miR-550-1 functions as a tumor suppressor in acute myeloid leukemia via the hippo signaling pathway

MicroRNAs (miRNAs) and N6-methyladenosine (m6A) are known to serve as key regulators of acute myeloid leukemia (AML). Our previous microarray analysis indicated miR-550-1 was significantly downregulated in AML. The specific biological roles of miR-550-1 and its indirect interactions and regulation of m6A in AML, however, remain poorly understood. At the present study, we found that miR-550-1 was significantly down-regulated in primary AML samples from human patients, likely owing to hypermethylation of the associated CpG islands. When miR-550-1 expression was induced, it impaired AML cell proliferation both in vitro and in vivo, thus suppressing tumor development. When ectopically expressed, miR-550-1 drove the G0/1 cell cycle phase arrest, differentiation, and apoptotic death of affected cells. We confirmed mechanistically that WW-domain containing transcription regulator-1 (WWTR1) gene was a downstream target of miR-550-1. Moreover, we also identified Wilms tumor 1-associated protein (WTAP), a vital component of the m6A methyltransferase complex, as a target of miR-550-1. These data indicated that miR-550-1 might mediate a decrease in m6A levels via targeting WTAP, which led to a further reduction in WWTR1 stability. Using gain- and loss-of-function approaches, we were able to determine that miR-550-1 disrupted the proliferation and tumorigenesis of AML cells at least in part via the direct targeting of WWTR1. Taken together, our results provide direct evidence that miR-550-1 acts as a tumor suppressor in the context of AML pathogenesis, suggesting that efforts to bolster miR-550-1 expression in AML patients may thus be a viable clinical strategy to improve patient outcomes.


Introduction
Acute myeloid leukemia (AML) is a form of cancer that arises when hematopoietic stem cells (HSCs) undergo oncogenic mutations. In the United States, 19,940 new AML cases are expected to be diagnosed, and 11,180 AML-associated deaths are expected to occur in 2020 (https://www.seer.cancer. gov/). While there have been countless efforts to develop novel therapeutic strategies suited to the treatment of AML, the majority of patients still suffer from poor outcomes, with recent reports estimating a 5-year survival rate of 40% among AML patients [1,2]. As such, there is a clear need to better understand the molecular basis for AML in order to expedite the Ivyspring International Publisher development of more efficacious therapeutic interventions.
MicroRNAs (miRNAs) are small 20-24 nucleotide non-coding RNA molecules that exhibit endogenous biological functionality via targeting specific downstream mRNAs [3]. These miRNAs mediate their activities through their interactions with the RNA-induced silencing complex (RISC), pairing with compatible bases in the 3' untranslated region (UTR) of target mRNAs. These interactions can result in either a suppression of mRNA translation, or a reduction in mRNA stability that lead to mRNA degradation, thus resulting in a marked reduction in target gene expression at the protein level [3,4]. There is now countless evidence that specific miRNAs serve essential regulatory roles in both the context of normal physiology and disease pathogenesis, including leukemogenesis [5][6][7][8].
Importantly, improved understanding of these miRNAs has led to their utilization for the treatment of certain disorders [9]. In hepatitis C mouse models, knockdown of miR-122 led to reduced liver damage and viral load via owing to its ability to regulate several targets, such as mannan binding lectin serine protease 1 (MASP1) and prolyl 4-hydroxylase subunit α1 (P4HA1) [10]. Using locked nucleic acids (LNAs)-modified antimiR-122, preclinical studies were performed in an effort to treat hepatitis C infection. The results indicated this LNAs could bring about a significant reduced liver injure and reduction in infection burden [10]. In nonsmall cell lung cancer (NSCLC), Wiggins JF et al. [11] chemically synthesized miR-34a and a lipid-based transport vehicle, and found that this combination effectively blocked cell proliferation by targeting cyclin-dependent kinase 4 (CDK4) in vitro and in vivo. In our previous research, we have identified a set of miRNAs with specific regulatory roles in the context of the proliferation, differentiation, and apoptosis of AML cells. These miRNAs include miR-9, miR-22, miR-26a, miR-150, miR-495, miR-181, miR-126, miR-196b and the miR-17-92 cluster [12][13][14][15][16][17][18][19][20][21]. In light of our research into miR-150, we ultimately developed a novel FLT3 ligand-binding (amidoamine)-miR-150 nanoparticle (G7-FLT3L-miR-150 nanoparticle) [22], which specifically delivered miR-150 to FLT3overexpressing AML cells by employing FLT3L as a guiding molecule. Through inhibiting the activation of PIM, AKT, ERK, and STAT5, this nanoparticle displayed a strong anti-leukemic effect in vitro and in vivo. Even so, however, the specific role of these miRNAs in AML is not completely understood.
WW-domain containing transcription regulator-1 (WWTR1), also known as the transcriptional co-activator with PDZ-binding motif (TAZ), was first detected based on its status as a 14-3-3 interacting protein [23]. WWTR1 and the paralogous Yes-associated protein (YAP) serve as central downstream regulatory factors in the Hippo signaling pathway, which modulates a wide range of cellular processes pertaining to cellular energy status, hypoxia, osmotic stress, tissue organ size, regeneration, homeostasis, and tumorigenesis [24,25]. Indeed, elevated WWRT1 mRNA and protein expression have been found to be associated with the development of gastric, colorectal, breast, and lung cancers [26][27][28][29][30]. Consistent with this, elevated WWTR1 protein level has been determined to be a risk factor for the development of both glioblastoma multiforme and colorectal cancer [31,32]. In one study, Justice et al. [33] also determined that there was an association in gastric cancer between increased WWTR1 expression and tumor TNM stage as well as incidence of lymph node metastasis. Wang et al. [27] further found that WWTR1 expression in NSCLC led to its regulation of Cyclin A and C transforming growth factor (CTGF), which in turn led to a disruption of apoptosis in neoplastic cells. Recent work has revealed that large tumor suppressor (LATS)-mediated YAP/WWTR1 phosphorylation was the key regulatory event controlling the activity of these proteins in cells [34]. Consistent with this, Jimenez-Velasco et al. [35] found LATS1 and LATS2 to be downregulated in leukemia as a consequence of their hypermethylation, and reduced LATS2 expression has been found to be associated with worse outcomes among leukemia patients. This suggests the possibility that a reduction in LATS1/2 activity may underlie the alterations in YAP/WWTR1 stabilization and activation in the context of leukemia. However, clarity is still needed regarding the mechanisms governing increased WWTR1 activity in AML.
In the present study, we for the first time provided evidence that miR-550-1 was significantly downregulated in AML. Moreover, when overexpressed, miR-550-1 was able to impair AML cell proliferation and oncogenesis both in vitro and in vivo owing to its ability to regulate the Hippo signaling pathway. We further found WWTR1 to be a direct miR-550-1 target, thereby at least partially explaining its role in regulating AML progression.

