MicroRNA-451 as a prognostic marker for diagnosis and lymph node metastasis of papillary thyroid carcinoma

Abstract

BACKGROUND AND OBJECTIVE:

To investigate the diagnostic potentials of microRNA-451(miR-451) in papillary thyroid carcinoma (PTC) diagnosis and lymph node (LN) metastasis, formalin-fixed, paraffin-embedded (FFPE) tissue specimens corresponding to PTC tumors ( $n=$ 60) and their normal counterparts (Normal tissues Adjacent to Tumor, NAT, $n=$ 60), along with sera from PTC patients with malignant tumors ( $n=$ 70) and benign lesions ( $n=$ 70) were analyzed for the expression of miR-451 by real-time PCR.

METHODS:

The usefulness of miR-451 expression as a prognostic marker for diagnosis of PTC malignancies was evaluated by Receiver Operating Curve (ROC). We reported that when compared to those in NAT, the levels of miR-451 in FFPE tissues from various stages of PTC patients ( $n=$ 60) were significantly lower (Mean $\pm$ SEM; 12.62 $\pm$ 1.73 vs 38.8 $\pm$ 3.51, $p<$ 0.0001). Receiver operating curve (ROC) analysis revealed that the area under curve (AUC) was 0.808; suggesting miR-451 expression was a reliable tissue biomarker for PTC malignancies. Further in depth analyses of these specimens revealed that miR-451 levels were significantly lower in PTC patients with lymph node (LN) metastasis than those without LN metastasis (3.96 $\pm$ 1.67 vs. 14.15 $\pm$ 1.95, $p=$ 0.006) with calculated AUC of 0.792, supporting the notion that miR-451 expression was also a good indicator for PTC lymph node involvements. Analyses sera from the cohorts of PTC patients indicated that miR-451 levels in patients with malignant lesions ( $n=$ 70) were significantly lower (10.72 $\pm$ 1.52 vs. 19.28 $\pm$ 2.73, $p=$ 0.010) than those with benign ones ( $n=$ 70). Parallel analyses of serum miR-451 levels in patients with LN metastasis also showed that they were significantly lower when compared to those without LN metastasis (6.79 $\pm$ 2.29 vs. 12.08 $\pm$ 1.86, $p=$ 0.017).

RESULTS:

ROC analyses revealed that AUC was 0.626 for malignancies and was 0.690 for lymph node involvement, respectively, suggesting that miR-451was a modest blood based biomarker for PTC malignancies and lymph node metastasis.

CONCLUSIONS:

We concluded that miR-451 expression is a reliable FFPE tissue biomarker for PTC malignancies and it may have potentials to become a noninvasive, blood-based biomarker for PTC diagnosis and evaluation of LN status.

1. Introduction

Thyroid carcinoma (TC) is the most common human endocrine malignancies, accounting for 95% of all endocrine tumors. Papillary thyroid carcinoma (PTC) has the most common histotype (approximately 80% of all TCs); its incidence has steadily increased over years [1]. Most PTCs can be effectively treated, with 5 years of survival rate over the 95%. However, a small fraction of patients who failed to response to the treatments could progress to a grave stage with local lymph node metastasis [2]. Despite to recent advances in tumor diagnosis and patient cares; it has not had a major breakthrough with respect to identification of reliable PTC-specific prognostic markers. Genomic analyses of large cohorts of PTC patients have identified quite a few tumor associated molecular markers, including $>$ 100 cellular RNA transcripts or proteins, a set of non-coding RNAs and genetic variations (mutations and rearrangements) [3, 4, 5]. Although aberrant expression of these molecular markers is believed involved in the tumorigenesis of PTC, the diagnostic and prognostic usefulness of these genetic markers remains to be further defined.

