Circ_0027446 induces CLDN1 expression to promote papillary thyroid cancer cell malignancy by binding to miR-129–5p

Abstract

Background

Previous data have shown that circular RNA (circRNA) is a key regulator in papillary thyroid cancer (PTC). However, the role and the detailed mechanism of circ_0027446 in PTC progression have not been reported.

Methods

Circ_0027446, miR-129–5p, claudin 1 (CLDN1), C-myc and MMP2 expression were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), Western Blot or immunohistochemistry (IHC) assay. Cell viability was evaluated by cell counting kit-8 (CCK-8) assay. Cell proliferation was investigated by 5-Ethynyl-2′-deoxyuridine (EdU) assay and cell colony formation assay. Cell apoptosis, invasion and migration were assessed by flow cytometry analysis, transwell assay and wound-healing assay, respectively. Dual-luciferase reporter assay was conducted to identify the associations among circ_0027446, miR-129–5p and CLDN1. The effect of circ_0027446 on PTC cell malignancy in vivo was revealed by a xenograft mouse model assay.

Results

Circ_0027446 and CLDN1 expression were significantly upregulated, while miR-129–5p expression was downregulated in PTC tissues and cells. High circ_0027446 expression was closely associated with the poor prognosis of PTC patients. Circ_0027446 depletion inhibited PTC cell proliferation, migration and invasion but increased cell apoptosis. In addition, circ_0027446 acted as a miR-129–5p sponge, and miR-129–5p bound to CLDN1. Moreover, miR-129–5p inhibitors attenuated circ_0027446 depletion-induced effects in PTC cells. CLDN1 also participated in the regulation of miR-129–5p in PTC cell tumor properties. Importantly, circ_0027446 mediated CLDN1 expression by interacting with miR-129–5p. In vivo data showed that the decreased expression of circ_0027446 led to delayed tumor formation.

Conclusion

Circ_0027446 contributed to PTC cell tumor properties by regulating the miR-129–5p/CLDN1 pathway, showing circ_0027446 might be a therapeutic target in PTC.

Introduction

Thyroid cancer (TC) is a prevalent tumor in countries with an iodine-excess diet [1]. Papillary TC (PTC) is an aggressive malignancy in the endocrine system and accounts for more than 80% of total thyroid malignancies [2]. PTC occurs at any age and its incidence increases with age [3]. The risk factors of this cancer include follicular PTC, stage 4 tumors, thyroid nodule involvement and older age [4]. Despite the fact that PTC cases have low rates of local invasion and recurrences, a small group of clinical specimens still exhibit more aggressive variants and pathological features [5]. Thus, an in-depth investigation on mechanisms related to PTC progression is necessary to improve prognosis of PTC patients.

Circular RNA (circRNA) is an endogenous RNA formed by back-splicing and has a higher tolerance exonuclease, featured by a circular structure [6]. CircRNA is distributed in mammals and plays key parts in cell biological processes [7]. CircRNA can combine with microRNAs (miRNAs) to regulate their expression and actions, further functioning in various diseases, such as cardiovascular disease, diabetes mellitus, and cancers [8]. Previous data have shown that circ_102171 contributes to PTC cell proliferation and motility through β-catenin pathway [9]. Circ_102002 accelerated PTC metastasis due to the downregulation of miR-488–3p [10]. Another reference revealed that circ_0002111 was increased in PTC tissues when compared with non-cancerous tissue samples and that its expression had a close association with lymph-node metastasis of PTC cases [11]. Circ_0027446 is located on chr12:66221780–66232349 + and is formed by back-splicing of high mobility group AT-hook2 (HMGA2), which can bind to DNA binding proteins and contributes to PTC cell migration and invasion [12], [13]. Nevertheless, the functions of circ_0027446 in PTC are unknown.

MiRNA is a noncoding RNA of ∼22 nucleotides long and plays essential regulatory roles in cancer development through interaction with mRNA 3′UTR [14]. Increasing evidence indicated that miRNAs were dysregulated in PTC cells and mediated thyroid cancer cell tumor properties [15]. In thyroid cancer, miR-129–5p was associated with histone deacetylase inhibitor-induced antitumor activity [16]. Moreover, miR-129–5p was decreased and inhibited thyroid cancer cell growth and migratory ability through ret proto-oncogene (RET) [17]. Our preliminary experiments predicted that circ_0027446 bound to miR-129–5p, but the relationship between them has not been analyzed in cancer progression.

In the study, we analyzed the function of circ_0027446 in PTC cell tumor properties and determined whether circ_0027446-induced effects involved miR-129–5p. Given that miRNA could be a bridge molecular between circRNA and mRNA [18], we further searched for mRNA associated with circ_0027446 and miR-129–5p in PTC development.