Circular RNA circ-CCAC1 Facilitates Adrenocortical Carcinoma Cell Proliferation, Migration, and Invasion through Regulating the miR-514a-5p/C22orf46 Axis

Adrenocortical carcinoma (ACC) is a rare but clinically aggressive endocrine malignancy. Circular RNAs (circRNAs) were found to play key roles in tumorigenesis. In the current study, we aimed to investigate the functions and mechanisms of a novel circRNA, circ-CCAC1, in ACC cells. circ-CCAC1 expression levels in ACC tissue specimens and cell lines were evaluated by RT-qPCR. Kaplan-Meier analysis was applied to explore the relationship between circ-CCAC1 and patients' prognosis. Cell counting kit-8 (CCK-8), colony formation, acridine orange/ethidium bromide (AO/EB) double fluorescence staining, and Transwell assays were performed to evaluate the functions of circ-CCAC1 in ACC cells. Bioinformatics analysis and a dual-luciferase reporter assay were utilized to explore the mechanisms of circ-CCAC1. As a result, circ-CCAC1 was overexpressed in ACC tissue samples and cell lines and correlated with poor prognosis. Gain- and loss-of-function tests demonstrated that circ-CCAC1 acted as an oncogene in ACC. What is more, circ-CCAC1 enhanced C22orf46 expression by sponging miR-514a-5p in ACC cells. A rescue assay illustrated that circ-CCAC1 facilitated ACC progression through miR-514a-5p/C22orf46 signaling. To sum up, we identified a novel circRNA, circ-CCAC1, which may be used as a potential therapeutic target for ACC.


Introduction
Adrenocortical carcinoma (ACC) is a rare but clinically aggressive endocrine malignancy [1][2][3]. The incidence of ACC is around 0.7/10000 to 2/10000. ACC frequently occurs in adolescent [4,5]. Although the treatment of ACC has made remarkable progression, the prognosis of ACC is still unfavorable due to local infiltration or extensive metastasis [6]. Moreover, ACC shows medium-low response rates to conventional chemotherapy and emergence of chemoresistance properties [7]. Therefore, understanding the molecular regulation mechanism of ACC progression and identifying an effective therapeutic target are the key issues for the treatment of ACC.
Previous research used whole genome sequencing, and findings indicated that greater than 98% of a genome was transcribed into noncoding RNAs (ncRNAs). Circular RNAs (circRNAs) are a novel type of ncRNAs. However, emerging evidence indicates that several circRNAs are able to encode peptides [8]. For instance, circ-SHPRH encodes a novel protein, SHPRH-146aa [9]. circRNAs are characterized by a closed-loop structure with limited protein coding capacity [10,11] and have been found to function as oncogenes or cancer suppressor genes in a wide variety of human cancers [12,13]. For example, circ_0001649 inhibits cholangiocarcinoma progression in vitro and in vivo [14]. Accumulating evidence has indicated that circRNAs play important roles in cancer with respect to cell proliferation, migration, invasion, and apoptosis, as well as can be important biomarkers that can predict patients' prognosis [15,16]. Furthermore, important interactions between mammalian circRNA and miRNA have been identified in assessments of various types of cancers [17]. For example, hsa_circ_ 101141 acts as a competing endogenous RNA (ceRNA) to facilitate progression of hepatocellular carcinoma by regulating miR-1297/ROCK1 signaling [18]. Previously, the researchers identified a novel circRNA, circ-CCAC1, which functions as an oncogene in cholangiocarcinoma [19]. circ-CCAC1 (circBase ID: hsa_circ_0043469; circBank ID: hsa_ circERBB2_014) is located on chr17: 37880978-37882106 and looped by exons 23-26 of ERBB2. The spliced variant of circ-CCAC1 is 565 nucleotides long. Interestingly, we found circ-CCAC1 was also remarkably increased in ACC, which is related to poor prognosis among ACC patients. Silencing of circ-CCAC1 inhibits ACC cell progression in vitro. Mechanistically, circ-CCAC1 is the sponge of miR-514a-5p, which promotes C22orf46 expression, thus promoting the proliferation and aggressiveness of ACC. Our findings provided a new insight for exploring a potential therapeutic strategy for the treatment of ACC.

