Downregulated miR-217 expression predicts a poor outcome in acute myeloid leukemia

Abstract

BACKGROUND:

Circulating microRNAs (miRNAs) play an essential role in the development and progression of acute myeloid leukemia (AML). However, the clinical value of serum miR-217 in AML remained poorly known.

OBJECTIVE:

This study aimed to explore the clinical significance of serum miR-217 in AML.

METHODS:

Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed to detect miR-217 levels in the blood samples obtained from 89 AML patients and 60 healthy controls.

RESULTS:

The results showed that miR-217 expression was significantly decreased in AML patients compared to controls. Likewise, serum miR-217 levels were greatly downregulated in the AML patients with poor risk cytogenetic. ROC analysis demonstrated that serum miR-217 could effectively differentiate AML patients from normal controls. Also, miR-217 expression was markedly increased in patients achieving complete remission after their treatment. In addition, low miR-217 expression was associated with aggressive clinical features. Moreover, Kaplan-Meier analysis showed that AML patients with low miR-217 expression tended to have shorter overall survival and disease free survival. In the multivariate analysis stratified for prognostic parameters, miR-217 was proved to be an independent prognostic indicator.

CONCLUSIONS:

Collectively, miR-217 was identified as an independent marker for the diagnosis and prognosis of AML.

1. Introduction

Acute myeloid leukemia (AML), the most common hematological fatal disease in adults, is characterized by differentiation arrest, increased self-renewal and malignant proliferation of clonal myeloid precursors [1, 2]. According to cytogenetic information, AML patients can be divided into three risk-based subgroups: favorable, intermediate and poor [3]. Although treatment of AML has greatly improved over the past years, with improvements in risk assessment, chemotherapy-based regimen and stem-cell transplantation, less than 30% patients can achieve long-term disease-free survival and most of patients mainly die of either persistent or relapsed AML. Early detection and timely intervention can reduce the mortality of patients with AML [4, 5]. Therefore, new biomarkers for the diagnosis and prognosis of AML are urgently required.

MicroRNAs (miRNAs) are a group of small, non-coding RNAs that regulate protein expression by binding to the 3’ untranslated region of their targetmRNAs, and play an important role in a variety of biological processes including cell proliferation, differentiation and apoptosis [6, 7]. MiRNAs can function as either oncogenes or tumor suppressors and several aberrant expressed miRNAs in AML have been reported by previous studies. In leukemia research, Xu et al. showed that a positive correlation was observed between increased miR-196b expression and short overall survival of patients with pediatric AML, and miR-196b overexpression was strongly correlated with aggressive clinical parameters of pediatric AML [8]. Conversely, miR-215 expression was significantly decreased in AML patients. Moreover, AML patients with higher white blood cells or higher incidence of FLT3-ITD mutation possessed lower miR-215 expression, suggesting miR-215 acted as a tumor suppressor in AML [9].

In the current study, we focused on miR-217 which was first reported to exert a tumor suppressive property by Szafranska and colleagues [10]. Accumulating evidence had revealed that miR-217 played an important role in carcinogenesis of various cancers, including AML [11]. However, little is known about the potential clinical significance of miR-217 in AML. The aim of our study was to explore the diagnostic and prognostic value of circulating miR-217 in patients with AML.

2. Materials and methods

2.1 Study population

The present study was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University and prior informed consent was obtained from all participants. A total of 89 newly diagnosed AML patients, including 40 men and 49 women, were enrolled in this study. The diagnosis and classification of AML subjects were based on the French-American-British (FAB) and World Health Organization criteria. Complete remission (CR) was defined as a cellular BM containing less than 5% blasts and normalization of the peripheral blood counts after one or two cycles of chemotherapy. Overall survival (OS) was defined as the time from diagnosis to any cause of death. Disease free survival (DFS) was defined as the time from diagnosis to the date of induction failure or relapse or death. In addition, 60 healthy adult donors comprised 30 men and 30 women were recruited as controls. Details of clinical characteristics of the AML patients were summarized in Table 1.

