Circular RNA circRHOBTB3 acts as a sponge for miR-654-3p inhibiting gastric cancer growth

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.

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.

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.

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 [18].

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).

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).

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 [22‐24]. 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.

Pull down assay was carried out as described [25]. 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.

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 [26]. 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 [27].

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.

To identify a candidate circRNA that may implicate in the tumorigenesis of GC, we firstly analyzed the high-throughput sequencing results of GC (accession code: GSE77661) from the open GEO database [18]. Then we selected five candidate circRNAs from the RNA-seq data by the criteria: fold change > 2 (Circbank numbers: circN4BPL2L, circRELL1, circFAM120A, circFNDC3B, circRHOBTB3). Moreover, the relative expression of these five circRNAs were detected by qRT-PCR in GC and paired non-tumorous tissues (cohort 1, n = 30). Results showed that the relative expressions of circN4BPL2L displayed no significant changes, but the other four circRNAs were low-expressed. Interestingly, we found that circRHOBTB3 was the most significantly downregulated circRNA among them (P < 0.0001 and fold-change (GC/ANT) < 0.274, Fig. 1a), which suggested that circRHOBTB3 might be a circular molecular correlated with progression of GC.

To further confirm the relationship between the expression level of circRHOBTB3 and the clinical progress of GC patients. qRT-PCR was used to detected the expressions of circRHOBTB3 in newly internalized GC tissues and adjacent tissues (cohort 2, n = 75). Results displayed that circRHOBTB3 was frequently repressed in GC tissues compared with non-tumorous tissues (Fig. 1b). When primary cancers were paralleled to corresponding normal tissues, the downregulation of circRHOBTB3 (less than a 0.5-fold change) was observed in 65.4% (49/75) of patients with GC (Fig. 1c, d). Consistently, we also found that the expression of circRHOBTB3 in GC cell lines was obviously downregulated compared with GES-1 cells (the normal gastric mucosal cells) (Fig. 1e). Subsequently, these patients were stratified into high and low groups based on the median value of circRHOBTB3 expression. The relationship between circRHOBTB3 expression in GC tissues and clinicopathological features. As shown in Table 1, low expression of circRHOBTB3 was dramatically correlated with tumor stage, whereas no significant correlation with other clinicopathologic parameters. In addition, survival analyses of these patients revealed that patients with low expression of circRHOBTB3 had poor prognosis in GC than patients in the high circRHOBTB3 expression group (Fig. 1f). These collective data suggested that circRHOBTB3 might play pivot role in the tumorigenesis of GC.

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.

CircRHOBTB3 was generated from exon 6 and exon7 of RHOBTB3 gene (CircBase ID: hsa_circ_00074444, splicing length: 479 nucleic acid base). To further confirm circular characteristics of circRHOBTB3, the transcripts of both circRHOBTB3 and RHOBTB3 mRNA was tested by qRT-PCR in three tumor tissues, AGS and HGC27 cell lines after treatment with or without RNase R. Results showed that the fragment of linear form of RHOBTB3 gene was digested by RNase R while cirRHOBTB3 was retained after RNase R treatment (Fig. 2a, b), which verified that circRHOBTB3 was resistant to RNase R due to its loop structure. Secondly, to rule out the possibility of head-to-tail sequencing produced by trans-splicing or genomic rearrangement, Divergent primers and convergent primers were designed to amplify circRHOBTB3 and RHOBTB3 mRNA, respectively. cDNA and gDNA (genomic DNA) from three GC tissues and AGS, HGC27 cell lines were used as templates. We found that circRHOBTB3 was only amplified by divergent primers in cDNA, but no amplification product was visualized in gDNA. Meanwhile, the head-to-tail junction sequences were validated by Sanger sequencing (Fig. 2c, d). Then, inhibiting transcription experiment was utilized to reveal the stability of circRHOBTB3, and illustrated that it was more stable than its linear mRNA (Fig. 2e). Additionally, the subcellular localization of circRHOBTB3 was determined in nucleoplasmic separation and FISH experiments. Results indicated that circRHOBTB3 was preferentially localized in cytoplasm (Fig. 2f, g and Additional file 1: Figure S1). Taken together, the above results indicated that circRHOBTB3 is an abundant, circular and stable transcript that mainly localized in cytoplasm of GC cells.

