hsa_circ_0000745 promotes cervical cancer by increasing cell proliferation, migration, and invasion

Abstract Circular RNAs (circRNAs) participate in gene regulation and malignant tumor progression, including uterine cervical cancer (CC). In this study, the expression profile of circRNAs in CC was detected using circRNA microarrays. Then, we selected hsa_circ_0000745 for further examination from the significantly dysregulated circRNAs. Proliferation assays, Transwell assays, quantitative reverse transcription polymerase chain reaction, western blot analysis and tumorigenesis tests in vivo were used to validate the role of hsa_circ_0000745 in CC. hsa_circ_0000745 was upregulated in CC, and its level positively correlated with the level of its linear messenger RNA isoform. Patients with poorly differentiated tumors or vascular/lymphatic invasion presented higher expression of hsa_circ_0000745. The role of hsa_circ_0000745 was illuminated by knocking down hsa_circ_0000745 in CC cells, and the results revealed that reducing hsa_circ_0000745 inhibited cell proliferation, migration, and invasion in CC by upregulating E‐cadherin (E‐cad) expression. In summary, as a tumor promoter in CC, hsa_circ_0000745 enhances the cell's ability to proliferate, migrate, and invade by reducing the expression of E‐cad. hsa_circ_0000745 is a candidate target for the treatment of CC in the clinic.

and 5′ end of the RNA creates the circular shape of the circRNAs (Suzuki & Tsukahara, 2014). There are several mechanisms by which circRNAs participate in cellular biology. circRNAs contain multiple binding sites for microRNAs (miRNAs). circRNAs bind miRNAs at their miRNA response elements to inhibit the activities of the miRNAs and regulate their targets (Cai et al., 2019;Han et al., 2017;Hansen, Jensen, et al., 2013). Research on this role is a hotspot of circRNA research. circRNAs directly regulate the transcription of linear RNAs through competitive splicing and regulate the expression of proteins by isolating RNA-binding proteins that normally make up RNA-protein complexes to control the expression of genes (Li et al., 2015;Wilusz & Sharp, 2013).
The abnormal expression of circRNAs is closely related to various cancers. circZKSCAN1, which may be a useful diagnostic marker, suppresses the growth and metastasis of hepatocellular carcinoma (Yao et al., 2017). circPSMC3 inhibits the development and metastasis of gastric cancer (GC) by inhibiting functions of miR-296-5p (Rong et al., 2019). Aberrantly expressed circRNAs have also been identified in CC. The expression profiles of ncRNAs, including circRNA, have been explored in CC using high-throughput RNA sequencing, and circRNAs represent potentially new diagnostic and therapeutic markers of CC (Wang, Zhao, Chen, & Cui, 2017). circ_0067934 participates in CC development by regulating miR-545 expression and promotes the progression of CC (Hu et al., 2019). A previous study  identified circRNAs that are dysregulated in the response to radiation in CC cells using high-throughput sequencing. Based on the results, these circRNAs may function in the response of CC to radiation in humans. But the specific role of circRNAs in CC still remains largely unknown.
In this study, a circRNA microarray analysis was conducted to detect the aberrantly expressed circRNAs in CC tissues. hsa_-circ_0000745 (hereafter referred to as circ-0745) was selected for in-depth study among the dysregulated circRNAs because of its significantly abnormal expression according to the microarray result. circ-0745 is transcribed from the sperm antigen with calponin homology and coiled-coil domains 1 (SPECC1) located on chromosome 17. circ-0745 expression was markedly upregulated in CC.
Functionally, downregulation of circ-0745 obviously suppressed the proliferation, migration, and invasion of CC cells. According to our findings, circ-0745 promoted CC cell proliferation and metastasis; therefore, circ-0745 represents a potential prognostic and therapeutic marker for CC patients.

| circRNA microarray analysis
All samples were ground into powder and RNAs were extracted with TRIzol (Invitrogen, Carlsbad). The purity and quantity of the RNAs in the specimens were evaluated using a NanoDrop ND-1000 (Implen, Munich, Germany).
We selected five pairs of SCC tissues and paracancerous tissues from patients. The circRNA microarray (Arraystar Inc., Rockville) was conducted following the manufacturer's protocol. The detected circRNAs in two groups were analyzed by the false discovery rate and paired t tests. Significantly differentially expressed circRNAs were defined as circRNAs with a fold change >2.0 and a p value <.05.
The clinicopathological data from the patients are shown in Table 1

| Cell migration and invasion assay
Transwell chambers (Corning) without and with a basement membrane coating (Corning) were used to detect the mobility of transfected CC cells, respectively. For migration test, 2.5 × 10 4 cells were placed into chambers of Transwell inserts without basement membrane coating; for the invasion test, 5 × 10 4 cells were seeded in chambers of Transwell inserts with a basement membrane coating.
After 24 hr, nonmigratory or noninvasive cells were wiped and the cells migrating through the membrane were counted under a microscope (Olympus, Tokyo, Japan).

