Epigenetic inactivation of miR-9 family microRNAs in chronic lymphocytic leukemia - implications on constitutive activation of NFκB pathway

Background The miR-9 family microRNAs have been identified as a tumor suppressor miRNA in cancers. We postulated that miR-9-1, miR-9-2 and miR-9-3 might be inactivated by DNA hypermethylation in chronic lymphocytic leukemia (CLL). Methods Methylation of miR-9-1, miR-9-2 and miR-9-3 was studied in eight normal controls including normal bone marrow, buffy coat, and CD19-sorted peripheral blood B-cells from healthy individuals, seven CLL cell lines, and seventy-eight diagnostic CLL samples by methylation-specific polymerase chain reaction. Results The promoters of miR-9-3 and miR-9-1 were both unmethylated in normal controls, but methylated in five (71.4%) and one of seven CLL cell lines respectively. However, miR-9-2 promoter was methylated in normal controls including CD19 + ve B-cells, hence suggestive of a tissue-specific but not tumor-specific methylation, and thus not further studied. Different MSP statuses of miR-9-3, including complete methylation, partial methylation, and complete unmethylation, were verified by quantitative bisulfite methylation analysis. 5-Aza-2′-deoxycytidine treatment resulted in miR-9-3 promoter demethylation and re-expression of pri-miR-9-3 in I83-E95 and WAC3CD5+ cells, which were homozygously methylated for miR-9-3. Moreover, overexpression of miR-9 led to suppressed cell proliferation and enhanced apoptosis together with downregulation of NFκB1 in I83-E95 cells, supporting a tumor suppressor role of miR-9-3 in CLL. In primary CLL samples, miR-9-3 was detected in 17% and miR-9-1 methylation in none of the patients at diagnosis. Moreover, miR-9-3 methylation was associated with advanced Rai stage (≥ stage 2) (P = 0.04). Conclusions Of the miR-9 family, miR-9-3 is a tumor suppressor miRNA relatively frequently methylated, and hence silenced in CLL; whereas miR-9-1 methylation is rare in CLL. The role of miR-9-3 methylation in the constitutive activation of NFκB signaling pathway in CLL warrants further study.


Introduction
DNA methylation refers to the chemical modification of the cytosine ring in a CpG dinucleotide by the addition of a methyl group (-CH3) to the number 5 carbon of the cytosine pyrimidine ring in the CpG dinucleotide, resulting in the formation of 5-methylcytosine [1]. Global DNA hypomethylation, but aberrant, gene-specific DNA hypermethylation of the promoter-associated CpG islands of tumor suppressor genes (TSGs), is a hallmark of many human cancers [2,3]. Methylation of multiple TSGs has involved in the dysregulation of signaling pathways in leukemia, lymphoma and myeloma, including cell cycle (CDKN2A/B), apoptosis (DAPK1/CDKN2A/APAF1), JAK/ STAT signaling and WNT signaling; thereby indicating the importance of TSGs methylation in the pathogenesis of hematological cancers [4][5][6]. Of note, the DNA hypermethylation in TSGs, such as DAPK1, ID4, SFRP1, TWIST2 and ZAP70, has been identified to play a role in the pathogenesis or prognosis of chronic lymphocytic leukemia (CLL) [7][8][9][10][11].
In humans, there are three independent miR-9 genes (miR-9-1 on chromosome 1; miR-9-2 on chromosome 5 and miR-9-3 on chromosome 15), with identical mature miR-9 sequence. In cancers, miR-9 could be either oncomir or tumor suppressor miRNA, depending on the type of cancers or tissues [19,20]. For instance, overexpression of miR-9 has been shown to enhance metastasis or invasion in breast cancer cells or glioblastoma, identifying its oncogenic role [21][22][23]. Conversely, miR-9 has been shown to target and repress NFκB1 translation in ovarian tumor cells by binding to the 3′ untranslated region (3′ UTR) of the NFκB1 mRNA, leading to inhibition of cell proliferation; hence demonstrating a tumor suppressor function [24].
In this report, we studied methylation of miR-9-3, in addition to miR-9-1 and miR-9-2 in a representative cohort of CLL to define its pathogenetic role.

Patient samples
Bone marrow samples were obtained from 78 CLL patients at diagnosis. The diagnosis of CLL was made according to the WHO Classification, based on classical morphology, low level of expression of light-chain-restricted surface immunoglobulin, and concomitant expression of CD5 and CD23 as demonstrated by flow cytometry [25,26] 49 and 111 months respectively (P = 0.006). Furthermore, the median OS for those with or without high/intermediate-risk karyotype were 28 months and 111 months respectively (P = 0.003). Samples were obtained with written informed consent, and the study was approved by the Institutional Review Board of Queen Mary Hospital and in accordance with the Declaration of Helsinki.