Measurement of cell viability
A MTT assay (Promega, Madison, USA) was used to measure viability based on provided directions. Briefly, MV4-11 and Kasumi-1 cells were plated into 96-well plates (10000 cells/100 μL), with dye solution added to wells at the indicated time points. After 4-hour 37°C incubation, stop buffers were added and cell absorbance was assessed the following day at 570 nm.

Flow cytometry
A BD LSRII Flow Cytometer was used in all analyses, and FlowJo v10 was used for data analysis. For measurements of apoptosis, 0.5×10 6 cells were stained with an Annexin V-APC Apoptosis Detection Kit (BD Biosciences, San Diego, USA) based on provided directions.
For cell cycle analyses, 0.5×10 6 cells were fixed overnight at 4°C in 75% ethanol, washed thrice in PBS, and stained using propidium iodide for 20 minutes.

Quantitative RT-PCR
A miRNeasy kit (Qiagen, Frederick, USA) was used for extracting total RNA from 1×10 6 cells based on provided directions. cDNA was then synthesized from 1 µg of this RNA via M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA). A 7900HT real-time PCR system (Applied Biosystems, Foster City, USA) was employed for qPCR analyses, with SYBR Green used to setup triplicate reactions assessing relative mRNA expression. For miRNA expression, TaqMan qPCR was conducted according to provided directions (Applied Biosystems). The 2 −ΔΔCt method was used to calculate miRNA and mRNA relative expression, which was normalized to endogenous levels of U6 and GAPDH, respectively.