Materials and Methods
2.1. Patients. Starting from January 2014 and proceeding through February 2016, we collected 48 paired samples of ACC tissues and corresponding noncancerous tissues from patients admitted to the First Hospital of Qiqihar by way of surgical resection (Table S1). All pathological specimens were independently diagnosed by three clinical pathologists. After sectioning, all ACC tissues and noncancerous tissues were immediately stored in liquid nitrogen and then preserved in a -80°C freezer. All aspects of experimental designs and protocols were reviewed and approved by the Committee for the Protection of Human Subjects at the First Hospital of Qiqihar. All patients signed written informed consent forms before surgical resection.

Cell Lines and Transfection.
Human ACC cell lines (H295R and SW-13) and an immortalized normal cell line (Y1) were acquired from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). We cultured all the cell lines in RPMI-1640 medium following the manufacturer's protocols and supplemented the medium with 10% fetal bovine serum (FBS, Gibco). Next, we incubated all samples in a humidified atmosphere with a constant temperature of 37°C and a 5% CO 2 atmosphere.
Nuclear and cytoplasmic fractions of H295R/SW-13 cells were partitioned using a PARIS Kit (Thermo Scientific). A total of 10 7 fresh cultured cells were collected, placed on ice, and resuspended with 500 μL ice-cold cell fractionation buffer. Cells were incubated on ice for 15 min. Samples were centrifuged at 500 g for 5 min, and then the cytoplasmic fraction was carefully aspirated away to a new tube from the nuclear pellets. RNA isolation from chromatin and nucleoplasm was performed using a TRIzol reagent (Invitrogen) according to the manufacturer's protocol.

Western
Blotting. The cultured cells were lysed in 2% SDS buffer plus protease and phosphatase inhibitor cocktails (Thermo Scientific, USA). BCA protein assay (Solarbio) was used to measure the protein concentrations. Equivalent samples were separated by SDS-PAGE and then transferred into a PVDF membrane (Millipore). After immersion in 5% skim milk at 22-25°C, the membrane was incubated with primary antibodies overnight at 4°C. After incubation with the HRP-conjugated secondary antibody for 2 h, the membrane was visualized using an ECL kit (Beyotime).
2.5. Dual-Luciferase Reporter Gene Test. starBase 2.0 and circBank databases were utilized for predicting the miR-NAs potentially interacted with circ-TOP2A. The binding relationship of 3 ′ -untranslated region (UTR) of C22orf46 and miR-514a-5p was predicted by the TargetScan database. For the dual-luciferase reporter gene test, we seeded ACC cells into a 24-well plate and cultured the plate for 24 h. To confirm the interaction between miR-514a-5p and circ-CCAC1/3'-UTR of C22orf46, we transfected the following into ACC cells: circ-CCAC1/3 ′ -UTR of C22orf46 wild reporter vector (pmir-GLO), circ-CCAC1/3 ′ -UTR of C22orf46 mutant reporter

circ-CCAC1 Is Upregulated in ACC and Correlates with
Poor Prognosis. circ-CCAC1 was more stable than ERBB2 mRNA under the circumstance of RNase R (Figure 1(a)).
Total RNA was isolated to measure the expression levels of circ-CCAC1 and ERBB2 mRNA after treatment with actinomycin D at different time points. We found that the half-life of circ-CCAC1 was longer than its linear isoform (ERBB2 mRNA) (Figure 1(b)). circ-CCAC1 expression was significantly higher in ACC tissues compared to noncancerous samples (Figure 1(c)). According to the median value of circ-CCAC1, we classified the enrolled ACC patients into two groups (high and low expression groups). We found that high circ-CCAC1 expression was linked to worse overall survival (P = 0:006) for the ACC patients after surgery (Figure 1(d)). Next, we evaluated the expression of circ-CCAC1 in ACC cell lines (H295R and SW-13) and a normal cell line (Y1). As a result, the two ACC cell lines exhibited significantly higher levels of circ-CCAC1 compared to Y1 cells (Figure 1(e)).