To better understand the role of circRHOBTB3 in GC cells. We selected si-circRHOBTB3–1 to insert into lentivirus carrier to establish stable silencing circRHOBTB3 (SH-circRHOBTB3) in AGS and HGC27 cell lines due to its higher inhibitory efficacy of circRHOBTB3. Data demonstrated that stable SH-circRHOBTB3 AGS and HGC27 cell lines were established successfully (Additional file 1: Figure S2A, B). Moreover, circRHOBTB3 were stably over-expressed by circRHOBTB3-overexpressed lentivirus vector in MKN45, AGS and HGC27 cells lines (Additional file 1: Figure S2C). Subsequently, functional assays were executed to reveal the effects of circRHOBTB3 on GC cell proliferation. The cck8 assay showed that knocked-down of circRHOBTB3 enhanced the growth ability of AGS and HGC27 cell lines significantly (Fig. 3a and Additional file 1: Figure S2D). Whereas, over-expression of circRHOBTB3 suppressed the proliferation of MKN45 cells (Fig. 3b). In addition, the colony formation assay indicated that SH-circRHOBTB3 AGS and HGC27 cell lines produced more clonogenicities compared with control group but elevation of circRHOBTB3 displayed opposite effect in MKN45 cells (Fig. 3c and Additional file 1: Figure S2E). Consistently, by using Edu assay, compared with control group, more proliferative cells were observed in AGS and HGC27 cell lines with down-regulation of circRHOBTB3 while less in MKN45 cells with up-regulation of circRHOBTB3 (Fig. 3d and Additional file 1: Figure S2F). Moreover, Flow cytometry analysis was performed to determine whether circRHOBTB3 affected the cell cycle profile. As presented in Fig. 3e and Additional file 1: Figure S2G, less cells stopped in G1 phase after silencing of circRHOBTB3 in AGS and HGC27 cell lines, which suggested that silencing of circRHOBTB3 induced G₁/S cell cycle progression. However, over-expression of circRHOBTB3 arrested cell cycle in G1 phase in MKN45 cells (Fig. 3f). These data collectively demonstrated that circRHOBTB3 inhibited the growth phenotype of GC cells and cell cycle progression.

Previous studies suggested that circRNAs mainly function as miRNA sponge to sequester miRNA and then regulate subsequent gene expression [30, 31]. To investigate whether circRHOBTB3 served as “miRNA sponge” in GC cells, we selected the eleven top potential binding miRNA (miR-244, miR-494, miR-548p, miR-570, miR-586, miR-600-3p, miR-619-3p, miR-654-3p, miR-1200-5p, miR-1265-5p, miR-1305) with a context score percentile≥90 predicted by CircInteractome database. The overexpression efficacy of circRHOBTB3 was quantified by qRT-PCR in AGS and HGC27 cell lines transfected with elevated circRHOBTB3 plasmid or empty (Additional file 1: Figure S3A). Subsequently, a 3′ terminal-biotinylated-circRHOBTB3 probe was designed to determine which miRNAs interacted with circRHOBTB3. As illustrated in Fig. 4a, the probe was confirmed to pull down circRHOBTB3 in AGS and HGC27 cell lines and overexpression of circRHOBTB3 increased pull-down efficacy. Then, qRT-PCR analyses revealed that several miRNAs including miR-548p, miR-570, miR-586, miR-654-3p and miR-1200-5p could all be pulled down in AGS and HGC27 cell lines. Among them, miR-654-3p was most abundantly pulled down by circRHOBTB3 in HGC27 and AGS cells (Fig. 4b, c). Meanwhile, miR-654-3p was reported and predicted by Target Scan website to target p21 protein, a well-known proliferative suppressor in various tumors [32‐34]. Therefore, we focused on miR-654-3p for further study. To adequately consolidate the binding between circRHOBTB3 and miR-654-3p, biotin-coupled miR-654-3p and its mutant were utilized to pull down circRHOBTB3 in MKN45 and HGC27 cell lines with stable circRHOBTB3 overexpression. Results showed that wild-type miR-654-3p captured more circRHOBTB3 compared with its mutant. GAPDH served as negative control and showed no significant changes between biotin-labeled miR-654-3p and mutant group (Fig. 4d). Furthermore, we conducted luciferase reporter assay and verified that up-regulation of miR-654-3p relatively reduced the luciferase activity of vector containing full circRHOBTB3 sequences, but didn’t influence the luciferase activity of vector including mutant binding sites of miR-654-3p in AGS, HGC27, and MKN45 cell lines (Fig. 4e and Additional file 1: Figure S3B, C). Moreover, we conducted FISH assay to assess whether there was co-location between circRHOBTB3 and miR-654-3p. Results indicated that co-location of circRHOBTB3 and miR-654-3p was mainly visualized in cytoplasm (Fig. 4f and Additional file 1: Figure S3D). Based on the above, data demonstrated that circRHOBTB3 could serve as a sponge molecule for miR-654-3p.

