Abstract Background Serum miRNA was once found as potential disease survival index,thus we investigated the role of miRNA in predicting prognosis in loco-regionally advanced NPC patients treated with CCRT. Methods This study included two phases: (i) We enrolled 3 NPC patients with recurrence or distant metastasis (experimental group, EG) and 3 NPC patients in clinical remission (control group, CG),who were treated with CCRT within 5 years. The paired serum was collected before and after treatment and biomarkers were discovered by LNA-TaqMan Human MicroRNA Arrays. (ii) we used the bioinformatic analysis, marker selection and an independent validation by qRT-PCR to analyse the serums of 29 NPC patients with recurrent disease or distant metastasis and 19 NPC patients in clinical remission treated with CCRT. Using the Kaplan-Meier method, log-rank test and Cox regression model to estimate the accuracy of the miRNAs to predict PFS and OS, and identified factors significantly associated with prognosis, respectively. Results Using fold change≥2.0 or ≤ 0.5 and p ≤ 0.05 as cutoff levels, we identified 1 up-regulated and 6 down-regulated miRNAs, 1 up-regulated and 9 down-regulated miRNAs in EG versus CG before and after CCRT, respectively. After these down-regulated miRNAs were dealed with bioinformatics analysis and normalization, only 5 different miRNAs were significantly reduced, which there were no significant difference in the expression of miRNA-26b, miRNA-29a and miRNA-125b before CCRT, and the expression of miRNA-143 and miRNA-29b after CCRT in the serum samples of 48 NPC patients. Based on this, we calculated a risk score with the expression of miRNA-26b、miRNA-29a、miRNA-125b、miRNA-29b、miRNA-143 and then classified patients as high or low risk group. Cox regression model suggested that combining miRNA-29a and miRNA-125b before CCRT with miRNA-26b after CCRT was independent prognostic factors for PFS (HR = 3.149, 95%CI:1.018–9.115, p = 0.034), whereas combining the former two is independent for OS (HR = 5.146, 95%CI:1.674–15.817, p = 0.04). Conclusions For loco-regionally advanced NPC patients treated with CCRT, especially high-risk patients- serum miRNAs, such as miRNA-29a, miRNA-125b and miRNA-26b etc., play an important role in predicting prognosis factors of PFS and OS, which will contribute to the strategic direction for future research. Keywords: Nasopharyngeal carcinoma, miRNA, Serum, Concurrent chemoradiotherapy Background Nasopharyngeal carcinoma (NPC) is endemic in the Far East, particularly in Southern China. NPC is more sensitive to radiotherapy than other head and neck cancers, which results in 5-year overall survival rates from 32 to 52% [[41]1]. However, local recurrences following radiotherapy and high affinity for distant metastasis are still two major causes of treatment failure. The search for non-invasive tools for the diagnosis and management of NPC after radiotherapy has long been a goal of cancer research, which has led research to focus on circulating nucleic acids in plasma and serum. MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs, approximately 22 nucleotides in length. They are known to negatively regulate gene expression or destroy the stability of genes via incomplete or complete matching with the 3′UTR of their target genes at the post-transcriptional level [[42]2]. Evidence suggests that miRNA expression profiles can cluster similar tumour types together more accurately than protein-coding mRNA genes profiles. Hence, the most promising application of miRNAs might permit to assess the outcome and modification of response in known and well established anti-tumour therapies (radiation and chemotherapy [[43]3]). Furthermore, a lot of studies have shown that tumour-associated miRNAs are in the serum or plasma of patients suffering from breast, colon, colorectal and nasopharyngeal cancers, etc. [[44]4]. In addition, the meta-analysis showed a possible impact of miRNA expression on NPC patients’ survival outcomes. They even pointed out to 65 miRNAs which have potential to function as prognostic markers in NPC. Further large-scale prospective studies about the clinical significance of the miRNAs may be necessary in order to obtain conclusive results [[45]5]. As a result, using miRNA as a novel noninvasive molecular