OTUD6B-AS1 Inhibits Viability, Migration, and Invasion of Thyroid Carcinoma by Targeting miR-183-5p and miR-21

Background: The long noncoding RNA (lncRNA) functions as a regulator of initiation, progression, and metastasis of thyroid carcinomas. lncRNA OTUD6B antisense RNA 1 (OTUD6B-AS1) is a tumor-suppressive noncoding RNA in clear cell renal cell carcinoma. The role of OTUD6B-AS1 in thyroid carcinomas has not been reported yet. We aim to investigate the expression and biological functions of OTUD6B-AS1 in thyroid carcinomas. Methods: The expression level of OTUD6B-AS1 was measured in 60 paired human thyroid carcinoma tissues and corresponding adjacent normal thyroid tissues. The correlations between the OTUD6B-AS1 expression levels and clinicopathological features were evaluated using the Mann-Whitney test. The effects of OTUD6B-AS1 on thyroid carcinoma cells were determined via the MTT and transwell assays. The potential targets of OTUD6B-AS1 were screened using the online programs OncomiR and StarBase 3.0, and the LncBase Predicted v.2. Luciferase reporter assay was used to confirm the interactions between OTUD6B-AS1 and its potential targets. Results: OTUD6B-AS1 was downregulated in thyroid carcinoma tissue samples. The expression of OTUD6B-AS1 correlated with tumor size, clinical stage, and lymphatic metastasis of thyroid carcinoma. Overexpression of OTUD6B-AS1 significantly decreased the viability, migration, and invasion of thyroid carcinoma cells. Online programs predicted miR-183-5p and miR-21 as potential targets of OTUD6B-AS1. Luciferase reporter assays showed miR-183-5p and miR-21 bound to OTUD6B-AS1. Moreover, overexpression of miR-183-5p and miR-21 compromised the inhibitory effects of OTUD6B-AS1 on viability, migration, and invasion of thyroid carcinoma cells. Conclusions: Taken together, our findings present in vitro evidence of lncRNA OTUD6B-AS1 as a tumor suppressor in thyroid carcinomas. OTUD6B-AS1 inhibits viability, migration, and invasion of thyroid carcinoma by targeting miR-183-5p and miR-21.


INTRODUCTION
Thyroid carcinoma is a malignant tumor derived from thyroid epithelial cells (1). It exhibits multicentricity in the thyroid gland and frequently metastasizes to the regional lymph nodes (2). It can damage liver and kidney function and the respiratory system (2). Although some achievements have been made in the study of thyroid cancer, its pathogenesis is still unclear.
Long noncoding RNAs (lncRNAs) refer to the type of noncoding RNA that is derived from genome-encoded transcripts with lengths longer than 200 RNA nucleotides (3). Recent studies suggest it may be directly involved in the regulation of development of tumor cells (4)(5)(6). LncRNAs are involved in the regulatory network of thyroid carcinomas. Several lncRNAs have been reported to promote or inhibit proliferation, migration, invasion, and angiogenesis of thyroid carcinomas (7,8). In addition, dysregulation of lncRNAs is associated with poor prognoses thyroid carcinomas (9). lncRNA OTUD6B antisense RNA 1 (OTUD6B-AS1) is oriented in an antisense direction relative to the protein-coding gene OTUD6B on the opposite DNA strand. OTUD6B-AS1 locates on chromosome 8q21.3 and has 2179 bp (NR_110439, ENST00000524003.1). OTUD6B-AS1 was initially reported as active in fibrosis in systemic sclerosis (10). It is downregulated in skin biopsies of systemic sclerosis and regulates the apoptosis of dermal fibroblasts (10). The role of OTUD6B-AS1 in cancer is largely unknown. A recent study reveals the role of OTUD6B-AS1 in clear cell renal cell carcinoma (ccRCC) (11). This study demonstrates that the lower expression of OTUD6B-AS1 is associated with poor prognosis of ccRCC (11). In vitro and in vivo evidence indicates that OTUD6B-AS1 functions as a tumor suppressor in ccRCC (11). However, the expressions and biological functions of OTUD6B-AS1 are still unknown in thyroid carcinomas. microRNAs (miRNAs) have emerged as key posttranscriptional regulators of tumourigenesis in thyroid carcinoma which able to bind to the 3 ′ -untranslated region of target mRNAs and inhibited the protein expression of target gene (12,13). Additionally, miRNA can be sponged by lncRNAs via competitive endogenous RNA (ceRNA) mechanism to regulate the abundance of miRNAs, then regulate miRNAs targets expression (14). In thyroid carcinoma, lncRNA FOXD2-AS1 promoted the tumorigenesis via sponging miR-7-5p to upregulate TERT expression (15). Currently, no OTUD6B-AS1 that act as ceRNAs have been reported in thyroid carcinomas.
In this work, we investigated the expression of OTUD6B-AS1 in human thyroid carcinoma tissues and corresponding adjacent normal thyroid tissue samples. We also evaluated the correlations between the expression levels of OTUD6B-AS1 and the clinicopathological parameters. Moreover, we screened the potential targets of OTUD6B-AS1 and explored the ceRNAs molecular mechanisms underlying the regulatory effects of OTUD6B-AS1 on thyroid carcinoma cells.