Plasmid and virus production
The pri-miR-550-1 sequence was amplified by PCR from healthy human BM mononuclear cells (MNCs). Primers with mutated sequences (Table 1) were then used to generate the indicated mutant miR-550-1 template, and these wild type (WT) and mutant miR-550-1 sequences were thereafter cloned into the MSCV-PIG vector (MSCV-Puromycin-IRES-GFP vector) (Cold Spring Harbour Laboratory, USA) in order to overexpress these two miRNA isoforms. These sequences were inserted between the XhoI (CTCGAG) and EcoRI (GAATTC) sites in this vector. For WWTR1-CDS vectors, the WT sequence was amplified from healthy human BM MNCs prior to insertion into the pCDH vector (SBI, Mountain View, USA). The MSCVneo-MLL-AF9 plasmid was kindly provided by Dr. Scott Armstrong.
One day prior to transfection, 5 ×10 5 HEK293T cells were plated into 60-mm dishes. Retroviruses were then produced via transfecting cells with vector DNA and a packaging vector (PCL-Eco or PCL-Ampho) with the Effectene Transfection Kit (Qiagen). The WWTR1 overexpressing lentivirus was generated via co-transfection of the WWTR1-pCDH plasmid and packaging lentivirus vectors (pRSV-Rev, pMDLg/ pRRE and pMD2.G). At 48 and 72 h post-transfection, cellular supernatants were harvested and filtered through a 0. 45

Dual luciferase reporter and mutagenesis assay
We conducted dual luciferase reporter and mutagenesis assays based on a modified version of a previously reported protocol [16]. The WWTR1 3'-UTR sequences containing putative miR-550-1binding sites were synthesized via PCR utilizing the following primers: forward 5'-GGGCACTAGTATTC GACCTGATTTACAGTTTC-3' and reverse 5'-TATT ACGCGTTGAGATCAGGAGTTTGAGAAC-3'. The resultant fragment underwent insertion into the pMIR-REPORT vector (Ambion, Austin, USA). In addition, a mutated version of this 3'-UTR fragment was generated using primers bearing the mutant sequence. A total of 6,000 HEK293T cells were plated per well of a 24-well plate in triplicate, and following overnight culture these cells were co-transfected with pMIR-REPORT-WWTR1 or mutant pMIR-REPORT-WWTR1 vectors and MSCV-PIG-miR-550-1, mutant MSCV-PIG-miR-550-1, or MSCV-PIG empty vectors (20ng each). The β-galactosidase vector (1ng) (Ambion) was additionally transfected into all experimental cells, and after a 48 h incubation all cells were lysed. Relative luciferase activity was then measured via a Dual-Light Combined Reporter Gene Assay System (Applied Biosystems).

Colony forming/replating assay
Colony formation assays were performed in accordance to a modified version of a previously reported protocol [16]. Donor murine BM cells were isolated from 6-week old B6.SJL (CD45.1) after injecting 5-fluorouracil (5-FU; 150mg/kg) for 5 days, and hematopoietic progenitor cells were isolated via a Mouse Lineage Cell Depletion Kit (Miltenyi Biotec Inc., Auburn, USA). These progenitor cells then underwent infection with the indicated viruses using a spinoculation protocol with the assistance of polybrene [36][37][38]. Following transduction, cells were incubated overnight at 37 o C in fresh media, and this was then repeated the following day. Thereafter, 2 × 10 4 cells were plated in methylcellulose medium (Stem Cell Technologies Inc, Vancouver, Canada) containing 10 ng/ml IL-3, IL-6, granulocytemacrophage colony-stimulating factor (GM-CSF), 30 ng/ml SCF, and 2 μg/ml puromycin and/or 1 mg/ml G418, as appropriate. After incubating for 7 days, colony formation in each of the experimental groups was assessed, and these colonies were then replated.
In the primary BMT assays, BM cells from healthy donor mice (B6.SJL) were transduced with the indicated retrovirus combinations (MSCV-PIG + MSCVneo-MLL-AF9, MSCV-PIG-miR-550-1 + MSCVneo-MLL-AF9, and mutant MSCV-PIG-miR-550-1 + MSCVneo-MLL-AF9). The resultant donor cells were then mixed with helper cells (BM cells from a healthy C57BL/6 mouse) at a ratio of 3 × 10 5 to 1 × 10 6 per recipient mouse. These cells were then injected into the tail vein of an 8-week old lethally irradiated (960 rads) recipient mouse. In secondary BMT assays, these lethally irradiated recipient mice were injected using leukemic BM cells that had been isolated from the initial primary recipient mice, and no helper cells were added.
Once recipient mice exhibited signs of systemic illness, peripheral blood (PB) samples were collected via tail bleeding in order to establish whole blood counts. Engraftment was evaluated via flow cytometry based on CD45.1 expression in PB samples. Moribund mice were euthanized, and liver, thymus, and spleen weight was determined. BM cells were collected from euthanized animals and prepared for cytospin slides, which then underwent Wright-Giemsa staining.

