circIFT80 Functions as a ceRNA of miR-1236-3p to Promote Colorectal Cancer Progression.

Circular RNAs (circRNAs), a recently identified new member of non-coding RNAs, are demonstrated to participate in diverse biological processes; however, the molecular mechanisms that link circRNAs with colorectal cancer (CRC) are not well understood. In the present study, we attempted to explore the roles of the exosomal circRNAs on CRC progression. We first compared the expression patterns of exosomal circRNAs between the plasma of CRC patients and healthy controls. We identified 448 significantly dysregulated exosomal circRNAs in CRC plasma. We focused on hsa_circ_0067835, which is located on chromosome 3 and derived from IFT80; thus, we named it circIFT80. Then, the expression of circIFT80 was detected in 58 CRC tissues and cell lines by qRT-PCR. Functional assays were performed to evaluate the effects of circIFT80 on tumor growth in vitro and in vivo. The relationship between circIFT80 and miR-1236-3p was confirmed by luciferase reporter assay. We found that circIFT80 was significantly upregulated in CRC serum exosomes, CRC tissues, and CRC cell lines compared with normal control. Silencing circIFT80 suppressed CRC cell growth both in vitro and in vivo. We further demonstrated that circIFT80/miR-1236-3p/HOXB7 axis plays an important role in regulating CRC progression. Dual-luciferase reporter system validated the direct interaction of circIFT80, miR-1236-3p, and HOXB7. Western blot verified that inhibition of circIFT80 decreased HOXB7 expression, while a miR-1236-3p inhibitor attenuated the effect of inhibition of circIFT80. In conclusion, these data suggest that circIFT80 is a central component linking circRNAs to the progression of CRC via a miR-1236-3p/HOXB7 axis.

Introduction

Colorectal cancer (CRC) is one of the most common causes of cancer death worldwide. Patients with early-stage CRC could be cured by surgery. Unfortunately, chemotherapy and surgical resection are not effective for patients with advanced CRC. Despite recent progresses in the therapeutic strategies for CRC, including targeted therapies and immunotherapy, the prognosis for advanced-stage CRC patients is still far from satisfactory. Thus, further understanding of molecular mechanisms of CRC may help identify potential tumor biomarkers and develop novel therapeutic targets.

Circular RNAs (circRNAs) are a novel class of non-coding RNAs, which are characterized by their covalently closed-loop structures, which gives them a higher tolerance to exonucleases. Emerging evidence demonstrates that circRNAs could serve as potential biomarkers and therapeutic targets for various cancers. Notably, a large body of recent evidence indicates that circRNAs are involved in the initiation and progression of CRC. High-throughput RNA sequencing (RNA-seq) revealed that circDDX17 functions as a tumor suppressor and could serve as a potential biomarker and a therapeutic target for CRC. Li et al. indicated a suppressor role of circITGA7 and ITGA7 in CRC and revealed that circITGA7 inhibits the proliferation and metastasis of CRC cells by suppressing the Ras signaling pathway and promoting the transcription of ITGA7. circRNAs can function as competitive endogenous RNAs (ceRNAs) to sponge microRNAs (miRNAs), regulate transcription and alternative splicing, interact with RNA-binding proteins (RBPs), and be translated into protein. However, as the functions of most circRNAs remain largely unknown, the investigations of circRNAs in CRC are still in their infancy.

Exosomes are small, membrane-derived vesicles with a diameter of approximately 30–150 nm. It has been well established that exosomes originate from multivesicular endosomes (multivesicular bodies) by inverse budding to form multivesicular bodies and are released into the extracellular space when the multivesicular body fuses with cell membrane exosomes.11, 12 Exosomes could act as message transmitters in intercellular communication of various cancers.13, 14 Recent studies have demonstrated that circRNAs are abundant and stable in exosomes; however, there is little evidence for the expression profile and potential function of exosomal circRNAs in CRC.

