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Circpip5k1a Facilitates Gastric Cancer Progression Via Mir-376C-3P

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Circpip5k1a Facilitates Gastric Cancer Progression Via Mir-376C-3P Ma et al. Cancer Cell Int (2020) 20:81 https://doi.org/10.1186/s12935-020-1122-5 Cancer Cell International PRIMARY RESEARCH Open Access CircPIP5K1A facilitates gastric cancer progression via miR-376c-3p/ZNF146 axis Yan Ma1†, Xiliang Cong1†, Yiyun Zhang2, Xin Yin1, Ziyu Zhu1 and Yingwei Xue1* Abstract Background: Recently, many emerging circular RNAs (circRNAs) have been studied in human malignancies, includ- ing gastric cancer (GC). Researches concerning cancers have revealed that aberrant expression of circRNAs play a big part in tumorigenesis and development of diverse malignant tumors. Although hsa_circ_0014130 (circPIP5K1A) has been confrmed to be closely related to non-small cell lung cancer (NSCLC) progression, the knowledge of its function on GC progression remains unclear. Therefore, it is of great interest to uncover the underlying role of circPIP5K1A in GC. Methods: The expression and characteristic of circPIP5K1A were separately analyzed by RT-qPCR, nucleic acid elec- trophoresis, RNase R and Actinomycin D treatment. CCK-8, colony formation, EdU, transwell, TUNEL, fow cytometry, luciferase reporter, RIP and RNA pull-down assays were employed to testify the regulatory role of circPIP5K1A in GC. Results: In current study, circPIP5K1A, featured with closed-loop structure, was proved to be highly expressed in tis- sues and cells of GC. Loss-of-function assays depicted that silencing circPIP5K1A suppressed GC development. Follow- up mechanism tests unveiled that circPIP5K1A bound with miR-376c-3p and inhibition of miR-376c-3p reversed circ- PIP5K1A downregulation-mediated efect on GC progression. Additionally, ZNF146 was verifed to be the downstream molecule of circPIP5K1A/miR-376c-3p axis in modulating GC progression. Conclusions: circPIP5K1A stimulates GC progression by sponging miR-376c-3p to upregulate ZNF146 expression. Keywords: Gastric cancer, CircPIP5K1A, miR-376c-3p, ZNF146 Background frequent recurrence and metastasis of malignancy, the Gastric cancer (GC), one of the most prevalent fatal neo- prognosis of GC patients at advanced stage is still poor, plasms, ranks the second factor that leads to the occur- characterized by a low 5-year overall survival rate [7]. rence of cancer-related death in human all over the Te tumorigenesis and development of GC are extremely world [1–3]. GC has been frequently diagnosed with complicated biological courses regulated by aberrant high incidence in many Asian countries, especially China expression of oncogenes or dysregulation of anti-tumor and Japan [4, 5]. In the past years, with rapid progress genes [8–11]. Tus far, the knowledge of potential molec- in diagnostic apparatus and therapeutic methods, the ular mechanisms in GC remains poorly understood. To morbidity and mortality ratio of GC appeared to be on identify efective targeted therapies, further exploration a stably downward trend [6]. However, because of the and elucidation on the underlying molecular mechanisms involved in GC are urgent events for investigators. Circular RNAs (circRNAs), a newly identifed type *Correspondence: [email protected] † of endogenous non-coding RNAs characterized with Yan Ma and Xiliang Cong—co-frst authors ′ ′ 1 Department of Gastroenterological Surgery, Harbin Medical University closed-loop structure without 5 to 3 polarity, are gen- Cancer Hospital, 150 Haping Road, Harbin 150081, China erated via end-to-end combining of RNA transcrip- Full list of author information is available at the end of the article tion fragments [12–14]. Circular transcripts were once © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ma et al. Cancer Cell Int (2020) 20:81 Page 2 of 12 regarded as abnormal RNA splicing or particular path- miR-376c-3p inhibitor and NC inhibitor, miR-376c-3p ogens despite the fact that they have been known for mimics and NC mimics were also synthesized by Gene more than 40 years [15]. Besides, circRNAs, diferent Pharma. All plasmids were transfected into cells by using from other non-coding RNAs, are very stable because Lipofectamine 2000 kit (Invitrogen, NY, USA). of their loop structure [16]. Multiple studies have sug- gested that circRNAs are related to a variety of biological Nucleic acid electrophoresis processes of diverse tumors, including GC. For instance, Utilizing agarose gels together with TE bufer (Termo circRNA hsa_circ_0052112 accelerates breast cancer Fisher Scientifc, Waltham, MA, USA), the cDNA and progression by sponging miR-125a-5p [17]. CircRNA gDNA PCR products of circPIP5K1A were analyzed. hsa_circ_0000064 exerts critical role in lung cancer cell Afterwards, DL600 (KeyGen, Nanjing) was used as the proliferation and metastasis [18]. CircRNA circ-LDL- DNA marker. Te electrophoretic voltage was 110 volts. RAD3 is associated with pancreatic cancer tumorigenesis DNA was separated for 30 min. UV irradiation was [19]. CircRNA-ZFR regulates GC progression via miR- adopted to test the results. 