CircPTK2 Suppresses the Progression of Gastric Cancer by Targeting miR-196a-3p/AATK Axis Ling Gao The First Aliated Hospital of Soochow University Tingting Xia The First Aliated Hospital of Soochow University Mingde Qin Soochow University Xiaofeng Xue The First Aliated Hospital of Soochow University Linhua Jiang The First Aliated Hospital of Soochow University Xinguo Zhu ( [email protected] ) The First Aliated Hospital of Soochow University https://orcid.org/0000-0001-9686-6797 Primary research Keywords: Gastric cancer, circPTK2, miR-196a-3p, AATK Posted Date: June 8th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-569952/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/20 Abstract Background Gastric cancer is a type of malignant tumor with high morbidity and mortality. It has been shown that circular RNAs (circRNAs) exert critical functions in gastric cancer progression via working as microRNA (miRNA) sponges to regulate gene expression. However, the role and potential molecular mechanism of circRNAs in gastric cancer remain largely unknown. Methods CircPTK2 (hsa_circ_0005273) was identied by bioinformatics analysis and validated by RT-qPCR assay. Bioinformatics prediction, dual-luciferase reporter and RNA pull down assays were used to determine the interaction between circPTK2, miR-196a-3p, AATK. Results The level of circPTK2 was markedly downregulated in gastric cancer tissues. Upregulation of circPTK2 suppressed the proliferation, migration and invasion of gastric cancer cells, while downregulation of circPTK2 exhibited opposite effects. Mechanically, circPTK2 could competitively bind to miR-196a-3p and prevent miR-196a-3p to reduce the expression of AATK. In addition, circPTK2 overexpression inhibited tumorigenesis in a xenograft mouse model of gastric cancer. Conclusion Collectively, circPTK2 functions as a tumor suppressor to suppress gastric cancer cell proliferation through regulating miR-196a-3p/AATK axis, suggesting that circPTK2 may serve as novel therapeutic target for gastric cancer. Introduction Gastric cancer is the fth most common malignancy worldwide and is the third leading cause of cancer- related deaths [1-3]. Although the current clinical diagnosis and treatment for gastric cancer are continuously improving, the 5-year survival rate of patients with gastric cancer is less than 30% [4, 5]. Gastric cancer is a complex cellular network, currently, the etiology and pathogenesis are not yet clear, which brings corresponding diculties to its early diagnosis and treatment [6, 7]. Circular RNAs (circRNAs) are a class of non-coding RNAs derived from back-spliced exons [8, 9]. Unlike linear RNA, circRNAs are covalently closed continuous loops that lack 5' (cap) and 3' (polyadenylation) ends [10]. Meanwhile, circRNAs are found to be relatively stable and evolutionally conserved in the cytoplasm [10, 11]. CircRNAs have been found to be implicated in the progression of cancers [12, 13]. Signicantly, circRNAs are aberrantly expressed in multiform types of cancer, including gastric cancer [14, 15]. However, the biological function of circRNAs in gastric cancer remains largely unclear. Page 2/20 MicroRNAs (miRNAs) are a kind of non-coding RNAs with 19-25 nucleotides in length [16, 17]. It has been shown that miRNA can regulate gene expression at the post-transcriptional level [18, 19]. Considerable studies reported that circRNAs could exhibit their biological roles through acting as “miRNA sponges” to regulate gene expressions [20, 21]. For instance, zhang et al showed that circNRIP1 could promote gastric cancer progression through sponging miR-149-5p [22]. Luo et al found that circCCDC9 inhibited gastric cancer cell proliferation via targeting miR-6792-3p/CAV1 axis [23]. In this study, we screened differentially expressed circRNAs (DEcircRNAs) in gastric cancer and found that circPTK2 was signicantly downregulated in gastric tissues. In addition, circPTK2 could inhibit proliferation, migration and invasion of gastric cancer cells through functioning as a miRNA sponge to upregulate the expression of tumor suppressor gene AATK. These data indicated that circPTK2 may be used as a potential target in gastric cancer therapy. Materials And Methods Identication ofdifferentially expressed circRNAs (DEcircRNAs) Gastric cancer-related datasets (GSE93541, GSE89143 and GSE78092) were downloaded from the GEO database. For GSE93541 dataset, R language was utilized to analyze the DEcircRNAs in plasma samples from gastric cancer patients and healthy controls. For GSE89143 and GSE78092 datasets, R language was utilized to analyze the DEcircRNAs between gastric cancer tissues and adjacent normal tissues. The threshold value of differentially expressed genes was set at 2 times of different multiple and p<0.05. The intersection of DEcircRNAs from three datasets was performed using the Venn Diagram package. Specimen collection Gastric cancer tissues and matched adjacent normal tissues were obtained from the First Aliated Hospital of Soochow University. The written consent was obtained from