In this study, we first compared the expression patterns of exosomal circRNAs between CRC patients and controls. We identified 448 significantly dysregulated exosomal circRNAs in CRC plasma. We further tested circIFT80 (hsa_circ_0067835) in 58 pairs of CRC samples by qRT-PCR, and the results showed that the expression of circIFT80 was markedly elevated both in CRC tissues and exosomes from CRC plasma. Silencing circIFT80 suppressed CRC cell growth both in vitro and in vivo. We further demonstrated that the circIFT80/miR-1236-3p/HOXB7 axis plays an important role in regulating CRC progression. Our findings will provide new insights into the regulatory mechanisms of circIFT80 in CRC progression.

Results

Expression Profiles of Peripheral Plasma Exosomal circRNAs in Human CRCs

Transmission electron microscopy showed that plasma exosomes have a diameter of 100–150 nm with a spherical shaped morphology (Figure 1A). Western blotting of plasma exosomes demonstrated the presence of Tsg101 and HSP70, which are exosome markers (Figure 1B). To identify exosomal circRNAs, we extracted plasma exosomal RNA from 5 CRC patients and 5 healthy individuals and analyzed the expression profile using the ceRNA array mentioned earlier. As a result, 1,889 circRNAs were upregulated, whereas 1,975 circRNAs were downregulated based on the log2 (fold changes) ≥ 2, p < 0.01, and false discovery rate (FDR) < 0.05. Among these differently expressed genes, we identified the most top 10 upregulated and downregulated circRNAs between CRC patients and negative-control individuals (Figure 1C), which were then subjected to validation by qRT-PCR. There was an increasing trend in hsa_circ_0067835 (chr3:160073800–160099506) levels from the serum exosomes of healthy individuals and CRC patients, with more than 10-fold change from microarray analysis. By browsing the human reference genome (GRCh37/hg19), we identified that hsa_circ_0067835 is derived from IFT80, which is located on chromosome 3, with a spliced mature sequence length of 734 bp; thus, we named it circIFT80.

Figure 1

circIFT80 Was Upregulated in Exosomes Derived from Plasma of CRC Patients, CRC Tissues, and Cell Lines

(A) Transmission electron microscopy images show that plasma exosomes have a diameter of 100–150 nm with a spherical shaped morphology. (B) Western blotting analysis of the exosomal markers Tsg101 and HSP70 in plasma exosomes. (C) The heatmap showed the top 10 most increased and decreased circRNAs in plasma exosomal RNA from 5 CRC patients and 5 healthy individuals analyzed by circRNAs Arraystar Chip. (D) The level of circIFT80 was significantly increased in tumor tissues as compared to that in the matched non-tumor tissues of 58 pairs of CRC patients. ***p < 0.001. (E) The level of circIFT80 was significantly increased in patients with distant metastasis compared to those without metastasis stage. **p < 0.01. (F) The qRT-PCR assay indicated that the expression level of circIFT80 in CRC cell lines was generally higher than that in the HCoEpiC normal colonic cell line. **p < 0.01.

circIFT80 Was Upregulated in CRC Tissues and Cell Lines

Among the upregulated exosomal circRNAs in CRC patients, we used the qRT-PCR assay to analyze their expression in our corhort of 58 pairs of CRC tumor tissues. We first found that circIFT80 was upregulated in CRC tissues when compared with normal tissues (p < 0.001; Figure 1D). Furthermore, the relative higher level of circIFT80 was determined in patients with distant metastasis compared to those without metastasis stage (p < 0.01; Figure 1E). Using the median expression level of circIFT80 as the cutoff value, patients who expressed circIFT80 equal to or greater than the average level were assigned to the “circIFT80 high” group. Analyses of the correlation between circIFT80 expression and clinicopathological traits showed that circIFT80 expression is much higher in tumors with advanced tumor stage (p = 0.003), in tumors with distant metastasis (p = 0.04), and in tumors > 5 cm than it is in tumors ⩽ 5 cm (p = 0.003). There is no significant correlation between circIFT80 expression with age (p = 0.283) or gender (p = 0.186) (Table 1), which indicates that circIFT80 plays a vital role in CRC progression.

