CircRNA casein kinase 1 gamma 1 (circ‐CSNK1G1) plays carcinogenic effects in thyroid cancer by acting as miR‐149‐5p sponge and relieving the suppression of miR‐149‐5p on mitogen‐activated protein kinase 1 (MAPK1)

Abstract Background The initiation and development of thyroid cancer may be associated with the deregulation of circular RNAs (circRNAs). The purpose of this work was to explore the role of circRNA casein kinase 1 gamma 1 (circ‐CSNK1G1) in thyroid cancer. Methods The expression of circ‐CSNK1G1, miR‐149‐5p, and mitogen‐activated protein kinase 1 (MAPK1) was concluded using quantitative real‐time PCR (qPCR), and the expression of MAPK1 protein was detected by Western blot assay. Cell viability was monitored by CCK‐8 assay. Cell proliferation was determined by colony formation assay and EdU assay. Cell apoptosis and cycle were checked by flow cytometry assay. Cell invasion was determined by transwell assay. The predicted binding relationship between miR‐149‐5p and circ‐CSNK1G1 or MAPK1 was verified by dual‐luciferase reporter assay. The role of circ‐CSNK1G1 in vivo was determined by establishing animal models. Results The present work discovered the upregulation of circ‐CSNK1G1 in tumor tissues of thyroid cancer. In function, circ‐CSNK1G1 knockdown inhibited proliferation, survival, and invasion in cancer cells, and tumor growth in mouse models. MiR‐149‐5p was a target of circ‐CSNK1G1, and the anti‐tumor effects of circ‐CSNK1G1 knockdown were abolished by miR‐149‐5p downregulation. In addition, miR‐149‐5p directly targeted MAPK1, and miR‐149‐5p restoration‐inhibited cell proliferation and invasion were recovered by MAPK1 overexpression. Conclusion Circ‐CSNK1G1 acted as miR‐149‐5p to relieve the inhibition of miR‐149‐5p on MAPK1, thus promoting the malignant development of thyroid cancer.

the usual standard of treatment, and many cases will relapse within 10 years, leading to death. 4 Thus, a comprehensive understanding of thyroid cancer pathogenesis is crucial.
Along with the boom of circRNA sequencing, the dysregulation of various circRNAs has been exposed in tumor tissues or cells.
Accumulating studies have published that circRNAs played wide functions in cancer cell growth, survival, and migration. 5 In thyroid cancer, the published literature showed that the increased expression of circRNA_102171, circZFR, and circNEK6 in thyroid cancer was related to promoting cell proliferation, invasion, and migration, [6][7][8] highlighting the vital role of circRNAs in the aggression of thyroid cancer. In view of the high stability, circRNAs are appropriate for clinical diagnosis and treatment as biomarkers in cancers. 9 However, research on the role of circRNAs in thyroid cancer is just the tip of an iceberg. There are still numerous circRNAs with unclear functions in thyroid cancer, such as circ_0001955. Circ_0001955 is produced from casein kinase 1 gamma 1 (CSNK1G1) by back-splicing, also terming as circ-CSNK1G1. It was shown to be highly regulated in papillary thyroid cancer tumor tissues compared with normal tissues through microarray analysis by the public GEO database (accession: GSE93522), hinting that circ-CSNK1G1 was involved in thyroid cancer development. Nonetheless, the detailed functions of circ-CSNK1G1 in thyroid cancer were still unclear.
It is widely demonstrated that circRNAs, as competing endogenous RNAs (ceRNAs), compete for microRNA (miRNA) binding sites to relieve the inhibition on downstream target genes. 10 Public bioinformatics database predicts that circ-CSNK1G1 harbors binding sites with miR-149-5p. MiR-149-5p was previously announced to be involved in medullary thyroid cancer as a tumor suppressor. 11 It was unknown whether circ-CSNK1G1 functioned by targeting miR-149-5p in thyroid cancer. In addition, public bioinformatics database also shows that miR-149-5p binds to the 3' untranslated region (3'UTR) of mitogen-activated protein kinase 1 (MAPK1), hinting the potential binding between MAPK1 and miR-149-5p. MAPK1 was a well-known oncogene in various cancers, including thyroid cancer. 12 It was unclear whether miR-149-5p played anti-tumor roles in thyroid cancer by depleting MAPK1.
Our current work was the first to investigate the detailed role of circ-CSNK1G1 in thyroid cancer. Mechanically, we clarified that circ-CSNK1G1 served as a ceRNA to compete with MAPK1 for miR-149-5p binding site. This work intended to determine the role of circ-CSNK1G1 in thyroid cancer and provide a mechanism to explain its function.

