Circ_0110940 Exerts an Antiapoptotic and Pro-Proliferative Effect in Gastric Cancer Cells via the miR-1178-3p/SLC38A6 Axis

Circular RNAs (circRNAs) are essential regulators in human cancers, including gastric cancer, by the miRNA/mRNA axis. A previous study identified the upregulation of circ_0110940 in human gastric cancer tissues. The present study performed in vitro assays to reveal the functions of circ_0110940 and its downstream miRNA/mRNA axis in gastric cancer cells. Traditional proliferation and apoptosis assays including colony formation, EdU staining, and Annexin V-PI staining assays were conducted. A luciferase reporter assay was performed to assess the binding between miR-1178-3p and circ_0110940 or SLC38A. We found the significant upregulation of circ_0110940 in human gastric cancer cells AGS and MKN45. Circ_0110940 was a stable circRNA and exerted an antiproliferative and proapoptotic effect in AGS and MKN45. Circ_0110940 binded with miR-1178-3p, which further targeted SLC38A6 3′UTR. Circ_0110940 degraded miR-1178-3p, and miR-1178-3p degraded SLC38A6. Thus, circ_0110940 has a positive effect on SLC38A6 expression. Furthermore, SLC38A6 rescued the effects of circ_0110940 knockdown on gastric cancer cell proliferation and apoptosis. In conclusion, circ_0110940 exerted an antiapoptotic and pro-proliferative effect in gastric cancer cells via the miR-1178-3p/SLC38A6 axis, which may provide basis for the targeted therapy of gastric cancer.


Introduction
Cancer is a malignant disease and causes a great burden on the global population. e traditional methods for treating cancer consist of surgery, radiotherapy, and chemotherapy. New treatments including targeted therapy, immunotherapy, cryoablation, and hormone therapy have been developed [1][2][3][4]. Targeted therapy has a unique strength because of its specificity to cancer cells while avoiding causing toxicity to off-target cells [2]. Gastric cancer is the sixth most common cancer with new cases of 1,089,103 and the second leading cause of cancer death with new deaths of 768,793 worldwide in 2020 [5]. ree drugs including trastuzumab [6], ramucirumab [7], and pembrolizumab [8] are currently approved by the FDA to treat gastric cancer. ese drugs have good effects on breast cancer and colon cancer. However, they are not effective enough for treating gastric cancer. us, to explore novel, effective targeted therapy for gastric cancer is needed.
Circular RNAs (circRNAs) are endogenous biomolecules that are regulated by specific cis-acting elements and trans-acting factors.
ese covalently closed biomolecules are expressed in specific cells and tissues in eukaryotes. Compared with linear RNAs, circRNAs lack 5′ and 3′ ends and are resistant to RNA exonuclease, which makes them serve as promising biomarkers and therapeutic targets. Advances in circRNA-specific computational tools and high-throughput RNA sequencing have promoted the detection, quantification, and functional characterization of circRNAs [9]. Many circRNAs act as microRNA (miRNA) inhibitors ("sponges") to exert their regulatory functions and are implicated in cancers including gastric cancer by modulating miRNAs. Circ_0008259, derived from LMO7 pre-mRNA, is highly expressed in gastric cancer, promoting the proliferation, motility, and regulating the glutamine metabolism of gastric cancer cells by "sponging" miR-30a-3p [10]. High expression of circ-RanGAP1 has a significant association with lymph node metastasis, advanced TNM stage, and worse survival in patients with gastric cancer and contributes to gastric cancer progression dependent on the miR-877-3p/VEGFA pathway [11]. Epstein-Barr virusencoded circLMP2A induces and maintains stemness phenotypes in gastric cancer by inhibiting miR-3908 [12].
According to a previous study by Wang et al. [13], circRNA located at chr1: 1572769-1635783 (annotated as circ_0110940 by circBase [14]) showed the most significantly abnormal expression in gastric cancer tissues compared with para-gastric cancer tissues (log 2 FC � 6.99, P � 2.35°Ε −06 ). Circ_0110940 is formed by back-splicing of the exons 2-10 of CDK11B pre-mRNA. According to the circBase annotation, circ_0110940 has a genomic length of 63014 and a spliced length of 48427. Despite the fact that there are some studies demonstrating the association of CDK11B and tumorigenesis [15,16], no published papers have revealed the role of circ_0110940. We detected expression of circ_0110940 in gastric cancer cells and identified its upregulation. Next, the functions of circ_0110940 and its downstream miRNA/ mRNA axis in gastric cancer cells were investigated.