each patient with gastric cancer. All samples were frozen in liquid nitrogen and stored at -80°C. This study was approved by the ethics committee of The First Aliated Hospital of Soochow University. Cell culture Human AGS and MKN45 cells were obtained from ATCC (Manassas, VA, USA). Cells were maintained in DMEM medium (Thermo Fisher Scientic, Waltham, MA, USA) containing 10% fetal bovine serum (FBS) in a 5% CO2 incubator at 37°C. Cell transfection MiR-196a-3p mimics and miR-196a-3p inhibitor were designed and synthesized by Ribobio (Guangzhou, China). After that, AGS and MKN45 cells were transfected with miR-196a-3p mimics or miR-196a-3p inhibitor using the Lipofectamine 2000 kit (Thermo Fisher Scientic). Page 3/20 Human circPTK2 or AATK cDNA was synthesized and cloned into pcDNA3.1 vector. After that, AGS and MKN45 cells were transfected with the pcDNA3.1 control plasmid, pcDNA3.1-circPTK2 (circPTK2-OE) or pcDNA3.1 AATK (AATK-OE) using Lipofectamine 2000, followed by selected with G418. Lentivirus-containing short hairpin RNA (shRNA) targeting circPTK2 or AATK plasmids were purchased from Hanbio (Shanghai, China). After that, 293T cells were transfected with above-mentioned lentiviral plasmids were transduced into 293 T cells to package lentivirus to infect AGS and MKN45 cells. Subsequently, the infected cells were selected by 2 µg/mL of puromycin. RT-PCR and RT-qPCR TRIzol Reagent (Thermo Fisher Scientic) was used to extract total RNA. Meanwhile, genomic DNA (gDNA) was isolated using the Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, China). After that, cDNA was synthesized using EntiLink™ 1st Strand cDNA Synthesis Kit (ELK Biotechnology). Later on, the DreamTaq DNA Polymerase (Thermo Fisher Scientic) was used for PCR. Then, the cDNA and gDNA PCR products were analyzed 2% agarose gel electrophoresis. In addition, qPCR was performed using the EnTurbo™ SYBR Green PCR SuperMix on a Verse ow cytometry system (BD Biosciences). β-actin and U6 were used as internal controls. The primers were listed in Table 1. Actinomycin D and RNase R treatment AGS and MKN45 cells were incubated with Actinomycin D (2 μg/ml; Sigma) for 0, 6, 12, 18 and 24 h to assess the stability of circPTK2 and its linear isoform. In addition, total RNA (10 μg) was treated with RNase R (5 U/μg; Epicenter Technologies) for 30 min at 37°C, then, the level of circPTK2 was detected using RT-qPCR assay. Cell viability assay Transfected AGS and MKN45 cells were seeded onto 96 well plates and cultured for the indicated times. Later on, cell viability was determined using the Cell Counting Kit-8 (CCK-8) reagent (Dojindo Laboratories, Kumamoto, Japan). Subsequently, the absorbance was measured at a wavelength of 450 nm. Colony formation assay Transfected AGS and MKN45 cells were plated onto 6 well plates. After 2 weeks of incubation, cells were xed with 4% paraformaldehyde for 20 min and then stained with 0.1% crystal violet at room temperature. After that, cell colonies were imaged and counted using a light microscope. Transwell assay Transfected AGS and MKN45 cells were suspended in 200 μL serum-free medium and placed into the upper chambers (Corning, NY, USA). Later on, the lower chambers were loaded with DMEM medium (600 μL) containing 10% FBS. After 24 h of incubation, the cells on the lower surface were xed with 4% formaldehyde, and then stained with 0.1% crystal violet solution. After that, the stained cells were imaged Page 4/20 using a light microscope. Transwell chambers that coated with Matrigel were used for cell invasion assay. Animal study BALB/c nude mice (5–6 weeks old) were purchased from the Jingda experimental animal Co., Ltd. (Changsha, China). This study was approved by the First Aliated Hospital of Soochow University and conducted according to the institutional guidelines. Animals were divided into 4 groups (shRNA NC; circPTK2 shRNA2; OE NC; circPTK2 OE). AGS or MKN45 cells (5 x 106 cells/mouse) were subcutaneously injected into the right ank of each mouse. The size of the tumor was measured every 5 days. The tumor volume was calculated by the formula: (length x width2)/2. In the end of the experiment, the tumor was removed, and tumor weight was measured. Luciferase reporter assay The sequences including miR-196a-3p binding sites in the circPTK2 3’ UTRs and AATK 3’ UTR were cloned into the luciferase reporter vector pGL6-miR (Beyotime). After that, AGS or MKN45 cells were co- transfected with the luciferase plasmids and miR-196a-3p mimics for 48 h. Later on, the rey and Renilla luciferase activities were measured by a dual-luciferase reporter assay system (Promega, USA). RNA-pull down assay The biotinylated circPTK2 or biotinylated miR-196a-3p probe were incubated with Streptavidin magnetic beads (Thermo Fisher Scientic) for 2 h at room temperature. Later on, AGS or MKN45 cells were incubated with the magnetic beads at 4°C overnight. After that, the complex was pulled down and analyzed by RT-qPCR assay. Immunohistochemistry (IHC) The tumor tissues were xed in 4% paraformaldehyde and then embedded in paran.