Pyrosequencing quantified methylation level of miR-124 predicts shorter survival for patients with myelodysplastic syndrome

Background Aberrant CpG island methylation has been increasingly recognized as a common event in myelodysplastic syndrome (MDS). To date, most of the previous studies of miR-124 in MDS have focused on epigenetic changes and little is known about the underlying mechanism through which miR-124 regulates CDK6 expression. Results In the present study, we employed pyrosequencing analysis to quantify the methylation levels of upstream regions of the miR-124 genes (miR-124-1, miR-124-2 and miR-124-3) in 56 primary MDS patients. We found the three miR-124 genes were methylated in MDS patients. Univariate analysis revealed that the World Health Organization (WHO) classification, marrow blast count, karyotype, International Prognostic Scoring System (IPSS), mean corpuscular volume, as well as high methylation of miR-124-1, miR-124-2 and miR-124-3 were significantly related to overall survival. In leukaemia-free survival, patients who were older and had an advanced WHO classification, high marrow blast counts, high IPSS risk and high methylation of miR-124-1 and miR-124-2 progressed rapidly to acute myeloid leukaemia. Multivariate analysis demonstrated that high methylation of miR-124-3 was an independent factor of overall survival. Median survival of patients with high miR-124-3 methylation was significantly shorter (7.6 months) than patients with low methylation (32.7 months; P = 0.010). A functional study revealed that silencing of miR-124 resulted in upregulation of its target gene, cyclin dependent kinase CDK6, which in turn promoted cell proliferation in the MDS cell line SKM-1. Conclusions High methylation of miR-124-3 predicts shorter survival for patients with MDS, which may be a useful prognostic marker in MDS. Electronic supplementary material The online version of this article (doi:10.1186/s13148-017-0388-5) contains supplementary material, which is available to authorized users.


Background
Myelodysplastic syndrome (MDS) is a clonal disorder characterized by dysplastic and ineffective haematopoiesis and a high risk of acute myeloid leukaemia (AML) progression. In recent years, aberrant CpG island methylation has been increasingly recognized as a common event in MDS, and the relation between prognosis and DNA methylation has been demonstrated [1][2][3][4].
MicroRNAs (miRNAs) are a group of small noncoding RNAs consisting of 18-22 nucleotides that negatively regulate mRNA transcripts, typically by base pairing with a complementary region in the 3′untranslated region (3′-UTR) of the target gene [5]. They play pivotal roles in a wide range of biological processes including proliferation, apoptosis and differentiation [6]. Increasing evidence indicates that miRNAs are involved in carcinogenesis, either oncogenically, when tumour suppressor genes are targeted, or by tumour suppression, when oncogenes are targeted [7]. Dysregulation of miRNAs has been found to be associated with the clinical outcomes of haematological malignancies, such as miR-212, miR-3151, miR-181 and miR-29b in AML [8][9][10][11], miR-26A1 in chronic lymphocytic leukaemia [12], and miRNA-194-5p in MDS [13].
In the present study, we used pyrosequencing analysis to precisely quantify the methylation levels of upstream regions of the miR-124 genes (miR-124-1, miR-124-2 and miR-124-3) in 56 primary MDS patients to determine whether they are methylated in this disorder. We also analysed the correlation between different methylation levels and clinical features to evaluate their prognostic significance in MDS patients. To further clarify the underlying mechanism through which miR-124 regulates its target, CDK6, we performed a functional study in the SKM-1 cell line in vitro.  [29]. The patients comprised 36 men and 20 women with a median age of 65 years (range 20-84 years). According to the 2016 WHO classification, there were 5 patients with single lineage dysplasia (SLD), 7 with multilineage dysplasia (MLD), 5 with ring sideroblasts (RS, including 2 with RS-SLD and 3 with RS-MLD), 15 with excess blasts-1 (EB-1), 21 with EB-2, 1 with MDS unclassifiable (MDS-U) and 2 with isolated del(5q-). Cytogenetic examination was carried out on samples from 55 patients. The prognostic score for each patient was calculated using the International Prognostic Scoring System (IPSS) [30]. Patients with lower blast counts (<5%), including SLD, MLD, RS, MDS-U and isolated del(5q-), received supportive care, including blood transfusion and the use of erythropoietin. Patients with EB-1 and EB-2 received AML-like chemotherapy at low doses. All patients were followed until death from any cause or until the last follow-up date (30 April 2008). At the last follow-up date, 34 patients (60.7%) had died and 15 (26.8%) had progressed to AML.