Specimens
Sixty thyroid carcinoma tissues (include 1 undifferentiated thyroid carcinoma, 3 follicular thyroid carcinoma, and 56 papillary thyroid carcinoma) and the corresponding normal tissues were collected from patients undergoing thyroidectomy at the Xiangya Hospital. This study obtained approval from the Medical Ethics Committee of Xiangya Hospital, Central South University (Approval number: 201612010). Informed written consent for the use of the tissue samples was obtained from all the patients. Clinicopathological data of the patients were showed in Table 1. All fresh tissues were frozen in liquid nitrogen immediately and stored at −80 • C until use.

qRT-PCR
Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Complementary DNA (cDNA) was used to determine the expression of All the experiments were performed in duplicate. The results are represented as the fold-induction using the 2-CT method (16). The primers used to determine the expression of OTUD6B-AS1 (11), miR-183-5p, and miR-21 are listed in Table 2.

Cell Viability Assay
Cell proliferation was assayed using an MTT assay at 48 h after transfected. The absorbance of the solution was measured with a spectrophotometer at 490 nm.

Transwell Assay
The transwell assay for migration was performed using chambers with polycarbonate filters (pore size 8µm) (Becton Dickinson Labware). The transwell assay for invasion was performed using chambers with Matrigel. Briefly, we harvested SW579 and TPC-1 cells and placed 5 × 10 4 transfected cells in the upper chamber of the filter. After 24 h of incubation and removal of the cells in the upper chamber using a cotton swab, the cells on the underside were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet in 20% ethanol, and counted in five randomly selected fields under a phase-contrast microscope. The migrated or invasive cells were monitored by photographing at 400× magnification using a LEICA microscope. The assays were performed in triplicate.

In silico Prediction of OTUD6B-AS1 Potential Targets
To investigate the mechanisms underlying the inhibitory effect of OTUD6B-AS1 on thyroid carcinoma cells, we screened potential targets of OTUD6B-AS1 using several online programs, OncomiR (17), StarBase 3.0 (18), and LncBase Predicted v.2 (19). StarBase 3.0 and LncBase Predicted v.2 were used to predict the bind sites between OTUD6B-AS1 and miRNAs according to the keyword "OTUD6B-AS1". Additionally, based on the tumor suppressive behavior of OTUD6B-AS1, we speculated that its targets may be cancer-promoting miRNAs. In OncomiR website,  (20). KEGG pathway enrichment analysis of target mRNAs were used DAVID website (21).

Luciferase Reporter Assays
Cells were seeded in 24-well plates. Cells were co-transfected with 150 ng of OTUD6B-AS1 reporter vector, 10 nM of miRNAs, were harvested 24 h after transfection, and luminescence was measured in triplicate using Promega Dual-Glo luciferase assay reagents according to the manufacturer's protocol, using an Optima series luminometer.

Statistical Analysis
Statistical data were analyzed using the Statistical Program for Social Sciences (SPSS) 19.0 software (SPSS, Chicago, IL, USA). GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA) was used to plot all the graphs. The differences between thyroid carcinoma tissue and adjacent normal tissues were analyzed using the Mann-Whitney test. An analysis of variance (ANOVA) was used for the overall comparison between three groups followed by the Newman-Keuls t-test used for multiple comparisons between the two groups. The correlations between the OTUD6B-AS1 expression and clinicopathological features were evaluated using the Mann-Whitney test. P < 0.05 was considered statistically significant.

