Hsa_circ_0012563 promotes migration and invasion of esophageal squamous cell carcinoma by regulating XRCC1/EMT pathway

Abstract Background Recent reports have indicated that circular RNA (circRNA) may regulate tumorigenesis development. However, the function of circRNAs in esophageal squamous cell carcinoma (ESCC) is unclear. Material and method The RT‐qPCR assay was performed to detect hsa_circ_0012563 expression in ESCC tissues and cell lines. Then, the MTT assay, colony formation assay, flow cytometric assay, and cell migration and invasion assay were performed to examine the function of hsa_circ_0012563. In addition, the RT‐PCR and Western blot were used to detect XRCC1 and epithelial‐to‐mesenchymal transition (EMT) related gene expression. Results The RT‐qPCR revealed that the hsa_circ_0012563 expression was remarkably upregulated in ESCC tissue and ESCC cell lines. Functionally, downregulation of hsa_circ_0012563 suppressed cell proliferation, migration, and invasion and promoted cell apoptosis. Mechanically, the knockdown of hsa_circ_0012563 inhibited XRCC1‐mediated EMT pathway to suppress cell migration and invasion. Conclusions Therefore, these results reveal hsa_circ_0012563 is a critical oncogene and may be a novel biomarker in ESCC.

cancer and inhibits the proliferation and invasion of tumor cells by targeting miR-532-5p. 14 Hsa_circ_0004370 promotes esophageal cancer progression through miR-1294/LASP1 pathway. 15 X-ray repair cross-complementing group 1 (XRCC1), one of the DNA repair genes encoding a scaffolding protein, participates in base excision repair (BER) pathway. 16 Early study indicated XRCC1 was associated with cell migration and invasion. For example, upregulation of XRCC1 suppressed tumor migration and invasion in glioma. 17 Nevertheless, the correlation of XRCC1 and epithelial-mesenchymal transition (EMT) in ESCC is unclear.
Recently, research has been showed ZYG11A is a potential oncogene that promotes NSCLC cell proliferation and migration in vitro and in vivo. 18 The hsa_circ_0012563 is located at chr1:53308182-53333449, and its associated-gene symbol is ZYG11A. This data were from circbase (http://www.circb ase.org/cgi-bin/simpl esear ch.cgi). Thus, hsa_circ_0012563 was chosen for this study. Notably, biological function and mechanism of hsa_circ_0012563 in tumor are unclear. Therefore, in this study, we detected expression of hsa_circ_0012563 in ESCC and examined its biological function and mechanism in vitro.

| Patients and tissue samples
All 60 pairs of ESCC tissues and adjacent normal tissues were obtained at the Department of Thoracic Surgery, Tongren Hospital of Wuhan University (Wuhan Third Hospital), between 2016 and 2018.
We confirmed that all methods accord to HIPAA guidelines and approved institutional protocols and written informed consent were obtained from all patients. The research was approved by the Ethics Committee of Department of Thoracic Surgery, Tongren Hospital of Wuhan University (Wuhan Third Hospital).

| Cell proliferation assay
Cell proliferation was examined by using the MTT assay (MTT; SigmaAldrich) following the manufacturer's instructions (Biosharp).
The cells were seeded in 96-well plates (5000 cells/well). Cell proliferation was examined after 24 hours, 48 hours, 72 hours, and 96 hours.
The solution was then measured spectrophotometrically at 490 nm.

| Cell cycle and apoptosis assay by flow cytometric
The cell cycle was detected by labeling cells with PI for 15 minutes, and the cells were then detected by using a FACScan analyzer. The transfected cells were harvested after 48 hours and were then stained with Annexin V and PI (BD Pharmingen) before analysis by flow cytometry (C6; BD Biosciences).

| Clonogenic assay
The 1000 cells were plated in 6-well plate. After 7 days, the cells were immobilized with 4% paraformaldehyde and stained with crystal violet. The visible colonies were then manually counted with ImageJ.

| Real-time quantitative PCR
Total RNA was extracted by using TRIzol reagent (Invitrogen).
For RT-qPCR analysis, cDNA was reverse transcribed by Reverse Transcription Kit (Toyobo). RT-qPCR was performed with SYBR Green kit (Qiagen). All sample were run by the ABI Prism 7500 Sequence Detector (Applied Biosystems). The mRNA expression level was calculated and normalized by using the 2 -ΔΔCt method relative to GAPDH. The primers for hsa_circ_0012563, E-cadherin, N-cadherin, Vimentin, snail, and GAPDH were showed in Table 1.

