Dysregulation of miR-200s clusters as potential prognostic biomarkers in acute myeloid leukemia

Background Increasing studies showed that miR-200 family (miR-200s) clusters are aberrantly expressed in multiple human cancers, and miR-200s clusters function as tumor suppressor genes by affecting cell proliferation, self-renewal, differentiation, division and apoptosis. Herein, we aimed to investigate the expression and clinical implication of miR-200s clusters in acute myeloid leukemia (AML). Methods RT-qPCR was performed to detect expression of miR-200s clusters in 19 healthy donors, 98 newly diagnosed AML patients, and 35 AML patients achieved complete remission (CR). Results Expression of miR-200a/200b/429 cluster but not miR-200c/141 cluster was decreased in newly diagnosed AML patients as compared to healthy donors and AML patients achieved CR. Although no significant differences were observed between miR-200s clusters and most of the features, low expression of miR-200s clusters seems to be associated with higher white blood cells especially for miR-200a/200b. Of the five members of miR-200s clusters, low expression of miR-200b/429/200c was found to be associated with lower CR rate. Logistic regression analysis further revealed that low expression of miR-429 acted as an independent risk factor for CR in AML. Based on Kaplan–Meier analysis, low expression of miR-200b/429/200c was associated with shorter OS, whereas miR-200a/141 had a trend. Moreover, multivariate analysis of Cox regression models confirmed the independently prognostic value of miR-200b expression for OS in AML. Conclusions Expression of miR-200a/200b/429 cluster was frequently down-regulated in AML, and low expression of miR-429 as an independent risk factor for CR, whereas low expression of miR-200b as an independent prognostic biomarker for OS. Electronic supplementary material The online version of this article (10.1186/s12967-018-1494-7) contains supplementary material, which is available to authorized users.

In this study, we investigated expression of miR-200s clusters in AML patients except for acute promyelocytic leukemia (APL), and found that low expression of miR-200s clusters acted as potential prognostic biomarkers in AML.

Patients and treatment
A total of 98 de novo AML patients except for APL and 19 healthy donors were enrolled in this study. Bone marrow (BM) was collected from all the patients at diagnosis time as well as 35 patients at complete remission (CR) time. AML was diagnosed based on the French-American-British (FAB) and 2016 revised World Health Organization (WHO) criteria [16,17]. All the patients received chemotherapy as reported [18]. Induction chemotherapy therapy was 1-2 courses of daunorubicin combined with cytarabine. Subsequent consolidation treatment after CR for younger patients included highdose cytarabine, mitoxantrone with cytarabine, and homoharringtonine combined with cytarabine, whereas for older patients received in an individualized manner decided by the physicians, such as CHG protocol (cytarabine, homoharringtonine, and G-CSF). This study was approved by the Ethics Committee of the Affiliated People's Hospital of Jiangsu University, and written informed consents were informed and signed by all participants in accordance with the Declaration of Helsinki Principles.

RNA isolation and reverse transcription
BM mononuclear cells (BMMNCs) were extracted as reported using Lymphocyte Separation Medium (Absin, Shanghai, China) [27]. According to the manufacturer's protocols, RNA was extracted from BMMNCs using the mirVana miRNA isolation kit (Ambion, Austin, TX, USA), and was synthesized to cDNA by reverse transcription using MiScript Reverse Transcription Kit (Qiagen, Duesseldorf, Germany).

Real-time quantitative PCR
The level of miR-200s clusters was detected by real-time quantitative PCR (RT-qPCR) using miScript SYBR green PCR kit (Qiagen, Duesseldorf, Germany). The primers were miR-200s specific (Additional file 1: Table S1) and the manufacturer-provided miScript universal primer (Qiagen, Duesseldorf, Germany). The programs for RT-qPCR reactions were performed as reported [28]. U6 small nuclear RNA was selected as the endogenous normalizer detected by RT-qPCR using 2× SYBR Green PCR Mix (Multisciences, Hangzhou, China). Relative miR-200s level was calculated by 2 −ΔΔCT method. The healthy donors that possessed the minimal ΔCT between miR-200s (each member) and U6 expression was selected as control, and was defined as 100% expression.