m 6 A dot blot assay
We isolated total RNA with the miRNeasy kit and quantified RNA levels. Next, RNA samples were spotted using a Bio-Dot Apparatus (Bio-Rad, Hercules, USA) on Amersham Hybond-N+ membranes (GE Healthcare, Chicago, USA), and a UV cross-linker was then used to cross-link them to this membrane. Membranes then underwent two washes using Milli-Q, followed by treatment for 10 min using 0.02% methylene blue (Sigma-Aldrich). Membranes were then rinsed until the dye was washed away from background regions, and dots of methylene blue were then imaged. Next, 5% nonfat dry milk was used for membrane blocking for 1 h, after which an antibody against m 6 A (1:2000 dilution, Synaptic Systems, Goettingen, Germany, #202003) was used to probe blots at 4°C overnight. Membranes were then washed thrice in TBST and probed at room temperature using HRP-conjugated goat anti-rabbit IgG for 1 h, prior to visualization with an ECL system.

Statistical analysis
SPSS v16 (IBM, Armonk, USA) was used to compare all experimental results via Student's t-tests or two-way ANOVAs. Data are given as means ± standard deviations (SDs) from at least three repeat experiments. The Kaplan-Meier approach was used to assess overall survival. P<0.05 was the threshold of statistical significance.

miR-550-1 is down-regulation in AML
We have previously detected a set of specific miRNAs that were down-regulated in AML samples with t(8; 21), inv(16), t(15; 17), or mixed lineage leukemia (MLL) rearrangements, relative to normal controls (NC) [21]. Of these previously identified miRNAs, miR-550-1 was among the most significantly down-regulated in the AML cohort. We therefore first confirmed that miR-550-1 was significantly down-regulated in AML patients using bone marrow (BM) mononuclear cell (MNC) samples from 12 patients with primary AML (from University of Chicago Hospitals), revealing a marked decrease in the expression of this miRNA as compared to NC samples (n=4) (P=0.003) (Fig. 1A). We then extended these findings to a larger 166 patient primary AML sample cohort (from the First Affiliated Hospital of Zhejiang University) ( Table 2 and Table S1), using qPCR to confirm that miR-550-1 was significantly down-regulated in AML patients relative to NC samples, which included 8 normal BM samples and 20 normal peripheral blood (PB) samples (P<0.001) (Fig.  1B). Similarly, miR-550-1 expression was markedly decreased in 9 leukemic cell lines (including MV4-11, Kasumi-1, MOLM-13, MONOMAC-6, OCI-AML3, NB4, THP-1, HL-60, OCI-AML2) relative to controls (P<0.001) (Fig. 1B). In addition, the expression level of miR-550-1 was significantly increased at complete remission (CR) compared to initial diagnosis (P=0.010) ( Figure S1). We did not find any significant differences with respect to miR-550-1 expression as a function of the French-American-British (FAB) subtype (Fig. 1C). Given its low expression in AML patients, we next sought to determine whether the level of miR-550-1 expression correlated with patient clinical outcomes. Using a 162 patient TCGA AML dataset, we divided patients based on whether they expressed high or low levels of miR-550-1, as determined based on the median expression level of this miRNA. In so doing we found that patients with lower miR-550-1 expression exhibited poorer overall survival (OS) than did those with higher expression (P=0.026; Fig. 1D). Together, these findings validated our previous results with respect to patterns of miR-550-1 in AML, and strongly suggested the possibility that this miRNA may serve as a tumor suppressive role in the context of leukemogenesis. Promoter methylation is known to be a key regulator of many miRNAs in the context of AML, including miR-126 and miR-375 [13,39]. Whereas we have previously found the miR-22 promoter to be hypomethylated [15], when we assessed this TCGA dataset we found that the pri-miR-550-1 promoter region was hypermethylated (Fig. 1E). Consistent with these findings, when the MV4-11 and Kasumi-1 AML cell lines were treated using the hypomethylating agent, decitabine, miR-550-1 expression rose significantly (Fig. 1F, G). In addition, the methylation degree of miR-550-1 CpG islands was apparently inhibited with decitabine ( Figure S2). These findings are therefore consistent with a model wherein miR-550-1 promoter hypermethylation in AML cells may lead to its reduced expression.
Notably, when miR-550-1 was overexpressed, this led to a marked induction of cell differentiation (Fig. 2E). In order to assess the role of miR-550-1 in the context of AML biology, we next used the MV4-11 and Kasumi-1 human AML cell lines to conduct gain-of-function experiments. We found that forced ectopic miR-550-1 expression led to a clear reduction in the viability and proliferation for both of these cell lines (Fig.  3A-F). Furthermore, miR-550-1 overexpression led to a marked increase in the apoptotic cell death and G0/1 phase arrest (Fig. 3 G  and H). Consistent with these results [42,43], western blotting confirmed that the levels of the G0/1-S checkpoint regulatory molecules CCND1 and CDK2 were reduced upon ectopic miR-550-1 expression, as well as the levels of the cell cycle regulatory proteins p-Rb, E2F1, and P27. We also observed clear decreases in the levels of the proliferation-associated proteins p-AKT and c-myc, as well as the anti-apoptotic BCL-2 protein upon ectopic miR-550-1 expression, whereas levels of the pro-apoptotic PARP protein were markedly increased in these same cells (Fig. 3I). Together these results strongly suggest that miR-550-1 significantly reduces rates of AML cell proliferation and leukemic cell transformation proliferation in vitro.
We additionally utilized secondary BMT assays as a means of assessing the importance of miR-550-1 in the maintenance of AML following its development. We found that mice in the miR-550-1 + MLL-AF9 group exhibited slower AML development than did those in the MLL-AF9 only group (median overall survival: 33 vs. 27 days, P=0.010; Fig. 4I), suggesting that suppressing miR-550-1 did contribute to the sustained maintenance of AML driven by the MLL-AF9 fusion gene. Secondary CFA further provided confirmation that miR-550-1 overexpression was linked to delayed leukemogenesis ( Fig. 4J and K). Our results together therefore provide clear evidence for the role of miR-550-1 as a tumor suppressor in the context of AML development and maintenance.

