Circulating miRNAs as diagnostic biomarkers for multiple myeloma and monoclonal gammopathy of undetermined significance

Abstract Background Multiple myeloma (MM) is still an incurable hematological malignancy evolved from asymptomatic monoclonal gammopathy of undetermined significance (MGUS). New evidence suggests that circulating microRNAs (miRNAs) can serve as stable diagnostic biomarkers for MM and MUGS. Methods Serum miRNAs in MM patients, MUGS patients, and healthy controls (HC) were performed by Agilent Bioanalyzer 2100. MicroRNAs in MM detected as promising biomarkers were validated by using quantitative real‐time PCR (qRT‐PCR). Receiver operator characteristic (ROC) curve and multivariate logistic analysis were used to evaluate the diagnostic value of miRNAs for MM and MUGS. Results In microarray analysis, the top ten differential expressed miRNAs in MM included miR‐134‐5p, miR‐107, miR‐15a‐5p, miR‐5159‐3p, miR‐1914‐3p, miR‐4723‐3p, miR‐5588‐3p, miR‐6893‐3p, miR‐7106‐3p, and miR‐6722‐5p. Three up‐regulated miRNAs (miR‐134‐5p, miR‐107, and miR‐15a‐5p) were further validated. The elevated expression levels of miR‐134‐5p, miR‐107, and miR‐15a‐5p in qRT‐PCR were increased consistent with microarray analysis. These miRNAs distinguished MM and MUGS from HC significantly. Multivariate logistic analysis showed combination miR‐107, miR‐15a‐5p with Hb, the AUC was 0.954 (95% CI: 0.890‐1.000), sensitivity of 91.3%, and specificity of 93.7% for distinguishing MM from MUGS. Conclusions These data demonstrate that miR‐134‐5p, miR‐107, and miR‐15a‐5p are potential diagnostic biomarkers in MM and MUGS. Moreover, the combination miR‐107 and miR‐15a‐5p with Hb can distinguish MM from MUGS.


| INTRODUC TI ON
Multiple myeloma (MM) is a B-cell malignancy characterized by clonal expansion of plasma cells in the bone marrow and present with typical clinical manifestations, including hypercalcemia, renal failure, anemia, and bone lesions. 1 Monoclonal gammopathy of undetermined significance (MGUS), a pre-malignant condition, which progresses to MM at rate of 1% per year. 2 Early treatment can decrease M-protein level in MUGS patients. 3 Although serum M-protein and bone marrow plasma cells are used for diagnosis and discrimination of MM and MGUS, these existing markers are limited.
Moreover, bone marrow aspiration does not reflect tumor burden and is generally considered unacceptable. So the search for minimally invasive and more effective biomarkers has been paid more attention.
MicroRNAs (miRNAs) are small non-coding single-stranded RNAs that negatively regulate specific target gene mRNAs by binding with 3′-end non-translation region of mRNA. They regulate the expression of target genes at the post-transcriptional concentration through base pairing to partially or fully complementary sites. 4 Individual miRNA can target multiple mRNAs and participate in and regulate cell proliferation, apoptosis and immune response. 5 Studies have shown that miRNAs regulate critical processes in tumor initiation and progress by targeting oncogenes or tumor suppressor genes. 6 They exist in a stable form in plasma and serum, so circulating miRNA expression profiles could be served as novel biomarkers for disease diagnosis, progression, and prognosis. 7 In the present study, we used microarray analysis to identify miRNAs that were differential expressed in MM, MUGS, and health individuals. Then, quantitative real-time PCR (qRT-PCR) was used to assess the abundance of miRNAs in each sample. Using these methods, we found three miRNAs as potential diagnostic biomarkers in MM and MUGS. Multivariate logistic analysis showed combination miRNAs and serum parameter can effective discriminate MM from MUGS.

| RNA extraction
Total RNA including miRNAs were isolated using the miRNeasy kit (Qiagen) according to the manufacturer's instructions for serum samples. The RNA was stored at −80°C until use. SnRNA U6 as internal control standard, the relative of the gene expression level was calculated by the 2 −ΔΔCt . Each sample was measured repeatedly.

| MiRNA profiling and analyzing
The

| Validation of miRNAs using qRT-PCR
Out of the top 10 dysregulated miRNAs in MM, three up-regulated miRNAs (miR-134-5p, miR-107, and miR-15a-5p) miRNAs were further validated, based on their fold change and P value.
Although there was a significant difference in expression of miR-548y, miR-6500-5p, and miR-5011-5p in MM and MUGS compared with HC (P < .05), the elevated expression was contrary to the microarray results. Therefore, these three miRNAs were excluded from further analysis.

| D ISCUSS I ON
A significant number of miRNAs are present in genomic regions associated with cancer and appear as circulating molecules in human peripheral blood. 10 Effective circulating miRNAs testing can offer potential tool for early tumor detection and allow treatment at a less advanced stage for remarkable clinical improvement.
In our study, we used the array platforms and employed normal- In ROC curves analysis, all of these three miRNAs showed clearly specificity and sensitivity in discriminating MM and MUGS from HC. MiR-134-5p and miR-15a-5p showed better sensitivity (87.0%, 87.0%, respectively) and miR-107 have greater specificity (88.9%) in discriminating MM from HC. Compared with HC, the sensitivity and specificity of miR-15a-5p in MUGS were better than miR-134-5p and miR-107, which were 81.3% and 77.8%, respectively.
MiR-134-5p involved in the pathogenesis and progression of hematologic malignancy has already been reported. 13 This is the first time that it serves as a diagnostic biomarker for MM. The predicted target genes of miR-134-5p include ITGB1 and PIK3R1, which are involved in insulin-like growth factor receptor (IGF-1) signaling pathway, cell proliferation, and apoptotic process, and are related to the occurrence and development of MM. 14,15 Our results revealed that miR-15a-5p, belonging to 15a/16 cluster located at chromosome 13q14, was up-regulated in MM. 16 Several studies have reported that cytokines such as interleukin-6 (IL-6), IGF-

1-Specificity
In summary, our study indicates that miR-134-5p, miR-107, and miR-15a-5p are up-regulated in MM and MUGS, which could be served as potential diagnostic markers. Moreover, combination of miR-107 and miR-15a-5p with Hb will distinguish MM from MUGS so as to provide early treatment and improve the prognosis.

ACK N OWLED G M ENTS
The authors gratefully acknowledge the support, advice, and help of the staff at their laboratory. We would also like to thank Huiyuan Kang Ph. D for technical advice and Juan Ma Ph. D for writing assistance.