| Western blot
Total protein was collected by using RIPA lysis buffer (Beyotime), and protein concentrations were examined with the bicinchoninic acid method (BCA, Beyotime). The proteins were used for 12% SDS-polyacrylamide gels and transferring onto PVDF membranes.
Primary antibodies against XRCC1, E-cadherin, N-cadherin, and GAPDH were obtained from Abcam. All the secondary antibodies were purchased from Cell signaling technology.

| Statistical analysis
Differences between groups were assessed by using paired sample t test, and all data are represented by the mean ± SD. A P-value of <.05 (*) was considered statistically significant; A P-value of <.01(**) was considered statistically very significant; A P-value of <.001(***) was considered statistically very much significant.

| Hsa_circ_0012563 is upregulated in both clinical specimens and ESCC cell lines
The expression level of hsa_circ_0012563 in ECSS tissue and normal tissue was initially evaluated by RT-qPCR. The result showed that the expression level of hsa_circ_0012563 was significantly increased in ESCC compared with normal tissues ( Figure 1A). Higher hsa_circ_0012563 expression in ESCC patients was related to lower survival rate by Kaplan-Meier survival analysis ( Figure 1B). It yet showed hsa_circ_0012563 expression was significantly increased in ECSS cell lines compared with HEEC cells ( Figure 1C). Therefore, these results demonstrate hsa_circ_0012563 is consistently expressed in both ESCC tumor tissues and ESCC cell.

| Hsa_circ_0012563 exerts a tumor-promotive function in ESCC
To explore hsa_circ_0012563 exerts a tumor-promotive function in ESCC, we designed siRNA to suppress hsa_circ_0012563 expression. RT-qPCR showed hsa_circ_0012563 expression was inhib-

| Downregulated of hsa_circ_0012563 inhibited TE-1 cell migration and invasion in vitro
As shown in Table 2, hsa_circ_0012563 expression was associated with metastasis and suggested that hsa_circ_0012563 may promote ESCC metastasis. In addition, the migration assays  Figure 3A). Invasion assay also showed that hsa_circ_0012563 knockdown remarkably inhibited the invasive capacity of TE-1 cells ( Figure 3B). Therefore, overexpression of hsa_ circ_0012563 promotes ESCC cell migration and invasion in vitro.

| Hsa_circ_0012563 promoted cell migration and invasion by regulating XRCC1/ EMT pathway
Our data have revealed that hsa_circ_0012563 was related to migration and invasion of ESCC, and we further explored metastasis mechanism. Then, early reports pointed out that XRCC1 was associated with cell migration and invasion. 17 Thus, we evaluated expres-

| D ISCUSS I ON
Esophageal cancer is a complex, dynamic, and biological processes, and multiple genes are related to ESCC. 19 In addition, metastatic relapse is one of the main causes of poor prognosis of ESCC, including cell adhesion, migration, and reaching target organs, and the lncRNAs and miRNAs are also related to F I G U R E 2 Hsa_circ_0012563 exerts a tumor-promotive function in ESCC. (A) Hsa_circ_0012563 expression was suppressed by transfected siRNAs in ESCC cells. The effect of hsa_circ_0012563 on cell proliferation was detected by using MTT (B) and colony formation (C) assays. Flow cytometry was performed to detect the cell apoptosis (D) and cell cycle phase (E) in TE-1 cell-si-hsa_circ_0012563. All the data shown represent the mean ± SD (n = 3). *P < .05, **P < .01, ***P < .001. All siRNA was si-hsa_circ_0012563 In summary, hsa_circ_0012563 is upregulated in ESCC tumor tissues and cells, and its expression is associated to tumor pathogenesis and metastatic. Further, hsa_circ_0012563 promotes migration and F I G U R E 3 Downregulated of hsa_circ_0012563 inhibited TE-1 cell migration and invasion in vitro. Knockdown of hsa_circ_0012563 inhibited cell migration (A) and invasion (B) in TE-1 cells. All data shown represent the mean ± SD (n = 3). *P < .05, **P < .01, ***P < .001. All siRNA was si-hsa_circ_0012563 invasion of ESCC via regulating XRCC1/EMT pathway. Therefore, this suggests hsa_circ_0012563 is an oncogene in ESCC and may represent a novel diagnostic and therapeutic target for treatment of ESCC.

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