Circ-CCDC66 upregulates REXO1 expression to aggravate cervical cancer progression via restraining miR-452-5p

Background Cervical cancer is one most common cancer types among females over the world. While its underlying mechanisms remain unclear. Circ-CCDC66 has been revealed to participate in multiple biological functions, and contribute to various diseases’ progression. In the current study, we aimed to demonstrate the role of circ-CCDC66 in cervical cancer progression. Methods Real-time quantitative PCR (RT-qPCR) was conducted to measure the expression of circ-CCDC66, miR-452-5p, and REXO1 mRNA. Cell fractionation assay and RNA fluorescence in situ hybridization (FISH) were performed to locate circ-CCDC66 in cells. Cell account kit 8 (CCK-8) was used to detect cell proliferation ability. Transwell assay was applied to evaluate cell migration or invasion ability. Bioinformatics analysis, biotinylated RNA pull-down, RNA immunoprecipitation, and dual-luciferase reporter assays were conducted to assess the association between miR-452 and circ-CCDC66 or REXO1. Western blot was applied to measure the protein expression of REXO1. The animal tumor model was used to assess the effect of circ-CCDC66 in vivo. Results The expression of circ-CCDC66 was upregulated in cervical cancer tumor tissues in comparison with normal tissues, and correlated with later tumor stage and larger tumor size. Downregulated circ-CCDC66 inhibited cervical cancer cell proliferation, migration, and invasion. Circ-CCDC66 was an efficient molecular sponge for miR-452-5p, and negatively regulated miR-452-5p expression. MiR-452-5p directly targeted to REXO1. Circ-CCDC66 regulated REXO1 expression to modulate cervical cancer progression via miR-452-5p. Moreover, downregulated circ-CCDC66 was found to suppress tumor growth in vivo. Conclusion Our results demonstrated the role of circ-CCDC66/miR-452-5p/REXO1 axis in cervical cancer progression, we might provide novel therapeutic targets for cervical cancer clinical intervention.


Background
Cervical cancer is one most common malignancy types of gynecological tumors, and a primary reason caused cancer-related death to female worldwide [1]. In china, cervical cancer is the sixth most common cancer type, which causes more than 34,000 death in 2015 [2]. Despite the application of vaccine has been widespread, and the diagnosis and intervention of cervical cancer have made great improvement, 25% of patients diagnosed with cervical cancer still fail to recover due to the high recurrence and distance metastasis [3,4]. Cervical cancer has brought huge health burden for world health care system. It is imperative to find novel targets for cervical cancer treatment.
Circular RNA (circRNA) is one type of non-coding RNAs, which is characterized by its covalently closed loop structure [5]. Due to its stable, conserve, and spatio-temporal specificity futures, circRNAs are abundantly expressed in human tissues, and involved in various cellular progression [6,7]. In the past decades, the functions of circRNAs in multiple biological progressions have been deeply studied, including cervical cancer [8][9][10][11]. Gao et al. revealed that has_circ-0018289 mediates cervical cancer cell proliferation, invasion, and migration via acting as a molecular sponge for miR-497 [12], Song T et al. elucidated the role of hsa_cir-cRNA_101996 in cervical cancer development through regulating miR-8075/TPX2 axis [13], Tang Q et al. demonstrated that hsa_circ_0000515 mediates cervical cancer progression via miR-326/ELK1 pathway [14]. Accumulating evidence suggest that circRNAs play essential role in the initiation or progression of cervical cancer. CircRNA circ-CCDC66 (hsa_circ_0001313) derives from chr3:56626997-56628056 and consists about 460 nts [15]. Circ-CCDC66 was found to participate in Hirschsprung's disease, Colon cancer, Gastric cancer, and Abdominal Aortic Aneurysm development via regulating multiple cellular progressions [16][17][18][19]. However, the role circ-CCDC66 in cervical cancer development is still uncovered.
In this study, we aimed to investigate the role of circ-CCDC66 in cervical cancer progression. Firstly, we measured the expression of circ-CCDC66 in thirty-six pairs of cervical cancer human samples. Next, we assessed the biological functions of circ-CCDC66 in cervical cancer cells. Subsequently, by using bioinformatic analysis, luciferase reporter assays, circ-CCDC66 was found to upregulate REXO1 expression via sponging miR-452-5p. Collectively, our study demonstrated the role of circ-CCDC66/miR-452-5p/REXO1 pathway in cervical cancer progression. We might provide new insights for cervical cancer basic research and novel therapeutic targets for clinical management.