Identification of potential miR-550-1 target genes
miRNAs are able to mediate their biological activities through the suppression of specific target genes. Given that our results suggested that miR-550-1 played a central role in suppressing the development of leukemia through regulating the cell cycle and inducing apoptosis, we next sought to identify potential miR-550-1 target genes linked to the promotion of apoptosis and G0/1 phase arrest. We therefore employed the TargetScan, PITA, miRanda, and miRBase programs in order to predict potential targets, identifying a total of 4,941 putative target genes identified by a minimum of 1 of these programs. Of the genes identified via this approach, WWTR1 was of potential interest as it scored highly among the predicted genes. WWTR1 has previously been found to serve as a key transcriptional co-activator in the Hippo signaling pathway, and there were multiple reports indicating that it could positively regulate both the cell cycle (specifically the G1/S transition) and mitochondrially-induced apoptosis in a range of cancer types [26,28,44]. We therefore first sought to assess WWTR1 mRNA expression in AML patient samples, revealing it to be significantly upregulation in these patients' samples relative to NC samples (P<0.05; Fig. 5A). Kaplan-Meier survival curves also confirmed that higher WWRT1 mRNA levels were correlated to poorer overall survival outcomes in these patients (P=0.030; Fig. 5B). This suggests that WWTR1 may serve as an oncogenic function in AML, and may also be a miR-550-1 target gene.  Using the TCGA and CALGB datasets, we performed in silico analyses revealing a negative correlation between miR-550-1 and WWTR1 expression (r=-0.173, P=0.021; r=-0.291, P=0.007, respectively). We additionally examined the expression of miR-550-1 and WWTR1 in 90 AML samples in our cohort, again revealing a significantly negative correlation between these two factors (r=-0.257, P=0.014; Fig. 5C). We also confirmed that the expression of WWTR1 was reduced at both the mRNA and protein level upon miR-550-1 overexpression in AML cells (Fig. 5D and E). Curiously, in the MV4-11 cells, the WWTR1 was downregulated only at the protein but not at the mRNA level upon miR-550-1 overexpression, suggesting that miR-550-1 might primarily affect the WWTR1 mRNA stability. Previous work suggested that Wilms tumor 1-associated protein (WTAP) targeting could lead to alterations in mRNA stability as a function of m 6 A modification [45,46]. Our results also suggested that WTAP was a direct miR-550-1 target gene (Fig. 5F and G). Induced ectopic expression of miR-550-1 also led to a reduction in global mRNA m 6 A levels (Fig. 5H), potentially contributing at least in part to the reduced WWTR1 stability observed in our results.
To further confirm that there was a direct interaction between miR-550-1 and WWTR1, we next utilized a luciferase reporter assay system, generating luciferase reporter constructs bearing either a WT or mutated form of the WWTR1 3'-UTR in the pMIR-REPORT vector (Fig. 5I). When utilized in cells, we found that miR-550-1 was able to significantly decrease the luciferase activity associated with promoters bearing the WT WWTR1 3'-UTR (P=0.002), whereas they had no effect on those bearing the mutated WWTR1 3'-UTR (Fig. 5J). These results thus provide strong evidence supporting the fact that WWTR1 is a miR-550-1 target.