2.2 Serum collection and RNA extraction

Blood samples were taken from all participants and processed within one hour after collection. The samples were centrifuged at 3000 g for 5 min, and followed by a second centrifugation at 12000 g for 5 min at 4 ${}^{\circ }$ C. Then, the supernatant was transferred to cryotubes and stored at $-$ 80 ${}^{\circ }$ C. Total RNA was extracted from the plasma using the miRVana PARIS kit (Ambion, Austin, TX, USA). RNA sample concentration was quantified by NanoDrop ND-2000 (NanoDrop Products, Wilmington, DE, USA). The quantity of RNA was assessed by measuring absorbance at A260/280.

Table 1

Correlation between miR-217 expression and clinical parameters of 89 AML patients

Variables	Cases	Low miR-	High miR-	$P$
		217, $n=$ 45	217, $n=$ 44
Age				0.2214
$<$ 60	62	34	28
$⩾$ 60	27	11	16
Sex				0.0718
Men	40	16	24
Women	49	29	20
PLT ( $\times$ 10 ${}^9$ /L)				0.5969
$<$ 50	45	24	21
$⩾$ 50	44	21	23
Blasts in bone marrow				0.0260
$<$ 50%	40	15	25
$⩾$ 50%	49	30	19
Cytogenetics				0.0070
Favorable	36	12	24
Intermediate	41	23	18
Unfavorable	12	10	2
FAB classification				0.3599
M0	17	6	11
M1	21	9	12
M2	5	2	3
M4	14	9	5
M5	32	19	13
WBC ( $\times$ 10 ${}^9$ /L)				0.6035
$<$ 10	36	17	19
$⩾$ 10	53	28	25
Complete remission				0.0428
Yes	37	14	23
No	52	31	21

PLT platelet; FAB French – American – British; WBC white blood cells.

Figure 1.

A. The miR-217 levels in AML patients were lower than those in controls. B. The miR-217 levels in AML patients with favorable risk cytogenetic were higher than those with intermediate/unfavorable risk cytogenetic.

2.3 Quantitative reverse-transcription polymerase chain reaction (RT-PCR)

Reverse transcription was performed with a Reverse Transcription kit (Takara, Dalian, China). Quantitative RT-PCR was processed on an Applied Biosystems 7500 Detection System (Applied Biosystems, Foster City, USA) using SYBR Premix Ex Taq TM II (Takara). Triplicates were performed for all qRT-PCR reactions. The relative miR-217 expression levels were calculated by the 2 ${}^{-\mathrm{\Delta }\mathrm{\Delta }\mathrm{Ct}}$ method using RNU6 as the endogenous control.

2.4 Statistical analysis

The comparison of miR-217 expression between groups were assessed using the Mann-Whitney U test or Kruskal-Wallis test. Chi-square tests was employed to evaluate the difference of categorical variables. The area under the curve (AUC) for serum miR-217 was determined using Receiver Operator Characteristic (ROC) analysis. Survival curves were plotted using the Kaplan-Meier method with log-rank test. Multivariate analyses were performed to analyze the association between odds ratio (OR) and OS/DFS. A $p$ -value less than 0.05 was considered statistically significant. All statistical analyses were processed with MedCalc 13.2.2 (MedCalc Software bvba, Ostend, Belgium) and GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA).

3. Results

3.1 MiR-217 was significantly down-regulated in AML patients and its diagnostic accuracy

The miR-217 expression levels were measured in blood samples from AML subjects and controls by qRT-PCR. The result showed that miR-217 levels in AML patients were markedly lower relative to those in controls ( $p<$ 0.05, Fig. 1A). Moreover, AML patients in favorable cytogenetic risk group had much higher miR-217 expression than that in intermediate or unfavorable cytogenetic risk group ( $p<$ 0.05, Fig. 1B).