Patients and SKM-1 cell line
The MDS SKM-1 cell line was derived from a peripheral blood sample of a 76-year-old Japanese male patient at the leukaemia stage who was initially diagnosed as MDS EB-2 [31]. The SKM-1 cell line was purchased from the Health Science Research Resources Bank, Japan. Cells were grown at 37°C under 5% CO 2 in RPMI-1640 medium with 10% foetal bovine serum.

DNA isolation, sodium bisulphite conversion and pyrosequencing analysis
Genomic DNA was isolated from the bone marrow samples of 56 MDS patients using a QIAamp DNA Blood Mini Kit according to the manufacturer's instructions (Qiagen). We also isolated genomic DNA from peripheral blood of 10 healthy donors. Sodium bisulphite modification of the DNA was performed using an EpiTect Bisulfite Kit (Qiagen). MiR-124 was represented at three genomic loci [miR-124-1 (8p23.1), miR-124-2 (8q12.3) and miR-124-3 (20q13.33)]. One CpG-rich region for each of these loci was identified using the CpG Island Searcher, and one pair of primers was designed to analyse the CpG sites. The primer sequences were as follows: miR-124-1: forward: GGGGAGAATAAAGAGTTTTTGGA, reverse: TACTCAACCAACCCC ATTCTTAA; miR-124-2: forward: GTGTGTTGTAAATGGTATGGAGATAT, reverse: CCCAACTCCTATCTCTACTCATCT; and miR-124-3: forward: AAA GGGAGAAGTGTGGGT, reverse: CCCAAAAAAACCCTCAAAACT. PCRs were performed with a PyroMark PCR Kit (Qiagen) under the following conditions: 95°C for 3 min; 50 cycles of 95°C for 15 s, 54°C for 30 s and 72°C for 30 s; and an elongation step of 72°C for 5 min. The success of amplification was assessed by 2% agarose gel electrophoresis. The DNA methylation level of the specific CpG nucleotides was then evaluated by pyrosequencing. The sequencing primers were as follows: miR-124-1: GAATAAAGAGTTTTTG-GAAG; miR-124-2: TCTCTAA CACATCTACCAAA; and miR-124-3: GGAGGATTGGGATAGTATA. Quantitative pyrosequencing analysis was performed using a PyroMark Q96 ID platform (Qiagen) according to the manufacturer's protocol. The pyrosequencing assays interrogated 4-9 adjacent CpG sites close to the primary (pri)-miR-124. Output data were analysed using PyroMark Q24 2.0.6 Software (Qiagen). The genomic locations of the bisulphite pyrosequencing assays and the number of investigated CpG sites in each assay are shown in Fig. 1.

RNA isolation and quantitative real-time PCR
Total RNA was extracted from the SKM-1 cells using TRIzol reagent (Invitrogen, San Diego, CA, USA), and small RNAs were isolated using a mirVana miRNA isolation kit (Ambion, Inc., Austin, TX, USA) according to the manufacturer's instructions. Reverse transcription was performed at 48°C for 15 min. PCR was performed for 40 cycles at 95°C for 15 s and 60°C for 60 s. TaqMan microRNA assays were used to quantify the expression levels of mature miR-124 according to the manufacturer's protocol (Applied Biosystems, San Diego, CA, USA). U6 was used as an internal control. All samples were normalized to internal controls, and the fold changes were calculated according to the relative quantification method (RQ = 2 −ΔΔCT ). The primer sequences were as follows: miRNA Universal R: GTGCAGGGTCCGAGGT; miR-124-F: GCCTAAGGCACGCGGTG; miR-124-RT: GTCGTATC CAGTGCAGGGTCCGAGGTATTCGCACTGGATACGA CGGCATT; U6-F: CTCGCTTCGGCAGCACA; U6-R: AACG CT TCACGAATTTGCGT.

Western blot analysis
For the western blot analyses, total proteins were extracted from the cultured cells and then quantitated using a bicinchoninic acid assay kit (Pierce, Rockford, IL, USA), with bovine serum albumin as a standard. Proteins were fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) transferred to a polyvinylidene fluoride (PVDF) membrane, were blocked in 5% dry milk at room temperature for 1 h and were immunostained overnight at 4°C using antibodies against CDK6 (Epitomics, Burlingame, CA, USA). An antibody to GAPDH (ProteinTech Group, Chicago, IL) was used as a loading control.

