Differential expression of miRNA199b-5p as a novel biomarker for sporadic and hereditary parathyroid tumors

MicroRNAs (miRNAs) are dysregulated in many tumors; however, miRNA regulation in parathyroid tumors remains poorly understood. To identify differentially expressed miRNAs between sporadic and hereditary parathyroid tumors and to analyze their correlation with clinicopathological features, a microarray containing 887 miRNAs was performed; then, the differentially expressed miRNAs were validated by qRT-PCR using 25 sporadic and 12 hereditary parathyroid tumors and 24 normal parathyroid tissue samples. A receiver operating characteristic curve (ROC) analysis was applied to evaluate the utility of the miRNAs for distinguishing parathyroid tumor types. Compared to the miRNAs in the normal parathyroid tissues, 10 miRNAs were differentially expressed between the sporadic and hereditary parathyroid tumors. Seven of these miRNAs (let-7i, miR-365, miR-125a-3p, miR-125a-5p, miR-142-3p, miR-193b, and miR-199b-5p) were validated in the parathyroid tumor samples. Among these miRNAs, only miR-199b-5p was differentially expressed (P < 0.001); miR-199b-5p was significantly downregulated and negatively associated with PTH levels (γ = −0.579, P = 0.002) in the sporadic tumors but was upregulated in the hereditary tumors. This miRNA showed 67% sensitivity and 100% specificity for distinguishing sporadic and hereditary parathyroid tumors. These results reveal altered expression of a miRNA between sporadic and hereditary parathyroid tumors and the potential role of miR-199b-5p as a novel biomarker for distinguishing these two types of parathyroid tumors.

SCIEntIfIC RepoRts | (2018) 8:12016 | DOI: 10.1038/s41598-018-30484-9 cancer types [13][14][15][16] . The potential uses of miRNAs for the effective diagnosis and optimal treatment of parathyroid tumors have also been investigated [17][18][19][20] . However, there is little data on parathyroid tumor miRNAs that are differentially expressed between the sporadic and hereditary forms. The purpose of the present study was to identify and analyze differentially expressed miRNAs and to determine their correlation with the clinicopathological features of sporadic and hereditary parathyroid tumors. We determined whether miRNA profiling could serve as a potential biomarker for distinguishing these tumor types.

Differences in clinical manifestations between sporadic and hereditary parathyroid tumors.
We compared the clinical and biochemical parameters of sporadic and MEN1 parathyroid tumors. The laboratory results showed higher PTH and calcium levels in patients with parathyroid tumors than in normal controls. Patients with sporadic parathyroid tumors had larger tumor sizes and higher levels of PTH and calcium than patients with hereditary parathyroid tumors (Table 1). Consistent with a previous report on the correlation between PTH and tumor volume 21,22 , PTH levels were correlated significantly with tumor size in patients with sporadic parathyroid tumors (γ = 0.592, P = 0.002) but not significantly in patients with hereditary parathyroid tumors.
Next, seven commercially available miRNAs were used to validate the results in sporadic and MEN1 parathyroid tumors compared with normal parathyroid tissues using quantitative real-time PCR (qRT-PCR) (Fig. 2). The expression levels of miR-193b and miR-365 were lower in sporadic parathyroid tumors than in normal parathyroid tissues, and miR-193b expression was higher in MEN1 parathyroid tumors than in sporadic tumors. Interestingly, only miR-199b-5p had significantly different expression between the two parathyroid tumor types; compared with that in normal parathyroid tissue, miR-199b-5p was downregulated in the sporadic form (median fold change of 0.2) and upregulated in the hereditary form (median fold change of 3.9).
Discriminating value of miR-199b-5p in parathyroid tumors. The diagnostic relevance of miR-199b-5p was analyzed using a ROC curve analysis (Fig. 3). miR-199b-5p had a large area under the concentration-time curve (AUC = 0.863, P < 0.001) with a sensitivity of 67% and a specificity of 100% for discriminating sporadic and hereditary parathyroid tumors.
Clinical implication of miR-199b-5p in parathyroid tumors. Given that miR-199b-5p was differentially expressed according to the parathyroid tumor type, we further analyzed the correlation between miR-199b-5p and PTH levels. Interestingly, different correlations between miR-199b-5p and PTH levels in sporadic and hereditary parathyroid tumors were identified: there was a negative association in the sporadic form (γ = −0.579, P = 0.002) and no significant correlation in the hereditary form (Fig. 4).  miRNA target genes and biological function analysis. To inspect the function of miR-199b-5p in parathyroid tumorigenesis, we collected 113 validated miR-199b-5p targets based on miRWalk and miRTarBase (data not shown). Gene ontology (GO) analysis of the candidate target genes showed that DNA-templated regulation of transcription, regulation of cell growth, angiogenesis, regulation of transcription from RNA polymerase II promoter, regulation of cell proliferation, and response to drug are the most significantly enriched GO terms (Table 2). Moreover, KEGG pathway enrichment analysis revealed that the miRNA-targets were significantly associated with pathways in cancer, focal adhesion, and central carbon metabolism in cancer (Table 3).