circ-CCAC1 Promotes ACC Cell Proliferation and
Aggressiveness. To facilitate explorations of the roles that circ-CCAC1 may have played in the progression of ACC, we designed two circ-CCAC1 siRNAs that would knock down circ-CCAC1. We found that the levels of circ-CCAC1 were notably downregulated in H295R cells that had been transfected with si-circ-CCAC1-1/-2 ( Figure 2(a)). Additionally, an overexpression study was conducted on SW-13 cells due to its lowest expression of circ-CCAC1, and the overexpression efficiency of circ-CCAC1 vector was favorable (Figure 2(a)). Furthermore, ERBB2 mRNA expression levels were unaffected after circ-CCAC1 knockdown/overexpression ( Figure 2(b)). We used CCK-8 and colony formation tests to detect ACC cell viability and clone-forming capacity affected by circ-CCAC1. The results confirmed that silencing of circ-CCAC1 significantly inhibited cell viability and cloneforming capacity (Figures 2(c) and 2(e)). Conversely, overexpression of circ-CCAC1 accelerated cell growth in vitro (Figures 2(d) and 2(f)). Furthermore, AO/EB double staining assay indicated that the cell growth-promoting role of circ-CCAC1 is partly attributed to its suppression on cell apoptosis in ACC cells (Figures 2(g) and 2(h)). Results from Transwell assays indicated that silencing of circ-CCAC1 attenuated the migratory and invasive potential of H295R cells (Figure 2(i)). By contrast, elevated circ-CCAC1 strengthened the migratory and invasive capacities of SW-13 cells (Figure 2(j)).

circ-CCAC1 Enhanced C22orf46
Expression by Sponging miR-514a-5p in ACC. As displayed in Figures 3(a) and 3(b), circ-CCAC1 was mainly localized to the cytoplasm of the two ACC cell lines. circRNAs can affect tumor cell malignant behaviors via sponging certain microRNAs [17,18]. Therefore, we hypothesized miRNAs would bind to circ-CCAC1 and examined this prediction by using web-based circRNA-miRNA prediction tools. As a result, five miRNAs (miR-182-5p, miR-1343-3p, miR-514a-5p, miR-3619-5p, and miR-6746-5p) were predicted by both circBank and starBase 2.0 databases (Figure 3(c)). Silencing of circ-CCAC1 increased miR-514a-5p expression in H295R and SW-13 cells. The expression levels of other miRNAs were unchanged (Figures 3(d) and 3(e)). In addition, miR-514a-5p expression 3 BioMed Research International was downregulated in ACC samples relative to their normal counterparts (Figure 3(f)). TCGA datasets indicated that the patients with low expression of miR-514a-5p had a worse prognosis (Figure 3(g)). Furthermore, miR-514a-5p was downregulated in H295R and SW-13 cells than Y1 cells (Figure 3(h)). A negative association between circ-CCAC1 and miR-514a-5p was identified in ACC tissues (Figure 3(i)). We constructed a luciferase reporter vector of Wt and Mut       BioMed Research International circ-CCAC1 (Figure 3(j)). The vectors were cotransfected with miR-514a-5p mimics or mimics-NC in H295R and SW-13 cells. We found that miR-514a-5p mimics remarkably suppressed the luciferase signal relative to the negative control (Figure 3(k)). The TargetScan database was used to predict the downstream targets of miR-514a-5p, and C22orf46 was chosen for further study. Pearson's correlation analysis demonstrated that circ-CCAC1 expression was positively correlated with the levels of expression of C22orf46 for our examinations of ACC human tissue samples (Figure 3(l)). TCGA datasets indicated that the patients with upregulated C22orf46 expression had a worse overall survival (Figure 3(m)). Next, we examined expression levels of C22orf46 and found significantly higher levels of C22orf46 in ACC cells (Figures 3(n) and 3(o)). Additionally, we found that knocking down of miR-514a-5p significantly enhanced the levels of C22orf46 mRNA in H295R cells, whereas ectopic-expressed miR-514a-5p attenuated C22orf46 mRNA expression (Figure 3(p)). Dual-luciferase reporter assays indicated that cotransfection with Wt-C22orf46 3 ′ -UTR reporter and miR-514a-5p mimics induced significant inhibition of luciferase activity in ACC cells (Figures 3(q) and 3(r)).