WWTR1 is a key miR-550-1 target in AML
As WWTR1 is known to have important roles in the context of cancer [26,47,48], we next performed loss-and gain-of-function experiments in order to ascertain as to whether WWTR1 contributed to the anti-leukemic activity of miR-550-1. At present, the role of WWTR1 in AML has not been specifically assessed. We therefore employed a small interfering RNA construct to silence WWTR1 expression at the mRNA and protein level ( Fig. 6A and D), and this led to a marked decrease in the proliferation of Kasumi-1 and MV4-11 cells, which instead underwent G0/1 arrest ( Fig. 6B and C). We found that WWTR1 knockdown was linked to decreased protein levels of CCND1, CDK6, p-Rb, E2F1, BCL-2, p-AKT, and c-myc (Fig. 6D), phenocopying the anti-leukemic activity of miR-550-1. When WWTR1 was instead overexpressed in Kasumi-1 cells, this led to an increase in cell viability and proliferation, largely reversing the inhibitory effects of miR-550-1 (Fig. 7A-E). When WWTR1 and miR-550-1 were co-expressed, this led to a reversal in miR-550-1-mediated suppression of cell proliferation and G0/1-phase arrest in Kasumi-1 cells (Fig. 7F and G). Together, these findings thus suggest that miR-550-1-mediated suppression of WWTR1 expression at least partially governs the anti-leukemic activity of this miRNA.