Clinical human samples
Thirty-six pairs human samples were collected from cervical cancer patients who underwent surgery in the department of obstetrics and gynecology, Renmin Hospital of Wuhan University from 2018 to 2019. The cervical cancer patients who received chemotherapy, immunotherapy, or radiotherapy were not enrolled. All specimens (2 cm away from the tumor tissues) were exanimated and approved by two pathologists, respectively. All samples were immediately froze and stored at −80 °C after operation. All patients or their families were informed the approach of sample collection, and informed consent were obtained. All experiments in this study were approved by the Ethics Committee of the Renmin Hospital of Wuhan University.

Animal experiment
The 8-week old nude mice (28 for study) were purchased from Vital River Inc. (Beijing, China). The ethics committee of the Renmin Hospital of Wuhan University approved animal experiments in this study. HeLa cells were stably transfected with Sh-circCCDC66 and Sh-NC. Then, about 1 × 10 7 treated cells (per tumor) were subcutaneously injected into nude mice. After 28 days, mice were sacrificed, and tumors were harvested. The animal experiments were followed with the ethical standard of Helsinki Declaration of 1975 (1983 reversion).

Real-time qPCR
Total RNAs from cervical cancer human samples and cells were collected by Trizol kit (Invitrogen, USA). A PrimerScript RT Reagent kit (TaKaRa, Japan) was used to reversely transcribed RNA into cDNA. The amplification was conducted using SYBR Green dye. GPADH was used as an internal reference. The reliability of PCR results was confirmed by the comparative CT(2 −ΔΔCt ) method.
The information about primers used in study is showed in the supplementary Table 1.

RNA fluorescence in situ hybridization (FISH)
Fluorescent In Situ Hybridization Kit (RiboBio, China) was used to carry out RNA FISH assay according to manufacturer's instruction. Cy3-labeled circ-CCDC66 probes were procured from GeneChem (Shanghai, China). 4% paraformaldehyde was adopted to fix cells. Subsequently, cells were treated by permeabilization with 0.5% Triton X-100, then subjected to hybridization solution. After that, treated cells were incubated with Cy3-labeled circ-CCDC66 probes overnight. The results were detected by Fluorescent In Situ Hybridization Kit and visualized with a confocal microscopy.

Cell proliferation experiment
Cell proliferation ability was detected by cell account kit 8 (CCK-8, Dojindo, Japan) according to a previous study [20]. Collectively, HeLa and SiHa cells (1 × 10 4 per well) were seeded into a 96-well plate and housed for 3 days. CCK-8 solution (10 μl) was added into well, two hours before detection. The absorbance at a wavelength of 450 nm was recorded, and repeated three times.

Cell migration and invasion experiment
Transwell chamber with 8.0 μm pores (Corning, USA) was used to detect cell migration and invasion ability. For migration, the upper chambers were seeded with cells (1 × 10 4 per well) with DMED, and the lower chambers were added with DMED with 10% FBS. After 2 days, the cells on the lower surface were fixed with 4% paraformaldehyde, and visualized under a microscope. For invasion, Matrigel (BD, USA) was used to cover upper chambers, cells were incubated on the upper chambers with Matrigel and DMEM, lower chambers were filled with DMEM with 10% FBS. After 2 days, cells attached on lower surface were fixed with 4% paraformaldehyde, and visualized under a microscope. Experiments were carried out three times.

RNA pull-down
The biotinylated RNA pull-down assay was carried out followed by previous studies [21,22]. Biotinylated CCDC66 and miR-452-5p probes were synthesized and commercially obtained from Sangon Biotech (Shanghai, China). Briefly probe-coated beads were generated using C-1 magnetic beads (Life Technologies, Carlsbad, CA, USA). Cell lysates were collected using lysis buffer (Sigma, Germany), subsequently, incubated with C-1 magnetic beads at 4 °C for 120 min. Then, elution buffer (Sigma-Aldrich) was used to elute precipitated RNAs from beads. RNA bands were analyzed by qRT-PCR.

AGO2-RIP
RNA immunoprecipitation assays using anti-AGO2 and anti-IgG antibodies were performed using a Magna RIP ™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA) according to manufacturer's protocol. Then, RNA complexes were subjected to qRT-PCR assay. Experiments were repeated three times.

