice
Forty female specific-pathogen-free BALB/c nude mice aged 4-6 weeks (gifted from Changzhou Kavens laboratory Animal Co. Ltd.) were maintained at 22-26°C with 50-60% relative humidity. After three days of adaptive feeding with free access to water and food, all mice were subcutaneously injected with 100 μl of U-937 cells (1 × 10 7 cell/ml) at the right axilla. When the tumor was clearly identifiable, tumor volume was measured every two days using the following formula: tumor volume (mm 3 ) = length × width 2 /2 [21]. The tumorigenesis rate was 75%. When the volume reached approximately 100 mm 3 , the nude mice were randomly divided into five groups (n = 6 per group): Control, miR-149-3p agomir (miR-agomir), miR-149-3p agomir negative control (Agomir-NC), miR-149-3p antagomir (miR-antagomir), and miR-149-3p antagomir negative control (Antagomir-NC). The mice in miR-agomir, Agomir-NC, miR-antagomir, and Antagomir-NC groups received multisite intratumoral injection of 30 μl of the respective substance at a concentration of 5 nmol (once every three days, four times). Mice in the control group received equal-volume saline treatment. At the end of the experimental period, the mice were sacrificed by an overdose of pentobarbital sodium (100 mg/kg). The tumors were removed, weighted, and fixed in 4% paraformaldehyde.

Hematoxylin-eosin (HE) staining
Pathological changes in the tumors were evaluated by HE staining. After embedding and slicing, the sections were dewaxed and stained with hematoxylin (Solarbio) for 3 min and eosin solution for 3 min (Solarbio). Pathological changes were assessed using a DM1000 apparatus (Leica, Germany).

Immunohistochemical staining
The protein expression of Ki-67 in tumor tissues was examined by immunohistochemistry. After embedding and slicing, tissue sections were dewaxed and hydrated before antigen retrieval using citrate buffer. The sections were blocked in 3% H 2 O 2 for 15 min and immersed in 10% goat serum for 20 min. They were incubated with Ki-67 primary antibody (Bioswamp) overnight and MaxVision TM HRP-Polymer anti-Mouse/Rabbit IHC secondary antibody (Fuzhou Maixin Biotechnology Development Co. LTD) for 45 min at room temperature. The samples were then visualized using diaminobenzidine (Solarbio) and counterstained using hematoxylin (Solarbio). Thereafter, the sections were observed using a microscope (Leica, Germany).

Statistical analysis
Data are presented as the mean ± standard deviation (SD). Statistical differences among data were analyzed using one-way analysis of variance followed by Tukey's tests. *P < 0.05 was considered to be statistically significant.

MiR-149-3p promotes apoptosis and suppresses EMT in U-937 cells and HL-60 cells
Apoptosis was assessed by Hoechst 33258 staining and flow cytometry. Hoechst results showed that miR-149-3p agomirs enhanced the apoptosis of U-937 cells, as demonstrated by the increase in the number of cells with bright blue staining (white arrow) and the percentage of apoptotic cells, respectively (Figure 2A). The flow cytometry results were similar to Hoechst 33258 staining, miR-149-3p agomirs significantly increased apoptosis in U-937 and HL-60 cells ( Figure 2B & C). Then, the mRNA and protein expression of apoptosis-related factors were evaluated by qRT-PCR and western blot, respectively. miR-149-3p agomirs increased the expression of pro-apoptotic indicators (caspase 3 and Bax, Figure 3A) and decreased that of anti-apoptotic indicators (survivin and Bcl-2, Figure 3B). The results of qRT-PCR and western blot are consistent with those of Hoechst 33258 staining and flow cytometry. In addition, the expression of EMT-related proteins in transfected U-937 cells was detected by western blot. miR-149-3p agomirs increased the expression of E-cadherin and decreased that of vimentin, whereas miR-149-3p antagomirs showed the opposite results ( Figure 3C).

MiR-149-3p suppresses U-937-induced tumor growth
To investigate the effect of miR-149-3p on lymphoma in vivo, we constructed a xenograft mouse model via the injection of U-937 cells. The mice were treated with miR-149-3p agomirs/antagomirs or their corresponding negative controls. The results showed that miR-149-3p agomirs suppressed tumor growth, while miR-149-3p antagomir aggravated tumor growth ( Figure 4A-D). HE detection of the pathological morphology of the tumors indicated that miR-149-3p agomirs attenuated tumor progression (Figure 4 E). In addition, the expression of Ki-67, a tumor proliferation marker [22], in tumor tissues was detected using immunohistochemical staining. The results indicated that Ki-67 expression was decreased by miR-149-3p agomirs but increased by miR-149-3p antagomirs. Collectedly, the results indicated that miR-149-3p is involved in regulating the growth of tumors induced by U-937 cells.