Methylation-specific polymerase chain reaction (MSP)
DNA was extracted from seventy-eight bone marrow samples of CLL at diagnosis, seven cell lines and eight normal controls (CD19 sorted peripheral blood B cells from healthy donors, N = 3; peripheral blood buffy coats from healthy donors, N = 2; and bone marrow buffy coats from healthy donors, N = 3) by the QIAamp DNA Blood Mini Kit (QIAGEN, Germany). The MSP for aberrant gene promoter methylation was performed as described in detail previously [30]. Each sample was amplified with two sets of primers, one set for methylated DNA (methylated MSP) and one set for unmethylated DNA (unmethylated MSP). Treatment of DNA with bisulfite for conversion of unmethylated cytosine to uracil (but unaffecting methylated cytosine) was performed with the EpiTect Bisulfite Kit kit (QIAGEN, Germany). Details of primers and conditions for MSP of miR-9-1, miR-9-2 and miR-9-3 were given in Additional file 1: Table S1.
Quantification of pri-miR-9-3 and miR-9 According to the manufacturer's instructions, total RNA was isolated using the mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA). For quantification of pri-miR-9-3, miRNA was reversely transcribed by the Quan-tiTect Reverse Transcription Kit (QIAGEN, Valencia, CA), and quantified using the TaqMan Pri-miRNA Assay (ABI, Foster City, CA). GAPDH was used as reference for data analysis using the 2 -ΔΔCT method [32]. Moreover, miR-9 was quantified by the TaqMan MicroRNA RT Kit, and TaqMan MicroRNA Assay Kit. RNU48 was chosen as reference using the 2 -ΔΔCT method [32].

Cell proliferation, viability analyses
The MTT method was used to determine cellular proliferation [34]. Cells were seeded in a 96-well microtitre plate at 2.5 × 10 4 /well in 100 μl of medium. At the assay test time point 48 hours after transfection, 10 μl of 5 mg/ml MTT reagent was added to each well and incubated for 4 hours. Then each well was added with 100 μl dimethyl sulfoxide (DMSO), followed by the measurement of absorbance at 550 nm with reference to 650 nm. Cellular viability assay was performed by the Trypan blue dye exclusion assay under microscope. Dead cells (%) = (total number of dead cells per microscopic field/ total number of cells per microscopic field) × 100. Five random microscopic fields were counted in each sample.

Cellular apoptosis analysis
Cell apoptosis was assessed by flow cytometry using FITC Annexin V and PI staining as described previously [35]. FITC Annexin V Apoptosis Detection Kit I (BD-Pharmingen) was used here. 1 × 10 6 I83-E95 or WAC3CD5+ cells were washed with cold PBS and resuspended in 100 μl binding buffer with 5 ul of Annexin V and 5 ul PI, and then incubated for 15 minutes at room temperature in the dark. After adding 400 μl binding buffer to each tube, samples were analyzed by flow cytometry (BD FACS Canto II). FITC Annexin V positive, PI negative or FITC Annexin V positive, PI positive cells were counted as apoptosis cells.

Statistical analysis
In CLL, the association of miR-9-3 methylation status with continuous variables (such as mean age, mean lymphocyte counts, diagnostic hemoglobin or platelet counts) and categorical variables (gender, Rai stage or high-risk karyotypes) were analyzed by student's t-test and chi-square test (or Fisher's exact test) respectively. OS is assessed from the date of diagnosis to the date of last follow-up or death. OS of patients with limited Rai stage (stages 0, I and II) was compared with those with advanced Rai stage (stages III and IV). Furthermore, OS of patients with high-risk karyotypes [del(17p), del(11q) or trisomy 12] was compared to those with standard-risk karyotypes [del(13q), normal karyotype or other karyotypic changes]. The mean values of MTT assay, Trypan blue exclusion assay, FITC Annexin V and PI staining apoptosis analysis in I83-E95 or WAC3CD5+ cells transfected with precursor miR-9 mimic were compared to those transfected with the scrambled oligo by student's t-test. Survival was plotted by the Kaplan-Meier method and compared by the log-rank test. All P values were two-sided.

