Low expression of circulating microRNA-328 is associated with poor prognosis in patients with acute myeloid leukemia

From February 2010 to September 2014, 176 newly diagnosed de novo AML patients from the Department of Hematology at Tangdu Hospital of Fourth Military Medical University were enrolled in this study; there were 86 males and 90 females, with a medium age of 39.7 (range 16.2–67.6) years. 70 unrelated healthy adult donors were collected as controls; all the control subjects were matched with patient population in terms of age and sex. None of these controls had previously been diagnosed with any type of malignancy or other benign disease. AML patients were diagnosed according to standard diagnostic methods including cytomorphological, cytochemical, immunological and cytogenetic evaluation. The diagnosis and classification of AML patients were based on the French-American-British (FAB) and World Health Organization (WHO) criteria combined to immunophenotyping and cytogenetic analysis [20‐23]. 124 patients received standard induction chemotherapy consisted of 1 or 2 courses of daunorubicin (45 mg/m² daily for 3 days) combined with cytarabine (100 mg/m²) by a 7-day continuous intravenous infusion. AML complete remission (CR) was defined as a normocellular BM containing less than 5 % blasts and showing evidence of normal maturation of other marrow elements; a neutrophil count of 1 × 10⁹/L and a platelet count of 100 × 10⁹/L. 76 patients achieved CR, and then given high- or medium dose cytarabine-based chemotherapy for consolidation according to their physical condition. Patients were followed up for a median 26 months (range 5–51 months); Patients without death or relapse by the time of last follow-up were censored on that date. Overall survival (OS) was defined as the time from the diagnosis of AML to any cause of death. Relapse-free survival (RFS) was defined as the time between the achievement of complete remission and the time of the hematological relapse or death. This study was approved by the Ethics Committee Board of Tangdu Hospital of Fourth Military Medical University. Informed consent was obtained from each participant according to the committee’s regulations. Details of clinical characteristics of the patients are provided in Table 1.

Blood samples were collected in EDTA-K₂ tubes and processed within 1 h of collection. Cell and nucleic acids free plasma was isolated from all blood samples using a 2-step centrifugation protocol (3000 g for 10 min and 12000 g for 5 min, all at 4 °C). The supernatant was transferred to RNase/DNase free tubes and stored at −80 °C. The plasma was first spiked with miScript miRNA mimic SV40 (Qiagen, Hilden, Germany, 2 μM, 1 μl per 100 μl plasma). Total RNA was isolated from the plasma using TRI reagent BD (MRC, USA) according to the manufacturer’s instructions and dissolved in 20 μl of RNase-free water. RNA sample concentration was quantified by NanoDrop ND-2000 (Thermo Fisher Scientific, USA). Quality of RNA was generally checked by the ratios of A260/A280 and A260/A230 and RNA integrity was assessed by electrophoresis through denaturing agarose gels.

Total RNA (1 μg) from each sample were converted into cDNA using PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Japan) and miRNA-specific stem-loop RT primer or SV-40 primers (Applied Biosystems, USA). Briefly, the reverse transcription reaction was performed in 20 μL mixture containing 10 μL of genomic DNA elimination reaction solution, 4 μL 5 × PrimeScript Buffer, 1 μL PrimeScript RT Enzyme Mix, 1 μL stem-loop RT primer or SV-40 primers, and 2 μL RNase Free water. For synthesis of cDNA, the reaction mixture was incubated at 42°C for 15 min, 85°C for 5 s, and then held at 4 °C. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed on ABI 7500 fast real-time PCR system (Applied Biosystems, USA) using SYBR Premix Ex Taq TM II (TaKaRa, Japan) according to the manufacturer’s instructions. PCR program conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s. A melting program was performed after each reaction to validate the specificity of the expected PCR product. The Ct values greater than 36 were considered as not expressed. Resultant miRNA levels were normalized using spiked-in SV40. The relative expression level of miR-328 was calculated by the equation of 2^-ΔCt (ΔC_t = C_{t miR-328} - C_{t spiked-in SV40}) [24]. The fold changes in miR-328 were calculated using the 2^-ΔΔCt method [25]. Each sample was analyzed in triplicate and the mean expression level was calculated.