Gastrin-related circRNA_0017065 promotes the proliferation and metastasis of colorectal cancer through the miR-3174/RBFOX2 axis

Abstract

Gastrin is a gastrointestinal peptide hormone that plays an important role in the progression of colorectal cancer (CRC). However, the molecular mechanism remains unclear. In this study, we identified gastrin-related circRNAs via high-throughput sequencing and selected circRNA_0017065 as the research focus. We further studied its specific role and molecular mechanism in the progression of CRC. Knockdown and overexpression of circRNA_0017065 were performed, and the biological function of circRNA_0017065 in CRC progression was studied via in vitro and in vivo functional experiments. The potential downstream target genes were subsequently identified via screening of databases and gene chip data. The expression of circRNA_0017065 in tumour tissues was significantly upregulated compared with that in adjacent normal tissues. In vitro and in vivo functional experiments revealed that the proliferation and migration of CRC cells were significantly suppressed after circRNA_0017065 knockdown, while apoptosis was promoted. After overexpression of circRNA_0017065, the proliferation and migration of CRC cells were significantly promoted, while apoptosis was inhibited. Mechanistic studies revealed that circRNA_0017065 can act as a sponge for miR-3174 and promote CRC progression via the miR-3174/RBFOX2 axis. In general, gastrin-related circRNA_0017065 plays a key role in the occurrence and development of CRC and is expected to be a potential molecular target for the treatment of CRC metastasis.

Introduction

According to the latest statistics from the International Agency for Research on Cancer (IARC) in 2020, the incidence rate of colorectal cancer (CRC) reached 10.0%, only following that of breast cancer and lung cancer, and it has become the third most common malignant tumour worldwide [1]. With the continuous improvement in medicine, the 5-year survival rate of patients with CRC has improved, but recurrence and metastasis are still primary causes of death in CRC patients [2]. Despite certain achievements in the study of CRC, we still face significant challenges in the development of effective treatments for its recurrence and metastasis. Therefore, further studies on the pathogenesis of CRC and the molecular mechanisms involved in tumour recurrence and metastasis are urgently needed.

Gastrin is a peptide hormone that plays an important role in the occurrence and development of CRC [3]. It has been shown to act on tumour cells through autocrine and paracrine mechanisms, thus endowing tumour cells with antiapoptotic properties [4]. It has been reported that the higher the serum gastrin level is, the greater the incidence of CRC [5]. A possible mechanism of the promotion of proliferation in colorectal cancer involves the spontaneous production or secretion of gastrin, which acts as a receptor on its own cell membrane, thus exerting biological effects. However, the specific downstream molecular mechanism remains unclear.

Circular RNAs (circRNAs), closed loops formed by covalent bonds without a 5’ cap or 3’ poly(A) tail, are resistant to exonucleases and are more stable than linear splicing products [6,7,8,9]. As high-throughput sequencing technology has advanced, the role of noncoding RNA has gained renewed recognition. The association between noncoding RNAs and human diseases is increasingly becoming a focal point of scientific research [10]. CircRNAs are widely involved in the pathological and physiological activities of the human body, including regulating the occurrence and development of various tumours [11]. A variety of factors can regulate the occurrence and development of tumours. For example, circRNAs can act as miRNA sponges to regulate the activity of miRNA-related target genes; they can also act at the transcriptional and splicing levels to regulate expression of target genes or interact with RBPs [12]. Studies on circRNAs and tumours have shown that abnormally expressed circRNAs may be tumour diagnostic markers or molecular therapeutic targets [13, 14].

In this study, we used high-throughput sequencing technology and identified a new gastrin-related circRNA, circRNA_0017065, in CRC. In vitro and in vivo functional experiments confirmed that the knockdown of circRNA_0017065 inhibited the proliferation and metastasis of CRC cells. It was found to sponge miR-3174 to regulate the proliferation and metastasis of CRC cells via the miR-3174/RBFOX2 axis. Additionally, circRNA_0017065 is expected to become a tumour marker and might be a molecular therapeutic target; thus, this study provides new ideas and insights for the diagnosis and treatment of CRC.

Results

RNA sequencing was used to analyse the differential circRNA expression profiles in CRC tissues

To obtain the expression profile of gastrin-related circRNAs and identify the differentially expressed circRNAs in CRC patients, we assessed the expression levels of gastrin in 40 patients with CRC undergoing surgical treatment at Yijishan Hospital. The tumour tissues and adjacent normal tissues of three patients with high gastrin expression and three patients with low gastrin expression were selected. Using high-throughput sequencing, we performed two analyses of circRNA expression profiles: one analysis assessed the differences between tumour tissues with high gastrin levels and their adjacent normal counterparts (Group 1), and the other analysis assessed the differences between tumour tissues with high gastrin levels and those with low gastrin levels (Group 2). Preliminary analysis through heatmaps and volcano plots revealed distinct circRNA expression patterns between high gastrin-expressing tumour tissues and adjacent normal tissues (Fig. 1a, b), as well as between tumour tissues with different gastrin expression levels (Fig. 1d, e). Group 1 included 356 differentially expressed circRNAs, 283 of which were significantly downregulated and 73 of which were significantly upregulated (Fig. 1c). Moreover, Group 2 presented 544 differentially expressed circRNAs (118 significantly upregulated circRNAs and 426 significantly downregulated circRNAs) (Fig. 1f). Cross-analysis of the two datasets revealed 62 circRNAs with differential expression in both groups; within this subset, only three circRNAs were consistently upregulated across both groups, whereas 21 were downregulated in both groups (Fig. 1g, h).