OTUD6B-AS1 Is Downregulated in Thyroid Carcinoma
We investigated the expression of OTUD6B-AS1 in 60 pair of thyroid carcinoma tissues and adjacent para-cancerous tissues. We found that OTUD6B-AS1 was downregulated in the thyroid carcinoma tissues compared to the para-cancerous tissues (p < 0.01, Figure 1). The correlations between the FIGURE 1 | OTUD6B-AS1 is downregulated in thyroid carcinoma tissues. The expressions of OTUD6B-AS1 in 60 pairs of thyroid carcinoma tissues and adjacent para-cancerous tissues were evaluated using qRT-PCR. The correlations between the OTUD6B-AS1 expression and clinicopathological features were evaluated using the Mann-Whitney test. **p < 0.01.
In order to ensure the reliability of the results, we took the overlap of the findings from the three programs. Two miRNAs, miR-183-5p and miR-21, were found to be potential targets of OTUD6B-AS1 ( Figure 5A). The sequences of the binding sites of lncRNA OTUD6B-AS1 with miR-21 and miR-183-5p were shown in Figure 5B. In order to verify the predictions of the online programs, luciferase reporter plasmids, psiCHECK-2-WT, containing wild type (WT) OTUD6B-AS1 were created using the psiCHECK-2 vector. psiCHECK-2-WT was co-transfected with miR-183-5p or miR-21 in 293T cells. A scramble RNA (NC) was used as a control for miR-183-5p and miR-21. Decreased luciferase activities were observed in cells co-transfected with psiCHECK-2-WT and its potential target miR-21 or miR-183-5p ( Figure 5C). Conversely, increased luciferase activities were observed in cells co-transfected with psiCHECK-2-WT and inhibitors of miR-21 or miR-183-5p ( Figure 5C). These data indicated that miR-21 and miR-183-5p are bound to OTUD6B-AS1. In order to determine the binding specificity of miR-21 and miR-183-5p, the binding sites of miR-21 and miR-183-5p were mutated in luciferase reporter plasmids psiCHECK-2-WT, named psiCHECK-2-mutant. No significant differences were observed in cells co-transfected with psiCHECK-2-mutant and miR-21 or miR-183-5p (Figure 5C), nor in cells co-transfected with psiCHECK-2-mutant and miR-21 inhibitor or miR-183-5p ( Figure 5C). These data indicate that mutations in the binding  sites of miR-21 and miR-183-5p compromised the binding of miR-21 and miR-183-5p to OTUD6B-AS1. In addition, we examined the expression of miR-183-5p and miR-21 in normal thyroid cells Nthy-ori, and thyroid carcinoma cells SW579 and TPC-1 using qRT-PCR. We found that miR-183-5p and miR-21 were upregulated in the SW579 and TPC-1 cells compared to Nthy-ori cells (Figure 5D). Moreover, decreased expression of miR-183-5p and miR-21 was observed in SW579 and TPC-1 cells that overexpressed OTUD6B-AS1 (Figure 5E). Taken together, these findings support the prediction that miR-21 and miR-183-5p are the direct target of OTUD6B-AS1.