Table 1

Correlation between circIFT80 Expression and Clinicopathologic Characteristics of CRC Patients

Clinicopathological Features	circIFT80 Expression			p Value
	Overall (n = 58)	High (n = 29)	Low (n = 29)
Age, in years (more than median)	23	14	9	0.283
Gender (male)	26	16	10	0.186
Tumor size (>5)	28	20	8	0.003
Tumor stages (T3 and T4)	26	19	7	0.003
Metastatic status (present)	11	9	2	0.04
Neoadjuvant therapy (yes)	9	7	2	0.144

To explore the expression of circIFT80 in CRC cells, we analyzed the expression of circIFT80 in different CRC cell lines (HT-29, SW480, RKO, HCT116, and DLD1) and a normal colonic epithelial cell (HCoEpiC) by qRT-PCR. HCT116 and DLD1 cells expressed relatively high levels of circIFT80, whereas HT-29 cells expressed relatively low levels of circIFT80 (p < 0.01; Figure 1F). These cells were then used to establish cell lines with knockdown or overexpression of circIFT80.

circIFT80 Knockdown Inhibits the Proliferation of CRC Cells by Inducing Cell-Cycle Arrest and Inhibiting Cell Apoptosis

To investigate the functional role of circIFT80 in CRC cells, we performed loss- or gain-of-function experiments. First, we knocked down the expression of both circIFT80 and IFT80 mRNA. We designed circIFT80-specific small interfering RNAs (siRNAs) targeting the back-splice sequence (si-circIFT80 #1, si-circIFT80 #2, and si-circIFT80 #3, respectively) or the sequence only in the linear transcript (si-IFT80). As expected, siRNA directed against the back-splice sequence knocked down only the circular transcript and did not affect the expression of linear species, and siRNA targeting the sequence in the linear transcript knocked down only the linear transcript and did not affect the expression of the circular transcript (p < 0.01; Figures S1A–S1D). The inhibition was successful with si-circIFT80 #2, which was used in the following experiments. Meanwhile, we induced ectopic overexpression of circIFT80 by transfecting HT-29 cell lines with pcDNA-circIFT80 expression vector (p < 0.01; Figures S1E and S1F). Furthermore, we found that circIFT80 was resistant to RNase R digestion (Figures S1G and S1H).

A CCK8 assay showed that circIFT80 knockdown significantly induced a proliferation-suppressing effect on HCT116 and DLD1 cells (p < 0.01; Figures 2A and 2B), whereas the results showed that the growth of HT-29 cells transfected with pcDNA-circIFT80 was significantly increased compared with that of control cells (p < 0.01; Figure 2C). Consistently, colony formation assays showed that circIFT80 knockdown decreased the colony formation ability of HCT116 and DLD1 cells (p < 0.01; Figures 2D and 2E). Furthermore, colony formation assays also indicated that clonogenic survival was significantly increased in HT-29 cells transfected with pcDNA-circIFT80 (p < 0.01; Figure 2F).

Figure 2

circIFT80 Inhibits CRC Growth In Vitro

(A) CCK8 assay showed that circIFT80 knockdown significantly repressed the cell proliferation of HCT116 cells. **p < 0.01. (B) CCK8 assay showed that circIFT80 knockdown significantly repressed the cell proliferation of DLD1 cells. **p < 0.01. (C) CCK8 assay showed that overexpression of circIFT80 promoted the proliferation of HT-29 cells. **p < 0.01. (D) Colony formation assays showed that circIFT80 knockdown decreased the colony formation ability of HCT116 cells. (E) Colony formation assays showed that circIFT80 knockdown decreased the colony formation ability of DLD1 cells. (F) Colony formation assays showed that circIFT80 overexpression increased the colony formation ability of HT29 cells.