MicroRNAs (miRNAs) are small non-coding RNAs (19–23 bp in size) well distributed in the whole spectrums of living organisms and are known to participate in the regulation of many physiological and pathological processes [6]. By targeting mostly on 3’ untranslational region (3’UTR) of various Messenger RNAs (mRNAs), miRNA affect gene expression through inhibition of gene translation [7]. Numerous studies have demonstrated that miRNAs play critical roles in oncogenesis of various cancers. Expression of a group of miRNAs, known as oncomiR [8], including but not limited to miR-17-92, miR-21, miR-155 and miR-372, are overexpressed in cancers, while another group of miRNAs (tumor-suppressive miRNAs), including but not limited to Let-7, miR-15a, miR-16-1 are underexpressed in cancers [8, 9]. There are yet another examples such as miRNA-125b, act as an oncomiR in the majority hematologic malignancies and as a tumor suppressor in many solid tumors [10]. These studies indicated a complex network system in which microRNAs play a key role in cell type specific oncogenesis; these findings also implied that comprehensive analyses of microRNA expression could result in the identification of biomarkers useful for diagnosis and prognosis of tumors.

Recent studies have implicated that deregulation of miRNAs was involved in the pathogenesis of thyroid cancers. A comprehensive miRNA profiling indicated that a handful of miRNA were deregulated in tissues of PTC patients when compared to that of normal counterparts [11, 12]. In particular, Lee et al. [13] and Wang et al. [11] have independently demonstrated that higher expression of miR-222 and miR-146b was associated with poor prognosis and lymph node involvement of thyroid cancers. Expression of other species of miRNAs in tissues of PTC, however, has not been well documented.

In the present study, we investigated if miR-451 can be a prognostic marker for malignancies of PTC by retrospective analyses its expression in two most commonly available specimens, formalin-fixed paraffin-embedded tissues (FFPE) and venous serum, from PTC patients prior to their surgeries. The correlation between miR-451 levels and the patients’ clinicopathological features was analyzed.

2. Materials and methods

2.1 Patents and ethics statement

This study was approved by the Ethics Committee of Xinhua Hospital, Shanghai Jiaotong University School of Medicine. Written informed consents were obtained from all participants of this study.

Two groups of patients were recruited for the current study. The first cohort of patients, who participated in the study of miRNA-451 in FFPE preserved tissues, was comprised of 60 patients (13 males and 47 females). These patients were diagnosed with papillary thyroid carcinoma (PTC) and have undergone thyroidectomy at Xinhua Hospital, Shanghai Jiaotong University School of Medicine, between July 2009 and July 2014. The data on patients’ clinical features including gender, age, tumor size, histological types, lymph node status and serum thyroglobulin (GT) levels at diagnosis, were retrospectively collected. Diagnosis of PTC were certified by a group of pathologists in accordance with the Guideline of WHO [14]. The second cohort of patients involved 70 PTC patients (28 males and 42 females) with various stages of PTC. These patients, along with patients determined to have benign nodules ( $n=$ 70) were enrolled in the second (blood based) cohort of study. All participants recruited in the present study received no radiotherapy, chemotherapy or any other treatment prior to surgical operation; venous bloods for the second cohort of this study were collected two days before surgery. Clinicopathological characteristics of PTC patients enrolled in the study are listed in Table 1.

Table 1

Clinicopatholgical characteristics of PTC patients in this study

Variables	FFPE patient cohort ( $n=$ 60)		Serum patient cohort ( $n=$ 70)
Gender
Male	13		28
Female	47		42
Age (years)
$⩽$ 45	47		46
$>$ 45	13		24
Primary tumor size*
$⩽$ 1 cm	15	$⩽$ 2 cm	23
$>$ 1 cm	45	$>$ 2 cm	47
Lymph Node metastasis
Yes	9		18
No	45		52
Tumor location
Left	25		24
Right	19		27
Isthmus	5		6
Bilateral	11		13
Serum TG level
Normal	ND		55
High	ND		15

${}^{*}$ Note: For FFPE patient cohort, tumor size cutoff was 1 cm; for serum patient cohort, tumor size cutoff was 2 cm. Abbreviations: ND, not determined; TG, thyroglobulin.

