Circular RNA circRHOBTB3 acts as a sponge for miR-654-3p inhibiting gastric cancer growth
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Circular RNAs (circRNAs) have recently emerged as a new family of noncoding RNAs that are involved in the causation and progression of various cancers. However, the roles of circRNAs in the tumorigenesis of gastric cancer (GC) are still largely unknown.
The expression profiles of circRNAs in GC were identified in open GEO database and were evaluated at the mRNA level in clinical GC samples compared with paired non-tumorous tissues. Kaplan-Meier survival curve was used to analyze the correlation of circRNA and patients’ prognosis. Subsequently, the circular structures of candidate circRNAs were validated by Sanger sequencing, divergent primer PCR, and RNase R treatments. Gain- and loss-of-function analyses were performed to evaluate the functional significance of it in GC initiation and progression. Dual-luciferase reporter and RNA pull-down assays were used to identify the microRNA (miRNA) sponge mechanism of circRNAs.
The expression of circRHOBTB3 was lower in GC tissues and cell lines. Downregulation of circRHOBTB3 was significantly correlated with poor differentiation and unfavorable prognosis in patients with GC. Overexpression of circRHOBTB3 in GC cells led to decreased proliferation and induced G1/S arrest in vitro, accompanied with inhibited xenograft tumor growth in vivo, while the opposite effects were achieved in circRHOBTB3-silenced cells. Furthermore, we demonstrated that circRHOBTB3 acts as a sponge for miR-654-3p and verified that p21 is a novel target of miR-654-3p.
Taken together, this study revealed that circRHOBTB3 might function as competing endogenous RNA (ceRNA) for miR-654-3p, which could contribute to growth inhibition of GC through activating p21 signaling pathway. Our data suggested that circRHOBTB3 would serve as a novel promising diagnosis marker and therapeutic target for GC.
KeywordscircRHOBTB3 miR-654-3p p21 Gastric cancer Growth
Competing endogenous RNA
Circular RNA RHOBTB3
Fluorescence in situ hybridization
Hematoxylin and eosin
Quantitative real-time polymerase chain reaction
Gastric cancer (GC) is one of the most common cancers and the third leading cause of cancer-related death worldwide  . Although in recent years, development of surgical treatment and adjuvant therapies significantly improves the prognosis of GC patients, the mortality rate remains high, mainly due to the heterogeneity and complicated regulatory relation at molecular level [2, 3, 4, 5]. Hence, better understanding of the underlying molecular mechanism in GC tumorigenesis and progression is urgently needed.
CircularRNAs (circRNAs) are a novel class of endogenous non-coding RNAs mainly formed by the mechanism of “direct back splicing” or “Exon skipping” through producing a covalently closed loop, which usually originates from exon of genes [6, 7]. Although being discovered for about 40 years, the pathological and physiological process of circRNAs remains largely unknown [8, 9, 10]. With the advent of high-throughput sequencing technique and novel bioinformatic analysis thousands of circRNAs have been successfully identified in multiple cell lines and across various species. These circRNAs contain many salient features including high stability, evolutionary conservation and tissue-specific or cell type-specific expression [7, 11, 12]. Moreover, several studies have found that many circRNAs were aberrant expressed and could involve in gene regulation rather than by-products of splicing or splicing errors. Also, evidences showed that circRNAs may act as sponges for microRNAs (miRNAs) or bind to proteins, and abnormal circRNAs expression could lead to alteration of gene products that may contribute to tumor biology including cell proliferation, apoptosis, angiogenesis and metastasis [13, 14, 15, 16]. In fact, evidence from recent literatures and our previous studies has implied that circRNAs play a pivotal role in the tumorigenesis of GC [17, 18, 19]. These findings suggested that circRNAs might be a novel biomarker for the diagnosis and treatment of cancer. However, these studies only exhibited the preliminary results on the circRNA-miRNA regulatory network in GC. The overall pathophysiological role of circRNAs in GC needs to be further investigated.
In the present study, we identified one circRNA originating from exon 6 and exon 7 of RHOBTB3 gene and termed it circRHOBTB3. Meanwhile, the expression of circRHOBTB3 was determined in paired GC tissues and the relationship between circRHOBTB3 with patients’ clinicopathologic characteristic was also analyzed. Besides, the functions of circRHOBTB3 in the growth of GC were explored both in vitro and in vivo. Finally, we confirmed that circRHOBTB3 could sponge miR-654-3p and promoted the expression of p21, a major effector molecule of the cell cycle inhibiting protein.