To assess whether the pro-proliferative effects of circIFT80 on the CRC cells are mediated by promoting cell-cycle progression, we examined cell cycling in CRC cells by flow cytometry. As shown by flow cytometry analysis, circIFT80 knockdown led to an arrest in the G1 phase (p < 0.01; Figures 3A and 3B) and increased apoptotic rates of HCT116 and DLD1 cells compared with those in the NC groups (Figures 3D and 3E). Correspondingly, overexpression of circIFT80 decreased G0/G1 phase percentage and inhibited apoptosis in HT-29 cells (p < 0.01; Figures 3C and 3F). Therefore, these data suggested that circIFT80 promotes cell proliferation of CRC by facilitating DNA synthesis and inhibiting cell apoptosis.

Figure 3

circIFT80 Knockdown Inhibits the Proliferation of CRC Cells by Inducing Cell-Cycle Arrest and Inhibiting Cell Apoptosis

(A) The flow cytometry analysis showed that circIFT80 knockdown led to an arrest in the G1 phase of HCT116 cells. **p < 0.01. (B) The flow cytometry analysis showed that circIFT80 knockdown led to an arrest in the G1 phase of DLD1 cells. **p < 0.01. (C) The flow cytometry analysis showed that overexpression of circIFT80 decreased G0/G1 phase percentage in HT-29 cells. **p < 0.01. (D) The flow cytometry analysis showed that circIFT80 knockdown increased the apoptotic rates of HCT116 cells. (E) The flow cytometry analysis showed that circIFT80 knockdown increased the apoptotic rates of DLD1 cells. (F) The flow cytometry analysis showed that overexpression of circIFT80 inhibited apoptosis in HT-29 cells.

circIFT80 Induces CRC Invasion and Migration via Epithelial-Mesenchymal Transition

Because both the invasion and migration of tumor cells contribute to metastasis in CRC, we next examined the invasion and migration of CRC cells. As shown in Transwell assays, inhibition of circIFT80 expression attenuated the invasion and migration of HCT116 and DLD1 cells (Figures 4A and 4B), whereas overexpression of circIFT80 dramatically promoted cell invasion and migration in the HT-29 cells, as indicated by comparisons with the negative-control groups (Figure 4C). To measure the effect of silencing circIFT80 expression on the epithelial-mesenchymal transition (EMT) of CRC cells, western blot analysis was performed to examine the expression of EMT-related markers in CRC cells after circIFT80. As expected, circIFT80 knockdown remarkably increased the expression of E-cadherin and, meanwhile, greatly decreased the expression of N-cadherin and vimentin in HCT116 and DLD1 cells (Figures 4D and 4E). Correspondingly, overexpression of circIFT80 decreased the expression of E-cadherin and, meanwhile, greatly increased the expression of N-cadherin and vimentin in HT-29 cells, indicating that downregulation of circIFT80 obviously blocked the EMT process (Figure 4F).

Figure 4

circIFT80 Induces CRC Invasion and Migration via EMT

(A) Transwell assays showed that inhibition of circIFT80 expression attenuated the invasion and migration of HCT116 cells. (B) Transwell assays showed that inhibition of circIFT80 expression attenuated the invasion and migration of DLD1 cells. (C) Transwell assays showed that overexpression of circIFT80 dramatically promoted cell invasion and migration in the HT-29 cells. (D) Western blot assays showed that circIFT80 knockdown remarkably increased the expression of E-cadherin and decreased the expression of N-cadherin and vimentin in HCT116 cells. (E) Western blot assays showed that circIFT80 knockdown remarkably increased the expression of E-cadherin and decreased the expression of N-cadherin and vimentin in DLD1 cells. (F) Western blot assays showed that overexpression of circIFT80 decreased the expression of E-cadherin and increased the expression of N-cadherin and vimentin in HT-29 cells.

