Circular RNA hsa_circ_0001368 suppresses the progression of gastric cancer by regulating miR-6506–5p/FOXO3 axis

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Highlights

  • In this study, we performed high-throughput circRNA microarray assays using gastric cancer (GC) patient samples to investigate the potential involvement of circRNAs in GC, and we revealed that lower levels of circRNA hsa_circ_0001368 were expressed in GC tissues than in normal adjacent tissues.

  • Functionally, knockdown of hsa_circ_0001368 dramatically promoted the ability of GC cells to proliferate and invasion in vitro and in vivo.

  • Regarding the mechanism, we found that hsa_circ_0001368 was mainly observed in the cytoplasm and was capable of sponging miR-6506–5p to increase the expression of the tumor-suppressive gene FOXO3.

  • Our study first revealed a novel signaling pathway of hsa_circ_0001368/miR-6506-5p/FOXO3 involved in GC progression.

Abstract

Gastric cancer (GC) is still a major aggressive malignancy worldwide. While the importance of circular RNAs (circRNAs) involved in carcinogenesis has gradually been acknowledged, their role in human cancers is not largely understood, including in GC. Here, we focused on hsa_circ_0001368 in GC, a novel circRNA that has not been previously reported. In the current study, we found a broad downregulation of hsa_circ_0001368 in GC tissues and cells, which correlates with a worse prognosis in GC patients. Functional experiments suggested that the knockdown of hsa_circ_0001368 promoted cell viability and motility by cell proliferation and invasion assays. In addition, the knockdown of hsa_circ_0001368 led to accelerated tumor growth in vivo. Mechanically, we demonstrated that hsa_circ_0001368 served as a competing endogenous RNA (ceRNA) to sponge miR-6506–5p. Subsequently, FOXO3 may act as the functional target of miR-6506–5p, and the knockdown of hsa_circ_0001368 decreased the expression of the tumor-suppressive gene FOXO3. Taken together, our study revealed that hsa_circ_0001368 plays a tumor-suppression role in GC via the miR-6506–5p/FOXO3 axis and may serve as a potential target for GC therapy.

Introduction

Gastric cancer (GC) is one of the most prevalent and aggressive malignancies worldwide [[1], [2]]. Although some significant improvements have been made in GC treatment in recent decades, the prognosis of patients is still dismal, mainly due to the high rate of relapse, distant metastasis, and the lack of effective biomarkers [[3], [4], [5]]. Therefore, screening for effective therapeutic targets and determining the potential molecular mechanisms of GC development are urgent endeavors.

With the rapid development of sequencing technology, circular RNAs (circRNAs) were identified as a new type of functional noncoding RNAs (ncRNAs), characterized by cell-type specific and stability [6]. Compared to linear RNAs, circRNAs exist as a covalently closed loop without 5′-3′ polarity and a polyadenylated tail [7]. Currently, increasing evidence has revealed that circRNAs can function as tumor suppressors or oncogenes in multiple cancers [[8], [9], [10]]. However, their roles in GC are still largely unknown [11]. In the present study, we focused on a novel circRNA, hsa_circ_0001368, which was down regulated circRNA in GC tissue screened by circRNA microarray.

Mechanically, there are several ways by which circRNAs function in mammalian cells, such as sponging microRNAs (miRNAs), interacting with proteins, regulating splicing, and transporting RNAs [10,12]. Among these functions, circRNAs usually act as miRNA “sponges” [13,14]. As has been reported, miRNAs usually bind to the 3’ UTR region of target mRNAs to suppress target gene expression [15]. Thus, circRNA binding to miRNA leads to the regulation of target gene expression and forms a functional circRNA-miRNA-mRNA network.

To our knowledge, hsa_circ_0001368 is a newly identified circRNA identified by circRNA microarray analysis and whose functional mechanism in GC progression has yet to be elucidated. Thus, we attempted to determine the role of hsa_circ_0001368 in GC. In this work, we discovered for the first time that hsa_circ_0001368 was downregulated in GC and was negatively correlated with patient survival. Functionally, we measured cell growth and invasion affected by hsa_circ_0001368 in vitro and in vivo. Mechanistically, we predicted and validated that the miR-6506–5p/FOXO3 signal pathway was the downstream target of hsa_circ_0001368. Above all, this study revealed the hsa_circ_0001368 tumor-suppressing effects by targeting miR-6506–5p and upregulating FOXO3 expression levels and may be a potential therapeutic target in GC.

Section snippets

CircRNA microarray analysis

Six pairs of gastric cancer tissue and adjacent nontumor tissue were used for the circRNA microarray assay to determine differentially expressed circRNAs. The microarray hybridization was performed based on the manufacturer’s standard protocols (Agilent Technology), which included purifying the RNA, transcribing it into fluorescent cRNA, and then hybridizing it onto the Human lncRNA Array v3.0 (Arraystar) and Human circRNA Arrays (Arraystar). Finally, the hybridized slides were washed, fixed

Identification of differentially expressed circRNAs in GC

We performed high-throughput circRNA microarray assays using six pairs of GC patient samples to investigate the potential involvement of circRNAs in GC (Fig. 1A). Hierarchical clustering revealed circRNA expression patterns, and Fig. 1B shows the top twenty differentially expressed circRNAs. The variation of circRNA expression was observed in the volcano (Fig. 1C) and scatter plots (Fig. 1D). As a result, 52 circRNAs were upregulated, whereas 80 circRNAs were decreased based on fold change ≥2.0

Discussion

Gastric cancer (GC) remains one of the most frequent malignancies worldwide. Due to its late presentation and insufficient sensitivity to chemoradiotherapy, the overall survival of GC patients is still not satisfactory. Thus, it is crucial to improve early diagnosis, develop effective prognostic markers, and search for novel molecular therapeutic targets for GC.

First thought to be a result of RNA splicing errors, people believed that only a few circRNAs existed. However, with the development of

Conflicts of interest

The authors declare no competing financial interests.

Acknowledgements

The present study was supported by Scientific and technological innovation joint capital projects of Fujian Province (2016Y9031). Construction Project of Fujian Province Minimally Invasive Medical Center (No. [2017]171). The second batch of special support funds for Fujian Province innovation and entrepreneurship talents (2016B013). QIHANG funds of Fujian Medical University (No.2016QH025).

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    Jun Lu and Peng-yang Zhang contributed equally to this work and should be considered co-first authors.

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