Circ-DONSON Knockdown Inhibits Cell Proliferation and Radioresistance of Breast Cancer Cells via Regulating SOX4

Background Circular RNAs have been validated as critical regulators in the development of breast cancer (BC). Circ-DONSON is involved in the progression of glioma and gastric cancer. However, the biological role of circ-DONSON in BC remains unclear, and the aim of this study was to explore the biological role of circ-DONSON in BC. Methods Human tissue samples and BC cell lines were collected in this study. siRNAs against circ-DONSON were transfected into BC cell lines for silencing of circ-DONSON. Quantitative real-time PCR was used to test the circ-DONSON expression. Cell counting kit-8 (CCK-8), 5-bromo-2′ deoxyuridine enzyme-linked immunosorbent assay (BrdU-ELISA), colony formation, and caspase-3 activity assays were used to assess cell proliferation, cell survival, and cell viability. Western blotting analysis was used to detect the protein expression levels. Results Our findings showed that circ-DONSON showed high expression in BC tissues and cell lines. CCK-8 and BrdU-ELISA assays showed that circ-DONSON knockdown inhibited BC cell proliferation. Moreover, cell survival, cell viability, and caspase-3 activity assays showed that circ-DONSON knockdown reduced the radioresistance of BC cells. Mechanistically, circ-DONSON regulated BC cell proliferation and radioresistance via SRY-box transcription factor 4 (SOX4). SOX4 overexpression significantly rescued the effect of circ-DONSON knockdown on BC cell proliferation and radioresistance. Moreover, circ-DONSON activated the Wnt/β-catenin pathway in BC cells via SOX4. Conclusion Our study concluded that circ-DONSON knockdown hindered cell proliferation and radioresistance through the SOX4/Wnt/β-catenin pathway in BC.


Introduction
Breast cancer (BC) is one of the most common cancers in women worldwide [1,2]. Although radiotherapy is a main treatment for BC patients, radioresistance leads to limited therapeutic efficacy [3,4]. erefore, it is urgently required to explore the molecular mechanisms of BC progression and radioresistance.
As a recently discovered member of the noncoding RNA family, circular RNAs (circRNAs) are characterized by a covalently closed continuous loop structure [5]. It has been reported that circRNAs are highly expressed in multiple tumors, such as colorectal cancer, lung cancer, gastric cancer, and BC [6,7]. For example, circRNA_102171 silencing suppresses cell proliferation, migration, and invasion while promoting apoptosis of papillary thyroid cancer cells through modulating CTNNBIP1-dependent activation of the β-catenin pathway [8]. Hsa_circRNA_103809 regulates the colorectal cancer cell proliferation and migration via the miR-532-3p/FOXO4 axis [9]. In BC, circTADA2As suppresses cell proliferation, migration, and invasion via targeting the miR-203a-3p/SOCS3 axis [10]. However, more circRNAs involved in BC progression and therapy are needed to be explored.
Circ-DONSON has been reported to be highly expressed in glioma tissues and promote cell proliferation and migration through modulating FOXO3 [11]. Circ-DONSON is upregulated in gastric cancer tissues and positively correlated with poor prognosis of this cancer [12]. Circ-DONSON promotes gastric cancer cell growth and invasion via NURF complex dependent activation of SRY-box transcription factor 4 (SOX4) [12]. Moreover, linear DONSON is predicted by bioinformatics analysis to be upregulated in breast cancer. However, the biological function of circ-DONSON in BC remains unclear.
Circ-DONSON and linear DONSON are derived from the same pre-mRNA. We hypothesized that circ-DONSON is also upregulated in BC and serves as an oncogene. In this study, we found that circ-DONSON showed high expression in BC tissues and cell lines. Circ-DONSON knockdown inhibited cell proliferation and reduced the radioresistance of BC cells. Mechanistically, circ-DONSON-mediated biological functions in BC were dependent on SOX4. Moreover, circ-DONSON modulated the Wnt/β-catenin pathway via SOX4 in BC. In summary, circ-DONSON contributed to cell proliferation and radioresistance through the SOX4/Wnt/β-catenin pathway in BC and might be a potential therapeutic target for this disease.

Tissue Samples.
irty paired human BC tissues and corresponding noncancerous tissues which are 5 cm away from tumor tissues were collected from patients who underwent surgical resection at the First People's Hospital of Lianyungang. e experiments were approved by the Ethics Committee of the First People's Hospital of Lianyungang. Written consent for this research purpose was obtained from each patient.

Cell Survival Assay.
Cell survival was determined with a colony formation assay as described previously [14]. In briefly, equal numbers of cells were seeded into 6-well plates and then exposed to 0, 2, 4, 6, or 8 Gy X-ray irradiation. After 15 days, the cell colonies were stained with crystal violet. e number of colonies with cells >50 was counted. e survival fraction was calculated as the surviving colony fraction (colonies counted/total cells seeded) of the treatment plates divided by that of the control group. e cell survival curves were then fitted using a single hit multitarget model.  Assay. CCK-8 (Dojindo, Kumamoto, Japan) was used to examine cell proliferation. Cells (5 × 10 3 ) were plated into 96-well plates and stained with 10 μL of sterile CCK8 solution at 2, 24, 48, and 72 h. e optical density (OD) was measured at a wavelength of 450 nm.

Cell Counting
CCK-8 was also used to test the sensitivity of cells to radiotherapy. Cells were plated into 96-well plates and exposed to 4 Gy X-ray irradiation. After 24 h, CCK8 solution was added to each well, and the optical density (OD) was measured at a wavelength of 450 nm.