| Clinical samples
Patients with thyroid cancer were newly diagnosed and surgically treated at the Yichun People's Hospital. Clinical tissues were frozen after excision and stored at −80°C conditions. Patients who had received any therapies (chemotherapy, radiotherapy, or others) prior to surgery and patients diagnosed with other types of cancer and severe systemic infectious diseases were excluded from this study.
A total of 47 pairs of tumor tissues and matched normal tissues were used in this study and respectively confirmed by pathological diagnosis. The clinicopathological parameters of patients with thyroid cancer enrolled in this study were displayed in Table 1, such as age, gender, and tumor stage. The use and study of these samples were permitted by patients with written informed consent. This study was approved by the Ethics Committee of Yichun People's Hospital. with 10% FBS. Cells were maintained in a 37°C incubator supplemented with 5% CO 2 .

| Quantitative real-time PCR (qPCR)
TRIzol reagent (Invitrogen,) was applied for RNA isolation. Then, total RNA was used for cDNA assemble using the TaqMan

| RNase R treatment
SW579 and TPC-1 cells-derived total RNA was treated with RNase R (2 U/μg; Epicentre,) for 20 min at 37°C conditions. Total RNA was then examined as abovementioned.

| Colony formation assay
Cells in complete medium were plated into 6-well plates (200 cells/ well) and continuingly cultured in a 37°C incubator supplemented with 5% CO 2 for 2 weeks. Then, cell colonies were fixed using methanol and stained with 0.1% crystal violet. The images of cell colonies were captured under a light microscope (Leica,).

| EdU assay
Utilizing Cell-Light EdU Apollo567 Kit (Ribobio) according to the protocol, the number of EdU-positive cells was identified. In brief, cells were cultured EdU medium for 4 h, fixed by 4% paraformaldehyde, and next stained by Apollo567 and DAPI. Images were taken with the use of a fluorescence microscope (Leica).

| Flow cytometry assay
Cells were cultured for 48 h and then collected. After re-suspending in binding buffer (1 × 10 6 cells/ml), cells were next stained with Annexin V-FITC (Beyotime) and propidium iodide (PI; Beyotime). Cell apoptosis was ensured by a FACS Calibur flow cytometer (BD Biosciences,).
Also by flow cytometry analysis, cell cycle was checked using a Cell Cycle Analysis Kit (Beyotime). Cells were suspended with PBS and then fixed with 70% cooled ethanol. Next, cells were exposed to PI staining buffer containing RNase A, followed by detection under a flow cytometer.

| Western blot
Total protein was extracted using RIPA reagent (Beyotime). Western blot was implemented in accordance with the method in a previous study. 13 The antibodies, including anti-CyclinD1 (dilution: 1/100;

| Animal models
The procedures of animal study were approved by Yichun People's Hospital. A total of 12 nude mice (Balb/c, 6-week-old) were bought from Vital River Laboratory Animal and raised in standard conditions.
Lentivirus suspensions of short hairpin RNA targeting circ-CSNK1G1 (sh-circ-CSNK1G1) or sh-NC were provided by Geneseed. TPC-1 cells were infected with sh-circ-CSNK1G1 lentivirus suspensions for stable circ-CSNK1G1 knockdown. Then, nude mice were subcutaneously injected with the infected cells (2 × 10 6 cells per mouse) to induce tumor growth (n = 6 per group). During tumor growth, tumor volume (length × width 2 × 0.5) was measured every three days. Tumors were allowed to grow 22 days, and all mice were killed for tumor excision.