Discussion
The differential diagnosis of sporadic and hereditary parathyroid tumors remains uncertain. To establish a precise differential diagnosis method for these parathyroid tumors, numerous parameters have been investigated from different clinicopathological conditions [23][24][25] , but the discrimination remains difficult. Even gene array data showed that hereditary parathyroid tumors are clustered with the sporadic form, indicating that these tumors may share a similar genetic pathway of tumorigenesis 26 . Consequently, new biomarkers are needed for the effective discrimination of these tumor types.
In the present study, miR-199b-5p showed good accuracy for distinguishing sporadic and hereditary parathyroid tumors. The differentiation of these tumor types is associated with improved therapeutic outcomes and decreased recurrence rates of hyperparathyroidism, which requires the reevaluation of uncertain prognoses. These results indicate the need for a more in-depth evaluation of miRNAs in parathyroid tumors. We also observed significantly lower expression of miR-199b-5p in sporadic parathyroid tumors, and miR-199b-5p expression was negatively associated with PTH levels, indicating a specific role for this miRNA in parathyroid tumorigenesis.
Little is known about the different molecular mechanisms between sporadic and hereditary parathyroid tumors that could explain the different disease progression profiles and phenotypes. Several responsible genetic germline changes associated with parathyroid tumors in familial syndromes, such as MEN1 in MEN 1, RET in MEN 2 A, and CDC73/HRPT2 in HPT-JS (hyperparathyroidism-jaw tumor syndrome), have been identified 27,28 . However, these genetic alterations have also been implicated in a subset of sporadic parathyroid tumors. Genetic alterations in the MEN1 gene have been reported in 20 to 30% of sporadic parathyroid tumors 29 . Therefore, the presence of constitutively mutated MEN1 alleles is not sufficient to explain the different tumor profiles between sporadic and hereditary parathyroid tumors.
Substantial advances in the study of miRNA involvement in parathyroid tumorigenesis have been achieved in recent years. Differentially expressed miRNAs between parathyroid carcinoma and adenoma were identified, including miR-139, miR-296, miR-222, miR-503 miR-26b, miR-30b, miR-126*, miR-517c, and miR-372 [17][18][19] . This subset of miRNAs was further verified by Hu et al. 20 . Emerging evidence has shown that even the partial inactivation of tumor suppressors can importantly contribute to tumorigenesis 11 . In this context, miRNAs can be good candidates for the subtle regulation of gene expression based on a continuum model of tumor suppressor gene function. Interestingly, this presumption was confirmed, in part, by Luzi et al., showing that miR-24-1 could bind to the MEN1 mRNA and inhibit menin expression, closing a feedback loop 30 . Grolmusz et al. also reported miR-24 and miR-28 were differentially expressed between sporadic and MEN1 parathyroid tumors 31 . Subsequently, miR-4258, miR-664, and miR-1301 were demonstrated to involve in the MEN1 associated parathyroid tumors 32 . Unfortunately, however, miR-199b-5p was not identified in previous studies. This inconsistent result might be due to small sample size because of rarity of MEN1 related samples and genetic heterogenesis of parathyroid tumors 20 .
Several studies related to miRNAs have shown that miR-199b-5p is a putative tumor suppressor that targets several signaling pathways: Hes1 involved in both Notch and Hedgehog pathways in medulloblastoma 33 and osteosarcoma 34 , PODXL and DDR1 in acute myeloid leukemia 35 , HIF-1α in hepatocellular carcinoma 36 , and HER2 and its downstream signaling ERK1/2 and AKT pathway in breast cancer 37 . Taken together, the overexpression of miR-199b-5p could significantly inhibit cell proliferation, migration, and clonogenicity. Interestingly, miR-199b-5p could be a fine tuner of target gene expression, suggesting its epigenetic control function during tumor development 33 . Although its exact functions in parathyroid tumors are unknown, miR-199b-5p was found to be negatively correlated with serum PTH which associated with tumor size in sporadic parathyroid tumors in our study. Moreover, GO analysis suggested that miR-199b-5p targeted genes may play roles in transcription regulation, regulation of cell growth and proliferation, and angiogenesis. The most significant pathway in KEGG analysis was pathways in cancer. Altogether suggests that miR-199b-5p could play a possible role in parathyroid tumorigenesis.
A significant direct association between miR-199b-5p and PTH levels in MEN1 parathyroid tumors was not observed in the present study, which may be due to the complex interactions between genetic backgrounds and other susceptibility factors affected by the MEN1 gene. To investigate the potential effects of miR-199b-5p on parathyroid tumorigenesis in different genetic backgrounds, we used bioinformatics to predict a network between miR-199b-5p and the MEN1 gene with Ingenuity Pathway Analysis (IPA) software (Ingenuity ® Systems version 8.0, www.ingenuity.com) (Fig. 5). Interestingly, one pathway was identified: gene expression, cellular development, cellular growth and proliferation. According to the IPA results, miR-199b-5p directly targets the transcription regulators HIF-1α and SIRT1, which play a key role in promoting cell proliferation 38,39 and have known interactions with MEN1 40 . However, it is unclear how altered miR-199b-5p expression occurs within the context of dysregulated MEN1 in parathyroid tumors. The ability of miR-199b-5p to mediate the expression of these two genes may also explain the relationship between miR-199b-5p and MEN1. These complex relations must be confirmed in further investigations. There are some limitations to this study. First, the sample size of MEN1-related parathyroid tumors is small due to the rarity of this condition. Second, the lack of experimental validation for functional studies of miR-199b-5p and its predicted target genes is a further limitation.
In conclusion, we identified that miR-199b-5p is differentially expressed between sporadic and MEN1 parathyroid tumors and could be a potential diagnostic marker for distinguishing these tumors in different genetic backgrounds. Considering these data on the association between PTH and miR-199b-5p in parathyroid tumors, it will be important to focus future studies on the role of miR-199b-5p in parathyroid tumorigenesis.