Materials and methods
Patients and tissue specimens
Seventy-five pairs of GC tissue and paired non-cancerous tissue were obtained from patients with GC who had endoscopically proven primary GC and received radical surgical treatment at Nanfang Hospital (Guangzhou, China) between 2017 and 2019. All patients enrolled in this study did not receive prior surgery, radiotherapy or chemotherapy. All specimens were immediately snap-frozen in liquid nitrogen before storage at − 80 °C. Fifty nine samples with clinico-pathological parameters was used to analyze the significance and prognostic value of circRHOBTB3. Written informed consent was obtained from all patients. The procedure of human tissue samples collection was conducted in accordance with the international Ethical guidelines for biomedical research involving in human subjects. This study was approved by Southern Medical University Ethics Committee.
Human GC cell lines (AGS, HGC27, MKN45) were obtained from the Committee of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). In addition, the gastric mucosal cell GES-1 was provided by the Department of Pathology, Nanfang Hospital, Southern Medical University. The cells were cultured in RPMI Medium 1640 (Gibco, Australiaorigin) supplemented with 10% FBS (BI, China) at 37 °C in a humidified atmosphere of 5% CO2.
RNA preparation, qRT-PCR and genomic DNA purity
Total RNA was extracted from tissues and cells by using TRIzol reagent (Takara, Otsu, Japan). Total RNA was reverse transcribed to cDNA and qRT-PCR were conducted by using a SYBR Green PCR Kit (Takara, Otsu, Japan) as described [20, 21]. For miRNA detection, reverse transcription was performed and expression of miRNA was measured by All-in-One™ miRNA qRT-PCR Detection Kit (Genecopoaie, Lot#QP015) according to the use manual. Genomic DNA (gDNA) was isolated from tissues or cultured cells according to easy pure genomic DNA kit (Transgen Biotech, Lot#L61221). All primers were listed in Additional file 2: Table S1.
Nucleic acid electrophoresis
The cDNA and gDNA PCR products were detected using 4% agarose gel electrophoresis with TAE running buffer at 100 V for 30 min. DNA marker used was DL2000 (Takara, Japan). The bands were examined by UV irradiation.
Treatment with RNase R and Actinomycin D
Total RNA (2 μg) was incubated for 15 min at 37 °C with or without 3 U/mg of RNase R (Epicentre Technologies, Madison, WI, USA). Then, the resulting RNA was directly reverse transcribed using the prime script RT master mix (Takara, Japan) for real-time PCR analyses. To assess the stability of circRHOBTB3 and its linear isoform, The culture medium was added to Actinomycin D or DMSO (sigma Aldrich, St. Louis, MO, USA) as described .
Cell proliferation and cell cycle
Cell proliferation was examined using cell counting kit-8 assay (Dojindo Laboratories, Kumamoto, Japan) and the cell-light™ Edu staining kit (RiboBio, Guangzhou, China) according to manufacturer’s instructions. Colony formation assay were performed to monitor cell cloning capability as followed: 1 × 103 cells was seeded into 6-wells plates and cultured at 37 °C in atmosphere of 5% CO2 for 2 weeks. Then the colonies were washed twice with PBS, fixed with 4% paraformaldehyde for 10 min and dyed with Wright-Giemsa Stain. The number of colonies was photographed and counted using a microscope. Cell cycle was analyzed by flow cytometry. In brief, cells were collected, fixed with cold ethanol for 2 h at 37 °C, washed by PBS, stained with propidium iodide (PI) (Keygentec, Nanjing, China) containing RNase A and then the cell cycle was detected by a flow cytometer (FACS Calibur, Becton Dickinson).
Fluorescence in situ hybridization (FISH)
Cy3-labled probes including circRHOBTB3 and 18 s and FAM-labled locked nucleic acid miRNA probes were designed and synthesized by RiboBio (Guangzhou, China) and GenePharm (Suzhou, China), respectively, and the probe sequences were obtained on request. The signals of the probes were detected by Fluorescence In Situ Hybridization kit (RiboBio, Guangzhou, China) according to the manufacturer instruction. The images were acquired on Nikon AISi Laser Scanning Confocal Microscope (Nikon instruments Inc., japan).
SiRNA or miRNA mimic was designed and synthesized by Suzhou GenePharma (Suzhou, china) and RioBio (Guangzhou, china), respectively. The sequences used were shown in Additional file 2: Table S1. The cells were transfected using lipofectamine2000 (Thermo Fisher, Shanghai, China).
