Identification of miR-320 family members as potential diagnostic and prognostic biomarkers in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis and the abnormal differentiation of hematopoietic stem cells. An increasing number of researches have demonstrated that microRNAs play crucial roles in the pathogenesis of myelodysplastic syndromes. Herein, we aimed to identify novel potential microRNAs bound up with the diagnosis and prognosis of MDS. MiRNA microarray analysis was used to screen deregulated microRNAs in the bone marrow of MDS patients. qRT-PCR was employed to confirm the microarray results. All members of miR-320 family (miR-320a, miR-320b, miR-320c, miR-320d, and miR-320e) were significantly increased in MDS patients compared to normal control. Although we found no correlation between miR-320 family and most clinical characteristics, high miR-320c and miR-320d expression seemed to be associated with high numbers of bone marrow (BM) blasts and worse karyotype. High expression of all the members of the miR-320 family seemed to be associated with a high prognostic score based on International Prognostic Scoring System (IPSS). The areas under the miR-320 family member ROC curves were 0.9037 (P < 0.0001), 0.7515 (P = 0.0002), 0.9647 (P < 0.0001), 0.8064 (P < 0.0001) and 0.9019 (P < 0.0001). Regarding Kaplan–Meier analysis, high miR-320c and miR-320d expression were related to shorter overall survival (OS). Moreover, multivariate analysis revealed the independent prognostic value of miR-320d for OS in MDS. The expression of miR-320 family members was up-regulated in MDS, and miR-320 family members could serve as candidate diagnostic biomarkers for MDS. High expression of miR-320d was an independent prognostic factor for OS in MDS.


Results
MiRNA expression profiles of MDS patients. Eight MDS patients whose samples were used for microarray analysis were divided into lower risk group (low and intermediate-1 risk, n = 4) and higher risk group (intermediate-2 and high risk, n = 4) based on the IPSS definition. Microarray results showed that the expression of each member of the miR-320 family was close to statistical significance (P < 0.05) and the fold change was higher or slightly lower than 2 between the lower risk group and normal control group. However, the expression of each member of the miR-320 family reached statistical significance (P < 0.05) in the higher risk group, and the fold change was higher than 5 between the higher risk group and normal control ( Table 1). Part of microarray results were shown in Fig. 1. Subsequently, miR-320 family members expression was validated by qRT-PCR in a large sample size.
Validation of miR-320 family members. miR-320 family members expression was validated in BM from 82 MDS patients and 24 normal controls using qRT-PCR. As displayed in Fig. 2, the expression of miR-320 family members was significantly up-regulated in each group of MDS compared with that in normal controls.

Association of miR-320 family members with clinical characteristics of MDS.
To evaluate the clinical implications of miR-320 family member expression, we divided MDS patients into two groups based on the median level of each member of the miR-320 family. The relationship between miR-320 family expression and clinical characteristics was evaluated. As displayed in Table 2, no correlation was found between miR-320 www.nature.com/scientificreports/ family members and most clinical characteristics. However, high miR-320c and miR-320d expression were associated with a high number of BM blasts (P = 0.003 and P = 0.047), advanced WHO type (P = 0.006 and P = 0.016), and worse karyotype (P < 0.001 and P = 0.024). Additionally, the high expression of all members of the miR-320 family was significantly related to advanced IPSS risk stratification.
Prognostic implications of miR-320 family in MDS. To investigate prognostic implications of miR-320 family members in MDS, we estimated the relationship between each member of miR-320 family and OS. Regarding on Kaplan-Meier analysis, we discovered that high miR-320c and miR-320d expression were related to shorter OS (P = 0.002 and P = 0.032) (Fig. 4). Additionally, multivariate analysis showed that high expression of miR-320d was an independent prognostic factor for OS (P = 0.020, Table 3).