Statistical analysis
Mann-Whitney's U test was carried to compare the difference of continuous variables between two groups, whereas Pearson Chi square analysis/Fisher exact test were applied to compare the difference of categorical variables between two groups. The impact of miR-200s clusters expression on overall survival (OS) was analyzed by Kaplan-Meier analysis, and Cox regression models (univariate and multivariate analyses) were further used to determine the independently prognostic value of miR-200s cluster expression. The effect of miR-200s clusters expression on CR was determined by Logistic regression analysis (univariate and multivariate analyses). All tests were two sided, and P < 0.05 was defined as statistically significant. SPSS software 20.0 and GraphPad Prism 5.0 was used to conduct the statistical analyses in this study.

Expression of miR-200s in AML
We analyzed miR-200s clusters expression in BM from 19 healthy donors, 98 AML patients, and 35 AML patients achieved CR by RT-qPCR. As presented in Fig. 1, expression of miR-200a/200b/429 clusters but not miR-200c/141 clusters was significantly decreased in AML patients as compared to healthy donors and AML patients achieved CR.

Relationship between miR-200s and clinical features in AML
To investigate clinical implication of miR-200s clusters expression, the whole-cohort patients were classified into two groups (high and low miR-200s clusters expression) based on the median level of each member of miR-200s clusters, respectively. We analyzed the association between each member of miR-200s clusters expression and clinic-pathologic features including gender, age,  white blood cell (WBC) counts, hemoglobin content, platelet counts, blasts (%), FAB subtypes, karyotypes, and common gene mutations. As shown in Table 1, no significant differences were observed between miR-200s clusters expression and most of the features. However, low expression of miR-200s clusters seems to be associated with higher WBC counts especially for miR-200a/200b (P = 0.001 and 0.041, respectively). In addition, low expression of miR-200a was related to male, whereas low expression of miR-141 was correlated with higher hemoglobin content (P = 0.013 and 0.024, respectively). Additionally, Logistic regression analysis was further performed to confirm and verify the effect of miR-200s clusters' expression on CR, and revealed low expression of miR-429 as an independent risk factor for CR in AML ( Table 2, P = 0.023).

Prognostic value of miR-200s in AML
We next evaluated the correlation of each member of miR-200s clusters expression with survival. Based on Kaplan-Meier analysis, low expression of miR-200b/429/200c was associated with shorter OS, whereas miR-200a/141 had a trend (Fig. 2). In addition, we also analyzed the impact of composite members of miR-200s clusters expression on OS by Kaplan-Meier analysis as shown in Fig. 3.
Since miR-200s clusters expression was associated with well-established prognostic factor such as WBC counts, we further conducted a Cox regression model adjusting for prognosis-related factors (age, WBC counts, karyotypic classifications, and gene mutations) for OS. Results showed that low expression of miR-200b acted as an independent prognostic biomarker for OS (P = 0.020, Table 2).

Discussion
In the current study, we for the first time investigated expression of miR-200s clusters in AML, and revealed that most of the members of miR-200s clusters were down-regulated in de novo AML patients. Recently, Li et al. revealed that introduction of a pre-miR-200c reduced the expression of ZEB2 protein and inhibited the proliferation of human leukemia cell lines (HL-60,  [14,30]. The miR-200s clusters were reported as key inhibitors of epithelial-to-mesenchymal transition by directly targeting transcriptional repressors of E-cadherin, ZEB1, and ZEB2 [13]. Moreover, miR-200s clusters also played crucial roles in the repression of cancer stem cells self-renewal and differentiation, modulation of cell division and apoptosis, and reversal of chemoresistance [14,30]. Notably, in some other hematological malignancies, expression or biological role of miR-200s clusters has been preliminary studied. For instance, Choi et al. reported that miR-200c was decreased in patients with myelodysplastic syndrome (MDS) [31]. González-Gugel et al. revealed that down-regulation of mmu-miR-30a and mmu-miR-141 as well as hsa-miR-193b clearly contributed to enhance the expression of Smoothened (SMO) gene in mouse and human lymphomas and, subsequently, to activate the GLI/Hh signalling [32]. In addition to basic research before, it has been noted that low expression of miR-200s clusters could correlate with adverse clinical outcome and serve as a prognostic biomarker for various cancer patients [15]. Although the potential prognostic value of miR-200s clusters in several human cancers remains controversial, a recent meta-analysis demonstrated that lower tissue expression of miR-200s clusters' members were associated with poor OS and progression-free survival, whereas lower expression of circulating miR-200s clusters' members were correlated with favorable prognosis [15]. From our study, we showed the negative effect of low expression of miR-200s clusters on AML chemotherapy response and survival. Moreover, multivariate analysis showed that low expression of miR-429 as an independent risk factor for CR, whereas low expression of miR-200b as an independent prognostic biomarker for OS in AML. Due to some