CircHIPK3 Acts as an Oncogene by Sponging miR-124 and Regulating AKT3 Expression in Esophageal Squamous Cell Carcinoma

Purpose: Circular RNAs (circRNAs) are an important type of RNA regulatory factor. Recent studies have demonstrated that circHIPK3 is closely related to the malignant behavior of cancer cells. However, the function of circHIPK3 in esophageal squamous cell carcinoma (ESCC) remains unclear. The aim of the current study was to investigate the value of circHIPK3 for the prognosis of patients with ESCC. Methods: The expression of circHIPK3 in 32 pairs of ESCC and normal tissues were detected by quantitative Real-time polymerase chain reaction (RT-qPCR); the correlation between circHIPK3 expression and the pathological features of patients was also analyzed. Cell biology experiments and bioinformatics were used to explore the function of circHIPK3 in the development of ESCC. Results: The expression of circHIPK3 in tumor tissues of ESCC patients was signicantly higher than that of adjacent tissues. Moreover, knockdown the expression of circHIPK3 retarded esophageal cancer cell proliferation in vitro and in vivo. Mechanistically, we found that circHIPK3 acted as a sponge to absorb miR-124 and promoted AKT3 expression. Conclusion: Our work revealed that circHIPK3, acting as an oncogene, promotes tumor progression in ESCC, and that the circHIPK3- AKT3 axis is a potential therapeutic target for patients with ESCC.


Introduction
Esophageal cancer is one of the most common cancers in human beings with a high mortality [1]. China is a high incidence area of esophageal cancer, and about 150,000 patients die of the disease every year [2]. The histological types of esophageal cancer mainly include esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma. The latter is common in European and American countries, while the incidence of esophageal cancer in China is mainly ESCC, accounting for about 90% [3]. Esophageal cancer is characterized by high metastasis and invasiveness, and lacks markers for early diagnosis. When diagnosed, it is mostly in the middle or late stage. Therefore, the 5-year survival rate of esophageal cancer is only 15% − 25%. Therefore, if we can further clarify the molecular mechanism of the occurrence and development of esophageal cancer and improve the early diagnosis of esophageal cancer, it is expected to improve the cure rate and reduce the mortality rate of esophageal cancer. Circular RNA (circRNA) is a class of non-coding single stranded RNA molecules. The median length of the circRNA was 530 nucleotides [4,5]. circRNAs play an important role in biological and pathological processes, affect cell apoptosis and metabolism, and work as oncogenes or tumor suppressor genes [6].
The closed-loop structure of circRNA determines its highly stable and conservative biological genetic characteristics. Currently, research on the circRNA has shown that the realization of its biological function mainly takes place as the "molecular sponge" of miRNA. By competitive adsorption of miRNA, it leads to its inactivation, and further affects the biological function of the cell. Zheng is the most abundant and has signi cant function in cells [7]. Many studies have found that the expression of circHIPK3 in human tumor cells and normal cells is very different [8][9][10]. Therefore, researchers speculate that circHIPK3 may affect the progression of tumors. At the same time, other studies have shown that circHIPK3 plays an important role in the development of bladder cancer, liver cancer and lung cancer. However, the functions of circHIPK3 in ESCC remain unclear.
As far as we know, this work is the rst to investigate the expression and function of circHIPK3 in ESCC.
We found that circHIPK3 was signi cantly upregulated in 32 pairs of ESCC tissues. Mechanistically, we found that circHIPK3 acted as a sponge to absorb miR-124 and promoted AKT3 expression. These observations indicate a possible novel therapeutic strategy involving circular RNAs in ESCC.

Patients and tissue specimens
The collection and manipulation of human patients' tissues were approved by the Human Research Ethical Committee of Shenzhen Second People's Hospital, The First A liated Hospital of Shenzhen University. Tissues used for research were collected from 32 patients with ESCC who underwent resection at Shenzhen Second People's Hospital from 2018 to 2020. All the patients signed informed consent forms. The tissues were obtained and instantly transported in an ice box to the laboratory. Half part of the tissue was frozen in liquid nitrogen for RNA and protein extraction, and the other part was xed in formaldehyde solution for RNAscope analysis.
Real time-quantitative PCR (RT-qPCR) analysis and In Situ RNA Detection Total RNA was extracted by Trizol (DP424, Tiangen, China) from tissues and cells. The cDNA and RT-qPCR assays were performed using SuperReal PreMix Plus (FP205, Tiangen, China) in the LightCyclerR480II System (Roche) following the manufacturer's instructions. The primers used in this study were in the supplementary table1. RNAscope manual procedure was conducted at Shenzhen Hospital laboratories following standard protocol as previously described with minor modi cations [11].
Nucleic Acid Electrophoresis and Treatment with RNase R.
The cDNA and gDNA PCR products were detected by 2% agarose gel electrophoresis. The DNA was separated by electrophoresis at 180 V for 15 min. The results were then illustrated by UV irradiation. were cultured according to ATCC guidelines at 37°C in a 5% CO2 incubator. KYSE-150 and ECA-109 cells were transfected with circHIPK3 knockdown lentivirus and negative control vectors, following the standard manufacturer's instructions (GENECHEM, Shanghai, China). The stable knockdown cell lines were selected with 4µg/ml of puromycin treatment after 72h of transfection. The e ciency of knockdown was tested by RT-qPCR.