Over-expressing plasmids construction and stable cells transfection
To establish circRHOBTB3 over-expression plasmids, human circRHOBTB3 full-length cDNA was synthesized by vigenebio (Shandong, China) and inserted into PKO-ciR vector including a front circular frame and a back circular frame. Transfection was carried out using lipofectamine2000 (Thermo Fisher, Shanghai, China) according to manufacturer’s instructions. Stable over-expression circRHOBTB3 cells were constructed using over-expressing circRHOBTB3 lentivirus carrier (Vigenebio, Shandong, China). We designed two small interfering RNA (siRNAs) targeting the junction sites of circRHOBTB3 to silence circRHOBTB3 expression in AGS and HGC27 cell lines. These siRNA could significantly knock down circRHOBTB3 levels without affecting its linear isoform, and we selected si-circRHOBTB3–1 to insert into lentivirus carrier to establish stable silencing circRHOBTB3 cell lines due to its higher inhibitory efficacy of circRHOBTB3.
Western blot analysis was performed as illustrated [22, 23, 24]. In brief, cells were harvested, lysed with radio immunoprecipitation assay buffer (RIPA, Beyotime, China), and quantified by bicinchoninic acid (BCA) analysis (Beyotime, China). Then, protein extractions were separated by 10% SDS-PAGE, transferred onto polyvinylidene fluoride (PVDF) membranes (millipore Corporation, Billercica, MA, USA), washed by TBST, and incubated with a high affinity anti-p21 antibody (1:1000), and anti-Tubulin antibody (1:1000) (Cell Signaling Technology, USA) overnight at 4 °C. Subsequently, the membranes were incubated with a secondary antibody (Cell Signaling Technology, USA) (1:5000). After washes, signals were visualized by the enhanced chemiluminescence (ECL) detection system (Pierce Biotechnology, Rockford, IL, USA) conducting in accordance with the manufacturer’s instructions.
Biotinylated RNA pull-down assay
Pull down assay was carried out as described . In brief, for circRHOBTB3 pull down miRNA, about 1 × 107 cells were harvested, lysed and sonicated. The circRHOBTB3 probe was incubated with streptavidin magnetic beads (Beaver, Suzhou, china) for 2 h to generate probe-coated beads, then incubated with cell lysates, followed by eluted with Trizol (Takara, japan), and qRT-PCR. for miR-654-3p pulled down, about 1 × 107 cells were collected, lysed, sonicated, and incubated with streptavidin magnetic beads (beaver, Suzhou, china) after transfected with biotinylated miR-654-3p mimics or mutant using lipofectamine2000 (Thermo Fisher, Shanghai, China), followed by washed, eluted and qRT-PCR.
Luciferase reporter assays
For circRHOBTB3 and miRNA luciferase assay, the circRHOBTB3 sequences containing wild-type or mutated miRNA binding sites were respectively synthesized and inserted into pEZX-MT06 luciferase vector (Genecopoaie, Guangzhou, China), and then co-transfected with miRNA mimics into GC cell lines using lipofectamine2000 (Thermo Fisher, Shanghai, China). After 48 h transfection, cells were harvested, lysed, and subjected to luciferase activity detection by the Luc-pair™ Duo-Luciferase HS assay kit (Genecopoaie, Guangzhou, China). Relative luciferase activity was normalized to the Renilla luciferase internal control.
All animal experiments were approved by the Animal Care Committee and Use Committee of Southern Medical University. The experiments were in accordance with the guidelines for the ethical treatment of animals. To investigate the circRHOBTB3 growth effect on GC cells in vivo, 4-week-old male BALB/C nude mice were randomly divided into two groups (n = 5 for each group). The stable over-expressed circRHOBTB3 MKN45 cells or control group were subcutaneously injected into flank region of legs (5 × 106 cells per mouse), respectively. Tumor size were measured from perpendicular axes and calculated as followed formula: volume = (length×width2)/2. One month later, mice were sacrificed by cervical vertebra dislocation. The primary tumors were removed and harvested, and weighed. Then, the tumors were fixed, paraffin-embedded and sectioned. Subsequently, the sections were visualized under a microscope following hematoxylin and eosin (H&E) staining.
Immunohistochemical staining was performed as described . Slides from nude mice were incubated overnight with primary antibodies against ki67 (#ab15580, Abcam), p21 (#ab15580, Abcam) at 4 °C. The complex was observed by DAB complex, and the nuclei were counterstained with haematoxylin. The immunoreactivity in each section was assessed by at least two experienced pathologists and scored by semi-quantitative H-score approach .