130a/107/PTEN axis [20]. CircPIP5K1A is an identifed circRNA and its critical role in non-small cell lung can- Quantitative real‑time PCR (RT‑qPCR) cer (NSCLC) has been mentioned in a previous research [21]. However, the underlying regulatory mechanism of Te experiment was applied to monitor the relative circPIP5K1A in GC needs further elucidation. expression of RNA in samples from cells or tissues [22]. Tis current study was designed to explore the specifc regulatory mechanism of circPIP5K1A in GC. And the Actinomycin D (ActD) and RNase R treatment assays results elucidated that circPIP5K1A facilitates GC pro- For RNase R assay, total RNA was cultivated for 20 min at gression via miR-376c-3p/ZNF146 axis, hinting that circ- 37 °C with 3U/ug of RNase R (Epicentre Biotechnologies, PIP5K1A can be applied as a new and efcient biomarker WI, USA). For ActD assay, ActD was bought from Sigma- in researches concerning GC treatment. Aldrich (Milan, Italy) and extracted RNA was treated with ActD (5 mg/ml) in indicated time points and then Methods RT-qPCR analyses were performed. Te two assays were Human tissue samples conducted in triplicate. 49 pairs of cancerous and paired non-cancerous tissues were collected from GC patients who underwent surgical Cell viability assay resections in Harbin Medical University Cancer Hospital MKN-45 cells were seeded into 96-well plates and the from Jan 2012 and Dec 2017. All patients did not receive CCK-8 reagent was added to every well at specifc time any treatment before operation. All fresh samples were points. Following cultivation for 4 h, OD was analyzed by stored in liquid nitrogen at 80 °C. Patients signed the − a spectrometer reader (Olympus, Tokyo, Japan). relevant informed consent and the study was allowed by the Ethics Committee of Harbin Medical University Can- cer Hospital. Colony formation assay After transfection, GC cells were maintained in 6-well Cell culture plates and incubated for 2 weeks. Later, cells in each GC cells (BGC-823, MGC-803, HGC-27, MKN-45) and well were fxed and dyed by methanol (Solarbio, Beijing, normal human gastric epithelial cell line (GES-1) were China) or crystal violet (Solarbio) for 10 min or 5 min, gained from Chinese Academy of Sciences (Shang- respectively. hai, China). Cells were cultivated in RPMI-1640 media (Gibco, Carlsbad, USA) supplied with 10% FBS (Gibco) in Luciferase reporter assay incubator including 5% CO2 at 37 °C. CircPIP5K1A fragment containing putative miR-376c-3p binding site was sub-cloned into pmirGLO vector (Pro- Cell transfection mega, Carlsbad, USA) and named circPIP5K1A-WT. Cells were placed into 6-well plates and developed to 70% Ten we made a circPIP5K1A-Mut which the putative confuence. For the knockdown of circPIP5K1A, specifc miR-376c-3p binding site was mutated. Te 2 reporter sh-circPIP5K1A#1/2 and corresponding control sh-NC plasmids were co-transfected with miR-376c-3p mim- were constructed by Gene Pharma (Shanghai, China). ics or NC mimics into MKN-45 or HGC-27 cells, sepa- Te pcDNA3.1/ZNF146, pcDNA3.1/circPIP5K1A and rately. Te activities of luciferase were measured by a the empty pcDNA3.1 (+) circRNA Mini Vector were dual-luciferase reporter system (promega) at 48 h after obtained from Gene Pharma. Meanwhile, specifc transfection. Ma et al. Cancer Cell Int (2020) 20:81 Page 3 of 12 In vivo experiment the cells were cultivated with 100 μL of 1 × DAPI reaction MKN-45 cells were injected into every nude mouse. solution followed by viability determination using a fuo- Finally, the mice were killed and pictures of their tumors rescent microscope (Olympus). were collected. Te tumor volume and weight were cal- culated. Tese experiments gained permission from the RIP assay Committee on the Ethics of Animal Experiments of Har- Tis assay was conducted by using a Termo Fisher RIP bin Medical University Cancer Hospital. kit (Termo Fisher Scientifc) following the supplier’s instructions. At a word, cells were lysed in RIP lysis Transwell assay bufer, and RNAs magnetic beads were conjugated with Transwell chamber which was coated with or without a human anti-Ago2 antibody or with anti-IgG.
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