Fig. 1

RNA sequencing analysis of gastrin-related circRNAs that are differentially expressed in CRC tissues and normal tissues. a-c Heatmaps and volcano maps of the first group of sequencing results and the corresponding statistical figure; d-f heatmaps and volcano maps of the second group of sequencing results and the corresponding statistical figure; g-h circRNAs that were significantly differentially expressed in both groups and their relevant statistical graphs

CircRNA_0017065 is a new gastrin-related circRNA

To verify the sequencing results, we selected the three circRNAs that were significantly upregulated in both groups and verified their expression levels in 20 pairs of CRC tissues and adjacent normal tissues. The results revealed that the expression levels of the three circRNAs in tumour tissue were significantly greater than those in adjacent normal tissues, and the expression differences for circRNA_0017065 were the most obvious (Fig. 2a). We further expanded the sample size and verified the differential expression of the three circRNAs in the tumour tissues and adjacent normal tissues of 40 CRC patients. Therefore, we chose to focus on circRNA_0017065 in our research(Fig. 2b), which was consistent with the sequencing results. The results of qRT-PCR experiments suggested that the expression of circ_0017065 was higher in tumor tissues than in adjacent normal tissues, so we collected clinical data and found that the high expression of circ_0017065 was associated with the depth of tumour invasion, lymph node metastasis, tumor size and vascular invasion(Table 1). To verify the expression level of circRNA_0017065 in cells, we detected the expression level of circRNA_0017065 in 6 CRC cell lines and normal colon mucosal epithelial cells. The results revealed that the expression level was the highest in SW480 and HCT116 cells (Fig. 2c). Sanger sequencing confirmed the circular structure of circRNA_0017065 (Fig. 2d). We then tested the stability of circRNA_0017065 after RNase R treatment; the results revealed that it was resistant to RNase R digestion (Fig. 2e). The circular structure of circRNA_0017065 was verified via two agarose gel electrophoresis methods (Fig. 2f, g). These results confirmed that circRNA_0017065 is more stable than its host gene.Therefore, we hypothesized that gastrin positively regulates the expression of circRNA_0017065. RNA fluorescence in situ hybridization (FISH) was performed to confirm that circRNA_0017065 was localized mainly in the cytoplasm (Fig. 2h). We subsequently overexpressed gastrin and circRNA_0017065 and detected changes in the expression levels of gastrin and circRNA_0017065 to verify the relationship between gastrin and circRNA_0017065. The results revealed that the level of circRNA_0017065 increased significantly after gastrin was overexpressed. However, after circRNA_0017065 overexpression, the level of gastrin did not change significantly (Fig. 2i).In this study, we focused on the role of circRNA_0017065 in the development of CRC.

Table 1 Associations between circ_0017065 levels and clinical paramenters in colorectal cancer patients

To verify the sequencing results, we selected the three circRNAs that were significantly upregulated in both groups and verified their expression levels in 20 pairs of CRC tissues and adjacent normal tissues. The results revealed that the expression levels of the three circRNAs in tumour tissue were significantly greater than those in adjacent normal tissues, and the expression differences for circRNA_0017065 were the most obvious (Fig. 2a). We further expanded the sample size and verified the differential expression of the three circRNAs in the tumour tissues and adjacent normal tissues of 40 CRC patients. Therefore, we chose to focus on circRNA_0017065 in our research(Fig. 2b), which was consistent with the sequencing results. The results of qRT-PCR experiments suggested that the expression of circ_0017065 was higher in tumor tissues than in adjacent normal tissues, so we collected clinical data and found that the high expression of circ_0017065 was associated with the depth of tumour invasion, lymph node metastasis, tumor size and vascular invasion(Table 1). To verify the expression level of circRNA_0017065 in cells, we detected the expression level of circRNA_0017065 in 6 CRC cell lines and normal colon mucosal epithelial cells. The results revealed that the expression level was the highest in SW480 and HCT116 cells (Fig. 2c). Sanger sequencing confirmed the circular structure of circRNA_0017065 (Fig. 2d). We then tested the stability of circRNA_0017065 after RNase R treatment; the results revealed that it was resistant to RNase R digestion (Fig. 2e). The circular structure of circRNA_0017065 was verified via two agarose gel electrophoresis methods (Fig. 2f, g). These results confirmed that circRNA_0017065 is more stable than its host gene.Therefore, we hypothesized that gastrin positively regulates the expression of circRNA_0017065. RNA fluorescence in situ hybridization (FISH) was performed to confirm that circRNA_0017065 was localized mainly in the cytoplasm (Fig. 2h). We subsequently overexpressed gastrin and circRNA_0017065 and detected changes in the expression levels of gastrin and circRNA_0017065 to verify the relationship between gastrin and circRNA_0017065. The results revealed that the level of circRNA_0017065 increased significantly after gastrin was overexpressed. However, after circRNA_0017065 overexpression, the level of gastrin did not change significantly (Fig. 2i).In this study, we focused on the role of circRNA_0017065 in the development of CRC.

