Elsevier

Gene

Volume 753, 30 August 2020, 144798
Gene

Research paper
CircFOXO3 functions as a molecular sponge for miR-143-3p to promote the progression of gastric carcinoma via upregulating USP44

https://doi.org/10.1016/j.gene.2020.144798Get rights and content

Highlights

  • CircFOXO3 is upregulated in GC cells and promotes GC malignancy in vitro and in vivo.

  • CircFOXO3 interacts with miR-143-3p and negatively regulates its expression.

  • MiR-143-3p interacts with USP44 and downregulates its expression in GC cells.

  • CircFOXO3 promotes the progression of GC by enhancing the output of miR-143-3p-USP44 axis.

Abstract

Gastric carcinoma (GC) ranks fifth in terms of cancer morbidity and third in cancer-related death worldwide and imposes enormous health and economic burdens. The molecular mechanisms underlying GC formation and progression remain unclear. Our aim was to identify the involvement of circular RNA circFOXO3 in GC, and to determine the underlying mechanisms. In this study, we revealed a stimulatory role of circular RNA circFOXO3 in tumor growth in vivo. CircFOXO3 enhanced GC cell proliferation and migration in vitro and promoted tumor growth of GC cells in vivo. Bioinformatic analysis revealed that circFOXO3 might regulate USP44 expression by specifically binding to microRNA (miR)-143-3p. Existence of circFOXO3-miR-143-3p-USP44 axis in GC cells was confirmed by RNA-binding protein immunoprecipitation, luciferase reporter assay, and an RNA pull-down experiments. All the data indicate that circFOXO3 promotes GC cell proliferation and migration by upregulating USP44 expression via targeting of miR-143-3p.

Introduction

Gastric carcinoma (GC) ranks among the five cancers with the highest morbidity and ranks third in terms of cancer-related deaths. GC also causes enormous economic problems for the global healthcare system (Nasrollahzadeh-Khakiani et al., 2017). The diagnosis of GC is often delayed, and clinical management can be inefficient. Therefore, most GC cases are initially evident as local diffusion or metastasis(Chen et al., 2012, Wu et al., 2017, Dan et al., 2018). However, little is known about early-stage molecular biomarkers of GC. It is imperative to improve our understanding regarding the essential mechanisms of GC initiation and progression for developing better targeted GC treatment.

Circular RNAs (circRNAs) are a type of noncoding RNA which characterized by covalently closed loops resulting from back splicing events(Ledford, 2013, Patop et al., 2019). Their functions are typically revealed by high-throughput sequencing and bioinformatic analysis. In the cell, circRNAs are primarily located in cytoplasm. And it has been found that circRNAs contain abundant miRNA competing binding sites indicating competing endogenous RNA function of circRNAs(Denzler et al., 2014, Thomson and Dinger, 2016). Furthermore, The crosslinking immunoprecipitation assays show that circRNAs are able to act as protein sponges due to the ability of interacting with abundant RBPs(Dudekula et al., 2016, Abdelmohsen et al., 2017). Therefore, circRNAs may be involved in crucial biological events(Chen and Yang, 2015, Barrett and Salzman, 2016, Beermann et al., 2016, Cortes-Lopez and Miura, 2016, Ebbesen et al., 2017, Li et al., 2018), including the formation and progression of cancers(Meng et al., 2017, Kristensen et al., 2018). CircRNAs were reported to play roles in the initiation and progression of GC. Zhang X et al found that circNRIP1 promotes GC via AKT1/mTOR pathway by targeting of miR-149-5p (Zhang et al., 2019b), Liu j et al demonstrated the molecular function of circ-SERPINE2 in the development of GC(Liu et al., 2019), Chen J et al found that circPVT1 is a proliferative factor and prognostic marker in GC(Chen et al., 2017), and Rong D et al revealed that circPSMC3 suppresses GC by sponging miR-296-5p(Rong et al., 2019). Recently, the molecular functions of circular RNA circFOXO3 in cancers have been investigated.

CircFOXO3 (hsa_circ_0006404) contains 1435 nucleotides and is derived from an exon of the FOXO3. CircFOXO3 involves in the progression of many cancers, including lung cancer(Zhang et al., 2018b), breast cancer(Lu, 2017), glioblastoma(Zhang et al., 2019a), prostate cancer(Shen et al., 2020), and esophageal squamous cell cancer(Xing et al., 2019). Nonetheless, the role of circFOXO3 in the progression of GC remains unelucidated, and neither are other molecular mechanisms of action of circFOXO3. The aim of this study was to address these issues.