| Circ-CSNK1G1 was overexpressed in tumor tissues and cell lines of thyroid cancer
The data from GSE3522 dataset (a circRNA expression profile) showed that has_circRNA_101555 (circ-CSNK1G1) was one of the forcefully expressed circRNAs in thyroid cancer tissues compared with normal tissues ( Figure 1A and Figure 1B). Then, we found that circ-CSNK1G1 expression was strikingly higher in tumor tissues of thyroid cancer than that in matched normal tissues ( Figure 1C). Besides, circ-CSNK1G1 expression was also significantly heightened in IHH-4, NIM, SW579, and TPC-1 cells relative to Nthy-ori 3-1 cells ( Figure 1D). Moreover, we found that circ-CSNK1G1 was stable compared with its linear transcript because it was resistant to RNase R digestion ( Figure 1E and 1F). Compared with linear CSNK1G1, circ-CSNK1G1 could not be amplified by oligo(dT) 18 primers, suggesting that circCSNK1G1 had no 3' and 5' tails in structure ( Figure 1G and 1H). Figure 1 mainly showed that circ-CSNK1G1 was abnormally upregulated in thyroid cancer tissues and cells.
The data from CCK-8 assay presented that cell viability was notably decreased in SW579 and TPC-1 cells after circ-CSNK1G1 knockdown ( Figure 2B). The data showed that circ-CSNK1G1 knockdown impaired the number of colonies and reduced the number of EdU-positive cells ( Figure 2C and 2D). Flow cytometry assay found that circ-CSNK1G1 knockdown enhanced the number of apoptotic cells ( Figure 2E and 2F) and arrested cell cycle at the G0/G1 stage ( Figure 2G). Transwell assay was conducted to assess cell invasion, and the capacity of cell invasion was significantly decreased in SW579 and TPC-1 cells after circ-CSNK1G1 knockdown ( Figure 2H).

| Circ-CSNK1G1 knockdown inhibited thyroid cancer cell malignant development by enriching miR-149-5p
To validate whether circ-CSNK1G1 interacted with miR-149-5p to play effects in thyroid cancer, rescue experiments were performed.
Through colony formation assay and EdU assay, we observed that the ability of cell proliferation was suppressed by circ-CSNK1G1 knockdown but partially restored by miR-149-5p depletion ( Figure 4D and   4E). In addition, circ-CSNK1G1 knockdown-induced cancer cell apoptosis and cell cycle arrest were largely relieved by miR-149-5p inhibition ( Figure 4F-4H). Circ-CSNK1G1 knockdown-suppressed cell invasion was promoted by the inhibition of miR-149-5p ( Figure 4I).

| MiR-149-5p restoration-inhibited thyroid cancer cell malignant development by sequestering MAPK1
To validate whether miR-149-5p played functions by targeting MAPK1, rescue experiments were performed. MAPK1 protein level was strikingly strengthened in SW579 and TPC-1 cells transfected with MAPK1 compared with pcDNA ( Figure 6A). For rescue experiments, the expression of MAPK1 was notably decreased in SW579 and TPC-1 cells transfected with miR-149-5p but recovered in SW579 and TPC-1 cells transfected with miR-149-5p+MAPK1 ( Figure 6B). In function, cell viability was markedly suppressed in SW579 and TPC-1 cells with miR-149-5p transfection but recovered in cells with miR-149-5p+MAPK1 transfection ( Figure 6C). The ability of cell proliferation was notably impaired by miR-149-5p restoration but restored by the reintroduction of MAPK1 in SW549 and TPC-1 cells ( Figure 6D and 6E). In addition, miR-149-5p restoration-induced SW549 and TPC-1 cell apoptosis and cell cycle arrest (at G0/G1 stage) were substantially relieved by the reintroduction of MAPK1 ( Figure 6F-6H). Moreover, the capacity of cell invasion in SW579 and TPC-1 cells was suppressed by miR-149-5p enrichment but partially promoted by MAPK1 overexpression ( Figure 6I). The expression levels of CyclinD1 and MMP9 were significantly lessened in SW579 and TPC-1 cells with miR-149-5p transfection but largely enhanced in cells with miR-149-5p+MAPK1 cotransfection ( Figure 6J and 6K). These data indicated that miR-149-5p restoration inhibited thyroid cancer cell malignant development by sequestering MAPK1.

| The downregulation of circ-CSNK1G1 inhibited tumor growth in animal models
We further confirmed the role of circ-CSNK1G1 in vivo. Animal models were established in nude mice by subcutaneously injecting with TPC-1 cells infected with sh-circ-CSNK1G1, using sh-NC as a control.
We found that tumor volume in the sh-circ-CSNK1G1 group was significantly lower than that in the sh-NC group at the day 19 postinjection ( Figure 8A). Besides, circ-CSNK1G1 knockdown inhibited tumor weight, leading to smaller tumor size ( Figure 8B). Moreover, the data from qPCR showed that the expression of circ-CSNK1G1 was decreased, while the expression of miR-149-5p was enhanced in sh-circ-CSNK1G1-administered tumor tissues ( Figure 8C). The expression of MAPK1 protein was pronouncedly declined in sh-circ-CSNK1G1-administered tumor tissues ( Figure 8D). Moreover, IHC analysis presented that the abundance of MAPK1, CyclinD1, and MMP9 was markedly decreased in tumor tissues from the sh-circ-CSNK1G1 group ( Figure 8E). All data suggested that circ-CSNK1G1 knockdown inhibited tumor growth and development through miR-149-5p-mediated MAPK1 inhibition.

