Prognostic value of miR-96 in patients with acute myeloid leukemia

From July 2010 to August 2012, 86 patients were diagnosed with de novo AML (non-M3) according to the French–American–British (FAB) criteria at Zhongshan Hospital of Xiamen University, China. The study was approved by the local ethics committee of Zhongshan Hospital of Xiamen University and informed consent was obtained from each patient or a family member. There were 42 male and 44 female patients, with a medium age of 46 (range 11–75) years. The median leukocyte count at diagnosis was 54,429/μL (range 710–315,000/μL). According to the FAB classification, three patients had AML M1, 32 had M2, 13 had M4, 34 had M5, three had M6, and one had M7. Sixty-five patients were subjected to cytogenetic classification according to karyotyping and detection of 16 types of common fusion genes, including AML1/ETO, PML/RARa, CBFβ/MYH11, MLL/AF10 and DEK/CAN and four types of mutations, namely, FLT3/ITD, NPM1, CEBPA and C-KIT. Clinical characteristics of the patients with AML are summarized in Table 1. Seventy-two patients received chemotherapy and were treated with standard cytarabine plus daunorubicin 7 + 3 induction chemotherapy. CR was defined by the criteria proposed by Cheson et al. [21]. Patients who achieved CR were then given high- or medium-dose cytarabine-based chemotherapy for consolidation according to their physical condition. Those who did not achieve CR were treated with medium-dose cytarabine-based chemotherapy. Forty-two patients with CR were followed up for a median 20 months (range 12–39 months). None of patients in CR had undergone stem cell transplantation until the end of follow-up because of the absence of suitable donors, poor condition, or other reasons. The data were censored when the patients relapsed or died. Bone marrow (BM) or peripheral blood (PB) specimens (peripheral white blood cells >50 × 10⁹) were obtained from patients at the time of their diagnosis, and from 14 patients in CR after two cycles of chemotherapy, after receiving informed consent. Five PB specimens from healthy donors for allogeneic hematopoietic stem cell transplantation were obtained as healthy controls, with informed consent.

Mononuclear cells from blood or BM specimens were isolated by Ficoll gradient (lymphoprep d = 1.077; Axis-Shield, Oslo, Norway) centrifugation for 20 min at 560 g at room temperature, and then washed and pelleted. Additionally, PB CD34⁺ cells from five healthy donors were enriched with immunomagnetic methods (Miltenyi Biotec, Bergisch-Gladbach, Germany; purity >90%). Total RNA was isolated using RNAprep Pure Blood Kit (Tiangen Biotech, Beijing, China), according to the manufacturer’s instructions, followed by treatment with RNase-free DNase I to remove contaminating genomic DNA. Finally, RNA was dissolved in RNase-free water. The RNA concentration was quantified by NanoDrop ND-1000 (Nanodrop, Wilmington, DE, USA) and stored at −80°C until use.

miR-96 and U6 snRNA-specific cDNA was formed using the Gene Amp System 9700 (Applied Biosystems, Foster City, CA, USA). U6 snRNA was used as an internal control. Reverse transcriptase primers for miR-96 and U6 were 5′-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACAGCAAAA-3′ and 5′-CGCTTCACGAATTTGCGTGTCAT-3′. Reverse transcription conditions contained 800 ng total RNA, 0.3 μl stem-loop RT primer (1 μM), 2 μl 10× RT buffer, 2 μl dNTP (2.5 mM each), 0.2 μl Moloney murine leukemia virus reverse transcriptase (Sangon, Beijing, China), and 0.3 μl RNase inhibitor (40 U/μl). The 20-μl reaction volumes were incubated at 16°C for 30 min, 42°C for 40 min, 85°C for 5 min, and then held at 4°C or stored at −20°C.

The 10-μl PCR mixture included 2 μl cDNA, 5 μl 2× Master Mix (SuperArray Bioscience, Frederick, MD, USA), 0.5 μl primer-F (10 μM), 0.5 μl primer-R (10 μM), and 2 μl nuclease-free water. Forward and reverse primers for U6 (89 bp) were 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and 5′-CGCTTCACGAATTTG-CGTGTCAT-3′. Primers for hsa-hsa-miR-96 (66 bp) were GSP: 5′-GGTTTGGCACTAGCACAT-3′; R: 5′-CAGTGCGTGTCGTGGAGT-3′. Real-time PCR was performed on an ABI PRISM7900 system (Applied Biosystems) with the following cycling conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 10 s and 60°C for 1 min. The cycle threshold was automatically given by SDS 2.4 software (Applied Biosystems) and was defined as the fractional cycle number at which the fluorescence passed the fixed threshold of 0.15. The relative expression levels of miRNAs were calculated using the comparative ∆∆Ct method as described previously [22]. The fold changes in miRNAs were calculated using the 2(-Delta Delta C(T)) method [23]. All experiments were performed at least in triplicate.

SPSS version 13.0 (IBM, Armonk, NY, USA) were used for statistical analysis. The Kruskal–Wallis nonparametric test was used to evaluate the significant difference of expression of miR-96 between the AML patients and NC. The t test was used to evaluate the difference of expression of miR-96 before and after chemotherapy. With regard to the correlation of leukemia clinical features with the expression level of miRNA-96, intergroup comparisons were performed using the χ² test. Kaplan–Meier survival curves were used to determine any significant relationship between the expression level of miRNA-96 and the status of the patients with respect to relapse-free survival (RFS) and overall survival (OS). RFS was defined as the time between the achievement of complete remission and the time of the hematological relapse or the last follow-up. OS was defined as the time between the moment of diagnosis and death or the last follow-up. Differences were considered statistically significant when P was <0.05.

miR-96 expression was detected in BM/PB samples from patients with AML and normal controls. Expression of miRNA-96 was normalized with U6, and the values obtained were compared. Consequently, the relative abundances of miR-96 were significantly downregulated in AML patients (mean expression value 19.26, n = 86) compared with those of healthy controls (mean expression value 195.43, n = 5), which was a significant difference (Figure 1, P < 0.001). AML patients expressing miR-96 at levels less than the median (19.26) were assigned to the low-expression group (mean expression value 4.34, n = 60), and those samples with expression equal to or above the median value were assigned to the high-expression group (mean expression value 52.06, n = 26). Moreover, 14 AML patients who achieved CR after one or two cycles of chemotherapy were monitored for miR-96 during the course of treatment. The mean expression value of these AML patients when diagnosed was 23.70 and markedly increased to 213.64 when CR was achieved after chemotherapy (Figure 2, P < 0.001).

The correlation of miR-96 expression with clinical characteristics at time of diagnosis is summarized in Table 1. Lower levels of miR-96 were associated with a higher white blood cell count, BM blast count (P < 0.001 and 0.022, respectively), and lower hemoglobin and platelet count (P = 0.036 and 0.033, respectively). Moreover, we failed to correlate the expression levels of miR-96 with other clinical parameters including sex (P = 0.541), age (P = 0.468), FAB subtype (P = 0.673) and cytogenetic abnormalities (P = 0.776).

Seventy-two newly diagnosed patients received chemotherapy. The CR rate after two cycles of chemotherapy was 53.85% (28/52) in the low-expression group, compared with 70.0% (14/20) in the high-expression group. Although it seemed that the low-expression group had a lower CR rate, there was no significant difference between the two groups. Forty-two CR patients were followed up for a median duration of 20 months (range 12–39 months). The low-expression group had a shorter RFS (P = 0.038) and OS (P = 0.022). The Kaplan–Meier curves for PFS and OS stratified according to miR-96 expression in BM from AML patients in CR are shown in Figure 3.