Figure 2.

The diagnostic value of miR-217 expression in AML patients.

The ROC curve analysis was used to evaluate the diagnostic value of plasma miR-217, and we found miR-217 could serve as potential marker for distinguishing AML patients from controls with an AUC of 0.836, and the sensitivity and specificity were 74.16% and 83.33%, respectively (Fig. 2).

3.2 Association of miR-217 expression with clinical variables of AML

The median serum miR-217 level was used as the cut-off point to define the high expression group or low expression group, and the high expression group had 44 subjects and the low expression group had 45 subjects. As shown in Table 1, a statistically significant difference was observed between serum miR-217 and blasts in bone marrow ( $p=$ 0.0260), cytogenetics ( $p=$ 0.0070) and complete remission ( $p=$ 0.0428). However, other clinical variables, including age, sex, PLT, FAB classification and WBC were not closely correlated with miR-217 expression (all $p>$ 0.05).

Table 2

Multivariate analyses of factors associated with OS and DFS in 89 AML patients

Variables	Overall survival			Disease free survival
	Odds ratio	95% CI	$P$	Odds ratio	95% CI	$P$
Cytogenetics	3.25	1.36–5.37	0.025	3.96	1.50–6.62	0.013
Blasts in bone marrow	2.18	1.03–3.32	0.042	2.54	1.14–4.13	0.034
MiR-217 expression	2.63	1.18–4.21	0.031	3.12	1.29–5.18	0.026

Figure 3.

A. Comparison of miR-217 levels in 37 AML patients achieving a CR before and after treatment. B. Comparison of miR-217 levels in 52 AML patients who did not achieve a CR before and after treatment.

Figure 4.

A. AML patients in low miR-217 expression group had shorter OS. B. AML patients in low miR-217 expression group had shorter DFS.

3.3 Association of miR-217 expression with treatment response

Interestingly, the expression levels of miR-217 were detected in all AML subjects before and after treatment. The results demonstrated that miR-217 levels were greatly increased in AML patients in CR ( $p<$ 0.05, Fig. 3A). However, no significant changes in miR-217 levels were found in AML patients without achievement of CR ( $p>$ 0.05, Fig. 3B).

3.4 Association of miR-217 expression with prognosis of AML

For the survival analysis, the AML patients in low miR-217 expression group had a significantly shorter OS ( $p=$ 0.014, Fig. 4A) and DFS ( $p=$ 0.005, Fig. 4B) compared to those in high miR-217 expression group. Next, multivariate analysis demonstrated that cytogenetics (OS: OR $=$ 3.25, 95% CI $=$ 1.36–5.37, $p=$ 0.025; DFS: OR $=$ 3.96, 95% CI $=$ 1.50–6.62, $p=$ 0.013), blasts in bone marrow (OS: OR $=$ 2.18, 95% CI $=$ 1.03–3.32, $p=$ 0.042; DFS: OR $=$ 2.54, 95% CI $=$ 1.14–4.13, $p=$ 0.034) and miR-217 expression (OS: OR $=$ 2.63, 95% CI $=$ 1.18–4.21, $p=$ 0.031; DFS: OR $=$ 3.12, 95% CI $=$ 1.29–5.18, $p=$ 0.026) were independent prognostic indicators for survival in AML patients (Table 2).