| Subcutaneous tumor formation in mice
The sequence of siRNA-2 was inserted a lentiviral vector for the stable knocked down of circ-0745 (named sh-circ-0000745). sh-circ-0000745 and an empty lentiviral vector as nonsense control (Genechem Co., Shanghai, China) were constructed containing the green fluorescent protein gene and a puromycin resistance gene.
CaSki cells were transfected and selected with puromycin (2 μg/ml; Sigma) to obtain stable cell clones.
All animal care and experimental procedures were conducted according to the standards of the National Institutes of Health and approved by the Animal Protection and Use Committee of Shandong University. First, 10 7 stably transfected CaSki cells were subcutaneously injected into the backs of female BALB/c mice (5 weeks old).
The tumors were monitored weekly. The mice were killed after 4 weeks, and tumors were excised and assessed.

| Differentially expressed circRNAs in SCC
Overall, 3,008 human circRNAs were detected in SCC tissue and normal cervical tissue cells. One hundred seventy-eight circRNAs

| circ-0745 is upregulated in SCC
In the circRNA microarray, circ-0745 was detected in cervical cells The linear SPECC1 mRNA was also upregulated in the SCC group (p < .001; Figure 2b). Furthermore, circ-0745 positively correlated with the expression of the linear SPECC1 mRNA in SCC tissues (r = 0.449, p < .001; Figure 2c). Interestingly, a higher circ-0745/linear SPECC1 mRNA ratio was observed in SCC tissues than in the control (p < .001; Figure 2d). Based on these findings, circ-0745 was involved in the development of SCC.

| circ-0745 is related to patients' clinicopathological parameters
The relationships between the levels of circ-0745 and several clinicopathological characteristics (Table 3)  In this study, we collected specimens from only CC patients with Stage I and II because patients with more advanced stages were treated with radiotherapy or chemotherapy rather than with

| Inhibition of circ-0745 suppresses CC cell proliferation
In SiHa and CaSki cells, specifically designed siRNAs were used to knockdown circ-0745 expression. circ-0745 expression was significantly reduced in cells transfected with siRNA-2; this construct was used in all subsequent analyses (Figure 3a). Compared with the negative-control treatment, circ-0745 knockdown obviously suppressed the proliferation ability of SiHa and CaSki cells, as examined using CCK-8 assays ( Figure   3b,c). Flow cytometry was conducted to detect cell apoptosis after circ-0745 expression was silenced. Changes in the expression of circ-0745 had no effect on the apoptosis of SiHa or CaSki cells (p > .05).
The role of circ-0745 in promoting CC cell proliferation was tested by generating a subcutaneous tumor model in nude mice. Tumor volume = Length × Width × Height × π/6. The volume of neoplasms in the sh-circ_0000745 group was markedly smaller than in the control (p < .05; Figure 3d), which indicated that inhibiting hsa_circ_0000745 obviously suppressed CC cells' proliferation in vivo.

SiHa and CasKi cells showed decreased migration through a
Transwell chamber after downregulation of circ-0745 (p < .001;   and is a promising marker in GC (Huang, He, Liang, Huang, & Zhu, 2017). In our study, circ-0745 was aberrantly upregulated in CC tissues, according to both circRNA microarray and qRT-PCR results.
These results suggest the same circRNA may be differentially invasion. Based on these findings, the inhibition of circ-0745 represents a direction for exploring new therapies for CC.
In epithelial cells, E-cad is essential for maintaining the homeostasis of the polarized epithelial monolayer. Downregulation of Ecad is common in tumors manifesting from epithelial tissues and promotes cell migration and invasion (Peng et al., 2018;Sarrio et al., 2004). The downregulation of E-cad during cancer progression is generally considered the result of the epithelial-to-mesenchymal transition (EMT). Reduced E-cad expression during EMT is often considered to be a major driver of cancer progression and metastasis (Kourtidis, Lu, Pence, & Anastasiadis, 2017).
As metastasis is considered the primary cause of death in tumor In summary, we highlight the expression of circRNAs in human CC.
circ-0745 is confirmed to be overexpressed in CC and may be related to the prognosis of patients with CC. Functionally and mechanistically, circ-0745 promotes the cellular ability to proliferate, migrate and invade by reducing the expression of E-cad, indicating its role as a tumor promoter in CC progression. Based on our data, circ-0745 plays an important part in promoting CC development and represents a candidate marker of patient prognosis and a target for clinical treatments for CC.

ACKNOWLEDGMENTS
We sincerely thank all the participants in the study.