Discussion
ACC is one of the most aggressive malignancies worldwide [2,3]. Although remarkable progression has been achieved recently in the diagnosis and treatment of ACC, the prognosis is still dismal in ACC patients [4]. Although numerous studies have revealed that alterations in the oncogene and tumor suppressor gene contribute to the progression and metastasis of ACC [3], the precise molecular mechanism remains vague. Accumulating lines of evidence indicates that circRNAs play important roles in the dynamics underlying epigenetic regulation, transcription, and posttranscriptional regulation and may facilitate development and progression of tumorigenesis [16][17][18]. For example, circRNA GLIS2 promotes colorectal cancer cell motility via activation of the NF-κB pathway [20]. A recent study indicated that cir-cRNA_100782 promotes proliferation and metastasis of gastric cancer by downregulating tumor suppressor gene Rb by adsorbing miR-574-3p in a sponge form [21]. Mounting studies have shown that circRNAs can regulate the biological behaviors of cancer cells via target genes, thus exerting a pivotal role in the progression of malignancies [22]. Zhang et al. found that overexpressed circ-PIP5K1A contributes to colon cancer progression by suppressing miR-1273a expression [23]. The other study indicated that circ-TSPAN4 facilitates lung adenocarcinoma metastasis by elevating ZEB1 via sponging miR-665 [24]. However, the functions and mechanisms of circRNAs in ACC remain unclear. A recent study indicated that circ-CCAC1 was enhanced in cholangiocarcinoma and indicated that circ-CCAC1 was likely to affect tumorigenesis and metastasis in human cancers [19]. circ-CCAC1 is located on chr17: 37880978-37882106 and is looped by exons 23-26 of ERBB2. We found that circ-CCAC1 expression was also overexpressed in ACC tissue specimens than normal tissues and correlated with worse overall survival. Nevertheless, the independent prognostic role of circ-CCAC1 was not investigated. We then evaluated the functions of circ-CCAC1 in tumorigenesis and progression of ACC cells. Gain-and loss-of-function assays illustrated that circ-CCAC1 significantly increased the viability, clone-forming, migration, and invasion of ACC cells. What is more, the cell growth-promoting role of circ-CCAC1 was partially dependent on its suppression on cell apoptosis. Increasing evidence showed that there were extensive interaction networks involving ceRNA, in which circRNAs could regulate the target RNA by binding to the miRNAs and titrating from the binding site on the protein coding messenger [25]. Target molecules regulated mutual expression by competing to bind miRNA's response element (MRE). Evidence suggested that the ceRNA regulatory model had been validated in other cancers [25,26]. The localization of circRNAs suggests how they exert their functions. circ-CCAC1 primarily localized to the cytoplasm rather than the nucleus, suggesting its mechanism in the posttranscriptional level. In this work, our results indicated that miR- 11 BioMed Research International 514a-5p expression was significantly lower in ACC tissues and cell lines. Meanwhile, a negative correlation was observed between miR-514a-5p and circ-CCAC1 expression. We found that circ-CCAC1 could sponge and negatively regulate miR-514a-5p expression in ACC cells. In fact, miR-514a-5p has been confirmed as a tumor suppressor gene in some types of human cancers [27]. Moreover, miR-514a-5p was regulated at the posttranscriptional level. For example, miR-514a-5p could be sponged by long noncoding RNA TRIM52-AS1 and SNHG7, thus releasing its suppression on MRPS18A and ELAVL1, respectively [28,29]. However, the functions and mechanisms of miR-514a-5p in ACC are still unclear. The above evidence suggested that miR-514a-5p functions as a tumor suppressor gene in tumors. Next, we verified that miR-514a-5p directly combined with the 3 ′ -UTR of C22orf46. Hence, we hypothesized that circ-CCAC1 induces the promotion of ACC malignancy by way of its interactions with miR-514a-5p to upregulate C22orf46 expression. Furthermore, a rescue assay demonstrated that the oncogenic role of circ-CCAC1 is partially dependent on its regulation of C22orf46 in ACC cells. C22orf46 was rarely studied before. The functions and mechanisms of C22orf46 are still unclear. To the best of our knowledge, this is the first study revealing the oncogenic role of C22orf46 in human cancers. In addition, we found it could be regulated at the posttranscriptional level in ACC cells. However, there are still some limitations within the study. For example, the detailed downstream targets of C22orf46 are worthy of investigation. The animal study is needed to validate the in vitro data. Additionally, a larger cohort of patients should be included in the study to further identify the clinical value of circ-CCAC1 in ACC patients.
To sum up, circ-CCAC1 functions as a sponge for miR-514a-5p to enhance C22orf46 expression, which subsequently contributes to ACC tumorigenesis and progression. These findings may provide additional insights into the molecular events responsible for ACC carcinogenesis.

Data Availability
The datasets used in the study are available from the corresponding author.

Conflicts of Interest
No potential conflicts of interest to disclose.