2.2 Specimen preparation

For every PTC patient ( $n=$ 60) in the first cohort, a pair of formalin fixed paraffin-embedded (FFPE) tissue blocks corresponding PTC tumors ( $>$ 85% by H&E staining) and that corresponding to the Normal tissues Adjacent to Tumor (NAT), which were at least 1 cm away from cancer margin and was confirmed normal by pathologists, were collected. Four fresh cut sections (20 min thickness) from paraffin blocks corresponding to PTC or NAT tissues were first dissolved in 1 ml xylene in a 1.5 ml microcentrifuge tube, heated at 50 ${}^{\circ }$ C for 3 min, centrifuged at a full speed to pellet tissues. After washed with 100% ethanol twice, pellets were dissolved in 150 ml proteinase K buffer and digested with the enzyme for 15 min at 37 ${}^{\circ }$ C prior to total RNA preparation.

For every PTC patient (malignant, $n=$ 70; benign, $n=$ 70) in the second cohort, a 5-ml peripheral venous blood sample was collected in a Gold Top (serum separation) tube. After 30 min coagulation at room temperature, whole blood samples from these two groups of patients were centrifuged at 1000 g for 30 min at 4 ${}^{\circ }$ C. Sera were separated, aliquoted and immediately stored at $-$ 80 ${}^{\circ }$ C prior to use.

2.3 Total RNA extraction

Total RNA was isolated from FFPE tissues using a miRNeasy FFPE Kit (QIAGEN) according the manufactory standard protocol without any modifications. For isolation of miRNAs from patient’s sera, sera stored at 80 ${}^{\circ }$ C were first thawed and centrifuged at 4 ${}^{\circ }$ C for 15 min to remove impurities. 200 ml aliquot of each serum was spiked with 3.5 l (1.6 $\times$ 10 ${}^8$ copies/l) C. elegans miR-39 miRNA Mimic (Dharmacon, Lafayette, Colorado, USA), used as a volume control. Total RNA was extracted from sera using a miRNeasy Serum/Plasma Kit (QIAGEN, Hilden, Germany). The purity and concentration of RNA was determined by NanoDrop 2000 (Thermo Scientific, USA). Only the RNA samples with a ratio of A260/A280 $>$ 1.8 were considered suitable for this study.

2.4 Quantitative real-time PCR

For quantitative detection of miRNA, purified serum or tissue total RNA was first used to convert to miRNA gene specific cDNA by reverse transcription reactions using a standard protocol provided by Applied Biosystems, (Grand Island, NY, USA). For PCR amplification, reaction mix was prepared in a 96 well plate; each reaction (5 ml) contained 2.5 ml TaqMan Universal PCR Master Mix II, 0.25 ml miRNA-specific primer/probe mixes, and 2.25 ml cDNA template prepared from above. The PCR reaction was prepared in triplicate. Amplification was performed in an ABI 7900HT thermocycler (Applied Biosystems) using following cycling parameters: 95 ${}^{\circ }$ C for 10 min, followed by 40 cycles of 95 ${}^{\circ }$ C for 15 sec and 60 ${}^{\circ }$ C for 1 min. Real-time PCR data were analyzed by SDS 2.2 software (Applied Biosystems). Relative levels of the tissue miRNA-451 were calculated using RNU-44, a small non-coding RNA, as an internal control. For measurement of miR-451 levels in sera, we used C. elegans synthetic miR-39, which was spike-in during sera preparation, as a serum volume (200 ml) control. Relative microRNA level of tested samples were calculated by 2 ${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{CT}}$ , where $\mathrm{\Delta }$ CT $=$ CT ${}^{\text{miRNA451}}$ -CT ${}^{\text{normalization control}}$ . CT was the number of PCR cycles for florescence signal to cross the threshold.

Figure 1.

Analysis of miR-451 expression in FFPE tissues from tumors and normal tissues adjacent to tumors (NAT) in 60 PTC patients. (A) Downregulation of miR-451 expression in tumors vs NAT. Total RNA were extracted from paired (tumor and NAT) FFPE samples from patients. Expression of miR-451 was performed by qRT-PCR and normalized by expression of an internal control microRNA RUN-44. The result was shown as Mean $\pm$ SEM. (B) ROC analysis of tissue miR-451 expression to determine its predictability as a biomarker for PTC. A value of 0.808 in AUC would indicate that it is a reliably “good” tissue marker for PTC.