To address whether circRHOBTB3 plays tumor-inhibiting role via circRHOBTB3/miR-654-3p/p21 pathway in GC, we performed the following experiments: Firstly, the expression levels of miR-654-3p were detected by qRT-PCR in 30 paired GC tissues and adjacent non-cancerous tissues. Results indicated that miR-654-3p was highly expressed in GC tissues compared with control group (Additional file 1: Figure S4A). In addition, we found that the expression of miR-654-3p was negatively correlated with circRHOBTB3 in GC tissues analyzed by Pearson’s correlation (Additional file 1: Figure S4B), which cued that miR-654-3p might severe as proliferation-promoting role in GC. Consistently, the expression level of miR-654-3p was up-regulated in GC cell lines in comparison to GES-1 cells (Additional file 1: Figure S4C). Subsequently, cck8 assay was conducted, and confirmed that miR-654-3p enhanced the growth of AGS and HGC27 cell lines transfected with miR-654-3p compared with NC-mimic (Additional file 1: Figure S4D, E). Then, we examined the protein levels of p21 in AGS and HGC27 cell lines transfected with miR-654-3p or NC-mimic, and found that p21 expression was significantly repressed in miR-654-3p group compared with NC-mimic, as shown in Additional file 1: Figure S4F. Additionally, we examined the protein expression of p21 using western blot in stable over-expression or down-regulation circRHOBTB3 GC cell lines. As expected, results showed that protein expression of p21 was upregulated in AGS and HGC27 cells with stably elevated circRHOBTB3 and downregulated in SH-circRHOBTB3 AGS and HGC27 cell lines (Additional file 1: Figure S4G, H). However, qRT-PCR analyses for the mRNA levels of p21 indicated that there were no significant changes in AGS and HGC27 cell lines treated with miR-654-3p or NC-mimic or stable overexpressed circRHOBTB3 GC cell lines (Additional file 1: Figure S3I), suggesting that miR-654-3p or circRHOBTB3 didn’t affect p21 mRNA levels. To further explore whether circRHOBTB3 acted as tumor-inhibitor in GC cell by attenuating activity of miR-654-3p to up-regulate protein expression of p21, rescue experiments was performed. cck8 assay displayed that AGS cells with elevated circRHOBTB3 plus miR-654-3p grown slower than miR-654-3p group (Fig. 5a, b), and consistent results was reproduced in HGC27 cells. Likewise, the colony assay showed that overexpressed circRHOBTB3 GC cells together with miR-654-3p exerted less cloned cells than GC cells transfected with miR-654-3p (Fig. 5c). Moreover, cell cycle distribution was analyzed by flow cytometry in GC cells and results suggested that miR-654-3p combined with circRHOBTB3 reversed miR-654-3p-induced cell cycle progression, resulting in more cells ceased in G1 phase (Fig. 5d, e). Then, to see whether circRHOBTB3 could restore the expression levels of p21 inhibited by miR-654-3p, p21 was examined using western blot, and the brightness of the protein band was observed to be stronger in circRHOBTB3 + miR-654-3p group than in miR-654-3p group (Fig. 5f). These results indicated that circRHOBTB3 reversed miR-654-3p-induced enhancement of GC cell growth, and could restore the expression of miR-654-3p target p21 at protein levels, forming the circRHOBTB3/miR-654-3p/p21 regulating axis (Fig. 5h).

To assess whether overexpression of circRHOBTB3 affects tumor growth in vivo, MKN45 transfected with elevated circRHOBTB3 lentivirus or control vector was injected subcutaneously into nude mice. Haematoxylin and eosin (H&E) staining was applied to confirm the histopathological features of tumors harvested. Tumor volume shrink and reduced average tumor weight were observed in overexpressed circRHOBTB3 group compared with control group (Fig. 6a-d). Immunohistochemistry (IHC) demonstrated that the expression of ki67 proliferation antigen was significantly weakened in overexpressed circRHOBTB3 xenografts. Moreover, the staining of p21 was observed stronger than control group (Fig. 6e). Therefore, circRHOBTB3 could present tumor-suppressing property to inhibit cell growth and produce cell cycle arrest.