Effect of miR-9 overexpression in I83-E95 cells and WAC3CD5+ cells
Complete methylation of miR-9-3 was found for the I83-E95 cells and WAC3CD5+ cells, leading to its under expression. Upon transfection of precursor miR-9 mimic into I83-E95 cells, overexpression of miR-9 was demonstrated by TaqMan stem-loop quantitative RT-PCR at 48 hours after transfection ( Figure 5A). Compared with the negative control transfected with a scrambled oligo at 48 hours, cells overexpressing miR-9 mimic showed a 26% reduction of cellular proliferation by MTT assay (P = 0.02, Figure 5B), a 12% increase of dead cells measured by Trypan blue exclusion assay (P = 0.03, Figure 5C) and a 13% increase of apoptosis cells by FITC Annexin V and PI staining (P = 0.02, Figure 5D). Similarly, miR-9 mimics reexpression in WAC3CD5+ cells also led to the inhibition of cell proliferation and increase of cell apoptosis (Additional file 1: Figure S3), indicating that miR-9-3 played a tumor suppressive role in CLL cells. Moreover, miR-9 overexpression in I83-E95 cells led to the downregulation of NFκB1 protein, including the 46% decrease of the cytoplasmic precursor P105 and 42% decrease of the corresponding processing product P50 respectively ( Figure 5E).

Discussion
Despite the retrospective nature, this cohort of patients had previously been shown to be typical of CLL, in that  ΔCt, Ct pri-miR-9-3-Ct GAPDH. GAPDH was used as reference for data analysis of pri-miR-9-3 expression by 2 -ΔΔCT method. 5-AzadC treatment led to the progressive demethylation of miR-9-3 promoter, and re-expression of the pri-miR-9-3 in I83-E95 and WAC3CD5+ cells. Columns represent mean ± standard deviation of triplicate qPCRs.
patients pursued an indolent course with prolonged survival, which was adversely impacted by advanced Rai stage and high-risk karyotypes.
Firstly, miR-9-3 and miR-9-1 methylation in CLL cell lines was tumor-specific as evidenced by the absence of methylation in normal peripheral blood buffy coat and CD19-sorted B-cells, and normal bone marrow cells. By contrast, miR-9-2 methylation appeared to occur both in normal CD19 + ve B-cells and CLL B-cells, and hence this pattern of methylation is tissue-specific but not tumor-specific [36]. Similarly, miRNAs such as miR-127 and miR-373 have been shown hypermethylated in both normal and tumor cells, and hence represent tissuespecific but not tumor-specific miRNA methylation [36]. On the other hand, a recent study of miRNAs deregulated by either gene hypo-or hyper-methylation in CLL showed methylation of miR-9-2 in CLL [37]. This disparity might be accounted by the use of quantitative Figure 5 Overexpression of miR-9 in CLL cells. I83-E95 cells, completely methylated for miR-9-3, were transfected with miR-9 mimic or scrambled control oligo. Cells were harvested for functional assays 48 hours after transfection. A, Stem-loop RT-qPCR analysis of miR-9 expression at 48 hours after transfection. ΔCt, Ct miR-9-Ct RNU48. RNU48 was chosen as reference using the 2 -ΔΔCT method. B, Cell proliferation of CLL cells in response to overexpression of miR-9 was measured by MTT assay. Column, mean percentage of cell proliferation from three experiments conducted in triplicate; C, Cellular death was measured by Trypan blue exclusion assay. Column, mean percentage of cell death from three experiments conducted in triplicate; D, Cell apoptosis of CLL cells after miR-9 transfection was assessed by flow cytometry using FITC Annexin V and PI staining (left panel: representative result; right panel: values represented mean percentage of cell apoptosis from three experiments performed in triplicate. E, Western blot analysis of NFκB1 (P105/P50) after miR-9 transfection. Total protein was extracted 48 hours after transfection and membranes were probed with antibodies to P105, P50 and Anti-actin. Anti-actin protein was regarded as the endogenous normalizer and the relative NFκB1 (P105/P50) protein level was shown. Error bars represent standard deviation.
MassARRAY in their study, in which aberrant methylation of miR-9-2 was defined as a certain level of methylation above that occurring in normal controls. However, as Chinese have a much lower incidence of CLL, whether miR-9-2 methylation in Caucasian patients arose from a primary biological difference between the Asian and Western could not be excluded [38,39].
Moreover, tumor suppressor activity of miR-9-3 was demonstrated in CLL. We showed that restoration of miR-9 in I83-E95 cells harboring complete methylation of miR-9-3 led to reduced cellular proliferation and enhanced apoptosis. In addition, overexpression of miR-9 led to downregulation of NFκB1 (P105/P50), consistent with previous data that NFκB1 was a direct target of miR-9 by translational repression in ovarian cancer cells [24]. On the other hand, while the tumor suppressor role of miR-9, due to epigenetically downregulation, has been reported in some cancers [19,[40][41][42][43]. miR-9 has also been implicated as an oncomir in other cancers. For example, in breast cancer cells, as a metastasis-promoting miRNA, miR-9 led to enhanced cell motility and hence invasiveness by targeting E-cadherin [21]. In glioblastoma cells, overexpression of miR-9/9* could inhibit the expression of the tumor suppressor gene CAMTA1, leading to enhanced cell survival, implying that it might be an oncomir [23].
Interesting, we noted the significant association of miR-9-3 methylation with advanced Rai stage (≥ stage 2) in CLL patients, which is a poor risk factor for survival. Similarly, miR-9-3 methylation has been shown to be associated with metastatic recurrence in renal cell carcinoma [40], in addition to shorter disease-free and overall survivals in squamous cell lung cancer [41]. However, apart from Rai stage, there was no association between the miR-9-3 methylation and other clinical parameters including age, gender, diagnostic hemoglobin, lymphocyte counts, high-risk karyotypic aberrations or survival. Given the small number of samples in our cohort, the possible role of miR-9-3 as a prognostic marker by impacting on survival requires validation in a larger scale study.
Constitutive activation of NFκB have played roles in carcinogenesis, including stimulating cell proliferation, inhibiting cell apoptosis and increasing tumor metastasis [44]. Upon canonical NFκB activation by cytokines such as IL-1, IL-2 or TNFα, NFκB (a dimer comprising P50:P65) is released from the NFκB-IκBα complex, allowing nuclear translocation of NFκB and signaling activation [45,46]. NFκB1 gene encodes 2 functional proteins, the cytoplasmic precursor P105 and the corresponding processed product P50. In HEK-293 cells and ovarian cancer ES-2 cells, NFκB1 has been shown to be a direct target of miR-9 by luciferase assay [24,47]. Moreover, miR-9 overexpression suppressed tumor cell proliferation in association with repression of NFκB pathway, thereby confirming the tumor suppressive role of miR-9 via the regulation of NFκB signaling in ovarian cancer [24]. Similarly, constitutive activation of NFκB activity has been demonstrated in CLL cells, conferring survival benefit through induction of a multitude of anti-apoptotic proteins including X-linked inhibitor of apoptosis protein (XIAP), FLICE-like inhibitory protein (FLIP) and members of the BCL2 family (BCL-XL and A1/BFL1) [48,49]. In our study, we also showed that miR-9 overexpression in I83-E95 cells resulted in downregulation of NFκB1 (P105/P50) protein. Therefore, miR-9-3 methylation may account for constitutive upregulation of NFκB1, and hence constitutive NFκB activation in CLL patients.