2.5 Statistical analysis

Statistical analyses were performed with the SPSS statistical package, version 16.0 (SPSS Inc., Chicago, IL, USA) for Windows. The images were made with GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA, USA). In the most cases, data were expressed as Whisker Plot to demonstrate the Medium and distribution of values. In some cases, data were also expressed as the Mean $\pm$ Sem (standard errors). $p<$ 0.05 was considered statistically significant. Mann Whitney test was used to analyze the differences between two groups. Receiver operating characteristics (ROC) analysis was used to evaluate the diagnostic potential of miR-451.

Figure 2.

Analysis of correlations between tissue miR-451 expression with clinicopathological features of PTC patients. (A) Expression of miR-451 in PTC tissue with respect to gender, age, tumor size, LN status and tumor location. Data were expressed by Whisker Plot to demonstrate the medium and the distribution of samples. Except for status of lymph node involvement ( $p=$ 0.006), there were no statistical differences among other variables. (B) Expression of miR-451 in LN ${}^{-}$ and LN ${}^{+}$ samples was replotted to show Mean $\pm$ SEM of these two groups. Note that miR-451 level was significantly (3.5 fold) downregulated. (C) ROC analysis of expression of tissue miR-451 as a good biomarker (AUC $=$ 0.792) of lymph node metastasis.

3. Results

3.1 Comparison of miR-451 expression between PTC and Normal Adjacent Tissues in FFPE cohort

To explore the expression and significance of miR-451 in oncogenesis of PTC, we analyzed expression of miR-451 in FFPE tissues from patients ( $n=$ 60) corresponding to either tumors or normal counterparts (NAT). In a qRT-PCR analysis (Fig. 1A) we showed that tissue miR-451 level was at least 3 folds lower (12.62 $\pm$ 1.75 vs 38.79 $\pm$ 3.51) in PTC tissues than those in NAT. Statistical analyses indicated that the change of expression is very significant with $P<$ 0.0001. This result indicated a strong correlation between occurrence of tumor and down-regulation of miR-451. Receiver operating characteristic (ROC) analysis further revealed that tissue miR-451 had high potential in the distinguishing PTC tissues from NAT, with an area under curve (AUC) of 0.808 (Fig. 1B). The curve of Youden index showed that the best cut-off value was 22.270, and the corresponding sensitivity and specificity were 0.717 and 0.783, respectively (data not shown). These results indicate that expression of miR-451 can reliably be used to identify the PTC tumors from normal tissues and therefore is a good biomarker for tumors of PTC.

Figure 3.

Analysis of miR-451 expression in sera from PTC patients. (A) Downregulation of serum miR-451 in patients with malignant lesions ( $n=$ 70) vs patients with benign lesions ( $n=$ 70). Expression of miR-451 was performed by qRT-PCR and normalized by spike-in serum volume control C. elegans miR-39. The result was shown as Mean $\pm$ SEM. Note that miR-451 in sera from malignant PTC patients was modestly (2 fold) lower but the difference was statistically significant ( $p=$ 0.010) (B) ROC analysis of serum miR-451 expression in malignant PTC patients. A value of 0.626 in AUC would indicate that it is a “modest” biomarker for malignancy.

