Long noncoding RNA LINC00460 promotes carcinogenesis via sponging miR‐613 in papillary thyroid carcinoma

Long intergenic noncoding RNA 460 (LINC00460) has been identified as a critical regulator for multiple types of cancers. However, the biological role and underlying mechanism in human papillary thyroid carcinoma (PTC) still remain unclear and need to be uncovered. This study was aimed to ascertain the biological role and molecular mechanism of LINC00460 in PTC progression. Our findings revealed that the level of LINC00460 was significantly upregulated in PTC tissues and cell lines, which was positively correlated with advanced tumor–node–metastasis (TNM) stage and lymph node metastasis. Cellular experiments exhibited that knockdown of LINC00460 decreased proliferative, migratory, and invasive abilities of PTC cells. Mechanism assays noted that knockdown of LINC00460 suppressed cell proliferation, migration, and invasion, and inhibited expression of sphingosine kinase 2 (SphK2, a target of miR‐613) in PTC cells, at least in part, by regulating miR‐613. These findings suggested that LINC00460 could function as a competing endogenous RNA to regulate SphK2 expression by sponging miR‐613 in PTC. Targeting LINC00460 could be a promising therapeutic strategy for patients with PTC.

LINC00460, a lncRNA from chromosome 13, has been shown to be upregulated and function as an oncogene in several types of cancers, including non-small-cell lung cancer , esophageal squamous cell carcinoma (Liang et al., 2017), nasopharyngeal carcinoma (Kong et al., 2018), head and neck squamous cell carcinoma (Cao et al., 2017). It has been shown that LINC00460 expression levels were upregulated in PTC tissues, and correlated with the pathologic stage of PTC (Zhao et al., 2018). However, the specific biological role of LINC00460 in PTC has yet to be elucidated.
The aim of this study was to explore the effect of LINC00460 on the progression of PTC. Our data suggested that the expression of LINC00460 was increased in PTC tissues and cell lines. Knockdown of LINC00460 inhibited PTC cell proliferation, migration, and invasion.
Mechanistically, we showed that LINC00460 inhibited cell proliferation, migration, and invasion in PTC by regulating miR-613/sphingosine kinase 2 (SphK2) axis. These results revealed functions of LINC00460/miR-613/ SphK2 axis in PTC, which might provide a new insight for the treatment of PTC.  Table 1.

| Patient and tissue specimens
All the patients were staged using the eigth edition, TNM classification of American Joint Committee on Cancer. All tissue samples were immediately frozen in liquid nitrogen after resection and sorted at −80°C until use. This study was conducted in strictly accordance with the guidelines and principles of the Declaration of Helsinki, and approved by the Ethical Committee of China-Japan Union Hospital of Jilin University. Written informed consent was obtained from all patients.
2.4 | RNA extraction and quantitative reversetranscription polymerase chain reaction (qRT-PCR)   (Table 1 and Figure 1b,c). However, no correlation was observed between LINC00460 expression and patient's age, gender, and tumor size (Table 1). We also examined the LINC00460 expression in PTC cell lines by qRT-PCR. The data revealed that LINC00460 was remarkably upregulated in three PTC cell lines (TPC-1, BCPAP, and IHH-4) compared with the normal thyroid epithelial cell line Nthy-ori 3-1 ( Figure 1d).

| MiR-613 was a direct target of LINC00460 in PTC cells
It has been shown that lncRNA could serve as competing endogenous RNAs (ceRNA) to exert its regulatory functions (Bayoumi et al., 2016). To further determine the underlying mechanism by which LINC00460 exerted oncogene role in PTC progression, we adopted a predication software miRcode to predict miRNAs that interacts with LINC00460. We found that miR-613 was a potential target of LINC00460 based on a potential binding site in LINC00460 with miR-613 (Figure 3a).
To validate it, dual luciferase reporter assays were conducted.
Results revealed that transfection with miR-613 mimic significantly inhibited the luciferase activity of Wt-LINC00460 in both TPC-1 and BCPAP cells, whereas miR-613 inhibitor increased the activity (both p < 0.05; Figure 3b,c). When the binding site was mutated, miR-613 mimics or inhibitors cannot regulate the luciferase activity, which implied that miR-613 directly bond to

| DISCUSSION
Dysregulation of lncRNAs has been proven to be involved in tumorigenesis and progression of PTC, suggesting the potential of lncRNAs to serve as novel target for PTC diagnosis and therapy (Jing et al., 2018;Murugan et al., 2018). In the present study, we LINC00460, located at chromosome 13q33.2, has been reported to be closely associated with progression in several types of cancers.
For instance, LINC00460 promoted nasopharyngeal carcinoma tumorigenesis through regulating miR-149-5p/IL6 signal pathway (Kong et al., 2018). LINC00460 promoted cell migration and invasion, and induces epithelial-mesenchymal transition in non-small-cell lung cancer by regulating hnRNPK . LINC00460 depletion suppressed esophageal squamous cell carcinoma cell growth through regulating cell proliferation and cell cycle and apoptosis (Liang et al., 2017). Although LINC00460 expression was reported to be upregualted and positively correlated with the pathologic stage of PTC (Zhao et al., 2018), the detail function and underlying mechanism of LINC00460 remain unclear. Here, we first examined the expression level of LINC00460 in PTC tissues and cell lines through qRT-PCR analysis. The data showed that LINC00460 was upregulated in the PTC tissues and cell lines, and increased LINC00460 expression was closely associated with TNM stage and lymph node metastasis, which was consistent with previous result (Zhao et al., 2018). And then we identified the function of LINC00460 by applying reduced expression approaches. It was found that knockdown of LINC00460 significantly inhibited proliferation, migration and invasion in TPC-1 cells and BCPAP cells.
These results suggested that LINC00460 played oncogene role in PTC progression.
We hypothesized that LINC00460 might also serve as a ceRNA exerting its biological function in PTC. To explore the correlation between LINC00460 and miRNA in PTC pathogenesis, we made a predication by bioinformatics analysis and found that the miR-613 had a higher score binding to LINC00460. MiR-613 was proven to function as a tumor suppressor in multiple types of cancers (Li et al., 2016;Sang, Liu, & Sun, 2018). Importantly, it has been showed that miR-613 overexpression significantly inhibited PTC cell proliferation, migration and invasion (Qiu et al., 2016). By luciferase reporter assays, we validated that LINC00460 directly bond to miR-613. We also found that over- Taken together, the present study first demonstrated that the expression of LINC00460 was upregulated in PTC tissues and cell lines. And the elevation of LINC00460 was correlated with advanced TNM stage and lymph node metastasis. Our results also revealed that the oncogene LINC00460 promoted PTC progression via partly regulating miR-613/SphK2 axis. These findings suggested that the LINC00460/miR-613/SphK2 might act as a novel therapeutic target for the treatment of PTC.

CONFLICTS OF INTEREST
All authors declare that there are no conflicts of interest.