RNase R Digestion Test.
e total RNA of gastric cancer cells was extracted and divided into two parts. One part was digested: 2.5 μg total RNA was added with 3 U RNase R and incubated at 37°C for 20 min; another part was added with 0.5 μL DEPC water as a control. After the reaction, RNA reverse transcription and PCR amplification were performed to detect the expression level of RNA.

Actinomycin D Test.
e logarithmic gastric cancer cells were inoculated into 6-well plates with 7 × 10 4 cells per well and cultured for 24 h. en, fresh complete medium containing 2 μg/ml actinomycin D was added. Cell RNA was extracted at 1 h, 2 h, 6 h, 8 h, and 10 h after treatment, and the circRNA and mRNA abundance were observed after actinomycin D treatment by RT-qPCR.

Proliferation Detection.
A colony formation assay and an EdU staining assay were used to assess cell proliferation. For the colony formation assay, cells were seeded in a 6-cm plate at a density of 500/well and cultured in a complete culture medium for two weeks. After discarding the culture solution, cells were fixed with methanol for 10 min, treated with crystal violet dye for 15 min, washed with running water, and dried. Colonies (>50 cells) were finally photographed and counted. For the EdU staining assay, cells were seeded into 96-well plates at a density of 6,000 cells/well and transfected for 24 h. Cells were incubated with the EdU reagent A for 2 h in an incubator at 37°C. Following the manufacturer's instructions of a Cell-Light ™ EdU Apollo643 2 Journal of Oncology In Vitro kit (C10310-2, RiboBio, Guangzhou), subsequent experimental steps were performed.

Annexin V-PI Staining.
AGS and MKN45 cells were seeded in a 6-well plate at a concentration of 5.0 × 10 4 cells/well and transfected for 24 h. Cells were centrifugated and resuspended in 195 μL Annexin V-FITC binding solution. Next, the cells were stained with 5 μL Annexin V-FITC and 10 μL PI using an apoptosis detection kit (C1062S, Beyotime) in the dark at room temperature for half an hour. Annexin + PI + and Annexin + PI − cells were considered as apoptotic cells. Cells were analyzed using a Beckman Cyan flow cytometer on the CellQuest 7.1 software (Beckman).

Statistical Analysis
. P values were derived by the Student's t-test and one/two-way analysis of variance using the GraphPad prism tool. Any value reported in graphs is an average of three independent experiments, and error bars represented SD. P < 0.05 indicates statistical significance.

Validation of the Circular Characteristics of circ_0110940.
Circ_0110940 is formed by back-splicing of the exons 2-10 in the CDK11B pre-mRNA and is annotated by circBase to have a genomic length of 63014 and a spliced length of 48427 (Figure 1(a)). It showed significant upregulation in AGS and MKN45 cells compared with the control GES-1 cell line (Figure 1(b)). e results of the RNase R exonuclease assay using RT-qPCR revealed that circ_0110940 was resistant to digestion, while CDK11B was not resistant to digestion, indicating the circular nature of circ_0110940 (Figure 1(c)). Moreover, circ_0110940 is more resistant to actinomycin D than CDK11B, further demonstrating the circular characteristics of circ_0110940 (Figure 1(d)).

Circ_0110940 Knockdown Exerts a Proapoptotic and
Antiproliferative Effect in AGS and MKN45 Cells. We used sh-circ_0110940 targeting circ_0110940 to knockdown circ_0110940 in AGS and MKN45 cells, and RT-qPCR verified the knockdown efficiency of circ_0110940 (Figure 2(a)). Sh-circ_0110940 reduced the number of colonies that were formed by AGS and MKN45 cells and decreased the percentage of EdU-positive cells, indicating that silenced circ_0110940 had a negative effect on gastric cancer cell proliferation (Figures 2(b) and 2(c)). Sh-circ_0110940 increased the percentage of Annexin + PI + and Annexin + PI − cells, revealing the promotive influence of silenced circ_0110940 on gastric cancer cell apoptosis (Figure 2(d)). Moreover, two apoptosis indexes, Bax and cleaved caspase-3, were detected using Western blotting. Sh-circ_0110940 increased the protein expression of Bax and cleaved caspase-3 (Figure 2(e)).