DISCUSSION
The dysregulation of lncRNA expression which acted as oncogenes or tumor-suppressor genes have great significance for the occurrence, development, metastasis, clinical stage and prognosis of thyroid cancer (7-9). OTUD6B-AS1 was first reported in systemic sclerosis (10). It is significantly downregulated in systemic sclerosis skin biopsies (10). In the field of cancer research, Wang and colleague reported OTUD6B-AS1 was downregulated in ccRCC tissue samples, and that patients with low OTUD6B-AS1 expression had shorter overall survival than patients with high OTUD6B-AS1 expression, which showed that the different expression levels of OTUD6B-AS1 indirectly correlated with patient survival (11). We reported downregulation of lncRNA OTUD6B-AS1 in thyroid cancer. The different expression level of OTUD6B-AS1 correlated with tumor size, clinical stage and lymphatic metastasis of thyroid carcinomas. The result suggested that lower OTUD6B-AS1 expression was closely related to the growth and metastasis of thyroid cancer. Consistent results in thyroid cancer and ccRCC indicate down-regulation of OTUD6B-AS1 as important for cancer development.
Under different physiological and pathological conditions, OTUD6B-AS1 is implicated in different cellular biological functions. In systemic sclerosis, OTUD6B-AS1 functions as a regulator of apoptosis (10). OTUD6B-AS1 regulates proliferation and apoptosis via cyclin D1 expression in a sense gene independent manner (10). In tumors, OTUD6B-AS1 regulates proliferation, migration and invasion. It inhibits ccRCC proliferation via the Wnt/beta-catenin signaling pathway (11). In the current study, we reported that OTUD6B-AS1 overexpression inhibited cells viability, migration, and invasion, similar with the effect of OTUD6B-AS1 in ccRCC. The results suggested thatOTUD6B-AS1 acts as a tumor suppressor in thyroid carcinomas. One of the important findings of this study is our identification of two downstream targets of OTUD6B-AS1, miR-183-5p, and miR-21. miR-21 is a well-studied oncogenic miRNA in a large variety of cancers. Up-regulation of miR-21 is implicated in cancer progression and metastasis (22,23). Several studies indicate that miR-21 expression is associated with the progression and recurrence of thyroid carcinomas (24)(25)(26). We showed that miR-21 is a direct target of OTUD6B-AS1. This result found that miR-21 overexpression promoted the viability, migration, and invasion, reversed OTUD6B-AS1 effect in thyroid carcinomas. OTUD6B-AS1 and miR-21 constitute a regulatory axis within thyroid carcinomas. miR-183-5p is another direct target of OTUD6B-AS1. The function of miR-183-5p in cancer is controversial. The role of miR-183 varies in different genetic and pathological contexts. Previous studies indicate that miR-183-5p is an oncogenic miRNA in breast cancer (27), hepatocellular carcinoma (28), colon cancer (29), colorectal cancer (30), bladder cancer (31), and pancreatic cancer (32,33). There are also several studies indicating that miR-183-5p functions as a tumor suppressor in lung cancer (34), acute myeloid leukemia (35), gastric cancer (36), and osteosarcoma (37). In thyroid carcinomas, miR-183-5p functions as an oncogene and promotes proliferation, migration, and invasion by targeting PDCD4 (38). Our work also found that miR-183-5p overexpression promoted the viability, migration, and invasion, reversed OTUD6B-AS1 effect in thyroid carcinomas, which supports the function of miR-183-5p as an oncogene in thyroid carcinomas. The result also showed that OTUD6B-AS1 inhibits the viability, migration, and invasion of thyroid carcinomas by targeting oncogenic miR-183-5p. OTUD6B-AS1 and miR-183-5p, constitute another regulatory axis of thyroid carcinomas. Additionally, miR-21 and miR-183-5p overexpression had stacked effect to reverse the effect of OTUD6B-AS1 on the viability, migration, and invasion abilities. The result suggested that OTUD6B-AS1-miR-183-5p axis and OTUD6B-AS1-miR-21 axis had coordinated with each other to regulate the viability, migration, and invasion of thyroid carcinomas. We revealed a novel mechanism underlying the inhibitory effect of OTUD6B-AS1 on tumor cells. Furthermore, OTUD6B-AS1-miR-21/miR-183-5p regulated downstream signaling pathway include MAPK signaling pathway, Regulation of actin cytoskeleton, Wnt signaling pathway, TGF-beta signaling pathway, Adherens junction, Chemokine signaling pathway, and Jak-STAT signaling pathway. Actin cytoskeleton and cell adhere, are closely related to epithelial to mesenchymal transition (EMT) and cell migration (39,40). Chemokine can recruit macrophage and induced macrophage M2 polarization which promoted tumor lymphatic metastasis (41). MAPK and TGF-beta signaling pathway has been known to mediate cell EMT and migration in thyroid carcinomas (42,43). Silenced Jak-STAT and Wnt/βcatenin signaling pathway reduced thyroid carcinomas cell viability, migration, and invasion (44,45). Previous study found that OTUD6B-AS1 silenced Wnt/β-catenin pathway in ccRCC (11). These results suggested that miR-21/miR-183-5p regulated downstream signaling pathway involve the regulation of thyroid carcinomas cell viability, migration, and invasion. However, the current study did not reveal the targets of miR-21 and miR-183-5p and the downstream signaling pathway.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

ETHICS STATEMENT
This study obtained approval from the Clinical Research Ethics Committee of Xiangya Hospital, Central South University. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
ZW and XL designed the study. ZW, FX, and TF collected the data. ZW, BJ, WW, and XL analyzed the data. ZW wrote the manuscript. WW and XL revised the manuscript. All authors read and approved the final manuscript.