Cell viability assays
Cells were seeded at 2000 per well in 96-well plates and cultured after transfection. Cell viability was assessed by Cell Counting Kit-8 (CCK-8) assay (Beyotime, Shanghai, China) following the manufacturer's instructions. Briefly, cells were seeded into a 96-well plate at a concentration of 2 × 10 3 cells per well. Each well contained 10 μl of CCK-8 in 90 μl of culture medium. The cells were incubated for 2 h at 37°C, and absorbance was measured at 450 nm. Three independent experiments were performed.

Cut-off values and statistical analysis
The MDS samples had different levels of DNA methylation. To explore the prognostic significance of the methylation level of each CpG site, we used ROC analysis to obtain the best cut-off values for classifying patients into "high-meth" and "low-meth" groups. To evaluate the predictive value of each gene, such as miR124-1, miR-124-2 or miR-124-3, we first used mean values from all pyrosequenced CpG sites as a measure of methylation of a given gene, as reported in two previous studies [18,32]. Then, we used ROC analysis to obtain the cut-off values for classifying patients into "highmeth" and "low-meth" groups. When we produced an ROC curve to estimate the sensitivity and specificity of each site or each gene as a prognostic marker for overall survival (OS) or leukaemia-free survival (LFS), we used median OS or median LFS as a criterion. The state variable of patients with a survival time or leukaemia-free survival time of more than the median OS or median LFS was defined as "1", while for those with less than the median OS or median LFS, it was defined as "0". The cut-off values of each site and each gene are shown in Tables 1 and 2. Analysis of variance and Student's t tests were used to determine the statistical significance of differences between samples, and the results were expressed as the mean ± s.d. OS was measured from the day of diagnosis until death from any cause or until the last follow-up date. LFS was calculated from the day of diagnosis until progression to acute leukaemia or end of follow-up. The Kaplan-Meier method was used to compare OS or LFS between patients in the low and high methylation groups. A log-rank test was used to estimate differences in survival. The Cox regression model was used for the multivariate survival analysis to identify the significant independent prognostic factors affecting OS or LFS. For all the analyses, a P value of <0.05 was considered statistically significant.

Prognostic value of miR-124 methylation and haematological factors Univariate analysis
To evaluate the prognostic value of different levels of methylation of miR-124-1, miR-124-2 and miR-124-3, we performed univariate analysis by using the Kaplan-Meier method and log-rank test. Prognostic factors affecting OS and LFS in the univariate analysis are listed in Table 3. Among the laboratory and clinical parameters, the World Health Organization (WHO) classification, marrow blast count, karyotype, International Prognostic Scoring System (IPSS) and mean corpuscular volume were found to be significant prognostic factors for OS in the univariate analysis. For LFS, patients who were older and in the advanced WHO classification stage, with high IPSS scores and high marrow blast counts had higher risk of progression to AML. High methylation levels of miR-124-1 and miR-124-2 were proved to be adverse prognostic factors with shorter OS and LFS in the MDS patients (Fig. 2a-d).

Multivariate analysis
To assess the prognostic value of factors found to be significant in the univariate analysis, we performed a multivariate Cox regression analysis of their effect on OS and LFS. As shown in Table 3, in addition to the WHO classification, marrow blast, karyotype and IPSS, high methylation of miR-124-3 was an independent factor of OS. The median survival of patients with high miR-124-3 methylation was 7.6 months, significantly shorter than patients with low methylation (median survival of 32.7 months, P = 0.010; Fig. 2e). According to Italics are statistically significant the multivariate analysis, age was the only independent factor that predicted worse LFS (Table 3).