Dual luciferase assay
The sequences of CCDC66 and REXO1 3′UTR containing wide-type or mutant-type miR-452-5p binding sites were subjected to pGL3-Basic luciferase vector (Promega, USA). Then, vectors were transfected into 293 T and HeLa cells with pRL-TK vector (Promega), miR-452-5p mimic and its normal control mimic. After 2 days, firefly activity was detected in comparison with the Renilla control by a Dual-Luciferase Reporter assay system (Promega).

Statistical analysis
All data from experiment were presented as mean ± Standard Deviation (SD). SPSS 17.0 software (SPSS, USA) was used to carry out results data. All experiments were repeated at least three times. The differences between two groups were calculated by student t-test. The differences between multiple groups were analyzed by Analysis of Variance (ANOVA). The statistical correlation between miR-452-5p and circ-CCDC66 or REXO1 were calculated by spearman analysis. p < 0.05 was considered statistically significant.

Circ-CCDC66 is upregulated in cervical cancer tissues
The expression of circ-CCDC66 in 36 pairs of cervical cancer human samples were measured. Circ-CCDC66 was significantly upregulated in tumor tissues compared with matched normal tissues (Fig. 1a).High expression of circ-CCDC-66 was found to associate with large tumor size and advanced tumor stage (Fig. 1b-c). Our findings indicated that circ-CCDC66 might be involved in the progression of cervical cancer.
Subsequently, it was found that circ-CCDC66 was upregulated in cervical cancer cell lines compared with normal cervical cell line H8, especially in Hela and SiHa cells (Fig. 1d), and mainly located in cell cytoplasm ( Fig. 1e and f ).

Circ-CCDC66 promotes the proliferation, migration and invasion of cervical cancer cells
To elucidate the biological function of circ-CCDC66, we constructed circ-CCDC66 knockdown cell models by stably transfecting Sh-NC or Sh-circ-CCDC66 into Hela and SiHa cells, the transfection efficiencies were detected (Fig. 2a). By performing CCK-8 and transwell assays, it was found that circ-CCDC66 knockdown significantly suppressed cervical cancer cell proliferation, migration, and invasion abilities (Fig. 2b-d). Furthermore, overexpression of circ-CCDC66 in cervical cancer cells were couple with promotive phenomenon in cell proliferation, migration and invasion (Additional file 1: Figure S1). These results suggested that circ-CCDC66 regulated cervical cancer cellular progression.

Circ-CCDC66 mediates REXO1 expression to promote cervical cancer progression via miR-452-5p
The biological effects of REXO1 on cervical cancer cells were evaluated. It was found that over expression of REXO1 promoted cell proliferation, migration, and invasion (Additional file 1: Figure S2). Subsequently, we investigated the role of circ-CCDC66/miR-452-5p/ REXO1 axis in cervical cancer cellular progression. Cell models were generated by transfecting Sh-NC, Sh-circ-CCDC66, Sh-circ-CCDC66 + OE-NC, Sh-circ-CCDC66 + OE-REXO1 into Hela and SiHa cells. The expression of cric-CCDC66 and REXO1 were measured (Fig. 5a). CCK-8 and Transwell assays showed that the inhibitory effects of downregulated circ-CCDC66 on Hela and SiHa cells were rescued by REXO1 overexpression (Fig. 5 b-f ), suggested that circ-CCDC66 regulated REXO1 expression to promote cervical cancer progression via sponging miR-452-3p. Fig. 3 Circ-CCDC66 acts as a molecular sponge for miR-452-5p. Putative downstream miRNAs of circ-CCDC66 were predicted by Miranda (http:// miran da.org.uk/) database, four miRNAs were selected for our further study. a Full length of circ-CCDC66 were biotinylated, and the complexes were subjected to qRT-PCR assays. Relative miRNAs expression were presented. b AGO-2 RIP assays were conducted, and the expression of circ-CCDC66 and miR-452-5p in protein bonds were detected by qRT-PCR. c The binding sites between circ-CCDC66 and miR-452-5p were showed. d Luciferase reporter assays were used to determine the interaction between circ-CCDC66 and miR-452-5p in 293 T and Hela cells. e The expression of miR-452-5p in Sh-NC or Sh-circCCDC66#1 transfected Hela and SiHa cells were detected by qRT-PCR. f the expression of miR-452-5p in cervical cancer tissues were measured by qRT-PCR. g Spearman analysis was employed to assess the correlation expression between circ-CCDC66 and miR-452-5p. Data were presented as mean ± SD, all experiments were performed at least three times. **P < 0.