3.2 Reduced miR-451 expression in patients with lymph node metastasis

Low expression of miR-451 in tumors prompted us to further investigate if its expression pattern is associated with any parameters with respect to clinicopathological features of PTC. We performed miRNA-451 analyses on all participants and retrospectively categorized patients according to different variables, including gender, age, tumor size, lymph node (LN) status and tumor location. Significance of changes of miR-451 level was analyzed with Mann-Whitney U test or one-way ANOVA (tumor location). As shown in Fig. 2A, there were no significant difference in miR-451 levels observed in the sub-group distributed by gender (12.50 $\pm$ 3.42 vs. 12.66 $\pm$ 2.02, $p=$ 0.342), age (11.43 $\pm$ 1.84 vs. 16.92 $\pm$ 4.42, $p=$ 0.203), tumor size (11.82 $\pm$ 3.30 vs. 15.04 $\pm$ 2.30, $p=$ 0.459) and tumor location ( $p=$ 0.576). On the other hand, miR-451 was significantly lower in LN positive patients than those in LN negative patients (3.96 $\pm$ 1.67 vs. 14.15 $\pm$ 1.95, $p=$ 0.006, Fig. 2B). These results suggest that while tissue miR-451 expression was not correlated with other clinicopathoglical features, it was clearly associated with lymph node metastasis. ROC analysis for miR-451 level with LN status revealed an AUC of 0.791, with a cut-off value of 2.805, a sensitivity of 0.667 and a specificity of 0.922 (Fig. 2C). The above results suggest that tissue miR-451 expression was strongly associated with aggressiveness of tumors (lymph node metastasis) and can be a reliable biomarker for such a clinicopathological feature.

3.3 Serum miR-451 level was significantly lower in patients with PTC than those with benign nodules

To evaluate the potential of miR-451 as a blood-based biomarker, sera from patients with malignant PTC ( $n=$ 70) and benign nodules ( $n=$ 70) were analyzed for expression of miR-451. As shown in Fig. 3A, we found that the level of miR-451 was significantly lower in PTC group than those in Benign group (10.72 $\pm$ 1.52 vs. 19.28 $\pm$ 2.73, $P=$ 0.010). ROC analysis revealed an AUC of 0.626 (Fig. 3B) with a cut-off value of 2.960, a sensitivity of 0.400 and a specificity of 0.857. This result indicated that, in the blood-based assay, low expression of miR-451 was associated with malignant PTC, when compared to that in patients with benign module and this association may be used as a modest biomarker for PTC malignancy.

3.4 Reduced expression of serum miR-451 correlated with lymph node metastasis

To investigate if serum miR-451 level can be an indicator of aggressiveness of tumor, we performed miR-451 expression in sera from all malignant PTC patients ( $n=$ 70) and retrospectively categorized them into various clinicopathological features. As shown in Fig. 4A, we found that serum miR-451 level was significantly lower in lymph node positive (LN ${}^{+}$ ) patients than those in lymph node negative (LN ${}^{-}$ ) patients (6.79 $\pm$ 2.29 vs. 12.08 $\pm$ 1.86, $P=$ 0.017). It is, however, was not associated with other parameters, including sex, age, tumor size, tumor location and serum TG levels. Comparing to that of LN ${}^{-}$ , downregulation of miR-451 in LN ${}^{+}$ was modest (77%), but were statistically significant ( $p=$ 0.017, Fig. 4B). ROC curve showed that serum miR-451 level with LN metastasis revealed an AUC of 0.690, with a cut-off value of 2.965, a sensitivity of 0.778 and a specificity of 0.731 (Fig. 4B). These results indicate that serum miR-451 level also had the potential to be a biomarker for PTC diagnosis and LN status evaluation.

Figure 4.

Analysis of correlations between expression of serum miR-451 and clinicopatholgical features of PTC patients. (A) Correlation of miR-451 level in PTC tissue with respect to gender, age, tumor size, LN status, tumor locations and serum TG levels. Data were expressed by Whisker Plot to demonstrate the medium and the distribution of samples. Except for the status of lymph node involvement ( $p=$ 0.017), there were no statistical differences between other variables. (B) Expression of miR-451 in LN ${}^{-}$ and LN ${}^{+}$ samples was replotted to show Mean $\pm$ SEM of these two groups. Although miR-451 level in LN ${}^{+}$ was only modestly (77%) lower than that in LN ${}^{-}$ , it was statistically significant ( $p=$ 0.017). (C) ROC analysis of serum miR-451 as a biomarker for evaluation of lymph node status. A value of 0.690 in AUC would indicate that it is a “modest” biomarker for lymph node metastasis.

4. Discussion

MiRNAs are key regulators of cell proliferation, differentiation, apoptosis and migration. Aberrant expression of miRNAs has been found associated with various types of tumors, including different types of thyroid cancers [15]. Whether these miRNAs can be independent predictors of aggressive clinicopathological features of PTC remains unclear. Searching for additional miRNA biomarkers, which correlate better with the progress of tumors, is urgently needed.