Cell proliferation and Colony formation assays
To conduct cell proliferation assays, CCK-8 kit (Dojindo, Kumamoto, Japan) and EdU Apollo® 567 In Vitro Imaging Kit (Ribobio, Guangzhou, China) were used according to the manufacturer's instruction. To conduct the colony formation assay, 2.5×10 3 cells were seeded into six-well plate. 10 days later, the colonies were xed and stained according to the manufacturer's instruction (Beyotime, Beijing, China).
Then, visible colonies were photographed (Nikon, Tokyo, Japan) and calculated. All experiments were repeated three times.

Transwell analysis
The KYSE-150 and ECA-109 cells were prepared into serum-free cell suspension. Each Transwell chamber was inoculated with 2 × 10 4 cells, and the lower Transwell chamber was added with 500 µ l serum containing DMEM. The cells were incubated in 5% CO2 incubator at 37 ℃ for 24 h, the cells were wiped with cotton swab, xed with 4% paraformaldehyde for 30 min, and stained with 0.1% crystal violet for 20 min at room temperature. Five visual eld cells were randomly selected under microscope, photographed and counted. The experiment was repeated three times.

Western Blotting
Western Blotting was performed as described previously [12,13]. The antibodies used for western blot are

Renilla luciferase
The fragment of AKT3 3' UTR containing the binding site of miR-124 was spliced to the 3'-end of the Renilla luciferase reporter gene. The wildtype or mutant circHIPK3 and miR-124 binding sites were subcloned into psiCHECK-2 system (Promega, Madison, WI, United States).

Animals
A total of 50 BALB/c nude mice were chosen and assigned to 2 groups: shCtrl group (injected with ECA-109 cells) and sh-circHIPK3 group (injected with ECA-109 cells with circHIPK3 knockdown). 200 ul of the above cell suspension containing 2 *10 5 cells was injected into the left or right back of each mice. Tumor sizes and tumor volume were measured as described previously [14]. We euthanized mice with carbon dioxide. The mice were anesthetized with ether.

Statistical Analysis
Statistical analyses were performed using excel or Graphpad Prism software version 8.0 (USA). Experimental data are described as mean ± SEM. The signi cance of the observed differences was measured via the Student's t-test or chi-square test. P < 0.05 was served to be statistically signi cant.

Results
The expression patten of circHIPK3 in ESCC tumor tissues.
To elucidate the functional roles of circHIPK3 in ESCC, we investigated the expression of circHIPK3 in 32 patients' tumor tissues and adjacent tissues by using RT-qPCR and RNAscope. Two sets of primers for circHIPK3 were designed. The rst set contained a divergent primer that ampli es only circHIPK3. The second set of primers contained an opposite-directed primer to detect the HIPK3 mRNA. Using cDNA and gDNA (genomic DNA) from ESCC tissues, circHIPK3 was only ampli ed by divergent primers in cDNA, and no ampli cation product was observed in gDNA (Fig. 1A). Sanger sequencing con rmed the cyclization site sequence (Fig. 1B). RNase R was used to digest the RNA in ESCC tissues. RT-qPCR results showed that the expression level of circHIPK3 did not change signi cantly before and after RNase R treatment, but the expression level of linear HIPK3 mRNA decreased signi cantly (Fig. 1C, P < 0.01). We then identi ed that the expression of circHIPK3 was markedly higher in ESCC tissues compared with the adjacent normal tissues. High expression of circHIPK3 was detected in 21/32 (66%) ESCC ( Fig. 1D and Table 1). Higher expression of circHIPK3 was also proved by RNAscope analysis (Fig. 1E). In addition, we also found a signi cant correlation between circHIPK3 expression and clinical features. The results revealed that patients with higher expression of circHIPK3 exhibited lymph node metastasis, larger tumor size and poorly tumor differentiation (Table 1). In order to choose the suitable cell lines for in vitro experiments, the expression of circHIPK3 was detected by RT-qPCR. Consistent with results in tumor tissue, the expression of circHIPK3 was higher in ESCC cell lines than the normal human esophageal epithelial cell line (HEEC) (Fig. 1F). Then, we selected KYSE-150 and ECA-109 cell lines with the highest circHIPK3 expression to investigate the functions of circHIPK3 in vitro.