Statistical analyses were performed using SPSS 20.0 software (IBM, Armonk, NY, USA) and Graphpad prism version 5.0 (Graphpad software, La Jolla, CA, USA). Data was expressed as mean ± SD. The statistical differences between groups were tested by two-tailed student’s t-test. The correlation between the circRHOBTB3 expression and clinicopathologic data in GC tissues and paired normal tissues were examined by χ2 test. The relationship between the circRHOBTB3 and linear RHOBTB3 or miR-654-3p was examined by Pearson’s correlation coefficients. Survival prognosis was analyzed by Kaplan-Meier curves and log-rank test for significance. p < 0.05 was considered statistically significant.
CircRHOBTB3 was significantly downregulated in GC tissues, cell lines and correlated with prognosis of patients with GC
Correlation between circRHOBTB3 expression and clinicopathologic characteristics in GC patients
Tumour size (cm in diameter)
It has been reported that some circRNAs may modulate the corresponding linear RNA transcripts expression and then execute function [28, 29]. Therefore, the regulatory relationship between circRHOBTB3 and its linear RNA transcript (RHOBTB3) was explored. Firstly, the expression level of RHOBTB3 was examined in the 19 paired GC and adjacent non-tumorous tissues (Fig. 1g). However, no significant changes of RHOBTB3 mRNA was observed. Pearson’s correlation analysis revealed a significant positive correlation between circRHOBTB3 and its linear RHOBTB3 in GC tissues (r = 0.54, P = 0.018, Fig. 1h). Nevertheless, RHOBTB3 did not change the mRNA expression levels when the expression of circRHOBTB3 was artificially changed in GC cells (Additional file 1: Figure S2A-C). These results indicated that RHOBTB3 is not the target gene of circ RHOBTB3.
Characteristics of circRHOBTB3
CircRHOBTB3 inhibited GC cell growth and cell cycle progression in vitro
CircRHOBTB3 acted as a molecule sponge for miR-654-3p
CircRHOBTB3 inhibits GC growth through the circRHOBTB3/ miR-654-3p/p21 pathway
Stable overexpression of circRHOBTB3 inhibits GC growth in vivo by targeting p21
CircRNAs has been long considered to be molecular flukes or by-products of splicing ever since they were observed in eukaryotic cells by electron microscopy almost 40 years ago [10, 35]. However, the understanding of circRNAs has gradually changed with the application and development of the next-generation sequencing. A large number of circRNAs were detected in various animal samples and multiple cell lines, and many of them were found to be highly stable and abundantly expressed, based on which, subsequent studies demonstrated that circRNAs were dysregulated in diverse cancers including colorectal cancer [13, 30], hepatocellular carcinoma , basal cell carcinoma , laryngeal cancer , bladder cancer . It has been reported that circRNAs could serve as the prognostic biomarkers for many cancers. For example, elevation of circHIPK3 was correlated with poor prognosis in bladder cancer and colorectal cancer [20, 30]. Patients with lower circ-ITCH expression had a poorer prognosis in bladder cancer , and higher levels of circPRCKI displayed as a worse progression marker in lung adenocarcinoma . Whereas, their roles in GC remained largely unknown. In this study, we found that circRHOBTB3 was frequently low-expressed in GC tissues compared with adjacent normal tissues. The low expression of circRHOBTB3 was correlated with tumor stage and provided the poor prognosis of GC patients. Astoundingly, clinical correlation analyses showed that tumor size was not associated with circRHOBTB3 expression, which was not consistent with its functional effects in GC. One possible reason might be that the number of GC specimens was too few. Thus, it’s necessary to analyze the relationship between circRHOBTB3 and clinicopathological parameter using more GC tissue samples in the future study.
CircRNAs could act as oncogenic or tumor suppressive factors in GC. CircFAT1 displayed inhibiting effects in GC , repressing proliferation and invasion of GC cells, but circPVT1/circDLST enhanced the malignancy of GC [19, 39]. Currently, we identified a functional role of circRHOBTB3 in GC cells and validated that re-expression of circRHOBTB3 suppressed the growth of GC in vitro and vivo and arrested cell cycle in G1 phase, while knockdown of circRHOBTB3 produced contrary effects. These results indicated that circRHOBTB3 might serve as a potential tumor suppressive marker in GC.