circIFT80 Has No Effect on Its Linear Transcript

Some circRNAs regulate the expression and function of the corresponding linear transcripts. Therefore, the regulatory relationship between circIFT80 and its linear transcript (IFT80) was investigated in this study. We first analyzed the colon adenocarcinoma (COAD) dataset from the Cancer Genome Atlas (TCGA) database and found that the level of IFT80 was not significantly higher in 275 COAD tissues than in 349 normal tissues (Figure 5A). Kaplan-Meier survival analysis from TCGA COAD datasets suggested that IFT80 expression is not significantly associated with worse overall survival (log-rank test, p = 0.14, Figure 5B) and disease-free survival (DFS) of COAD patients (log-rank test, p = 0.71, Figure 5C). The protein levels of IFT80 did not change when the expression of circIFT80 was artificially changed in CRC cells (Figure 5D). These data indicated that IFT80 is not the target gene of circIFT80.

Figure 5

circIFT80 Has No Effect on Its Linear Transcript

(A) IFT80 expression in CRC and normal samples from the TCGA COAD dataset. (B) Kaplan-Meier analyses of the correlations between IFT80 expression and overall survival of COAD patients from the TCGA COAD dataset. (C) Kaplan-Meier analyses of the correlations between IFT80 expression and DFS of COAD patients from the TCGA COAD dataset. (D) The western blot assay indicated that circIFT80 knockdown did not change the protein level of IFT80.

Knockdown of circIFT80 Inhibits CRC Growth In Vivo

HCT116 cells were used to establish a stable CRC cell line with low circIFT80 expression via lentiviral circIFT80-RNAi. HCT116 cells with low or normal circIFT80 expression induced by transfection with lentiviral or control vectors were subcutaneously implanted into nude mice. Compared with the vector control, the tumor growth in the low-circIFT80-expression group was significantly slower than that in the control group (p < 0.01; Figure 6A). After 28 days, the weights of the tumors were measured. Tumors derived from the low-circIFT80-expression group had lower weight than those from the negative controls (p < 0.01; Figure 6B). The expression level of KI67 was examined by IHC. The results showed that, compared to negative controls, expression of KI67 significantly decreased (Figure 6C). These results demonstrate that circIFT80 is significantly associated with the proliferation of CRC cells in vivo.

Figure 6

circIFT80 Promoted CRC Cell Growth In Vivo

(A) circIFT80 knockdown inhibits tumor growth in vivo. The tumor volume curve of nude mice was analyzed. (B) The tumor weights of nude mice were measured. (C) IHC analysis was performed to examine the expression levels of proliferation marker Ki-67 in tumors of nude mice. Knockdown of circIFT80 significantly decreased the percentage of Ki-67-positive cells in tumors of nude mice compared with that of sh-NC xenografts. **p < 0.01.

circIFT80 Acts as a Molecular Sponge for miR-1236-3p in CRC Cells

We detected the intracellular location of circIFT80 and found that this circRNA was predominantly localized in the cytoplasm (p < 0.01; Figure 7A), which indicates that it might function as a miRNA sponge. Therefore, we used the StarBase v2.0 target prediction tool to find 36 potential miRNAs that could bind to circIFT80. To figure out the miRNA implicated in CRC, all candidate miRNAs were subjected to microarray analysis. Among them, 10 miRNAs were significantly downregulated in response to circIFT80 overexpression (Figure 7B). For further selection, we chose the top five downregulated miRNAs to conduct a circRIP assay. The results demonstrated the interaction of only miR-1236-3p with circIFT80. Therefore, we used the Circular RNA Interactome (https://circinteractome.nia.nih.gov/index.html) to predict the potential circRNA/miRNA interaction. In addition, binding sites for miR-1236-3p were found within the circIFT80 sequence (Figure 7C). A subsequent luciferase reporter assay revealed that the luciferase intensity was reduced after the cotransfection of the wild-type luciferase reporter and miR-1236-3p mimics, while the mutated luciferase reporter exerted no such effect (p < 0.01; Figure 7D). In a further RIP experiment, circIFT80 and miR-1236-3p simultaneously existed in the production precipitated by anti-AGO2 (p < 0.01; Figure 7E), which indicates that circIFT80 directly interacts with miR-1236-3p and could act as a sponge for miR-1236-3p. To functionally confirm that circIFT80 promotes CRC progression by sponging miR-1236-3p, we transfected miR-1236-3p mimics into circIFT80-overexpressing cells to examine whether the tumor-promoting effect of circIFT80 overexpression could be reversed by miR-1236-3p upregulation. The results showed that miR-1236-3p upregulation could significantly reverse the circIFT80-overexpression-mediated promotion of proliferation, migration, and invasion of HCT116 cells (p < 0.01; Figures 7F and 7G). The aforementioned results collectively demonstrated that circIFT80 promoted CRC progression by sponging miR-1236-3p.