Downregulation of circ-CCDC66 inhibits cervical cancer cell growth in vivo.
The function of circ-CCDC66 in vivo was investigated through constructing circ-CCDC66 knockdown mouse models. As shown in Fig. 6 a and b, circ-CCDC66 knockdown significantly suppressed cervical cancer cell growth in vivo, the representative image of xenotransplantation tumors showed the inhibitory effect of circ-CCDC66 (Fig. 6c).

Discussion
Cervical cancer has been identified as a gynecologic malignancy cancer type with high morbidity and mortality rate in various cancers, and presents great health threat to female worldwide [25,26]. Due to its inefficiency in diagnosis and clinical intervention, finding novel therapeutic targets for cervical cancer treatment become urgent.
In this study, we revealed that circ-CCDC66 acted as an oncogene in cervical cancer progression. Firstly, circ-CCDC66 expression in cervical cancer human samples were assessed, our findings suggested that circ-CCDC66 was highly expressed in tumor tissues, and associated with later tumor stage and lager tumor size, indicated that circ-CCDC66 might be involved in cervical cancer progression. Previous study has demonstrated that circ-CCDC66 mediates tumorigenesis via regulating multiple cellular functions, such as proliferation, migration, invasion and EMT levels [18,19,27], suggested the important role of circ-CCDC66 in tumorigenesis progression. Here, knockdown of circ-CCDC66 was found to attenuate cervical cancer cell proliferation, migration and invasion abilities in vitro, and inhibit cervical cancer cell growth in vivo.
CircRNAs have been widely reported that mediate virous biological progressions through transcriptionally regulating gene expression via sponging micro-RNA [23,24,28,29]. To investigate the underlying molecular mechanisms of circ-CCDC66 in cervical cancer progression. Bioinformatics analysis, RNA-pull down, RIP, and dual-luciferase reporter assays was conducted. It was found that circ-CCDC66 was an efficient molecular sponge for miR-452-5p, and miR-452-5p directly targeted to REXO1 in cervical cancer cells. MiR-452-5p has been revealed that participates in multiple cancer progression, such as: renal cancer, prostate cancer, hepatocellular caner, and lung squamous cell carcinoma [30][31][32][33], and showed its specific role in tumorigenesis. While, the function of miR-452-5p in cervical cancer remains unelucidated. Our study found that circ-CCDC66 sponged to miR-452-5p and negatively regulated its expression in cervical cancer cells. Furthermore, it was found that miR-452-5p was downregulated in cervical cancer tumor tissues in comparison with matched normal tissues. Subsequently, our study found that miR-452-5p directly targeted to REXO1, and negatively regulated REXO1 expression in cervical cancer cells. CCK-8 and Transwell assays' results showed that overexpression of REXO1 reversed the inhibitory effects of circ-CCDC66 on cell proliferation, migration, invasion abilities.
Despite the role of circ-CCDC66/miR-452-5p/ REXO1 pathway has been partially revealed by our study. On the one hand, our results were limited by small human sample size. One the other hand, the downstream molecular mechanisms of REXO1 in cervical cancer progression need further exploration.

Conclusions
Our study revealed that the expression of circ-CCDC66 was upregulated in cervical cancer tumor tissues, and circ-CCDC66 mediated the proliferation, migration, and invasion abilities of cervical cancer cells. Moreover, our results demonstrated that circ-CCDC66 promoted cervical cancer progression via miR-452-5p/REXO1 axis, indicating that circ-CCDC66 might be a novel therapeutic target for cervical cancer.  6 Downregulation of circ-CCDC66 inhibits cervical cancer cell growth in vivo. Hela cell (10 6 per tumor) pre-transfected with Sh-NC or Sh-circCCDC66 were subcutaneously injected into node mouse (7 mice for each group). After 28 days, tumors were harvested after mice were sacrificed. a and b tumor volumes and tumor end weights were recorded. c Representative image of xenotransplantation tumors were showed. Data were presented as mean ± SD, all experiments were performed at least three times. *P < 0.05