4.1 Cellular miR-451 as a signature biomarker for thyroid malignancies

Studies by other groups have shown that expression of miR-451 negatively correlates with aggressiveness of hepatocellular carcinoma [16], Glioblastoma [17] and non-small cell lung cancer [18]In vitro studies showed that overexpression of miR-451 inhibited c-Myc mediated cell proliferation and migration via targeting Akt/mTOR signaling pathway [16, 19]. These studies suggest that miR-451 may function as a tumor suppressor that targets pathways important for oncogenesis. To address a possible role of miR-451 in the oncogenesis of PTC, we performed qRT-PCR analyses on 60 PTC patients FFPE paired samples (tumor and NAT, Normal Adjacent Tissues). Our results showed that expression of miR-451 in thyroid cancer tissues was significantly lower ( $p<$ 0.0001) than that in NAT. ROC analysis indicated that AUC was 0.808 (Fig. 1B), strongly suggesting that miR-451 expression can be graded as a reliably “good” biomarker for distinguishing PTC from the normal counterpart. Thus, our results are in good agreement with recent findings by Minna et al. [20] who demonstrated a similar downregulation of miR-451 in snap-frozen samples from PTC patients with aggressive clinical-pathological features. Our results also support the possibility that miR-451 may function as a tumor suppressive microRNA that targets genes involving in tumorigenesis of thyroid tumors.

We further analyzed the correlations between miR-451 expression and various clinicopathological parameters of our first PTC patient cohort, some of which have been suggested to be prognosticatively relevant [21]. Our results (Figs 2A and B) indicated a strong correlation between lower miR-451 levels in PTC patients with LN metastasis, suggesting that tissue miRNA-451 level is also a “good” biomarker (AUC $=$ 0.792) for lymph node metastasis. Thus, our results provide strong evidence that tissue miR-451 expression is reliable diagnostic marker for determination of malignancies of PTC tumors.

4.2 Circulating miR-451 as a biomarker for thyroid malignancies

Establishment of a non-invasive and low-cost assay for disease screening and diagnosis is the ultimate goal for the management of cancer patient cares. To investigate if miRNA-451 expression can be developed as a blood based diagnostic/prognostic marker for PTC, we analyzed serum miR-451 level in PTC patients with malignant features and patients with benign features. We found that miR-451 levels in patients with malignant forms was significantly lower than those with benign forms ( $p=$ 0.010, Fig. 3A) and that serum miR-451 was a fair biomarker (AUC $=$ 0.626) for distinguishing patients with PTC from benign lesions (Fig. 3B). Moreover, similar to that in tissues (Fig. 2A), miR-451 levels in patient sera were not correlated with gender, age, tumor size and location, but with PTC patients with LN metastasis (Fig. 4A), indicating serum miR-451 could be helpful in evaluation of LN status, although the diagnostic accuracy was probably limited (AUC $=$ 0.690). Larger sizes of samples and/or more stringent patients sample selections may be needed for validation if serum miR-451 can be a reliable biomarker for malignancies of PTC.

In summary, we have clearly demonstrated that miR-451 was down-regulated in both FFPE tissues and sera of PTC patients with malignant features. We have also shown that tissue miR-451 is a good predicting biomarker for malignant PTC. Together with other risk factors, such as overexpression of miR-146b and miR-222 [11, 13] and somatic mutation BRAF ${}^{\text{V600E}}$ [22], it may be possible to develop a reliable, genomic based, prognostic scoring system for precise prognosis/diagnosis of PTC and provided tailored treatments for an individual suffered a particular type of thyroid cancers.

5. Compliance with ethical standards

5.1 Funding

This study was funded by Science and Technology Fund Project of Shanghai Jiaotong University School of Medicine (grant number 13XJ22016).

5.2 Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study was approved by the Ethics Committee of Xinhua Hospital, Shanghai Jiaotong University School of Medicine.

Informed consent

Informed consent was obtained from all individual participants included in the study. Written informed consents were obtained from all participants of this study.