marker for prognosis prediction of cancer treatment is possible. On the one hand, the current study aimed at finding out if specific circulating miRNAs can be detected in serum,and meanwhile if specific miRNA expression level differ in NPC patients (treated by concurrent chemoradiotherapy) with recurrence or metastasis and without. To our knowledge, this study is the first to evaluate the feasibility of using serum miRNAs as a noninvasive prognostic prediction test in loco-regionally NPC patients treated with CCRT. Methods Study design and patient samples Written informed consent, was obtained from all patients. It included the permission to use blood for research purposes. The study was approved by institutional review boards and the hospital Clinical Research Ethics Committee. All patients were sporadic cases on the basis of family history of NPC. All patients first underwent neoadjuvant chemotherapy as described previously [[46]6]. The treatment consisted of neoadjuvant chemotherapy and concurrent radiotherapy. Chemotherapy was made of 2 cycles of 5- fluoroucilat(5-FU) 700 mg/m^2/a day, performed on day 1 and day 4 (intraveneous injection). In addition, on day 1 after fluoroucilat, nedaplatin was infused (100 mg/m2 over 2 h). Nedaplatin treatment was repeated every 3 weeks, on days 1, 22 and 43, respectively, which was given 60 min before concurrent intensity modulated radiotherapy. Radiotherapy consisted of external-beam radiotherapy to the nasopharynx (70–80 Gy), the lymph node–positive area (60–70 Gy), and the lymph node–negative area (50–60 Gy). Tumours were staged according to the 1997 American Joint Committee on Cancer (AJCC) Staging system. Inclusion criteria Patients were included when they were between 40 and 70; radiotherapy was indicated for them; they had no endocrinologic or metabolic disorders and no uncontrolled hypertension or infections. They needed to have a normal liver, heart and kidney function. Exclusion criteria Patients could not be included when they were intolerant to radiotherapy;they had not finished a prescribed treatment;they were unable to accept treatment. This study was divided into two phases * Phase I (Marker discovery): In this phase, 6 patients with loco-regionally advanced NPC underwent intensity-modulated radiotherapy (IMRT) concurrently performed with induction chemotherapy based on nedaplatin (NDP) and 5-FU. Serum samples were collected within the week before radiotherapy initiation and three months after radiotherapy completion. Among these 6 patients, 3 experienced recurrence or distant metastasis in the 5 years after chemoradiotherapy, who belonged to the experimental group (EG);while the other 3 NPC patients experienced complete clinical remission in the 5 years after chemoradiotherapy,who belonged to the control group (CG). Differences in miRNA profiles between EG and CG groups collected before and after chemoradiotherapy were assessed in serum samples. Two miRNA expression patterns were established by comparing miRNA profiles in these two groups using miRCURY LNA™ miRNA Arrays. The most frequently down-regulated miRNAs in CG group compared with EG group in both time points were identified by bioinformatics and used for further analysis in phase II. * Phase II (Marker selection and validation): The down-regulated miRNAs identified above were considered as possible candidates. Two batches of serum samples were collected from an independent cohort of 48 NPC patients within 7 days before the start of radiotherapy and three months after radiotherapy completion.29 NPC patients with recurrence or distant metastasis and 19 NPC patients in clinical remission within 5 years after CCRT composed the cohort. Putative miRNA markers identified in phase I were verified in these independent sets of serum samples via real-time quantitative RT-PCR. miRNA array analysis Trizol LS reagent (Invitrogen, Paisley, UK) was used to extract RNA. miRNAs were generated from the total RNA groups mentioned above. miRCURY locked nucleic acid (LNA) microarray platform (Exiqon, Denmark) was used [[47]7]. Total RNA was labeled Hy3™ or Hy5™ fluorophores, using miRCURY™ Array Power Labeling kit (Exiqon, Denmark). Then, the reaction was spinned and left at 4 °C. The two samples from the Hy3™ and Hy5™ labeling reactions were combined in