CircHIPK3 promotes proliferation and migration of ESCC cell lines in vitro and in vivo
To further investigate if circHIPK3 was correlated with ESCC progression, shRNA speci cally targeting circHIPK3 was transfected into KYSE-150 and ECA-109 cells by lentivirus infection, respectively. The results from RT-qPCR displayed that shRNA speci cally downregulated the expression of circHIPK3 ( Fig. 2A). To explore the role of circHIPK3 knockdown on cell proliferation, CCK-8 and EdU proliferation assays were conducted. CCK-8 and EdU analysis showed that cell proliferation in both KYSE-150 and ECA-109 cell lines were blocked after circHIPK3 knockdown (Fig. 2B-D). Additionally, knockdown circHIPK3 expression decreased growth ability as a result of fewer colonies formed after 9 days than the shCtrl group in both cell lines (Fig. 2E-F). We further tested whether the expression of circHIPK3 in uenced cell migration or not. While knockdown the expression of circHIPK3 signi cantly reduced the migration of KYSE-150 and ECA-109 cells (Fig. 2G). In addition, knockdown the expression of circHIPK3 clearly reduced the expression of E-cadherin and Vimentin (Fig. 2H); To explore whether knockdown the expression of circHIPK3 could reduce tumor growth and migration in vivo, normal expression of circHIPK3 and knockdown expression of circHIPK3 ECA-109 cells were seeded into the nude mouse, respectively. The tumor growth and migration were monitored (Fig. 2I-J). The in vivo experiments showed that knockdown expression of circHIPK3 reduced tumor growth and migration.
CircHIPK3 interacts with miR-124 to meditate ESCC cell lines proliferation and migration It has been found that circHIPK3 functions as "miRNA sponge" in various cancers. To address whether circHIPK3 could sponge miRNAs in ESCC cells, we selected candidate miRNAs from the experiment results of miRNA recognition elements in circHIPK3 datasets by Circ2Disease (http://bioinformatics.zju.edu.cn/Circ2Disease/index.html). The Circ2Disease database suggested that miR-124 was the target gene of circHIPK3 that had been veri ed most times. To investigate the relationship between miR-124 and circHIPK3, we performed luciferase reporter assays in KYSE-150 and ECA-109 cells. As shown in Fig. 3, we observed that co-transfection of miR-124 and circHIPK3 markedly suppressed the luciferase activity compared with that from the co-transfection of miR-124 and circHIPK3-MUT ( Fig. 3A and B). Moreover, the expression of miR-124 was up-regulated after knockdown of circHIPK3 (Fig. 3C). To explore the role of miR-124 in ESCC, we performed CCK-8 assays to detect the relationship between the expression of miR-124 and cell proliferation. As illustrated in Fig. 3, miR-124 overexpression markedly reduced cell proliferation which could be rescued by co-transfection with circHIPK3 in ESCC cell lines (Fig. 3D). Transwell analysis proved that miR-124 markedly reduced the migration of KYSE-150 and ECA-109 cells, whereas it was reversed by circHIPK3 overexpression (Fig. 3E). Western blot analysis illustrated that miR-124 overexpression-reduced the expression of E-cadherin and Vimentin (Fig. 3F). A reverse correlation was discovered between miR-124 and circHIPK3 in the real-world tumor tissues (Fig. 3G).

AKT3 is a target of miR-124 and is meditated by circHIPK3
Notably, the RTK-MAPK-PI3K pathway is frequently dysregulated by multiple molecular mechanisms in ESCC, suggesting that the genes involved in this signaling pathway may be the target genes of miR-124. Based on bioinformatics prediction using TargetScan (http://www.targetscan.org/vert_72/), Akt3 was screened out as a prime target, with a highly conserved complementary miR-124-binding site in its 3' UTR across vertebrates from Lizard to Human (Fig. 4A). To con rm miR-124 function at the AKT3 3' UTR, we performed luciferase reporter assays in KYSE-150 and ECA-109 cells. As shown in Fig. 4, we found that miR-124 up-regulation markedly suppressed the luciferase activity when co-transfected miR-124 with wild-type AKT3 3' UTR vectors (Figs. 4B and C). Moreover, miR-124 overexpression repressed the expression of AKT3, whereas it was reversed by circHIPK3 overexpression (Figs. 4D). Knockdown the expression of AKT3 obviously suppressed the cell proliferation and migration (Figs. 4E and F). In order to further verify the synergy of circHIPK3/miR-124/AKT3 in ESCC, we detected the correlation of circHIPK3/miR-124/AKT3 expression in ESCC tumor tissues. It is of note that the expression of AKT3 was positively correlated with the high expression of circHIPK3 (P < 0.01, Figs. 4G and H), which suggested that circHIPK3 might regulate the expression of AKT3 by sponging miR-124.