Fig. 2

CircRNA_0017065 is a new gastrin-related circRNA. a The expression level of circRNAs significantly upregulated in both groups was verified in patient tissues; b circRNA_0017065 expression level in tumour tissues and adjacent normal tissues of 40 patients with CRC; c the expression level of circRNA_0017065 in cells; d the sequence around the junction site; e-g the stability of circRNA_0017065 was verified by agarose gel electrophoresis and RNA enzyme digestion; h the location of circRNA_0017065 in the cytoplasm was determined via fluorescence in situ hybridization; i gastrin can positively regulate the expression of circRNA_0017065 in cells

Knockdown of circRNA_0017065 inhibits CRC cell proliferation and migration

To explore the function of circRNA_0017065 in CRC cells, we first designed three types of siRNAs that target the back-splice region. Loss-of-function assays were then performed in SW480 and HCT116 cells with relatively high expression of circRNA_0017065. After transfection with the 3 siRNAs, si-1 significantly reduced circRNA_0017065 expression in the two cell lines (Fig. 3a). CCK-8 and EdU assays confirmed that the downregulation of circRNA_0017065 significantly inhibited the proliferation of SW480 and HCT116 cells (Fig. 3b, c). Transwell and wound healing assays confirmed that after the expression of circRNA_0017065 was knocked down, the migration abilities of SW480 and HCT116 cells were significantly inhibited (Fig. 3d, e). We further studied whether circRNA_0017065 affects the apoptosis of CRC cells via flow cytometry. Annexin V and PI double staining revealed that circRNA_0017065 downregulation with si-1 significantly promoted apoptosis (48 h after transfection) compared with that in the si-NC group (Fig. 3f). These results suggest that circRNA_0017065 can affect the proliferation and migration of CRC cells in vitro.

Fig. 3

Knockdown of circRNA_0017065 inhibits CRC cell proliferation and migration. a Transfection efficiency of three types of siRNAs in colorectal cancer cells. b-c EdU and CCK-8 assays revealed that inhibition of circRNA_0017065 expression significantly reduced the proliferation ability of colorectal cancer cells. d-e Transwell assays and wound healing assays confirmed that inhibition of circRNA_0017065 expression significantly reduced the migration ability of colorectal cancer cells. f Flow cytometry confirmed that inhibition of circRNA_0017065 expression significantly increased the percentage of apoptotic colorectal cancer cells

CircRNA_0017065 can promote tumour growth in vivo

To explore the role of circRNA_0017065 in vivo, SW480 cells with circRNA_0017065 knockdown and overexpression, as well as negative control cells, were selected and subcutaneously injected into the right groin region of BALB/c nude mice. Changes in tumour volume and weight in the nude mice were observed and recorded every three days. On the 30th day, the nude mice were sacrificed, and the tumours were harvested. The results revealed that the tumour volume and weight were significantly lower in the circRNA_0017065-knockdown group than in the control group. The tumour volume and weight were significantly greater in the nude mice injected with circRNA_0017065-overexpressing cells than in the control group (Fig. 4a, b, c). Immunohistochemical staining was used to detect the expression levels of PCNA and the target protein RBFOX2 in tumour tissues. The results revealed that the expression levels of PCNA and RBFOX2 were significantly increased in the tumour tissues overexpressing circRNA_0017065, whereas the expression levels of PCNA and RBFOX2 were significantly decreased in the tumour tissues with circRNA_0017065 knockdown compared with those in the normal negative control group. (Fig. 4d). Thus, inhibition of gastrin-related circRNA_0017065 expression significantly inhibited the growth of colorectal cancer cells in vivo.

Fig. 4

CircRNA_0017065 can promote tumour growth in vivo. a-cIn vivo experiments confirmed that the overexpression of circRNA_0017065 significantly promoted the proliferation of colorectal cancer cells, whereas the inhibition of circRNA_0017065 significantly suppressed the proliferation of colorectal cancer cells. d Immunohistochemistry was used to detect changes in related proliferative indices in nude mouse tumours

CircRNA_0017065 functions as a sponge for miR-3174

The biological function of circRNAs as miRNA sponges has been recognized by most researchers. To explore the potential mechanism of circRNA_0017065 in the proliferation of CRC cells. We predicted six miRNAs that might bind to circRNA_0017065 via a biological prediction website, five of which were consistent with the results of gastrin-related miRNAs screened via our previous gene chip (Fig. 5a). Cytoscape software was used for bioinformatics analysis, and a network diagram was constructed on the basis of circRNA‒miRNA‒mRNA interactions (Fig. 5b). To verify whether circRNA_0017065 can act as a miRNA sponge, we designed a RIP experiment. Many studies have shown that miRNAs mainly inhibit translation and degrade mRNAs in an AGO2-dependent manner [15]. Therefore, to test this hypothesis, anti-AGO2 activity was assessed in SW480 and HCT116 cells. As shown in the figure, the enrichment rate of circRNA_0017065 in the AGO2 group was greater than that in the IgG group (Fig. 5c). We performed miRNA pull-down assays using a biotin-labelled probe targeting circRNA_0017065 to determine the binding potential of the five predicted miRNAs with circRNA_0017065. The results revealed that, among the miRNAs, miR-3174 was the most significantly enriched in the pull-down product of SW480 cells (Fig. 5d). Through bioinformatics analysis via the TargetScan website (http://www.targetscan.org/), we identified potential binding sites between circRNA_0017065 and miR-3174 (Fig. 5e). On the basis of this site, we designed a luciferase construct containing the potential miR-3174 binding site and one with a mutation in this site of circRNA_0017065. We performed dual-luciferase reporter gene tests in SW480 cells, and the results revealed that miR-3174 mimics significantly reduced the luciferase activity in the circRNA_0017065-WT group but had no effect on the circRNA00_17065-MUT group (Fig. 5f). On this basis, we detected the expression level of miR-3174 in 40 pairs of CRC patient samples. The results revealed that miR-3174 expression was significantly lower in tumour tissues than in adjacent normal tissues (Fig. 5g). Spearman correlation coefficient analysis also revealed that the expression of miR-3174 and circRNA_0017065 in CRC tissues was significantly negatively correlated (R = − 0.3229, P = 0.0421) (Fig. 5h). All of the above experiments confirmed that circRNA_0017065 could play a biological role as a sponge of miR-3174.