Caspase-3 Activity Assay.
e caspase-3 activity of cells was assayed using the kit (Beyotime, Shanghai, China) according to the manufacturer's instructions.

Cell Cycle Analysis.
A cell cycle detection kit (Keygen, Nanjing, Jiangsu, China) was used for cell cycle analysis according to the manufacturer's instructions. Cells were then analyzed using the ACEA NovoCyte system (ACEA Biosciences, Santa Clara, CA, USA).

Statistical Analysis.
e data are presented as the mean ± standard deviation (SD). Student's t-test was used for statistical analysis. P value < 0.05 was considered statistically significant.

Circ-DONSON Was Highly Expressed in BC Tissues and
Cell Lines. According to ENCORI database (https://starbase. sysu.edu.cn/), DONSON shows significant high expression in BC tissues compared to that in control tissues (Figure 1(a)). As shown in Figure 1 A previous report showed that circ-DONSON regulates SOX4 expression and facilitates gastric cancer cell growth and invasion [12]. Hence, we hypothesized that circ-DONSON promotes cell proliferation and radioresistance through the regulation of SOX4 in BC. As shown in Figure 2(g), results of western blot analysis showed that circ-DONSON siRNA treatment significantly reduced the protein expression of SOX4 in MCF-7 and MDA-MB-231 cells.
Circ-DONSON knockdown inhibited BC cell proliferation and reduced the radioresistance of BC cells via SOX4.
We used the pcDNA3.1-SOX4 vector to explore the rescue effect of SOX4 on circ-DONSON in BC cells. As shown in Figure 3 (Figure 3(e)). Moreover, silencing of circ-DONSON increased the percentage of cells in the G1 phase and decreased that in the S phase. Such effect was rescued by SOX4 ( Supplementary Figure 2(a)). Supplementary Figure 2(b) revealed that circ-DONSON knockdown reduced cell cycle proteins including cyclin B1, cyclin E1, and CDK2. SOX4 rescued the suppressive effects of circ-DONSON knockdown on these proteins.
e Wnt/β-catenin pathway has been reported to be involved in cell proliferation and radioresistance of BC cells [16,17]. erefore, we investigated whether circ-DONSON activates the Wnt/β-catenin pathway in BC cells via SOX4. As shown in Figures 4(a) and 4(b), circ-DONSON knockdown significantly reduced the protein expression of Wnt1  (Figures 4(a) and 4(b)).

Discussion
Several circRNAs have been reported to regulate BC progression. Tang [21]. In this study, we found that circ-DONSON was highly expressed in BC tissues and cell lines. Moreover, circ-DONSON knockdown inhibited BC cell proliferation. ese results suggest that circ-DONSON plays an oncogenic role in BC progression.
Previous studies also showed that circRNAs are involved in the radioresistance of cancers [22]. Circular RNA PRKCI knockdown inhibits esophageal cancer progression and elevates cell radiosensitivity through regulating the miR-186-5p/PARP9 axis [23]. Guan et al. showed that circPITX1 silencing represses glycolysis to enhance the radiosensitivity of glioma cells through the miR-329-3p/NEK2 axis [24]. e knockdown of circRNA_000543 sensitizes nasopharyngeal carcinoma to irradiation by targeting the miR-9/plateletderived growth factor receptor B axis [25]. Liu et al. reported that circRNA_100367 attenuates radioresistance of esophageal squamous cell carcinomas cells through the miR-217/ Wnt3 pathway [26]. In this study, we found that circ-DONSON knockdown reduced the clonogenic survival of BC cells and inhibited the cell viability of BC cells after irradiation. Moreover, circ-DONSON knockdown increased apoptosis of BC cells after radiation exposure. ese results suggest that circ-DONSON knockdown reduces the radioresistance of BC cells. As a member of the sex-determining region Y-related HMG box (SOX) transcription factor family, SOX4 is abnormally expressed in multiple cancers and exerts an oncogenic function [27]. e mRNA and protein levels of SOX4 are highly expressed in BC tissues compared with adjacent normal mammary tissues and positively correlated with the clinical stage [28]. Overexpression of SOX4 promotes epithelial-mesenchymal transition and stem cell characteristics of gastric cancer cells [29]. Koumangoye et al. showed that SOX4 promotes cell proliferation and invasion by silencing miR-31 via activation and stabilization of a corepressor complex with EZH2 and HDAC3 in esophageal  cancer [30]. Ding et al. showed that circ-DONSON could regulate SOX4 expression and facilitate gastric cancer growth and invasion [12]. erefore, we hypothesized that circ-DONSON promoted cell proliferation and radioresistance through regulating SOX4 in BC. Circ-DONSON knockdown significantly reduced the protein expression of SOX4 in MCF-7 and MDA-MB-231 cells. Moreover, SOX4 overexpression significantly rescued the effect of circ-DONSON knockdown on BC cell proliferation and radioresistance. ese results suggest that circ-DONSON regulates cell proliferation and radioresistance of BC cells through SOX4.
To sum up, our findings demonstrated that circ-DONSON knockdown inhibits proliferation and reduces the radioresistance of BC cells through SOX4 and Wnt/β-catenin signaling.

Data Availability
All data collection and analysis were conducted under double-blind and supported by the First People's Hospital of Lianyungang. e original data will be provided at any time if necessary.
Ethical Approval e treatment and grouping of the subjects in this study were informed by the Ethics Committee of the First People's Hospital of Lianyungang were informed about the treatment and grouping of the subjects in this study.

Conflicts of Interest
e authors declare no conflicts of interest.

Authors' Contributions
Xiufang Zhu wrote the paper and performed the experiments; Lei Li designed and analyzed the experiments.