Figure 7

circIFT80 Acts as a Molecular Sponge for miR-1236-3p in CRC Cells

(A) qRT-PCR for the expression of circIFT80 in the nuclear and cytoplasmic fractions. U6 snRNA and 18S rRNA were used as positive controls for nuclear and cytoplasmic fractions, respectively. The results showed that circIFT80 was predominantly localized in the cytoplasm. Data are listed as means ± SD of at least three independent experiments. **p < 0.01. (B) The microarray analysis showed that 10 miRNAs were significantly downregulated in response to circIFT80 overexpression. (C) The binding sequence between miR-1236-3p and circIFT80. (D) Dual luciferase reporter showed significant reduction of luciferase activity of the wild-type and luciferase activity is restored by the mutant sequence. (E) The RIP experiment showed that miR-1236-3p and circIFT80 simultaneously existed in the production precipitated by anti-AGO2. (F) miR-1236-3p upregulation significantly inhibited the proliferation of HCT116 cells. miR-1236-3p mimics significantly reversed circIFT80-overexpression-mediated promotion of proliferation of HCT116 cells. (G) miR-1236-3p upregulation significantly inhibited the migration and invasion abilities of HCT116 cells. miR-1236-3p mimics significantly reversed circIFT80-overexpression-mediated promotion of migration and invasion of HCT116 cells.

HOXB7 Was a Direct Target of miR-1236-3p

To validate whether circIFT80 sponges miR-1236-3p and liberates the expression of its downstream target, we searched TargetScan for potential target genes of miR-1236-3p, and HOXB7 was predicted (Figure 8A). A subsequent luciferase reporter assay revealed decreased luciferase intensity after cotransfection of miR-1236-3p mimics and wild-type luciferase reporter, while the mutated luciferase reporter exerted no such effect (Figure 8B). The immunohistochemistry results showed that HOXB7 expression in CRC specimens was significantly upregulated compared with that in the adjacent normal tissues. HOXB7 overexpression was observed in 45 of 58 (77.59%) CRC specimens when compared with adjacent non-neoplastic tissues (16 of 58; 27.59%); the difference of HOXB7 expression was statistically significant (p < 0.001; Figure 8C). Additionally, we analyzed the COAD dataset from the TCGA database and found that the level of HOXB7 was significantly higher in 275 COAD tissues than in 349 normal tissues (p < 0.05; Figure 8D). Furthermore, circIFT80 knockdown could suppress HOXB7 expression, while a miR-1236-3p inhibitor attenuated the effect of inhibition of circIFT80 (Figure 8E). These data further demonstrated the regulatory network of circIFT80/miR-1236-3p/HOXB7.