Fig. 5

CircRNA_0017065 functions as a sponge for miR-3174. a Prediction results of circRNA_0017065 downstream target genes in the database. b CircRNA‒miRNA‒mRNA interaction model predicted by Cytoscape software. c Enrichment of AGO2 in colorectal cancer cells. d In colorectal cancer cells, miR-3174 was most significantly pulled down by circRNA_0017065. e Predicted binding targets of circRNA_0017065 and miR-3174. f MiR-3174 expression affects the dual-luciferase activity of circRNA_0017065. g Expression level of miR-3174 in colorectal cancer patients. h Correlation analysis diagram of circRNA_0017065 and miR-3174

Overexpression of miR-3174 inhibited the proliferation and migration and promoted the apoptosis of CRC cells

To verify the biological function of miR-3174 in the development of CRC, we transfected SW480 and HCT116 cells with miR-3174 mimics and conducted cell function experiments after 48 h. We performed EdU and CCK-8 assays to verify the effect of miR-3174 on the proliferation of CRC cells. The number of proliferated cells in the transfected miR-3174 mimic group was significantly lower than that in the NC group (Fig. 6a, b, c, d). Transwell and wound healing assays revealed that the migration ability of CRC cells was significantly lower in the miR-3174-overexpressing group than in the NC group (Fig. 6e, f, g and h). Moreover, cell apoptosis assays confirmed that overexpression of miR-3174 increased the apoptosis rate of CRC cells (Fig. 6i, j, k).

Fig. 6

Overexpression of miR-3174 can inhibit the proliferation and migration and promote the apoptosis of CRC cells. a-c EdU assays in colorectal cancer cells revealed that the proliferation of colorectal cancer cells was significantly suppressed after overexpression of miR-3174. d CCK-8 assays confirmed that cell activity was significantly decreased after overexpression of miR-3174. e-h Transwell assays and wound healing assays confirmed that the migration of colorectal cancer cells was significantly suppressed after overexpression of miR-3174. i-k Flow cytometry confirmed that overexpression of miR-3174 significantly increased the percentage of apoptotic colorectal cancer cells

RBFOX2 is directly targeted by miR-3174 and indirectly regulated by circRNA_0017065

To identify and confirm the downstream targets of miR-3174, we used miRDB (http://mirdb.org/cgi-bin/search.cgi) and TargetScan (http://www.targetscan.org/) forecasting software for downstream target genes, and four targets were predicted (Fig. 7a). After we subsequently knocked down the expression of circRNA_0017065, only the levels of RBFOX2 were significantly downregulated, whereas those of the other three target proteins did not significantly change (Fig. 7b, c). Therefore, we assumed that RBFOX2 was our downstream target gene and predicted binding sites of miR-3174 and the downstream target protein RBFOX2 (Fig. 7d). To verify our hypothesis, we designed dual-luciferase reporter assays, which revealed that miR-3174 mimics significantly reduced the relative luciferase activity of the RBFOX2 3’UTR WT plasmid but had no significant effect on the dual-luciferase activity of the MUP plasmid (Fig. 7e). We subsequently confirmed that the expression of miR-3174 was significantly negatively correlated with the expression of RBFOX2 via qRT‒PCR (Fig. 7f). The expression level of RBFOX2 was detected again in 40 pairs of CRC patients, and the results revealed that the expression level of RBFOX2 in tumour tissues was significantly greater than that in normal tissues and that there was a significant positive correlation between the expression levels of RBFOX2 and circRNA_0017065 (Fig. 7g, h).