Materials and Methods
Parathyroid tissue samples. We obtained a total of 61 parathyroid tissue samples and the associated clinical and histopathological data. Thirty-seven parathyroid tumor tissues were obtained from parathyroidectomy procedures. Twelve samples were obtained from MEN1 patients and confirmed to have germline mutations in the MEN1 gene. Twenty-four normal parathyroid tissues were used as controls; these tissues were incidentally removed during thyroidectomy in hyperthyroidism patients who had no evidence of PHPT. The present study was approved by the Institutional Review Board of Severance Hospital (4-2011-0613), and written informed consent was obtained from all patients. All experiments were performed in accordance with the relevant guidelines and regulations. RNA isolation. Total RNA was extracted from the formalin-fixed and paraffin-embedded (FFPE) samples using TRIzol reagent (GIBCO, BRL, Gaithersburg, MD, USA) according to the manufacturer's protocol. Following extraction, total RNA was quantified by an ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE, USA).

Figure 5.
A network predicted to be regulated by miRNA-199b-5p and the MEN1 gene. One predicted network regulated by miRNA-199b-5p and the MEN1 gene was "Gene expression, cellular development, cellular growth and proliferation". Statistical analysis. Statistical analyses were performed using SPSS 18.0 software (SPSS, Inc. Chicago, IL, USA). Differences in continuous variables between the three groups were tested by one-way ANOVA with Tukey's post hoc test, and the differences between two groups were determined by the independent sample t-test or Mann-Whitney U-test. Spearman's rank correlation coefficients were used to assess the associations between the relative miRNA expression and PTH levels. Receiver operating characteristic curve (ROC) analysis was applied to obtain the utility of the miRNA for distinguishing between sporadic and hereditary parathyroid tumors. A value of P < 0.05 was considered statistically significant.