Recently, increasing evidences have shown that circRNAs functions as sponge for miRNAs to affect tumorous biological process . For instance, circSMARCA5 could act as sponge for miR-17-3p and miR-181b-5p to inhibit the growth and metastasis in hepatocellular carcinoma . CircPVT1 interacted with miR-125b to exert proliferative effect in GC . Herein, we found that circRHOBTB3 contained the binding sites of miR-654-3p predicted by bioinformatic website circinteractome and verified there was direct binding relationship between circRHOBTB3 and miR-654-3p using biotinylated-nucleic probe pull down assay and luciferase reporter experiments, which suggested that circRHOBTB3 might serve as a sponge for miR-654-3p to inhibit the proliferation of GC.
MiRNAs have been shown to be a large family of gene regulator that negatively regulate their target mRNAs in a sequence-specific manner and its role in cancers have been extensively studied [31, 42, 43]. Numerous evidences have confirmed that miRNAs play essential roles in multiple biological process related to cancer, including cell differentiation, proliferation, tumorigenesis, angiogenesis, invasion, and metastasis [44, 45]. As described, miR-654-3p was confirmed to target p21 mRNA and induced its protein downregulation in HEK293 cells . Previous studies have demonstrated that p21 could suppress multiple tumorous proliferation by targeting cyclin-dependent kinase (CDK) complexes [46, 47]. However, whether miR-654-3p could regulate the expression of p21 and biological function in GC cells remained unknown. In the present study, we proved that miR-654-3p was up-regulated in GC tissues and cells, and promoted the proliferation of GC cells. Additionally, we also confirmed that miR-654-3p could inhibit the protein expression of p21 in GC cells using western blot, whereas, circRHOBTB3 enhanced the protein expression of p21. Further reversed experiments were conducted and we found that circRHOBTB3 inhibited the activity of miR-654-3p, and thus, reversed the miR-654-3p induced proliferation of GC cells and up-regulated the protein expression of p21 indirectly. Hence, we validated that circRHOBTB3 could inhibit the proliferation of GC by sponging miR-654-3p to up-regulate the protein expression of p21. One thing we have to pay attention is that not all circRNAs can function as “miRNA sponges” . Intronic circRNAs and exon-intronic circRNAs, which predominantly localize in nucleus with lacking enrichment for miRNA target sites, have been reported to modulate parental genes expression via RNA-RNA interaction [29, 49]. Meanwhile, some circRNAs, such as circMbl, circFmn, circDMD, can strongly bind to cognate linear transcripts to prevent mRNA from translation and finally lead to the reduction in protein levels [48, 50]. This process is also termed as “mRNA trap”. Thus, diverse functions of circRNAs in GC need to be further explored.
Our study confirmed that circRHOBTB3 was frequently down-regulated in GC and low expression of circRHOBTB3 was correlated with tumor stage and patients’ unfavorable prognosis. Mechanistically, circRHOBTB3 could serve as a sponge molecule for miR-654-3p, and then rescued the expression of miR-654-3p-inbiting protein p21, finally suppressed GC cell growth. Our findings might provide new insight into GC development and provided a novel potential strategy for GC treatment.
LGX and WS lead this study. DGX, MTY and CD carried out the experiments. Data analyses were performed by DGX, HJY, and LDJ. LH, YJ assisted in harvesting tissues samples and providing clinical data. Funding was obtained from LGX. The manuscript was written by DGX and MTY, commented and revised by LGX and WS. All authors participated in preparing the manuscript and approved the final submitted and published version.
This work was supported by the grants from the State’s Key Project of Research and Development Plan (2017YFC0108300), National Natural Science Foundation of China (81672446), Guangdong Provincial Science and Technology Key Project (2014A020215014), Research Fund of Public Welfare in the Health Industry, the National Health and Family Planning Commission of China (201402015), the Southern Medical University Clinical Research Start-Up Project (LC2016ZD003), the Key Clinical Specialty Discipline Construction Program (121), and Presidential Foundation of Nanfang Hospital, Southern Medical University (2016C034) (2018B002).
Ethics approval and consent to participate
The collection of human specimens was approved by the Ethics Committee of Nanfang Hospital and the written informed consent were obtained from each patient.
Consent for publication
The authors declare that they have no competing interests.
- 13.Bachmayr-Heyda A, Reiner AT, Auer K, Sukhbaatar N, Aust S, Bachleitner-Hofmann T, et al. Correlation of circular RNA abundance with proliferation--exemplified with colorectal and ovarian cancer, idiopathic lung fibrosis, and normal human tissues. Sci Rep. 2015;5:8057.PubMedPubMedCentralCrossRefGoogle Scholar
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