Fig. 7

RBFOX2 is directly targeted by miR-3174 and indirectly regulated by circRNA_0017065. a The prediction results of the downstream target protein of miR-3174. b-c After knockdown of the expression of circRNA_001706, RBFOX2 was significantly downregulated, whereas the expression of the other three target proteins did not change significantly. d Predicted binding sites of miR-3174 and the downstream target protein RBFOX2. e Overexpression of RBFOX2 changed the dual-luciferase activity of miR-3174. f Overexpression and knockdown of the expression of miR-3174 in colorectal cancer cells also changed the level of RBFOX2. g The expression levels of RBFOX2 in colorectal cancer patients. h Correlation analysis diagram of RBFOX2 and circRNA 0017065

Inhibition of miR-3174 expression rescues the inhibitory effect of circRNA_0017065 knockdown on CRC cells

Rescue experiments were conducted to study the role of gastrin-related circRNA_0017065/miR-3174/RBFOX2 in the progression of CRC. The inhibition of si-circRNA_0017065 was reversed by using a miR-3174 inhibitor in SW480 cells. EdU and CCK-8 assays revealed that the miR-3174 inhibitor reversed the inhibitory effect of circRNA_0017065 knockdown on the proliferation of CRC cells (Fig. 8a, b, c). Wound healing assays and Transwell assays revealed that the miR-3174 inhibitor rescued the migration ability of CRC cells after knockdown of circRNA_0017065 (Fig. 8d, f). Apoptosis experiments revealed that the miR-3174 inhibitor weakened the apoptotic effect on CRC cells after circRNA_0017065 knockdown (Fig. 8g). Western blotting analysis revealed that knockdown of circRNA_0017065 reduced the protein expression levels of RBFOX2 and the apoptosis-related proteins Bax, Caspase3, CyclinD1 and MMP2 in CRC cells. This effect was reversed by the miR-3174 inhibitor (Fig. 8e). In conclusion, gastrin-related circRNA_0017065 could promote the malignant behaviour of CRC through miR-3174-mediated RBFOX2.

Fig. 8

Inhibition of miR-3174 expression attenuates the inhibitory effect of circRNA_0017065 knockdown on colorectal cells a-c CCK-8 and EdU assays confirmed that miR-3174 inhibitors attenuated the inhibitory effect of circRNA_0017065 knockdown on the proliferation of colorectal cancer cells. d–f Wound healing assays and Transwell assays confirmed that miR-3174 inhibitors attenuated the inhibition of colorectal cancer cell migration induced by circRNA_0017065 knockdown. e WB verified the effect of the miR-3174 inhibitor on colorectal cancer-related proteins after circRNA_0017065 knockdown. g Flow cytometry was used to verify the effect of the miR-3174 inhibitor on the apoptosis of colorectal cancer cells after circRNA_0017065 knockout

Overexpression of RBFOX2 rescues the inhibitory effect of circRNA_0017065 knockdown on colorectal cells

To verify that miR-3174 can rescue the effects of circRNA_0017065 knockdown on cell proliferation, migration and apoptosis, we designed a rescue experiment in which RBFOX2 was used against circRNA_0017065 in SW480 cells. EdU and CCK-8 assays revealed that overexpression of RBFOX2 rescued the inhibitory effect of circRNA_0017065 knockdown on the proliferation of CRC cells (Fig. 9a, b). Wound healing and Transwell assays confirmed that the overexpression of RBFOX2 rescued the migration ability of CRC cells after the knockdown of circRNA_0017065 (Fig. 9c, e). Western blotting analysis revealed that circRNA_0017065 knockdown reduced the protein expression levels of RBFOX2 and the apoptosis-related proteins Bax, Caspase3, CyclinD1 and MMP2 in CRC cells. This effect was reversed by the overexpression of RBFOX2 (Fig. 9d). Apoptosis experiments revealed that RBFOX2 overexpression weakened the apoptotic effect on CRC cells after circRNA_0017065 knockdown (Fig. 9f). The above experiments confirmed that gastrin-related circRNA_0017065 could regulate CRC cell proliferation through the miR-3171/RBFOX2 pathway.

Fig. 9

Overexpression of RBFOX2 attenuates the inhibitory effect of circRNA_0017065 knockdown on colorectal cells. a-b CCK-8 and EdU assays confirmed that RBFOX2 overexpression attenuated the inhibitory effect of circRNA_0017065 knockdown on the proliferation of colorectal cancer cells. c-e Wound healing and Transwell assays confirmed that RBFOX2 overexpression attenuated the inhibition of colorectal cancer cell migration induced by circRNA_0017065 knockdown. d WB verified the effect of RBFOX2 overexpression on colorectal cancer-related proteins after circRNA_0017065 knockdown. f Flow cytometry was used to verify the effect of RBFOX2 overexpression on colorectal cancer apoptosis after circRNA_0017065 knockdown

In conclusion, we discovered that circRNA_0017065 mediates CRC progression by increasing RBFOX2 expression by sponging miR-3174 (Fig. 10). Our results revealed the role and potential mechanism of circRNA_0017065 and provided evidence that circRNA_0017065 could promote the occurrence and development of CRC.

Fig. 10

Schematic diagram of the mechanism by which circRNA_0017065 promotes the proliferation and metastasis of colorectal cancer cells through the miR-3174/RBFOX2 axis

Discussion

CRC is one of the most common malignancies in the world. In recent years, the incidence of CRC has gradually increased, and it has become the third most common malignant tumour. It is also the third leading cause of cancer-related deaths worldwide [16]. Many domestic and foreign reports have confirmed that the progression of some CRC cases is significantly positively correlated with gastrin expression [17, 18], which may be related to the fact that hypergastrinemia can directly promote the mitosis of CRC cells or normal cells [19]. However, the molecular mechanism and potential signalling pathway involved are unclear. This study explored the molecular mechanism of gastrin in the progression of CRC from three aspects: gastrin, circRNAs and CRC. We confirmed that gastrin could cause differential expression of circRNAs in tumour tissues, thus exerting biological effects to promote the progression of CRC. This study provides potential molecular targets and new directions for the treatment of CRC.