Discussion
ESCC is one of the most lethal malignant tumors in the world, and its treatment is limited [3]. In addition, the biological mechanism of the occurrence and development of ESCC is still largely unknown. Therefore, it is urgent to explore the biological mechanisms of ESCC to identify molecular biomarkers for early diagnosis and prognosis. CircRNA is a long noncoding RNA (ncRNA), whose transcript length is usually more than 200 and contains no coding region [15]. Emerging evidence has revealed that circRNA plays a critical role in different types of human cancers [16]. As a miRNA sponge, circRNA regulates many biological processes by regulating the function of miRNA, affecting RNA splicing, chromatin structure and mRNA stability [17,18].
Current studies have found that circHIPK3 plays a dual role in a variety of human cancers. The overexpression of circHIPK3 can effectively reverse the miR-7-induced decrease in the progression of colorectal cancer cells by up-regulating the expression of several key miR-7 target genes (including EGFR, IGF1R, FAK and YY1) [8]. In gallbladder cancer cells, knockdown the expression of circHIPK3 could increase the expression of miR-124, thereby reducing tumor cell proliferation and survival [19].
Overexpression of circHIPK3 sponges miR-124, inhibits its activity and increases the expression of its target genes, such as IL6R and DLX2, thereby causing tumor cell growth. However, in some kinds of cancer, circHIPK3 also displays the function of inhibiting tumor growth. Overexpression of circHIPK3 signi cantly inhibited the growth of bladder cancer in vivo [20], while knockdown the expression of circHIPK3 promoted the proliferation of ovarian cancer cells (A2780 and SKOV3) and normal ovarian epithelial cells [21]. These studies proved that circHIPK3 participated in complex regulatory networks, and confers cell-type-speci c regulation of cell function in different cancers.
In this study, we investigated the expression of circHIPK3 in ESCC tumor tissues and ESCC cell lines. We further identi ed that circHIPK3 negatively regulated the expression of miR-124 in ESCC cell lines. Our in vitro experiments showed that miR-124 suppressed cell proliferation and migration in ESCC cells. Moreover, the expression of miR-124 was remarkably down-regulated in ESCC tumor tissues.
Therefore, we suggested that miR-124 functioned as a tumor suppressor gene that was dependent on circHIPK3 in ESCC.
Moreover, our data for the rst time proved that AKT3 was suggested to be a target gene of miR-124. AKT3 encodes a serine/threonine protein kinase that belongs to the AGC kinase family [22]. AKT-kinases play roles in signaling pathways involved in cell proliferation, oncogenic transformation, cell survival, cell migration, and intracellular protein tra cking [23,24]. AKT3 alterations have been related with tumor growth and migration. Previously, gain of function mutations in AKT3 were found in several cancer types, such as breast cancer and endometrium cancer, which were relied on the mediation of PI3K signaling pathways [25,26]. In this study, using luciferase and western blot analysis, we found that miR-124 negatively modulated AKT3 expression. In ESCC tumor tissues, AKT3 expression was highly increased, which was in line with the TCGA database. Intriguingly, promoting the expression of circHIPK3 enhanced AKT3 expression in ESCC cells, while the opposite result was observed in circHIPK3-knockdown cells. Moreover, enhancing AKT3 expression signi cantly enhanced ESCC cell proliferation and migration, which were in line with those promoting tumorigenesis in various cancers [25].

Conclusion
In summary, for the rst time in the present study, our results indicated that circHIPK3 functioned as an oncogenic circRNA that enhanced ESCC tumorigenesis and progression through the miR-124/ AKT3 pathway. The ndings here indicate that a circHIPK3-miR-124/ AKT3 axis may be a potential therapeutic target for ESCC.

Declarations
Ethics approval and consent to participate

Consent for publication
Participants have given their consent that data from this study can be published in an anonymized form.

Data availability statement
All data generated or analyzed during the present study are included in this published article.

Con ict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or nancial relationships that could be construed as a potential con ict of interest.