| DISCUSS ION
Our current data mainly found that circ-CSNK1G1 was overexpressed in tumor tissues of thyroid cancer relative to normal tissues.
The downregulation of circ-CSNK1G1 inhibited cancer cell colony formation, proliferation, survival, and invasion, which supported that circ-CSNK1G1 was a carcinogenic driver at least in thyroid cancer. Moreover, mechanism analysis suggested that circ-CSNK1G1 played carcinogenic effects in this cancer by acting as miR-149-5p sponge and relieving the inhibition of miR-149-5p on MAPK1. These F I G U R E 7 Circ-CSNK1G1 knockdown inhibited the expression of MAPK1 by enriching miR-149-5p. (A and B) The expression of MAPK1 mRNA and protein in SW579 and TPC-1 cells transfected with si-circ-CSNK1G1 or si-circ-CSNK1G1+anti-miR-149-5p was detected by qPCR and Western blot. **p < 0.01, ***p < 0.001 and ****p < 0.0001 present findings deepened the insights into the realizing of the role of circ-CSNK1G1 in thyroid cancer.
By reviewing the previous studies, we discovered that circ-CSNK1G1 served as an oncogenic role to facilitate the malignant progression of diverse cancers. [14][15][16] For example, circ-CSNK1G1 level was increased in hepatocellular carcinoma (HCC) tumor tissues, and circ-CSNK1G1 knockdown repressed tumor growth in animal model. 14 In vitro assays showed that circ-CSNK1G1 efficiently promoted HCC cell proliferation, invasion, and migration. 16 The expression of circ-CSNK1G1 was also aberrantly strengthened in colorectal cancer tissues. 15 The evidence supported that cancer aggressive development might be associated with increased circ-CSNK1G1 expression, whereas the role of circ-CSNK1G1 in thyroid cancer was poorly investigated. Only a circRNA microarray profile presented that circ-CSNK1G1 was one of the circRNAs that were significantly upregulated in papillary thyroid cancer tissues. 17 We thus explored the detailed role of circ-CSNK1G1 in thyroid cancer and found that circ-CSNK1G1 downregulation suppressed on certain miRNAs, such as miR-516a-5p and miR-145-5p. 14,16 Following this idea, we investigated the potential miRNAs targeted by circ-CSNK1G1 to explore the functional mechanism of circ-CSNK1G1.
It is well known that miRNAs mediate translational inhibition of target genes in post-transcriptional gene expression. 22 Our study discovered that miR-149-5p targeted MAPK1 3'UTR, suggesting that MAPK1 was a target of miR-149-5p. Previous studies exhibited that MAPK1 overexpression promoted cell proliferation, glycolysis, and motility in papillary thyroid cancer. 12,23,24 Our study found that the inhibitory proliferation, F I G U R E 8 Circ-CSNK1G1 knockdown inhibited tumor growth in animal models.
(A and B) Tumor volume and tumor weight were measured to assess tumor growth.
(C) The expression of circ-CSNK1G1 and miR-149-5p in the excised tumor tissues from animal models was measured using qPCR. (D) The expression of MAPK1 protein in the excised tumor tissues from animal models was measured using Western blot. (E) The abundance of MAPK1, CyclinD1, and MMP9 in tumor tissues was investigated by IHC assay. **p < 0.01, ***p < 0.001 and ****p < 0.0001 survival, and invasion of cancer cells caused by miR-149-5p restoration were largely restored by MAPK1 overexpression, indicating that miR-149-5p inhibited thyroid cancer progression by depleting MAPK1. The carcinogenic effects of MAPK1 were widely proposed in diverse cancers, such as gastric cancer and breast cancer. 25,26 These studies revealed that MAPK1 was an oncogene in cancers, and miR-149-5p bound to MAPK1 3'UTR, and thus inhibited MAPK1 expression. However, circ-CSNK1G1 acted as a ceRNA to compete for miR-149-5p with MAPK1, thereby relieving the suppression of miR-149-5p on MAPK1.
Taken together, this study was the first study to exploit the de-

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.