Conclusions
Taken together, our results revealed that miR-9-3 was a tumor suppressor miRNA frequently methylated in CLL. miR-9-3 was a tumor suppressor miRNA hypermethylated in CLL, which was associated with down-regulation of NFκB1 protein, and hence might contribute to constitutive activation of NFκB signaling pathway in CLL.

Additional file
Additional file 1: Figure S1. Methylation of miR-9-2 in controls. (A). M-MSP of miR-9-2 showed that the positive control (PC) and 3 normal CD19 + ve B-cell controls (N1-N3) were completely methylated (B). Sequence analysis of the miR-9-2 M MSP product from bisulfite treated PC DNA and 3 normal CD 19 + ve B cell controls showed that in normal controls, the cytosine (C) residues of partial CpG dinucleotides were methylated compared with the PC sequence. (C). Schematic diagram to display the six CpG dinucleotides illustrated in the sequence. PC showed methylated (black box) of all six CpGs, while normal CD19 + ve B-cell control (1-3) displayed partially miR-9-2 methylation. Table S1: miR-9-1, miR-9-2 and miR-9-3 MSP Primer sequences and the reaction condition. Table S2: Average percent methylation for miR-9-3 in 7 CLL cell lines by pyrosequencing. Figure S2: Quantitative bisulfite pyrosequencing analysis of miR-9-3. The pyrograms showed the methylation intensity on a stretch of 9 neighboring CpG dinucleotides of (A) Normal control without methylation and positive control with methylated DNA, (B-C) CLL cell lines with defined MSP methylation status (MM, MU and UU) and (D) WAC3CD5+ cells before and after 5-azadC treatment. Figure  S3: Overexpression of miR-9 in WAC3CD5+ cells. WAC3CD5+ cells, completely methylated for miR-9-3, which were transfected with miR-9 mimic or scrambled control oligo. (A). miR-9 expression at 48 hours after transfection was measured by Stem-loop RT-qPCR analysis. (B). Cell proliferation of CLL cells in response to overexpression of miR-9 was assessed by MTT assay, whereas (C) cellular death was measured by Trypan blue exclusion assay and (D) the percentage of apoptosis CLL cells was assessed by flow cytometry using FITC Annexin V and PI staining. Error bars represents standard deviation.