Downregulation and the Diagnostic Value of RMST in Patients with Papillary Thyroid Cancer

Background: Previous studiess have demonstrated that rhabdomyosarcoma 2-associated transcript (RMST) is an indispensable factor in promoting neuronal differentiation. Gene Expression Omnibus datasets showed that long noncoding RNA RMST was downregulated in papillary thyroid cancer (PTC). Further experiments were conducted to detect the expression and diagnostic value of RMST in PTC. Materials and methods: Quantitative reverse-transcription polymerase chain reaction was applied to uncover the expression of RMST in PTC tissues. Chi-square (χ2) analysis was employed to evaluate the association between RMST and clinical features. The area under the curve (AUC) and receiver-operating characteristic curves were used to evaluate the feasibility of using RMST to predict PTC, lymph node metastases and the tumor–node–metastasis (TNM) stage. Result: RMST expression was signicantly in to PTC tissues than in adjacent noncancerous thyroid tissues (P<0.001). We also found that low tissue RMST expression was related to the TNM stage (P=0.046), and lymph node metastasis (P=0.002). The AUC value of PTC vs. adjacent noncancerous tissues was 0.7243 [95% condence interval (CI)=0.6411-0.8076, P<0.0001] and AUC values of patients with lymph node metastasis vs. patients with out lymph node metastasis and stage I/IIPTC vs. stage III/IV PTC were 0.7148 (95%CI =6018-0.8351, P=0.0012) and , 0.7024 (95%CI= 0.5817-0.8231, P=0.0066), respectively. Conclusion: study, we rst uncovered downregulated expression of RMST in PTC tissues compared that in adjacent noncancerous tissues. Further analysis revealed that down-regulated RMST was associated with the TNM stage and lymph node metastasis. Moreover, we demonstrated that RMST could predict PTC, lymph node metastasis and the TNM stage using ROC curves and AUCs.

Result: RMST expression was signi cantly in to PTC tissues than in adjacent noncancerous thyroid tissues (P<0.001). We also found that low tissue RMST expression was related to the TNM stage Conclusion: In conclusion, RMST is associated with PTC progression and may serve as a diagnostic biomarker for predicting PTC, lymph node metastasis, and TNM stage.

Background
Thyroid cancer (TC) is one of the most common endocrine malignancies; moreover, the incidence of TC has been increasing in the past three decades (1,2). Currently, the incidence and mortality rates of TC in China are 0.32% and 0.03%, respectively (3). Fortunately, most of the cases of TC are of papillary thyroid cancer (PTC), which is a relatively indolent thyroid malignancy with excellent long-term survival rates [4].
Although most patients with PTC have a good prognosis, the risks of cancer recurrence and metastasis make it impossible to ignore these patients [5]. Although the aggressive characteristics and mechanism of PTC are unclear, the molecular regulatory mechanisms may provide novel insights for patients with PTC. Therefore, new molecular markers may contribute to the diagnosis of aggressive PTC.
Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs that are more than 200 nucleotides in length and play critical roles in epigenetic, transcriptional and post-transcriptional regulation (6,7).
Increasing evidence has demonstrated that lncRNAs are involved in the development, migration, and apoptosis of many malignancies, including PTC (8). For example, Yoon et al. identi ed a novel noncoding RNA, NAMA, which is downregulated in PTC and is associated with growth arrest (9). Increased expression of NEAT1_2 in PTC is associated with aggressive clinicopathological characteristics, such as the tumor-node-metastasis (TNM) stage and tumor size. Moreover, knocking down the expression of NEAT1_2 inhibits aggressive PTC behavior by downregulating microRNA(miR)-106b-5p to regulate the expression of ATAD2 (10). Previous studies have indicated that lncRNA LINC01061 upregulation is a predictor of poor prognosis and promotes PTC progression (11). Using integrative computational methods, You et al. found a three-lncRNA signature (PRSS3P2, KRTAP5-AS1, and PWAR5) which could effectively predict the prognosis of PTC (12). Therefore, it is worth while to explore lncRNAs in PTC.
Rhabdomyosarcoma 2-associated transcript (RMST), a long non-coding RNA, plays a crucial role in neurogenesis by aiding in the association between the Sox2 transcription factor and its target promoters, which is indispensable for neuronal differentiation (13,14). A recent study demonstrated that downregulated RMST may promote triple-negative breast cancer (TNBC) cell proliferation, invasion, and migration (15). However, few studies have been conducted to determine the functions of RMST in PTC. In the current study, we measured RMST expression in PTC samples and paired noncancerous samples of Gene Expression Omnibus (GEO) datasets and clinical patients.We then evaluated the association between RMST expression and clinicopathological characteristics in patients with PTC. Finally, we performed receiver-operating characteristic (ROC) curve to analysis the diagnostic value of RMST in predicting PTC, lymph node metastasis, and TNM stage.