Metastasis is still the leading cause of death in CRC patients. The onset of CRC is usually insidious, with no obvious early manifestations, and more than half of CRC cases are diagnosed before lymph node metastasis occurs [20]. Therefore, finding effective tumour markers is very important. CircRNAs are a new class of noncoding RNAs without 5’-3’ polarity and a poly A tail structure. They are covalently linked to form a closed continuous structure with high stability [21,22,23]. In addition, a number of studies in recent years have shown that circRNAs are widely involved in human pathology and physiology, including regulating the occurrence and development of various tumours. For example, circRNA_100367 is upregulated in oesophageal cancer and promotes tumour invasion and metastasis [24], circRNA_404686 plays an important role in the occurrence and development of thyroid cancer [25], and the overexpression of circRNA Cdr1as significantly promotes the proliferation and migration of HCC cells [26]. CircNFIB1 is also closely related to the invasion and metastasis of pancreatic cancer [27]. Therefore, circRNAs are also expected to become new tumour markers.

In our research, we discovered a novel circRNA associated with gastrin, designated circRNA_0017065, within colorectal cancer (CRC) tissues. This circRNA has been shown to increase CRC cell proliferation and migration while concurrently suppressing apoptosis. Notably, its expression is markedly elevated in tumour cells. Upon analysis of clinical data, we observed a substantial positive correlation between the expression of circRNA_0017065 and both tumour stage and lymph node metastasis in CRC patients. These findings suggest that circRNA_0017065 has potential as an emerging biomarker for colorectal cancer.

Growing evidence suggests that exon-derived circRNAs can function as miRNA sponges [28], thereby influencing various biological processes. In our study, we utilized miRNA gene chips, starBase, and circBank to predict the potential miRNA targets of gastrin-associated circRNA_0017065. Subsequent RIP experiments confirmed that circRNA_0017065 indeed acts as a miRNA sponge in a cellular context. To elucidate the interactions between circRNA_0017065 and its predicted miRNA partners, we employed Cytoscape to create an interaction network diagram featuring circRNA_0017065 and the five candidate miRNAs. Further investigation through RNA pull-down assays specifically confirmed that circRNA_0017065 effectively sponges miR-3174, one of the predicted miRNAs. This interaction suggests a regulatory mechanism by which circRNA_0017065 could modulate the progression of colorectal cancer by acting as an miRNA sponge.

To verify this hypothesis, we conducted a series of in vitro and in vivo functional experiments and found that circRNA_0017065 can affect the proliferation, migration and apoptosis of CRC cells. Similarly, miR-3174 mimics affected the proliferation, migration and apoptosis of CRC cells, and the inhibitory effect of si-circRNA_0017065 on CRC cells was attenuated by a miR-3174 inhibitor. Subsequent correlation analysis revealed that circRNA_0017065 was significantly negatively correlated with miR-3174. These findings support the hypothesis that circRNAs can serve as miRNA sponges, competitively binding miRNAs and thereby indirectly mitigating the suppressive effects of miRNAs on their target genes [29]. Having established that miR-3174 is sequestered by circRNA_0017065, we focused on identifying downstream target genes. By mining multiple databases, we identified several likely candidate genes. Subsequent experimental validation confirmed that miR-3174 specifically interacts with RBFOX2, influencing its expression in colorectal cancer cells. This discovery contributes to our understanding of the molecular interplay in CRC and highlights the potential regulatory role of circRNA_0017065 as an miRNA sponge.

RBFOX2, a class of RNA-binding proteins that regulate alternative splicing, is highly expressed in brain, muscle, and embryonic stem cells and is recognized for its critical role in the progression of lymphoma [30, 31]. Furthermore, the literature corroborates that alterations in RBFOX2 expression can influence CRC metastasis through the regulation of microexon splicing [32], which is also consistent with the results of our study.

Increasing evidence has revealed that circRNAs are potential cancer biomarkers and therapeutic targets. The most extensively documented role of circRNAs is their capacity to function as miRNA sponges within the circRNA–miRNA‒mRNA regulatory network. Our study introduces a newly identified gastrin-associated circRNA, circRNA_0017065, discovered through high-throughput sequencing. We have demonstrated, via database predictions and extensive experimental validation, that circRNA_0017065 modulates CRC cell proliferation, migration, and apoptosis through the miR-3174/RBFOX2 axis. The influence of the circRNA_0017065/miR-3174/RBFOX2 axis on CRC cellular dynamics was further substantiated via a series of techniques, including Western blotting, immunohistochemistry, and quantitative real-time PCR, underscoring its potential as a novel molecular mechanism in CRC pathogenesis and as a target for therapeutic intervention.

Conclusions

Overall, our findings reveal marked disparities in the expression levels of circRNAs among CRC patients with varying gastrin levels. The gastrin-associated circRNA_0017065 has a pivotal influence on CRC progression and holds promise as a prospective molecular target for controlling CRC metastasis. Additionally, it shows potential as a biomarker for the early detection of CRC, offering a new avenue for diagnostic and therapeutic strategies in the management of this malignancy.