Figure 8

HOXB7 Was a Direct Target of miR-1236-3p

(A) Bioinformatics analysis reveals the predicted binding sites between HOXB7 and miR-1236-3p. (B) Luciferase reporter assay demonstrates that miR-1236-3p mimics significantly decreased the luciferase activity of HOXB7-WT (wild type) in 293T cells. (C) IHC assay indicated that the expression of HOXB7 was significantly upregulated in CRC tissues compared with adjacent non-tumorous tissues. (D) HOXB7 expression in CRC and normal samples from the TCGA COAD dataset. (E) circIFT80 knockdown could suppress HOXB7 expression, while a miR-1236-3p inhibitor attenuated the effect of inhibition of circIFT80.

Discussion

In this study, peripheral plasma exosomal circIFT80 was found to be aberrantly upregulated in CRC by high-throughput microarray assay. We first found that circIFT80 was significantly upregulated in the CRC tissues and CRC cell lines. In addition, increased expression of circIFT80 in CRC patients is associated with increased tumor size and advanced stage. Our subsequent studies demonstrate that circIFT80 knockdown decreased cell proliferation and caused a dramatic decrease in CRC cell colony formation, whereas circIFT80 overexpression has the opposite results. In addition, circIFT80 knockdown promoted significant arrest in the G0/G1 phase and an obvious increase in CRC cell apoptosis. These observations of tumor growth were verified in a mouse xenograft model. Based on bioinformatics analysis, it was assumed that the circIFT80/miR-1236-3p/HOXB7 axis plays a pivotal role in CRC progression. Specifically, we also showed mechanistically that circIFT80 promotes the progression of CRC by functioning as a miR-1236-3p sponge to modulate HOXB7 expression. These findings suggest that circIFT80 might be a novel clinical molecular marker for the prognosis of CRC patients.

A number of recent papers have revealed that circRNAs are highly dysregulated in many types of cancers and exhibit high tissue and disease specificity.6, 15 The deregulation of circRNAs may result in increased proliferation, invasion, or angiogenesis and decrease the level of apoptosis or dedifferentiation, eventually leading to tumor formation.16, 17 Cancer-associated circRNAs and investigation of their molecular and biological functions are important to provide new insights into the diagnosis of cancer, including CRC. However, no preclinical reports on circRNAs as targets or therapeutic vectors for cancer treatment have been published thus far. Mounting evidence indicates that exosomes are critical mediators of communication and information transfer between tumor cells and surrounding cells and that cancer-derived exosomes can play important roles in tumor progression.13, 18

Inspired by these studies, we hypothesized that extracellular circIFT80 promoted CRC progression through incorporation into exosomes. To validate this hypothesis, we identified that circIFT80 was mainly in exosomes and found that circIFT80 was increased in CRC samples and cell lines. In our study, CCK8 assays combined with flow-cytometric analysis revealed that the circIFT80-mediated increase in the proliferation of CRC cells may be related to the promotion of DNA synthesis and inhibition of cell apoptosis.

In recent years, emerging evidence proposed that circRNAs are an important subtype of ceRNAs and that circRNA can be used as a miRNA molecular sponge to attenuate the effect of miRNAs on gene expression by combining with miRNA response elements (MREs).19, 20 Accordingly, we performed the RIP and luciferase assays and found that the mechanism by which circIFT80 promotes tumor progression of CRC in vitro is mediated by inhibiting miR-1236-3p expression, thus, influencing downstream genes’ HOXB7 expression. The miR-1236-3p inhibitor could attenuate the anti-tumor effects mediated by circIFT80 knockdown. Moreover, miRNAs are the most widely studied non-coding RNAs and also can act as oncogenes or tumor suppressor genes. In this study, bioinformatics analysis showed that miR-1236-3p interacted with the 3′ UTR of HOXB7 and suppressed HOXB7 expression at the post-transcriptional level, which was confirmed by the results of the luciferase reporter assay. Altered expression of HOXB7 genes could be important for tumorigenesis and progression of CRC. We found that HOXB7 expression was significantly higher in CRC tissues. According to the aforementioned data, we confirmed that circIFT80 can exert function in CRC by sponging miR-1236-3p to upregulate HOXB7 expression.