Quantitative real-time polymerase chain reaction
Total RNA was isolated from tissue samples using TRIzol reagent (TAKARA) according to the manufacturer's protocol. The quality of RNA was evaluated using a NanoDrop Spectrophotometer (Shimadzu Biotech, Beijing China). Only when the A260/A280 ratio was 1.8 to 2.1, we puri ed the extracted RNA. The isolated RNA was then reverse-transcribed into cDNA using the PrimeScript™ RT reagent kit with gDNA Eraser (RR047A; Takara, Dalian, China).Real-time quantitative reverse-transcripition PCR (qRT-PCR) was performed using the Light Cycle®480 II system(Roche, Basel, Switzerland). The qRT-PCR ampli cation procedure was performed as follows: 95°C for 5 s, 55°C for 30 s and 72°C for 30 s for a total of 45 cycles. The relative expression of RMST was normalized to the endogenous gene expression of β-actin and calculated using the 2 − ΔΔCt method. All reactions were performed repetitively. The primer sequences of RMST and β-actin were synthesized by Sangon Biotech (Shanghai, China).

Statistical analysis
SPSS version 21.0 software (IBM Corp, Chicago, IL, USA) was used to analyze the data, and GraphPad Prism V.7.00 software (GraphPad Software, La Jolla, CA, USA) was used to draw gures. Comparisons between groups were performed using the Student t-test or non-paired Mann-Whitney U test. The chisquare (χ2) test was applied to determine the association between RMST expression and the clinicopathological features of PTC. Multivariable logistic regression analysis was conducted and data are expressed as odds ratios (ORs) and 95% con dence intervals (95% CIs). ROC curves were used to evaluate the diagnostic value of RMST. P-values < 0.05 were considered statistically signi cant.

Downregulation of RMST expression in tissues from patients with PTC
As shown in Fig. 1A, the expression of RMST in PTC tissue was signi cantly downregulated compared to that in the paired noncancerous tissues (P < 0.001). To further con rm the expression of RMST in PTC samples, two GEO datasets (GSE33630, GSE66783) were used to obtain the expression of RMST. As shown in Fig. 1B and 1C, the expression of RMST was signi cantly decreased in PTC tissues compared to that in non-tumoral thyroid tissues in these datasets (P < 0.001). These results were consistent with the analysis of clinical PTC samples. Moreover, we found that the expression of RMST was signi cantly decreased in stage III/IV TC compared with that in I/II TC and noncancerous tissues in 83-paired samples (Fig. 2).

Correlation between RMST and clinicopathological features
The clinical characteristics of the 83 patients with PTC are listed in Table 1. Among all the patients with PTC, 49 had lymph node metastasis (59.04%), and 20 patients had in advanced-stage PTC (25.10%).
Other clinical features, such as age, sex, and tumor size, are also listed in Table 1. To analyze the correlation between RMST expression and clinicopathological features, patients with PTC were divided into two groups based on the median expression value (0.68) of RMST. As shown in Table 2, our data demonstrated that the expression of RMST was signi cantly associated with the TNM stage (P = 0.046) and lymph node metastases (P = 0.002). However, no signi cant correlation was found between RMST expression and other clinical features such as gender, age, and tumor size (P > 0.05). Furthermore, to investigate whether RMST is an independent protective factor of lymph node metastases (B < 0, OR < 1), multivariable analysis was employed. As shown in Table 3, RMST was an independent protective factor of lymph node metastases after adjustments for age, sex, body mass index, serum thyroid-stimulating hormone levels, and tumor size (OR = 0.094, P = 0.002).