Discussion
MiR-149-3p is a new type of tumor-related factor, which delays the development of tumors by targeting and regulating certain factors in the body. In this work, we investigated the effect of miR-149-3p on AML cell proliferation and the expression of various marker proteins and genes, and overall described the effect of miR-149-3p on AML cell apoptosis. Yang et al. found that miR-149-3p inhibited the invasion, migration, and proliferation of bladder cancer cells by targeting S100A4 [23]. Si et al. suggested that miR-149-3p overexpression promoted pancreatic cancer cell apoptosis by suppressing the level of Bcl-2, enhancing those of Bax and cleaved caspase 3/9, and contributing to cytochrome c release, which were associated with miR-149-3p-mediated inactivation of Akt1 signaling [24]. Cao et al. demonstrated that miR-149-3p inhibited cell proliferation and induced apoptosis by targeting Wnt-1, thereby preventing the initiation and progression of gastric tumors [25]. Yao et al. showed that miR-149-3p enhanced the apoptosis and suppressed the proliferation, migration, and invasion of chordoma cells in vitro and impaired chordoma tumorigenesis in vivo by downregulating Smad3 [26]. Okato et al. revealed that miR-149 decreased the migration and invasion of renal cell carcinoma cells by targeting FOXM1 [27].
This study indicated that miR-149-3p overexpression inhibited the proliferation of AML cells. In addition, miR-149-3p promoted U-937 cell apoptosis by decreasing the levels of surviving and Bcl-2 and increasing those of caspase 3 and Bax, which agrees with the results of previous studies [24]. Apoptosis can be classified as either endogenous or exogenous [28]. The main feature of the difference is that apoptosis caspases are activated in different ways. Exogenous apoptosis activates intracellular caspases through extracellular signals, while endogenous apoptosis activates caspases through mitochondrial release of apoptotic enzyme activating factors. Endogenous apoptosis, also known as mitochondrial apoptosis, is identified by the release of caspase activators such as cytochrome c into the cytoplasm. This results in the activation of caspase 9 and the cleavage of downstream caspases such as caspase 3, in turn inducing apoptosis [29]. The release of cytochrome c is associated with the increased permeabilization of the outer mitochondrial membrane caused by the upregulation of Bax and downregulation of Bcl-2 [30]. All indications suggest that miR-149-3p overexpression-induced apoptosis in U-937 cells and HL-60 cells may occur through endogenous apoptosis.
ETM plays an important role in embryonic development, chronic inflammation, tissue reconstruction, cancer metastasis and multiple fibrotic diseases. This study demonstrated that miR-149-3p inhibited EMT in U-937 cells by upregulating E-cadherin and downregulating vimentin, consistent with a previous study showing that miR-149-3p mimics suppressed EMT in colorectal cancer [31]. EMT is a cellular process wherein epithelial cells transform into motile mesenchymal cells [32]. In the process of EMT, junctions and apical-basal polarity are lost, prompting epithelial cells to undergo cytoskeletal rearrangement. This rearrangement leads to changes in the mechanotransduction of signaling pathways that affect cell shape and gene expression. In turn, the cells take on an invasive phenotype with increased motility [32]. EMT is related to wound healing, embryogenesis, and malignant progression [33], and the upregulation of vimentin, an EMT marker, has been shown to be correlated with poor clinical outcome in AML [34]. Clinical experiments have also shown that the expression of E-cadherin, which acts as a prognostic biomarker in AML patients, was substantially downregulated in AML [35].

Conclusions
In conclusion, the current study demonstrated that miR-149-3p inhibited cell proliferation and EMT in AML cells and promoted their apoptosis, suggesting that miR-149-3p is a potential therapeutic candidate for AML. However, only the first type of EMT in AML cells was studied in this research, and the specific mechanism of miR-149-3p-mediated inhibition of AML will be investigated in subsequent studies.

Disclosure statement
No potential conflict of interest was reported by the author(s).