Correlation between different levels of miR-124-3 methylation and clinical parameters
Based on the results of multivariate analysis, which showed that high miR-124-3 methylation was the independent risk factor of OS, we analysed the correlation between different levels of miR-124-3 methylation and clinical factors. As shown in Table 4, high methylation of miR-124-3 was independent of sex, white blood cell count, haemoglobin level, platelet count, mean corpuscular volume and karyotype and cytopenias at initial diagnosis. However, patients with the advanced WHO classification or high marrow blast count had a significantly higher frequency of miR-124-3 high methylation. There is a slight trend that patients with intermediate or high IPSS risk had higher incidence of miR-124-3 high methylation, it did not reach the statistical significance (Table 4).

miR-124 suppresses cell proliferation in the SKM-1 cell line
We first examined the methylation status of miR-124-1, miR-124-2 and miR-124-3 in the SKM-1 cell line, and the results showed that methylation levels significantly decreased after DAC treatment (Fig. 3a). Then we tested the expression of miR-124 in SKM-1 cell line and found that after DAC treatment, there was a significant increase in the expression of miR-124 (Fig. 3b). We also evaluated the effects of miR-124 on cell proliferation in MDS. In vitro cell proliferation assays revealed that increased expression of miR-124 induced by DAC significantly inhibited MDS cell proliferation (Fig. 3c). Furthermore, miR-124 mimics or miR-124 inhibitors were transfected to SKM-1 cells. After transfection, miR-124 expression was determined by real-time PCR and normalized to U6 (Additional file 1: Figure S2). We found that in miR-124 mimics transfected SKM-1 cells, cell proliferation was significantly suppressed (Fig. 3d), while a miR-124 inhibitor, which significantly decreased endogenous miR-124 expression, promoted cell proliferation (Fig. 3e). Taken together, these data indicate a proliferation-inhibitory role of miR-124 in MDS.

miR-124 targets CDK6 expression in the SKM-1 cell line
To investigate the potential mechanism of how miR-124 regulates its target genes, we focused on the gene encoding CDK6 because it has been confirmed   Different cut-off values of methylation levels were used to predict OS and LFS (Tables 1 and 2); therefore, a means the number of patients with low or high methylation levels to predict OS, and b means the number of patients with low or high methylation levels to predict LFS Italics are statistically significant as a target in previous studies [19,23,28]. Using western blot analyses, we observed that the SKM-1 cells, which had low miR-124expression levels, strongly expressed CDK6, while the cells treated with the demethylating agent DAC showed CDK6 downregulation (Fig. 3f). To further confirm the targeting of CDK6 by miR-124, SKM-1 cells were transfected with miR-124 mimic, miR-124 inhibitor or the respective controls. The result showed that CDK6 was decreased in cells transfected with miR-124 mimics, but increased in cells with the miR-124 inhibitor (Fig. 3g, h). These data indicate that CDK6 was targeted by miR-124, and that epigenetic silencing of miR-124 in SKM-1 cells led to CDK6 upregulation.