Materials and methods

Patient population and clinical data

A total of 40 CRC tissues and normal adjacent tissues were obtained from patients who underwent radical surgery at the Department of Gastrointestinal Surgery, the First Affiliated Hospital of WanNan Medical College, between January 2020 and February 2021. All the tissue samples were collected from patients who underwent colorectal surgery at our hospital. The tissue samples were put into cryopreservation tubes with RNAlater and quickly stored in liquid nitrogen until RNA extraction. All the patients received no radiotherapy or chemotherapy before surgery and signed an informed consent for surgery. The procurement of these specimens was conducted with full disclosure to the patients and their families, who provided informed consent. The use of these specimens for the study was approved by the Ethics Committee at Wannan Medical College, ensuring compliance with ethical standards.

RNA extraction and quantitative real-time PCR (qRT‒PCR)

Total RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s instructions. The expression of miRNAs was determined via SYBR Green quantitative real-time PCR (qPCR). For circRNA and mRNA, total RNA was reverse transcribed into cDNA (RT), and then, qPCR was performed via the SYBR Green PCR Kit (Takara, Otsu, Japan). All primer sequences were designed and synthesized by RiboBio (Guangzhou, China) (see the appendix for all primer sequences). GAPDH was selected as the reference gene for circRNAs and mRNAs. U6 was selected as the miRNA control. Gene expression was quantified via the 2^–∆∆Ct method. Three auxiliary wells were set for each group of data, and the average value was taken. The primers for GAPDH, RBFOX2 and circRNA_0017065 were as follows:

h-GAST_F1 ATGCAGCGACTATGTGTGTATG.

h-GAST_R1 GCCCCTGTACCTAAGGGTG.

h-RBFOX2_qPCR_116 bp_F1 ACCAGGAGCCGACAACAACT.

h-RBFOX2_qPCR_116 bp_R1 GTCTTGAGTGTGTGGCACCC.

h-hsa_circ_0017065_qPCR_149 bp_F1 TCTCCACAAAGTGACAGTGAATGA.

h-hsa_circ_0017065_qPCR_149 bp_R1 GAAATAAGGCCAACTGATCCTGA.

RNA sequencing

The tissue samples were extracted via the TRIzol method according to the manufacturer’s instructions, and the samples were inspected via a K5500 spectrophotometer and an Agilent 2200 TapeStation. rRNA and linear RNA were first removed, and the circRNAs were enriched. After the sample was segmented, the first strand cDNA and the second strand cDNA were synthesized and purified successively, and then the repair end and the 5′caps were added with 3′poly(A) tails. After the above process was completed, qRT‒PCR was used for amplification and purification, and an Agilent 2200 TapeStation was used for library quality inspection. The library that passed inspection was loaded on the computer according to the method described in the Illumina platform instrument corresponding to the User Guide, and the Pair End standard sequencing program was run. After the sequencing program was run, bioinformatics analysis was conducted on the obtained data.

Cell culture

Four kinds of human CRC cells (HT29, HCT116, SW480 and LOVO), as well as a normal colonic mucosal epithelial cell line (NCM460), were purchased from Genechem (Shanghai, China). These cells were cultured in DMEM containing 100 µg/mL streptomycin, 100 U/mL penicillin and 10% foetal bovine serum (FBS; Gibco, NY, USA), and the cells were maintained at 37 °C in a humidified atmosphere with 5% CO2.

Transwell assay

In accordance with the manufacturer’s instructions (BD Biosciences, Bedford, MA, USA), 5 × 10⁴ single-cell suspensions and 200 µL of serum-free medium were seeded into the upper compartment, and 500 µL of culture medium containing 10% FBS (bovine serum albumin) was added to the lower compartment. After 36 h of incubation, the cells located on the upper surfaces of the Transwell compartment were removed with cotton swabs, and the cells located on the lower surfaces were washed twice with PBS and fixed with 4% paraformaldehyde for 30 min. Then, the cells were stained with crystal violet (Sigma, MO, USA) for 10 min at room temperature, and the cells were photographed under a microscope. Each group included 3 replicate wells, and we calculated the mobility by counting cells in at least 5 random fields.

Wound healing assay

Wound healing assays were used to verify the migration ability of CRC cells. In brief, the transfected cells and the controls were cultured in 6-well plates. When the cells reached 100% confluence, the cell monolayer was subsequently scratched with a 1000 µL pipette tip. The scratch healing ability of the transfected cells and the control cells was observed 48 h later. The experiment was repeated three times for each group.

Apoptosis analysis

An Annexin V-FITC apoptosis detection kit (KeyGen Biotech, Nanjing, China) was used according to the manufacturer’s instructions. 48 h after transfection, the cells were collected and immobilized in 75% cold ethanol and then stored overnight at 4 °C. CRC cells were stained with Annexin V-FITC and PI. The number of apoptotic cells was quantified via fluorescence-activated cell sorting flow cytometry (BD Biosciences), and the percentages of apoptotic cells in the control group and the experimental group were analysed. The experiment was repeated three times.