SLC38A6 Rescued the Effects of Silenced circ_0110940 on Gastric Cancer Cell Proliferation and Apoptosis.
Finally, we cotransfected sh-circ_0110940 and ov-SLC38A6 in MKN45 cells. As shown in Figure 5(a)-5(d), silenced circ_0110940 presented antiproliferative and proapoptotic effect in MKN cells. However, the influence on MKN45 cell proliferation and apoptosis induced by sh-circ_0110940 should be partially counterbalanced by cotransfection with ov-SLC38A6. Collectively, the above-mentioned results indicated that circ_0110940 modulated the proliferation and apoptosis of gastric cancer cells by upregulating SLC38A6 (Figure 5(a)-5(d)).        Journal of Oncology

Discussion
Several circRNAs participate in the modulation of gastric cancer cell biological activities [10][11][12][13]. In this study, we focused on circ_0110940 in gastric cancer cells. Circ_0110940 is formed by back-splicing of the exons 2-10 in the CDK11B pre-mRNA. CDK11 B is a member of CDKs that significantly mediate tumor cell proliferation and growth [21]. CDK11 B has the potential to promote the selfrenewal capability of hepatocellular carcinoma stem cells and enhance their oncogenicity in vivo [16] and is associated with the poor prognosis of colon cancer [15]. After the validation of the circular feature of circ_0110940, its functions were evaluated. It was found that shRNA-mediated circ_0110940 knockdown exerts a proapoptotic and antiproliferative effect in AGS and MKN45 cells, indicating the oncogenic role of circ_0110940 in gastric cancer. Moreover, the downstream axis of circ_0110940 was searched using bioinformatics analysis and verified by experiments. MiR-1178-3p was identified to bind to circ_0110940 and showed downregulation in gastric cancer cells, which indicated that miR-1178-3p is an antioncogene in gastric cancer. However, several studies have demonstrated the tumor promoter role of miR-1178-3p in cancers. MiR-1178-3p has been reported to be "sponged" by circFNDC3B in bladder cancer and contributes to malignant phenotypes of bladder cancer cells [22]. MiR-1178-3p exerts an oncogenic role to facilitate nasopharyngeal carcinoma cell proliferation and motility by binding to STK4 [23]. MiR-1178-3p is "sponged" by hsa_circ_0077837 and rescued its suppressive effects on non-small-cell lung cancer cell proliferation, viability, and motility [24]. On the contrary, miR-1178-3p regulates the PI3K/Akt pathway by targeting TBL1XR1 to suppress hepatocellular carcinoma cell growth in vitro and inhibit xenograft tumor growth in vivo [25]. Next, we found that miR-1178-3p potentially targets SLC38A6 based on bioinformatics analysis. As a member of the SLC38 family, SLC38A6 is selectively expressed in the excitatory neurons of the brain [26] and is deemed as an orphan transporter with an unknown substrate profile [27]. A previous study revealed the upregulation of SLC38A6 in pancreatic adenocarcinoma [28]. In our study, upregulation of SLC38A6 in gastric cancer cells was verified after we identified that it exhibited higher expression in gastric cancer tissues than control tissues based on bioinformatics analysis. SLC38A6 serves as a direct target of miR-1178-3p in gastric cancer cells. MiR-1178-3p can cause SLC38A6 degradation by binding to the 3′UTR of SLC38A6. Considering that circ_0110940 can degrade miR-1178-3p, positive regulation of circ_0110940 on SLC38A6 was obvious.
However, some limitations of this study must be addressed. First, we lacked an in vivo assay to verify the oncogenic role of circ_0110940. Second, expression of circ_0110940/miR-1178-3p/SLC38A6 in human gastric cancer tissues needs to be validated. Moreover, since gastric cancer exhibits high metastasis, the effects of circ_0110940/miR-1178-3p/SLC38A6 on cell migration and invasion deserve further exploration.
In conclusion, circ_0110940 promoted cell proliferative ability and inhibited cell apoptotic capacity in gastric cancer cells via the miR-1178-3p/SLC38A6 axis, which may provide a basis for the targeted therapy of gastric cancer.

Data Availability
All data generated or analyzed during this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
e authors declare that there are no conflicts of interest.

Authors' Contributions
Xiaonan Miao conducted the experiment, analyzed the data, and wrote this manuscript. Yong Su proofread the manuscript and supervised this study. All authors had perused and reached a consensus on the final manuscript.