Discussion
In this study, we found that miR-124 genes (miR-124-1, miR-124-2 and miR-124-3) were highly methylated in MDS patients, and increased methylation levels of several CpG sites are associated with shorter OS or LFS. In addition, we found that miR-124-3 was associated with adverse clinical parameters, including high blast count and advanced WHO classification. These data suggested that miR-124 genes, which are, at least, mediated through an epigenetic mechanism, might be involved in the pathogenesis of MDS. This view was further supported by our findings that high methylation of miR-124-3 was significantly associated with decreased OS in both the univariate and in multivariate analyses. The clinical relevance of miR-124 methylation as an independent prognosticator has been investigated in several previous studies. In renal cell cancer, miR-124-3 high methylation was shown to be associated with disease recurrence [33]. In pancreatic cancer, high methylation of miR-124-2 and miR-124-3 was correlated with decreased survival time [18] and, in another study, the methylation level of miR-124-3 was shown be to be associated with increased risk of developing gastric cancer [34]. In haematological malignancies, Castoro et al. first reported the methylation status of miR-124-1 and miR-124-3 in MDS and AML, and identified miR-124-1 methylation as a prognostic marker for OS [21]. Roman-Gomez reported a shortened disease-free survival for patients with ALL using a multi-miRNA loci approach that included miR-124-3 [35]. Subsequently, methylation of the miR-124-3 locus was identified as an independent prognosticator for both disease-free survival and OS in ALL [23]. In addition, Castoro et al. demonstrated that dynamic changes in miR-124-1 and miR-124-3methylation may predict the clinical response after DAC therapy in MDS patients [21]. MiR-124-2 has been found to be methylated in both healthy lymphocytes and various types of cancer [17,18,23,35]. Although shorter OS and LFS in MDS patients with high miR-124-2 and miR-124-3 methylation were found in the univariate analysis, these correlations were not significant in the multivariate analysis. Recent studies have investigated the biological function of miR-124 in various cancers, and increasing evidence  [21]. Another recent study also demonstrated that high-risk MDS or AML patients who responded to epigenetic treatment showed significant induction of miR-124 and inhibition of CDK4 and CDK6 expression [39]. However, the role of miR-124 in cell proliferation and the regulation of its target have not been clearly elucidated in vitro. In the present study, we tested the effect of miR-124 on SKM-1 cells.
Our results showed that miR-124 re-expression after DAC treatment could suppress MDS cell growth. We also showed that synthetic miR-124 mimics inhibited cell growth, which suggests its role as a tumour suppressor in MDS. Furthermore, we validated CDK6 as a target of miR-124 by western blot analysis in vitro. Our findings suggest that post-transcriptional of CDK6 by miR-124 is a vital mechanism underlying cell proliferation, and miR-124 may serve as a potential treatment target for regulating CDK6 to inhibit cell proliferation in MDS.
There are several limitations to our study. First, the patients cohort of 56 cases included in the current study were diagnosed between June 2003 and April 2007, and during this period, demethylating agents were not approved by the China Food and Drug Administration. Therefore, no patients in our study received a demethylating agent such as decitabine as standard therapy. Second, we tested the methylation levels of the miR-124 loci only at the time of initial diagnosis and not after treatment; therefore, whether miR-124 methylation levels could predict responsiveness to demethylating agents has not been established. Third, the present study is retrospective and had a small sample size. Finally, because of the lack of frozen cells or mRNAs, miR-124 functional study was performed only in the SKM-1 cell line, with no further studies being performed in clinical patient samples. Several previous studies reported the silencing of the expression of miR-124 regulated by methylation in MDS, AML and ALL [20,21,23]. Our present study confirmed that miR-124-3 high methylation was an independent risk factor for OS in MDS patients. All three upstream regions of the miR-124 genes (miR-124-1, miR-124-2 and miR-124-3) express miR-124, and miR-124 negatively regulates its target mRNA transcripts, typically through base pairing with a section in the 3′-untranslated region (3′-UTR). Therefore, whether miR-124 expression at diagnosis was correlated with patient prognosis needs to be verified in future study. In the DAC era, dynamic examination of miR-124 methylation, miR-124 expression and its targeted genes in MDS patients both before and after DAC treatment may be crucial to resolve this issue.

Conclusions
In conclusion, we found that miR-124 methylation was a common molecular event in MDS, and that high (See figure on previous page.) Fig. 3 miR-124 suppresses cells proliferation and downregulates the expression of CDK6 in SKM-1 cell line. a Methylation status of miR-124 inSKM-1 cells before and after 4 μM DAC treatment for 48 h. b SKM-1 cells were treated with 2 and 4 μM DAC for 48 h, and miR-124 expression was determined by real-time PCR and normalized to U6. The results are presented as the means ± s.d. of values obtained in three independent experiments. Statistical significance was calculated using the Student's t test. * P < 0.05. c The effect of miR-124 on cell proliferation was measured by CCK-8 assay. Increased expression of miR-124 after DAC treatment inhibited cells proliferation. d The effect of miR-124 overexpression on cell proliferation. miR-124 means SKM-1 cells transfected with miR-124 mimics, and NC means negative control of SKM-1 cells transfected with miR-124 mimics. e The effect of miR-124 suppression on cell proliferation. Anti-miR-124 means the cells transfected with miR-124 inhibitors, and anti-NC means the negative control of cells transfected with miR-124 inhibitors. f Western blot and a quantitative analysis of bands intensity of CDK6 in SKM-1 cells treated with 2 and 4 μM DAC for 48 h. GAPDH served as the internal control. g The effect of miR-124 overexpression on endogenous CDK6 expression was measured by western blot and a quantitative analysis of band intensity. GAPDH served as an internal control. miR-124 means SKM-1 cells transfected with miR-124 mimics, and NC means negative control of SKM-1 cells transfected with miR-124 mimics. h The effect of miR-124 suppression on endogenous CDK6 expression was measured by western blot and a quantitative analysis of band intensity. GAPDH served as an internal control. Anti-miR-124 means the cells transfected with miR-124 inhibitors, and anti-NC means the negative control of cells transfected with miR-124 inhibitors