ice. The samples were hybridized in an hybridization station using miRCURY™ LNA miRNA Array (v.11.0) containing Tm–normalized probes for 847 human miRNAs. Microarrays with labeled samples were hybridized at 56 °C overnight using a heat-shrunk hybridization bag and washed with miRCURY Array Wash buffer kit (Exiqon, Denmark). After hybridization, the chip slides were immediately washed, dried and scanned. Each miRNA spot was replicated four times on the same slide and two microarray chips were used for each group. Scanning was performed with the Axon GenePix 4000B microarray scanner. GenePix pro V6.0 was used to read the raw intensity of the image. Signal intensities for each spot were scanned to produce the best within-slide normalization and minimize the intensity-dependent differences between the dyes. Then, they were calculated by subtracting local background (based on the median intensity of the area surrounding each spot) from total intensities using locally weighted scatter plot smoothing (Lowess, Locally Weighted Scatter plot Smoothing) Normalization (MIDAS, TIGR Microarray Data Analysis System). After normalization, the average values of each miRNA spot were used for statistics. The ratio between the green and red signals was calculated. Fold change > 2.00 or fold change < 0.5 (adjusted p-value< 0.50) were used to screen both up and down regulated miRNAs. Hierarchical clustering to differentiate expressed miRNAs was generated using standard correlation to measure their similarity. Real-time quantitative PCR Real-time PCR was done using GeneAmp Fast PCR Master Mix (Applied Biosystems) and ABI 7900HT real-time PCR machine. Table [48]1 summarizes the Oligonucleotides used in this study. To quantify the miRNAs expression in serum, U6 or miRNA-16 was respectively adopted as internal control. Appropriate internal normalization control was required to normalize sample-to-sample variations and relative quantification was applied in this qPCR. As no consensus on the use of internal normalization control in serum was defined for miRNA qPCR, we used expressions of miRNA-16 as the internal normalization control for miRNA quantification in serum. Although miRNA-16 turns less abundant than other miRNAs in the serum, miRNA-16 was selected as the normalization control as it proved higher stability and less variability. The threshold cycle (Ct) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. Each sample was run in triplicates for analysis. The relative amount of each miRNA was calculated using the eq. 2^-ΔCt, in which ΔCT = (CT^miRNA-CT^U6) or ΔCT = (CT^miRNA-CT^miRNA-16). Table 1. Oligonucleotides used in this study Primer set name Reverse transcripatase reation primer Real-time quantitative PCR primer Tm (°C) Length (bp) U6 5’CGCTTCACGAATTTGCGTGTCAT3’ Forward:5’GCTTCGGCAGCACATATACTAAAAT3′ Reverse:5’CGCTTCACGAATTTGCGTGTCAT3’ 60 89 miR-16 5’GGCGTAGGCAGTGCAGGGTCCGAGGTCTGCCTACGCCCGCCAATA3’ 16-F:TCGGCTAGCAGCACGT; 16-R:TGATTGCAGGGTCCGAG 60 60 miR-29a 5’GCGTGGTCGGTAACTCGGACCCTTCTACCGACCACGCTAACCGA 3’ 29a-F:GAACCCCTTAGCACCATCT; 29a/b-R:AGCGTAACTCGGACCCTT 60 66 miR-29b 5’GCGTGGTCGGTAACTCGGACCCTTCTACCGACCACGCAACACTG3’ 29b-F:GAACCCCTTAGCACCATTT; 29a/b-R:AGCGTAACTCGGACCCTT 60 66 miR-26b 5’GCCGTGACCGTCAGTGGAGGCAAGCCAGACGGTCACGGCACCTAT 3’ 26b-F:ACGACGGTTTCAAGTAATTCA; 26b-R: TCTCGTCAGTGGAGGCAA 60 65 miR-125b 5’GCCGTACCGTCAGTGGAGGCAAGCCAGACGGTACGGCTCACAAGT 3’ 125b-F:CTGGACTCCCTGAGACCCT; 125b-R:ATCCGTCAGTGGAGGCA 60 66 miR-143 5’GGCGTCAGCCAGAGTGGAGGCAAGCCACTGGCTGACGCCGAGCTAC 3’ 143-F:GCCAGTCCTGAGATGAAGCAC; 143-R:TGAAGAGTGGAGGCAAGC 60 62 [49]Open in a new tab Analyzing miRNA targets and correlative article The targeted mRNAs that have the potential binding sites for these miRNA to express in EG versus CG were searched in public databases endowed with prediction algorithms, such as TargetScan ([50]http://targetscan.org), PicTar ([51]http://pictar.mdc-berlin.de) and miRBase Target ([52]http://www.mirbase.org). The target genes we have chosen were significantly associated with different pathways. We also performed a PubMed search with various down-regulated miRNAs in our results from miRNA array analysis. Relevant publications dealing with miRNAs and their possible molecular mechanisms were obtained. Further, relevant articles were found by screening the references of