Here, we hypothesized that circFOXO3 involves in the progression of GC and relates to the tumor growth and metastasis of GC. By performing a serial in vitro and vivo experiments. CircFOXO3 was shown to upregulate the expression of ubiquitin-specific peptidase 44 (USP44) in GC cell lines by interacting with microRNA (miRNA,miR)-143-3p. CircFOXO3 also enhanced GC cell proliferation and migration through the miR-143-3p-USP44 pathway and promoted tumor growth in vivo. These finding provide a new perspective regarding the function of circFOXO3 and highlight a potential target for studies directed at developing GC therapies.

Section snippets

Cell culture and transfection

All GC cell lines (GES1, AGS, MKN45, SNU5, MKN74, MGC803, NCI-N87) and 293 T cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Those cells were cultured with Dulbecco’s Modified Eagle medium (DMEM) (Gibco) medium supplemented with 10% fetal bovine serum (FPS)(Gibco), and were cultivated according to the vendor’s recommendations at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air. A circFOXO3-expressing plasmid - the human circ-FOXO3 cDNA

CircFOXO3 is upregulated in GC cells

Initially, we investigated the expression of circFOXO3 in GC cells. we hypothesized that circFOXO3 might be involved in the progression of GC. Subsequently, we quantified circFOXO3 levels in GC cell lines (Fig. 1A). The expression of circFOXO3 was found to be significantly upregulated in MGC803 cells and downregulated in NCI-N87 cells. Then, we tested the efficiency of circFOXO3- PcDNA transfection and its NC as well as si-circFOXO3 transfection and its NC in NCI-N87 cells and MGC803 cells (

Discussion

In the present study, we found that circFOXO3 is overexpressed in GC tumors and that it is involved in the progression of GC via the miR-143-3p-USP44 axis. We performed a serial in vitro and vivo experiments to determine the role of circFOXO3 in GC progression, and the molecular mechanisms underlying it. In the past few years, the biological functions and pathological roles of circRNA in the formation and progression of cancers were determined by performing high-throughput sequencing and

Conclusions

• The results collectively reveal the role of circFOXO3 in GC.

• We clarified the mechanisms by which circFOXO3 participates in GC progression in vivo and in vitro.

• Our findings highlight the crucial function of circRNAs in GC progression, and reveal a new potential therapeutic target in GC.

CRediT authorship contribution statement

Tian Xiang: Validation, Formal analysis, Visualization, Software, Writing - review & editing. Hong-sheng Jiang: Validation, Formal analysis, Visualization, Software, Writing - review & editing. Bi-tao Zhang: Resources, Writing - review & editing, Supervision, Data curation. Gao Liu: Conceptualization, Methodology, Software, Investigation, Writing - original draft, Resources, Supervision, Data curation, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

N.A.

Data availability statement

The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Funding

This work was supported by the National Natural Science Foundation of China [grant number 81760540]. The sponsor had no role in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

References (35)

  • K. Abdelmohsen et al.

    Identification of HuR target circular RNAs uncovers suppression of PABPN1 translation by CircPABPN1

    RNA Biol

    (2017)
  • S.P. Barrett et al.

    Circular RNAs: analysis, expression and potential functions

    Development

    (2016)
  • J. Beermann et al.

    Non-coding RNAs in Development and Disease: Background, Mechanisms, and Therapeutic Approaches

    Physiol Rev

    (2016)
  • L.L. Chen et al.

    Regulation of circRNA biogenesis

    RNA Biol

    (2015)
  • W. Chen et al.

    miRNA expression profile in primary gastric cancers and paired lymph node metastases indicates that miR-10a plays a role in metastasis from primary gastric cancer to lymph nodes

    Exp Ther Med

    (2012)
  • M. Cortes-Lopez et al.

    Emerging Functions of Circular RNAs

    Yale J Biol Med

    (2016)
  • D.B. Dudekula et al.

    CircInteractome: A web tool for exploring circular RNAs and their interacting proteins and microRNAs

    RNA Biol

    (2016)
  • Cited by (0)

    1

    Co-first authors.

    View full text