Diagnostic value of RMST in patients with PTC
To investigate whether RMST may serve as a diagnostic marker for predicting PTC, lymph node metastasis and the TNM stage, ROC curves and areas under the ROC curves (AUCs) were employed. Our results illustrated signi cant difference between PTC and adjacent noncancerous tissues, with an AUC of 0.7243 (95%CI = 0.6411-0.8076, P < 0.0001; Fig. 3A) for RMST in 83-paired samples when the cutoff value was 1.398, with a sensitivity and speci city of 61.64% and 78.08%, respectively. The AUC of thyroid cancer vs. healthy controls for GSE33630 was 0.7859 (sensitivity, 93.88%; speci city, 55.56%; Fig. 3B). Moreover, RMST may serve as a diagnostic biomarker for predicting lymph node metastasis and the TNM stage with AUCs of 0.704 (sensitivity, 80.65%; speci city, 65.22%; Fig. 3C) and AUC 0.7024 (sensitivity ,70% ; speci city, 68.25%), respectively.

Discussion
Recently, the diagnostic value of lncRNAs in PTC has been evaluated in many studies; for example, GAS8-AS1 and LPAR4 may serve as potential diagnostic and therapeutic targets (18). However, the diagnostic value of RMST in PTC remains unclear.
In the present study, we rst uncovered downregulated expression of RMST in PTC tissues compared that in adjacent noncancerous tissues. Further analysis revealed that down-regulated RMST was associated with the TNM stage and lymph node metastasis. Moreover, we demonstrated that RMST could predict PTC, lymph node metastasis and the TNM stage using ROC curves and AUCs.
Increasing evidence has indicates that the differential expression of lncRNAs is closely related to the development and progression of cancers, including PTC (19). For instance, lncRNA MALAT1, which actes as a key regulator of proliferation and invasion in several cancers, is upregulated in PTC and promotes the proliferation and invasion of TC cells (20). A previous study showed that LncRNA HOXA-AS2 is upregulated in PTC and modulates the miR-15a-5p/HOXA3 axis to promote tumorigenesis and the progression of PTC (21). Ding et al. (22) revealed that lncRNA NONHSAT129183 is signi cantly upregulated in PTC tissues when compared with adjacent noncancerous thyroid tissue. Moreover, silencing NONHSAT129183 signi cantly suppressesd cell proliferation, migration, and invasion in PTC cell lines. The downregulation of lncRNA PTCSC3 has been demonstrated to contribute to drug resistance in AanaplasticTC (23). These studies indicate that differentially expressed lncRNAs are involved in carcinogenesis and proliferation in PTC and may provide novel diagnostic and prognostic biomarkers for patients with PTC.
In this study, we identi ed that lncRNA RMST is downregulated in PTC tissues; moreover, its expression is correlated with lymph node metastasis, and the TNM stage in patients with PTC. Ng et al. (14) pointed out that RMST is indispensable for neuronal differentiation. Yu et al. (24) found that transspliced RMST inhibits embryonic stem cell differentitation by targeting the epithelial-to-mesenchymal transition pathway. Wang et al. (25) found that RMST expression is low in TNBC tissues and cells; moreover, they demonstrated that RMST overexpression restrains the invasion and migration abilities of TNBC cells. The present study found that RMST was downregulated in PTC tissues compared to that in adjacent noncancerous tissues and associated with some aggressive features, such as lymph node metastasis. Finally, we found that RMST may serve as a diagnostic biomarker for predicting lymph node metastasis and the TNM stage,with AUCs of 0.7243, 0.704,and 0.7024, respectively. However, some limitations need to be addressed in further studies. For instance, the underlying mechanism of the involvement of RMST in tumor suppression in PTC must be elucidated. In addition, large-scale studies and long-term follow-up for veri cation are also needed.
In conclusion, we demonstrated low expression of RMST in PTC tissues. RMST may be employed as a potential diagnostic biomarker for PTC detection.

Ethics approval
This study was approved by the Ethics Committee of Hunan Cancer Hospital and the A liated Cancer Hospital of Xiangya School of Medicine, Central South University.

Consent
Written informed consent for publication of their clinical details was obtained from the patient.
Competing interests