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

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]. Of the 78 CLL patients, there were 51 male (65.4%) and 27 female (34.6%) patients at a median age of 65 years (range: 37–91 years). The median presenting lymphocyte count was 18 × 10⁹/L (range: 10–540 × 10⁹/L). Apart from 8 patients with insufficient Rai stage data, there were 42 (60.0%) limited Rai stage (<stage II) and 28 (40.0%) advanced Rai stage (≥stage II) patients. Among the 53 patients with cytogenetic information, 13 (24.5%) carried high/intermediate-risk cytogenetic aberrations [del(17p), N = 2; del(11q), N = 2; trisomy 12, N = 9] and 40 (75.5%) carried low/standard-risk cytogenetic alterations [del(13q), N = 12; normal karyotype, N = 21; other karyotypic changes, N = 7]. The median overall survival (OS) of this cohort was 69 months. The median OS of those with advanced Rai stage and limited Rai stage were 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.

The human CLL cell lines CLL-AAT and MEC1 were purchased from American Type Culture Collection (Manassas, USA) and Deutsche Sammlung von Mikroorganismen und Zellkulturen Deutsche GmbH (DMSZ) (Braunschweig, Germany) respectively. MEC2, I83-E95 and WAC3CD5+ were kindly provided by Dr John C. Byrd, Department of Medicine, Ohio State University [27, 28]. HG3 and 232B4 were kind gifts from Prof. Anders Rosén, Department of Clinical & Experimental Medicine, Linköping University [28, 29]. Cell cultures were maintained in RPMI medium 1640, supplemented with 10% fetal bovine serum, 50 U/ml penicillin, and 50 μg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) in a humidified atmosphere of 5% CO₂ at 37°C.

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.

DNA was treated with bisulfite and used as template. Primers for pyrosequencing were used to amplify the promoter region, which was overlapped with the amplicon of MSP. Primers were designed using PSQ Assay Design software (Biotage). Forward primer: 5′-GAAGGGGGTTGGGATTTGA-3′; Reverse primer: 5′-ATTTCTCCCCTACTCCCC-3′; condition: 2mM/61°C/50X. A stretch of DNA with 9 adjacent CpG dinucleotides was pyrosequenced by sequencing primer: 5′-ATGGGAGTTTGTGAT-3′.

I83-E95 and WAC3CD5+ cells at log-phase were cultured in six-well plates at a density of 1 × 10⁶ cells/ml, with 0.5 μM of 5-AzadC (Sigma-Aldrich, St. Louis, MO, USA) for 5 days respectively, as described [31]. Fresh 5-AzadC was replaced every 24 hours. I83-E95 and WAC3CD5+ cells on day 0 and day 5 of 5-AzadC treatment were harvested respectively.

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 QuantiTect 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^-∆∆CT 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^-∆∆CT method [32].

According to the manufacturers’ instructions, precursor miR-9 mimic (final concentration 100 nM) (Ambion, Austin, TX, USA) was transfected into 1 × 10⁶ I83-E95 cells and WAC3CD5+ cells respectively, using X-tremeGENE siRNA Transfection Reagent (Roche, Basel, Switzerland), as described [33]. Non-targeting oligonucleotide mimic was used as the negative control.

The MTT method was used to determine cellular proliferation [34]. Cells were seeded in a 96-well microtitre plate at 2.5 × 10⁴/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.

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 × 10⁶ 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.

After 48 hours transfection, I83-E95 cells were harvested and lysed in RIPA buffer (50 mM Tris- HCl, pH 7.4, 150 mM NaCl, 0.2% SDS, 1% Triton X-100, 2 mM EDTA). Protein lysates were separated on 10% SDS-PAGE and blotting were performed on a 0.2 μm nitrocellulose membrane (Bio-Rad, Hercules, CA). The membranes were incubated with NFκB1 (1:1000; Santa Cruz, CA) or anti-actin (1:5000; Sigma-Aldrich, USA) primary antibody at 4°C overnight. Then membranes were washed and incubated with anti-rabbit horseradish peroxidase conjugate secondary antibody at room temperature for 1 hour. Protein signals were detected by ECL plus Western blotting detection reagents (Amersham Biosciences, Buckinghamshire, UK) and exposed to X-ray film.

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.

Methylation of miR-9-3 was found in thirteen of seventy-eight (17%) patient samples at diagnosis (Figure 3A), while miR-9-1 methylation was not detected in any of CLL patients (Figure 3B). There was no significant correlation between miR-9-3 methylation and the diagnostic hemoglobin level (P = 0.14), lymphocyte count (P = 0.07), age (P = 0.83), gender (P = 0.36) or high-risk karyotypes (P = 0.67). However, a significant association of miR-9-3 methylation with platelet count (P = 0.03) and advanced Rai stage (≥ stage 2) (P = 0.04) was found. The mean OS of CLL patients with and without miR-9-3 methylation were 103 and 87 months respectively (P = 0.72).

I83-E95 and WAC3CD5+ cells were completely methylated for miR-9-3. 5-AzadC treatment of I83-E95 and WAC3CD5+ cells resulted in demethylation of miR-9-3 and the emergence of U-MSP signal on day 5 respectively (Figure 4; Additional file 1: Figure S2D), with the re-expression of pri-miR-9-3 as shown by TaqMan stem-loop quantitative RT-PCR (Figure 4).

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).