Discussion
In present study, we determined the expression of miR-320 family members in the bone marrow of de novo MDS patients. We discovered that miR-320 family members were markedly up-regulated in MDS patients compared with normal control. Additionally, miR-320 family members have been reported to play crucial roles in various cancers. For instance, miR-320a is obviously down-regulated in gastric cancer 15 , breast cancer 16 , lung adenocarcinoma 17 , and osteosarcoma 18 . Furthermore, down-regulation of miR-320a promoted growth and metastasis of cancer cell. However, in pancreatic cancer, miR-320a was evidently up-regulated and enhanced the drug-resistance and proliferation of pancreatic cancer cell 19 . Similarly, miR-320b downregulated in various cancers, including colorectal cancer 20 , nasopharyngeal carcinoma 21 , osteosarcoma 22 and glioma 23 , indicating that miR-320b might inhibit the progression of these cancers. Moreover, miR-320b was reported to inhibit tumorigenesis by targeting BMI1 in non-small cell lung cancer 24 . Similar with miR-320a/320b, miR-320d acts as an antioncogene in various cancers, such as glioma 25 , prostate cancer 26 , and breast cancer 27 . However, miR-320d was upregulated in pancreatic cancer 28 and rectal cancer 29 , and high expression of miR-320d was bound up with poor prognosis of rectal cancer. Regarding miR-320c, Iwagami et al. discovered that miR-320c enhanced gemcitabine-resistance by targeting SMARCC1 in pancreatic cancer 30 . Wang et al. demonstrated that miR-320c was significantly elevated in colon cancer and could be regarded as a diagnostic biomarker of early-stage colon cancer 31 . Another study found that miR-320c was decreased in bladder cancer and inhibited tumorous behaviors by targeting CDK6 32 . In regard to miR-320e, Perez-Carbonell et al. demonstrated that miR-320e was significantly elevated in colorectal cancer patients with recurrence compared with colorectal cancer patients without recurrence 33 , indicating that miR-320e expression was associated with the progression of colorectal cancer. Drahos demonstrated that miR-320e was up-regulated in esophageal adenocarcinoma compared to Barrett's esophagus 34 . In summary, miR-320 family members play anti-tumor roles in most solid tumors except miR-320a, miR-320c and miR-320d, which are increased in pancreatic cancer. In regard to hematological malignancies, the role of miR-320 family is complex. Circulating miR-320d was confirmed to be markedly increased in AML and has been regarded as a novel diagnostic biomarker for AML 35 . However, miR-320d was significantly decreased in diffuse large B-cell lymphoma (DLBCL) 36 . Low expression of miR-320d was associated with poor prognosis of DLBCL. miR-320a was found to be markedly elevated with a 17.502-fold change in classical Hodgkin lymphoma (cHL) compared with reactive lymphadenopathy 37 . miR-320e was increased with a 3.91-fold change in leukemia stem cells (LSCs) after ponatinib treatment 38 . In multiple myeloma, miR-320 family members acted as tumor suppressors. For instance, miR-320a inhibits tumor growth and increases apoptosis by targeting PBX3 in multiple myeloma 14 . The oncogene EZH2 was able to directly inhibit the expression of miR-320c to promote tumorigenesis in multiple myeloma 39 . Additionally, Mittal et al. reported that miR-320a inhibited erythroid differentiation by targeting SMAR1 in K562 cells (chronic myelocytic leukemia cell lineage) 40 . In our study, the expression of each member of miR-320 family was significantly elevated in MDS patients compared to normal control and showed diagnostic value for MDS. Additionally, high miR-320c and miR-320d expression were associated with poor prognosis of MDS. The exact mechanism and function of miR-320 family members in MDS needed to be study further.
In the present study, we identified miR-320 family members as potential diagnostic and prognostic biomarkers for myelodysplastic syndromes. We demonstrated the that expression of miR-320 family members was up-regulated in MDS, and multivariate analysis showed that high expression of miR-320d was an independent prognostic factor for OS. It was suggested that miR-320 family members might serve as oncogenes in MDS. Targeting miR-320 family members may provide a new strategy for MDS therapy in the future.   Table 4. All the participants had been given informed consent according to the Declaration of Helsinki. The study was approved by the Human Ethics Committees Review Board at Guangxi Medical University, Nanning, China (Approval Number: 2017(KY-E-098)).   Table 5. The microarray data was generated as previously reported (PMID: 32629683) 42 . Significant differentially expressed microRNAs between the two groups were defined as P < 0.05 and |logFC|> 1.

Statistical analysis. Microarray analysis is based on Student's t test (8 MDS patients vs. 6 controls), whereas
qRT-PCR validation is based on Mann-Whitney's U test (82 MDS patients vs. 24 controls). Mann-Whitney's U test and Pearson Chi square test were used to evaluate relationship between miR-320 family members and clinical parameters. Receiver Operator Characteristic (ROC) analysis was used to detect sensitivity, specificity, and the area under the curve (AUC) for miR-320 family members. Kaplan-Meier method used in univariate analysis and Cox proportional hazard regression model in multivariate analysis were used to determine the prognostic value of miR-320 family expression. P < 0.05 indicates statistical significance.