4. Discussion

To the best of our knowledge, this study was the first report on the clinical implications of serum miR-217 in AML. We found that miR-217 was significantly reduced in AML patients compared with controls. Moreover, serum miR-217 levels were greatly downregulated in the AML subjects with unfavorable risk cytogenetic compared to those with intermediate/favorable risk cytogenetic. ROC analysis revealed that serum miR-217 could effectively screen AML patients from healthy controls. Then, serum miR-217 levels were remarkably increased in CR AML patients after treatment, and downregulation of miR-217 was closely associated with aggressive clinical characteristics as well as poorer survival. Also, serum miR-217 was identified as prognostic factor for AML patients. These data indicated that miR-217 might function as a tumor suppressor in AML. In line with our findings, Xiao et al. confirmed KRAS as a direct target of miR-217, and elevated miR-217 expression suppressed AML cell proliferation and increased cell chemosensitivity to doxorubicin by the cell apoptosis pathway [11]. One limitation of our study is that not all the patients in this cohort had the complete mutation information. For the AML patients having the mutation information (12 patients with FLT3 mutation, 7 patients with NPM1 mutation, 5 patients with MLL mutation, 4 patients with TET2 mutation and 2 patients with DNMT3A mutation), no association was found between serum miR-217 levels and above gene mutation. One possible reason is due to the small sample size. Further studies are warranted to reveal the potential correlation between serum miR-217 levels and gene mutation.

Other than AML, miR-217 was found to act as a potential tumor suppressor in many cancers. In gastric cancer (GC), Chen et al. reported miR-217 was significantly underexpressed in cancerous tissues, and its reduced expression was correlated with aggressive clinical parameters and shorter survival. Moreover, miR-217 overexpression inhibited tumorigenicity both in vitro and in vivo by targeting EZH2 [12]. Likewise, loss of miR-217 was observed both in GC cells and cancer tissues, forced miR-217 expression restrained cell proliferation and invasion through directly regulating the oncogene Glypican-5 [13]. In colorectal cancer, Zhang et al. showed that miR-217 was decreased in cancer tissues, and patients with lower miR-217 expression had significantly dismal overall survival. Furthermore, miR-217 inhibition stimulated cell growth and decreased cell apoptosis in vitro through targeting MAPK 1 [14]. Similarly, Zhu and colleagues revealed that miR-217 was reduced in glioma tissues and its upregulation repressed cell growth and motility by suppression of Runx2. Moreover, loss of miR-217 was evident in advanced clinical stage patients compared to low stage patients [15]. In osteosarcoma, Shen et al. showed miR-217 was decreased both in cancer cell lines and tissues, and its downregulation was strongly correlated with metastasis. The miR-217 directly targeting WASF3 and its overexpression greatly inhibited cell proliferation, migration and invasion [16]. Guo and colleagues showed that miR-217 was significantly decreased in lung cancer tissues. Forced miR-217 expression suppressed tumor growth, induced cell apoptosis and increased cell sensitivity to cisplatin [17]. In chronic pancreatitis (CP) and pancreatic cancer (PC), a reduction in miR-217 expression was found in CP, PC and TGF- $\beta$ 1 treated PC cells. MiR-217 underexpression markedly promoted mesenchymal to epithelial transition in PC cells and significantly associated with aggressive clinical variables [18]. Another study revealed that miR-217 was remarkably reduced in pancreatic ductal adenocarcinoma (PDAC) tissues compared to healthy pancreatic tissues, indicating miR-217 could be used as a tumor marker for PDAC [19]. In triple negative breast cancer, Zhou et al. proved that increased miR-217 expression dramatically suppressed cell growth, migration, and invasion by silencing KLF5 expression [20].

More importantly, the opposite observation of miR-217 expression had been revealed by Zhang and colleagues. This miRNA was found to be remarkably elevated in breast cancer tissues, and its overexpression was significantly associated with high histological grade and advanced tumor stage. Furthermore, loss of miR-217 inhibited cell proliferation, induced G1 phase arrest, decreased cyclin D1 expression in vitro and repressed tumorigenesis in vivo by negatively regulating DACH1, suggesting miR-217 might play as an oncogene in breast cancer [21]. Thus, the role of miR-217 in different cancers seems to be complicated and need to be further investigated. Gene mutation is closely associated with the prognosis of AML.

Taken together, we demonstrated that downregulation of miR-217 was strongly associated with aggressive clinical outcome as well as unfavorable prognosis of AML. The results showed that serum miR-217 could be used as a diagnostic and prognostic indicator for AML patients.