Discussion
Herein, we produce evidence that miR-550-1 plays a role in suppressing the development of AML and the proliferation of AML cells, instead promoting their apoptotic death. In humans, miR-550-1 is encoded in the 7p14.3 locus, which is a non-coding section of the ZNRF2 gene. Landgraf et al. first identified this miRNA via small RNA library sequencing in 2007, but it has not previously been studied in depth in the context of cancer [49]. To our knowledge, no previous reports have shown that miR-550-1 plays a vital role in leukemogenesis. We determined that miR-550-1 expression was decreased in two independent primary AML patient cohorts, and elevated miR-550-1 expression was associated with higher hemoglobin (Hb) levels (Table 2), potentially contributing to improved patient prognosis. However, at present there is no strong evidence regarding a link between miR-550-1 and Hb, and as such further research is required. Consistent with our findings regarding the methylation of the miR-550-1 region, we found that decitabine was able to partially reverse miR-550-1 downregulation, suggesting methylation of this region in AML patients contributed to miR-550-1 dysregulation.
In order to explore the specific mechanisms governing the link between miR-550-1 and reduced leukemia severity, we first conducted an in silico target analysis approach. We then, using luciferase reporter and mutagenesis assays, confirmed that WWTR1, which is an oncogene in solid tumors [28,30,31], was one key miR-550-1 target. Noguchi et al. [28] recently found that elevated WWTR1 expression was associated with poorer overall survival in those with NSCLC, and that it was confirmed to be an independent inferior element in NSCLC patients (HR=1.34, 95%CI 1.01-1.76, P=0.040). Similarly, breast cancer patients with elevated WWTR1 expression also exhibit poorer outcomes [30]. Furthermore, there is a positive correlation between WWTR1 expression and poorer prognosis, increased tumor invasion, and metastasis in those with gastric cardia adenocarcinoma [50]. Consistent with these past results, we observed significant upregulation of WWTR1 mRNA expression in AML, and this expression was negatively correlated with that of miR-550-1. YAP and TAZ have been reported to be transcriptional coactivators capable of recognizing cognate cis-regulatory elements via interactions with additional transcription factors, such as TEA domain family members (TEAD) [51,52]. Li et al. [26] and Noguchi et al. [28] found WWTR1 to be capable of regulating the cell-cycle and apoptosis via regulating CCND1, CCND3, c-myc, BCL-2, and DKK1 expression, with WWTR1 knockdown resulting in a G1/S transition block in the cell cycle. YAP and TAZ have also been found to upregulate BCL-2-family member transcription, thereby suppressing the mitochondrial pathway of apoptosis [44]. Consistent with this, we found that both miR-550-1 overexpression and WWTR1 knockdown reduced CCND1, CDK6, p-Rb, E2F1, BCL-2, and p-AKT levels, and increased p27, and PARP levels. When ectopic WWTR1 lacking the 3'-UTR region was overexpressed, this significantly rescued the miR-550-1-induced G0/1-phase arrest observed in vitro. To date, the function of WWTR1 in AML was not previously determined, but our study thus highlights for the first time that WWTR1 is a key mediator related to the anti-leukemic activity of miR-550-1. m 6 A is the most common methylation event modifying mRNA molecules in mammals, regulating a range of processes such as heat shock, differentiation, DNA damage responses, tissue development, and miRNA processing [53][54][55]. There is clear evidence that a disruption in m 6 A is linked to the pathogenesis of AML [56,57]. WTAP is a m 6 A methyltransferase complex component, regulating m 6 A methyltransferase activity. Recent work indicates that WTAP is able to improve CDK2 stability via binding to the 3'UTR region, thereby enhancing cell proliferation in renal carcinoma [45]. Bansal et al. [58] found that WTAP knockdown reduced the proliferation of AML cells, suggesting it serves as an oncogenic function. Our work highlights, for the first time, that miR-550-1 mediates its anti-leukemic activity at least in part via decreasing WWTR1 stability by targeting WTAP. Although we found that impairing WWTR1 mRNA stability, rather than promoting its degradation, was the primarily regulatory role of miR-550-1 in MV4-11 cells, further clarification is needed to determine why this effect was not identical in both cell lines. Whether miR-550-1/YAP/WWTR1 interact in a manner so as to form a negative feedback loop in AML remains unknown. Interestingly, a report by Chaulk et al. [59] suggested that nuclear YAP and WWTR1 together induced DICER complex activity, thus suggesting that these proteins might promote the maturation of certain pre-miRNAs into their mature forms in certain contexts. Liu et al. [60] also found circRNA-000425 to be a YAP/WWTR1 target using a circRNA microarray analysis, and revealed that YAP/WWTR1 were able to promote the oncogenic activity of miR-17 and miR-106b via inhibiting circRNA-000425 transcription. Additional experiments, however, are needed to determine whether YAP and WWTR1 regulate miR-550-1 expression. Our findings indicate that miR-550-1 is able to inhibit cell proliferation and promote apoptosis of AML cells via, at least in part, vying the direct targeting of the WWTR1 3'-UTR. Ultimately our findings both identify a novel tumor-suppressor miRNA, and also characterize previously unknown regulatory pathways governing WWTR1 expression in AML.
In summary, our study reveals the following: (1) elevated miR-550-1 expression is a favorable prognostic indicator in AML, and in AML patients it is at least partially dysregulated due to the hypermethylation promoter; (2) miR-550-1 is able to promote apoptosis and inhibit proliferation via regulation of the WTAP/WWTR1/BCL-2 and WTAP/WWTR1/CDK6/Rb/E2F1 pathways in AML; (3) m 6 A modifications are important for regulating the ability of miR-550-1 to target WWTR1. Our results thus demonstrate that miR-550-1 is a latent factor which suppresses AML, and as such enhancing expression of this miRNA may be a valuable therapeutic strategy in those with AML.