EdU assay

In accordance with the manufacturer’s instructions, 48 h after transfection, 8 × 10³ cells/well were inoculated into the experimental and control group wells of a 96-well plate and were subsequently incubated with EdU labelling reagent for 2 h, fixed with 4% paraformaldehyde for 30 min, and fixed with 2 mg/mL glycine. Osmotic agent (0.5% Triton X-100 PBS) was added, and the samples were incubated on a shaker for 10 min. Apollo staining was performed, and the samples were incubated in a shaker for 30 min at room temperature in the dark. The samples were subjected to 4′,6-diamidino-2-phenylindole (DAPI) staining, incubated at room temperature for 30 min, and observed under a fluorescence microscope. The experiment was repeated 3 times in each group, and 5 fields were randomly selected.

Cell cycle measurement

CRC cells were pretreated according to the manufacturer’s instructions, then attached and floating cells were collected, and PI DNA staining was used to analyse the changes in the cell cycle distribution of cells in the control and experimental groups via FACS.

CCK-8 assay

Cell proliferation was detected via a CCK-8 assay (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. A total of 100 µL of DMEM containing 2 × 10³ CRC cells was inoculated into 96-well plates. At 24 h, 48 h, 72 h, 96 h and 118 h, 10 µL of CCK-8 solution was added to the 96 empty plates. After incubation for 2 h at 37 °C, the absorbance at 450 nm was measured and recorded. The experiment was repeated three times.

RNA pulldown and RNA‒protein immunoprecipitation (RIP)

RIP experiments were carried out in SW480 and HCT116 cells. First, 1 × 10⁷ cells were completely lysed with RNA lysate and incubated with magnetic beads conjugated with an anti-AGO2 antibody (Abcam, #AB186733) in either immunoprecipitation buffer (Millipore, USA) or negative control mouse IgG (Millipore, USA). The RIP samples were supplemented with protease K and incubated at 55 °C for 30 min. The enrichment of circRNA_0017065 was analysed via qRT‒PCR after the immunoprecipitated RNA was obtained.

Fluorescence in situ hybridization (FISH)

RNA fluorescence in situ hybridization was performed via a fluorescence in situ hybridization kit (Genepharma, Shanghai, China). The circRNA_0017065 and miR-3174 probes were designed and synthesized by GenePharma (Shanghai, China). The circRNA_0017065 probe was labelled with Cy3, and the miR-3174 probe was labelled with Dig. After the experimental steps were completed according to the instructions of the fluorescence in situ hybridization kit, the fluorescence was observed with a confocal microscope and photographed.

Luciferase reporter gene assay

The target gene of miR-3174 was predicted and analysed via the TargetScan and PicTar sites. The reporter plasmid was commissioned by RiboBio (Guangzhou, China) for design and synthesis. In accordance with the manufacturer’s instructions, CRC cells (5 × 10⁵ cells per well) were first inoculated into a 24-well plate, and then the corresponding reporter plasmid and miRNA mimics or negative controls were added to the 24-well plate for transfection using Lipofectamine 3000 reagent. 48 h after cotransfection, a dual-luciferase reporter assay system was used (Promega, Madison, MA, USA). Luciferase activity was detected. The experiment was carried out in triplicate.

Antibodies and western blotting

SW480 and HCT116 cells were lysed in RIPA and PMSF (100:1 mixture) lysis buffer. Equal quantities of protein were then separated via SDS‒PAGE and electrotransferred to PVDF membranes (Millipore, Schwalbach, Germany), which were then blocked with 5% skim milk powder for 2 h and incubated overnight with primary antibody against the following targets at 4 °C: RBFOX2 (#AB57154, Abcam), Caspase3 (# AB39675, Abcam), cyclin D1 (#4267, Cell Signaling Technology), Bax (#66281-IG, Proteintech), GAPDH (#AB181602, Abcam), and MMP2 (#40994, CST). The membranes were then incubated with HRP-conjugated secondary antibodies at room temperature for 1 h, after which the blots were observed via an enhanced chemiluminescence kit (Pierce, Waltham, MA, USA).

IHC

The tumour samples were fixed in paraformaldehyde, embedded in paraffin, sectioned, dewaxed, and stained according to the instructions. The primary antibodies used were anti-PANC antibody (# GB11010, Gibco) and anti-FOX2/RBM9 antibody (# AB57154, Abcam). The complexes were stained with DAB, and the nuclei were stained with haematoxylin. The nuclei stained with haematoxylin are blue. DAB-positive expression was visualized as a brownish yellow colour. All the sections were scored via the semiquantitative H-score method and then observed under a microscope and photographed.

Xenograft tumour model

BALB/C nude mice (female, 3 to 4 weeks of age) were subcutaneously injected with 1 × 10⁷ SW480 cells. The tumour dimensions were measured with callipers every 3 days, and the length (a) and width (b) were used to calculate volume via the following formula: volume (mm 3) = AB 2/2. The animals were sacrificed 30 days after injection, and the tumour tissue was removed for further evaluation of tumour weight and pathological staining.

Statistical analysis

GraphPad Prism 8.0 (GraphPad Software Inc., CA, USA) was used for statistical analysis. Student’s t test and one-way ANOVA were used to compare differences between groups as appropriate. Correlations between groups were analysed by Pearson correlation analysis. The data are presented as the means ± standard deviations (SDs), and P < 0.05 was considered statistically significant. Statistical significance is denoted as follows: **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.