Taken together, the evidence indicates that circIFT80 performed a pivotal function in tumor progression of CRC. In addition, we also confirmed that circIFT80 can promote CRC cell growth and proliferation, migration, and invasion through the miR-1236-3p/HOXB7 axis. Therefore, we provide a novel insight that inhibition of circIFT80 might be a potential therapeutic strategy for CRC.

Materials and Methods

Patients and Sample Collection

Pairs of CRC tissues and adjacent normal tissues were collected from 58 CRC patients at the First Affiliated Hospital of Huzhou University between January 2010 and January 2018. All these samples were formalin fixed and paraffin embedded. In addition, fresh resected tumors and adjacent noncancerous mucosa were harvested and then immediately frozen in liquid nitrogen and stored at −80°C until total RNA was extracted. For exosome purification, whole blood samples were collected from these 58 CRC patients and healthy controls. Fresh plasma samples (3 mL) were collected in tubes of EDTA from each of the subjects. These samples were centrifuged at 3,000 × g for 10 min at 4°C and then stored at −80°C. The specimens were evaluated according to the World Health Organization’s classification criteria. Disease progression was classified using the CRC guidelines outlined in the Seventh Edition of the American Joint Committee on Cancer Staging Manual. Patients who underwent chemotherapy, radiotherapy, or any other adjuvant treatment before surgery were excluded from the study. The study was approved by the Research Ethics Committee of the First Affiliated Hospital of Huzhou University, and written informed consent was obtained from all patients.

Plasma Exosome Isolation

First, the samples were centrifuged twice at 3, 000 × g and 10, 000 × g for 20 min at room temperature to remove cells and other debris in the plasma. The supernatants were then centrifuged at 100,000 × g for 30 min at 4°C to remove microvesicles that were larger than exosomes, harvested, and again centrifuged at 10,000 × g for 70 min at 4°C. Subsequently, the supernatants were gently decanted, and the exosome sediments were re-suspended in PBS. Concentration of exosomes was determined using BCA method as recommended by the manufacturer (Thermo Scientific, Waltham, MA, USA).

Transmission Electron Microscopy

The exosome suspension was diluted to 0.5 mg/mL with PBS and then spotted onto a glow-discharged copper grid placed on a filter paper and dried for 10 min by exposure to infrared light. Next, the exosome samples were stained with one drop of phosphotungstic acid (1% aqueous solution) for 5 min and dried for 20 min by exposure to infrared light. Finally, the exosomes were visualized under a transmission electron microscope (HT7700, Hitachi, Tokyo, Japan) at 100 keV.

Nanoparticle Tracking Analysis

Briefly, the exosomes were resuspended in PBS and filtered with a syringe filter (Millipore). Then, the samples were diluted until individual nanoparticles could be tracked. The size distribution of the exosomes was evaluated using a NanoSight NS300 instrument (Malvern Instruments, Worcestershire, UK).

Western Blotting

Western blotting was performed to assess the levels of these markers. Total proteins were extracted from exosome samples using lysis buffer. Each sample (40 μg) was loaded onto a 12% SDS-polyacrylamide gels and then transferred to a polyvinylidene fluoride membrane (Roche, Mannheim, Germany). These membranes were immersed in 2% BSA at room temperature for 1 h and incubated with the following primary antibodies: anti-HSP70 (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-TSG101 (1:2,000, Abcam, Cambridge, UK), followed by subsequent incubation with appropriate secondary antibodies after washing with PBS. Lastly, protein bands were visualized by incubating with an electro-chemiluminescence reagent.

Agilent Methods for ceRNA Array

Total RNA of exosomes was extracted from plasma using TRIzol LS Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and quantified using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Total RNA from exosomes was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and purified using a RNeasy Mini Kit (QIAGEN, Hilden, Germany). RNA samples of each group were then used to generate fluorescently labeled cRNA targets for the human ceRNA array v1.0 (4 × 180K; