CircHN1 affects cell proliferation and migration in gastric cancer

Abstract Background Increasing evidence indicates that circular RNAs (circRNAs) are dysregulated in human cancers. The biological roles of circRNAs in gastric cancer (GC) have not been well‐characterized. Methods The GEO database was used to analyze circRNA expression profile in GC. The expression level of target circRNA in tumor tissues and adjacent non‐tumor tissues was detected by reverse transcription‐quantitative PCR. Gene transfection was used to manipulate the expression of circRNAs. The biological roles of circRNAs in cell proliferation, migration, and invasion were determined by cell counting, colony formation, transwell migration, Matrigel invasion, and mouse xenograft tumor assays. The interactions between circRNAs and miRNAs were verified by RNA immunoprecipitation and luciferase reporter assays. Results We found that circHN1 was upregulated in GC tissues and cell lines compared to adjacent non‐tumor tissues and normal gastric epithelial cells. Additionally, circHN1 silencing significantly promoted GC cell growth, colony formation, migration, and invasion, whereas circHN1 overexpression had the opposite effects. CircHN1 overexpression also suppressed gastric cancer growth in the mouse xenograft tumor model. CircHN1 was mainly localized in the cytoplasm of GC cells and could bind to AGO2. MiR‐1248 and miR‐375 were predicted to interact with circHN1 by bioinformatic analyses. MiR‐1248 and miR‐375 overexpression inhibited the activity of the circHN1 luciferase reporter. Conclusion CircHN1 is aberrantly expressed in GC and affects the proliferation and migration of gastric cancer cells by acting as miRNA sponge.

and bioinformatics methods, many circRNAs have been shown to be involved in the pathogenesis of various diseases such as diabetes, heart diseases, psychiatric diseases, and cancers. [5][6][7][8] CircRNAs contain abundant miRNA binding sites. 9 Recent evidence has indicated that circRNAs can serve as efficient miRNA sponges. [10][11][12][13] For instance, circRNA PRMT5 has been documented to function as miR-30c sponge to induce epithelial-mesenchymal transition in urothelial bladder carcinoma. 14 In addition, circRNA MTO1 can sponge miR-9 to inhibit the progression of hepatocellular carcinoma. 15 However, the role and molecular mechanisms of circRNAs in GC are not fully understood.
In this study, we first studied the circRNA profile of GC in a cir-cRNA database and focused on a new circRNA, circHN1. We then evaluated the expression pattern of circHN1 in GC tissues and investigated the biological roles of circHN1. Moreover, we investigated whether circHN1 could act as miRNA sponge in GC.

| Analysis of microarray data
The GSE83521 human circRNA microarray dataset was downloaded from the GEO database (http://www.ncbi.nlm.nih.gov/geo/). The transcriptome data of 6 pairs of gastric tumor tissues and non-tumor tissues were included in the GSE83521 dataset. Quantile normalization and subsequent data processing were performed using the R software package.

| Patients and tissue samples
Paired GC tissues and adjacent non-tumor tissues (n = 101) were obtained from the Department of General Surgery, the Affiliated People's Hospital of Jiangsu University between June 2016 and April 2017. This study was reviewed and approved by the Jiangsu University Institutional Ethical Committee. Informed consent was obtained from all participants before sample collection.

| Cell counting and cell colony formation assays
For cell counting assay, 1 × 10 4 cells were seeded into each well of a 24-well plate and counted every day. Cell growth curves were plotted using the results. For colony formation assay, 1 × 10 3 cells were seeded into each well of a 6-well plate. After ten days, the colonies were fixed with 4% paraformaldehyde, stained with violet crystal, and counted under a microscope.

| Transwell migration and Matrigel invasion assays
Transwell migration and Matrigel invasion assays were conducted in transwell chambers (Corning, Inc). Serum-free media was added to the top chamber, and complete medium was added to the lower chamber. In the transwell migration assay, 1 × 10 5 cells were placed in the top chamber. The cells that had migrated to the other side of the membrane were counted after 24 hours. Matrigel was added to the top chamber for the Matrigel invasion assay. Next, 2 × 10 5 cells were placed in the top chamber and incubated for 36 hours.
The cells that had invaded to the other side of the membrane were counted.

| RNA immunoprecipitation (RIP)
A Magna RIP kit (Millipore) was used to conduct the RIP assay. The cells were lysed, and the cell lysate was immunoprecipitated on magnetic beads by human anti-AGO2 antibodies. An IgG antibody was used as the control. To detect RNA directly binding to AGO2, the immunoprecipitated RNA was purified and quantified by qRT-PCR.

| Animal studies
The animal study was approved by the Animal Use and Care Committee of Jiangsu University. Four-week-old male BALB/c nude mice were subcutaneously injected with MGC-803 cells transfected with circHN1 or control vector. Four weeks later, the mice were sacrificed, and the tumors were harvested.

| Statistical analyses
Data were analyzed using SPSS 20.0 software (SPSS Inc) and of circRNA was calculated as the ΔCt (Ct value of target −Ct value of GADPH). Fold changes were calculated using the 2 −ΔΔCt method.

Data were compared between two groups by Student's t test or
Wilcoxon rank-sum test. A P value less than .05 was considered as statistically significant.

| CircHN1 is upregulated in GC
We first downloaded the GSE83521 human circRNA microarray dataset from GEO and analyzed the differentially expressed circR-NAs using the R software package. We selected the top 5 upregulated circRNAs ( Figure 1A) according to the following criteria: fold change ≥2.0 and P value <.05. We next designed specific primers for qRT-PCR detection. The melting curves of qRT-PCR for hsa_ circ_0006089, hsa_circ_0004339, and hsa_circ_0002019 did not show specific single peaks; however, the primer melting curves of qRT-PCR for hsa_circ_0045602 and hsa_circ_0008768 showed single peaks, indicating that the primers for these two circRNAs were specific. Given that the fold change of hsa_circ_0045602 between GC and adjacent non-tumor tissues in the microarray was higher than that of hsa_circ_0008768, we selected hsa_circ_0045602 as our target circRNA. We further verified the PCR product of hsa_ circ_0045602 by agarose gel electrophoresis ( Figure 1B,C). We selected hsa_circ_0045602 for further study and termed it "circHN1" as it is derived from HN1 gene. We then detected the expression of circHN1 in both GC cell lines and a cohort of 101 paired GC and adjacent non-tumor tissues. The expression levels of circHN1 in HGC-27 and MGC-803 cells were significantly higher than that in GES-1 cells ( Figure 1D). Compared to that in non-tumor tissues, circHN1 expression was substantially upregulated in GC tissues ( Figure 1E,F).
We also explored the correlation between circHN1 expression and the clinicopathological parameters of patients with GC. The results showed that circHN1 expression was correlated with tumor invasion (

| CircHN1 is generated from exons 3 to 5 of HN1 by back-splicing
CircHN1 is formed by reverse splicing of the linear transcript of exons 3-5 of HN1 gene with a length of 260 nucleotides (Figure 2A).
PCR and sequencing analyses were conducted to detect the backspliced junction sequence of circHN1. The PCR results revealed that only the cDNA fragment could be amplified using circHN1-specific divergent primers ( Figure 2B). The sequencing result showed that the back-spliced junction sequence of circHN1 was formed by backsplicing of exon 5 and exon 3 of HN1 gene ( Figure 2C). To further characterize circHN1, RNase R was used to treat total RNA from HGC-27 cells. The results of PCR showed that circHN1 was more resistant to RNase R digestion compared to linear RNA ( Figure 2D).
These findings suggest that circHN1 is a circular RNA.

| CircHN1 knockdown promotes the proliferation, migration, and invasion of GC cells
To

| CircHN1 overexpression inhibits the proliferation, migration, and invasion of GC cells
We overexpressed circHN1 in HGC-27 and MGC-803 cells to further investigate its function ( Figure 4A). CircHN1 overexpression remarkably decreased the proliferation ( Figure 4B) and colony formation ( Figure 4C) of HGC-27 and MGC-803 cells. In addition, both the migration ( Figure 4D) and invasion ( Figure 4E) abilities of HGC-27 and MGC-803 cells were inhibited by circHN1 overexpression. We further used a xenograft tumor model to verify the role of circHN1 in vivo. BALB/c nude mice were subcutaneously inoculated with control and circHN1 overexpressing cells. The results showed that both the size ( Figure 5A) and weight ( Figure 5B) of tumors in the circHN1 group were considerably smaller than those in the control group.

| CircHN1 may function as miRNA sponge in GC
RNA fractionation results indicated that circHN1 was mainly localized in the cytoplasm ( Figure 6A). Given that circRNAs can serve as miRNA sponge and circHN1 is abundant in the cytoplasm, we further examined whether circHN1 could bind to specific miRNAs. RIP The results revealed that endogenous circHN1 was enriched in the AGO2 immunoprecipitates ( Figure 6B), suggesting potential interactions between circHN1 and miRNAs. Using the CircInteractome and StarBase v2.0 miRNA prediction programs, we selected 9 miRNAs (miR-1184, miR-1248, miR-198, miR-370, miR-375, miR-619, miR-942, miR-27a, and miR-27b) which have binding sites for the circHN1 sequence. Dual-luciferase reporter assays were used to assess which miRNA might bind to circHN1. The circHN1 luciferase reporter plasmid was co-transfected with different miRNA mimics into 293T cells ( Figure 6C). Compared to that in control group, miR-1248 and miR-375 inhibited the luciferase reporter activities of circHN1 luciferase reporter ( Figure 6C,D). These findings suggest that circHN1 may function as a sponge for these two miRNAs.

| D ISCUSS I ON
The roles of circRNAs in cancer biology have attracted wide attention. 16 Recent studies have demonstrated that some circRNAs are abnormally expressed in GC and involved in the development and progression of GC. [17][18][19][20] CircNRIP1 promotes GC progression by sponging miR-149-5p and regulating the AKT1/mTOR pathway. 17 CircFAT1(e2) prevents the development of GC by targeting miR-548g in the cytoplasm and interacting with the YBX1 protein in the nucleus. 18 The expression level of hsa_circ_0067582 is significantly downregulated in GC tissues. 19 Furthermore, hsa_circ_0000419 is lowly expressed in both GC tissues and plasma samples, making it a potential biomarker in GC screening and predictor of patient prognosis. 20 These results strongly indicate that circRNAs play important In this study, we identified a new circRNA, circHN1, by using available public circRNA expression profile data for GC. CircHN1 is formed by reverse splicing of the linear transcript of exons 3-5 of HN1 gene. HN1 mRNA has been shown to be upregulated in certain cancers. [21][22][23][24] Increased HN1 expression has been reported to upregulate c-Met to promote cell growth and migration and is linked to a poor prognosis for patients with hepatocellular carcinoma (HCC). 21 In breast cancer, HN1 can enhance MYC expression to contribute to cancer progression. 22 In prostate cancer, HN1 promotes the migration of cancer cells by negatively regulating the interaction of β-catenin/E-cadherin. 24 Our study showed that a circular RNA was generated from this gene locus, suggesting a critical role for HN1 gene in cancer. However, circHN1 and HN1 mRNA seem to play opposite roles in tumor development.
Generally, highly expressed mRNAs are considered as oncogenes, which affect the development, metastasis, and prognosis of tumors, either directly or indirectly. In our study, we found that circHN1 was overexpressed in GC tissues. However, a high level of circHN1 was negatively associated with tumor invasion. Moreover, circHN1 inhibits the proliferation, migration, and invasion of GC cells in vitro as well as suppresses tumor growth in vivo, which appears contradictory. The difference between mRNAs and circRNAs may explain this inconsistency. For example, it has been reported that cS-MARCA5 (circular RNA of SMARCA5) acts as a sponge for miR-17-3p and miR-181b-5p to inhibit the proliferation and migration of HCC cells, whereas SMARCA5 (mRNA of SMARCA5) activates the Wnt/ beta-catenin signaling pathway to promote HCC proliferation. 25 In addition, circPOK of zbtb7a functions as a proto-oncogenic RNA by co-activating the ILF2/3 complex, which operates separately and antithetically with the linear mRNA which serves as a tumor suppressor. 26 By analyzing the GISTIC and cBioportal databases, we found that the locus of HN1 was amplified in GC tissues, and HN1 mRNA expression was positively related to the copy number alterations of HN1 locus, indicating that amplification of its corresponding chromosomal region may cause overexpression of circHN1.
The contradiction between circHN1 expression and function may also be explained by different miRNAs bound by circRNA.
One circRNA can bind to multiple miRNAs, and different functions of miRNAs may cause different effects of circRNAs on tumors. 12 In this study, we found that circHN1 bound to miR-1248 and miR-375. MiR-1248 has been reported to be upregulated in osteosarcoma, and patients with high miR-1248 expression have poorer survival. 27 MiR-1248 can enhance the chemotherapy resistance of osteosarcoma cells by inhibiting AGTR1. 27 In nephroblastoma, the patients with high expression of miR-1248 also have poor prognosis. 28 MiR-375 plays a dual role in the development and progression of cancer. It acts as an oncomiR in some cancers 29 However, miR-375 was downregulated in GC tissues (P < .01). This result is also consistent with that reported in some other studies, suggesting that the oncogenic role of miR-375 is mainly related to drug resistance, but not to the onset of cancer. 33,34 Therefore, circHN1 may exert complicated effects on GC development and F I G U R E 6 CircHN1 sponges miR-375 and miR-1248. (A) Subcellular distribution of circHN1 in GC cells was detected by qRT-PCR. (B) RIP assay was performed to detect the binding of AGO2 protein to circHN1. (C) Luciferase reporter assays for potential circHN1-interacting miRNAs. (D) Bioinformatic prediction of the putative binding sites in circHN1 for miR-375 and miR-1248 progression by sponging different miRNAs, including miR-1248 and miR-375.
In conclusion, this is the first study to show that circHN1 expression is altered in GC and circHN1 regulates the proliferation and migration of GC cells by sponging miRNAs. CircHN1 may serve as a potential therapeutic target for GC.

ACK N OWLED G M ENTS
This work was supported by the National Natural Science

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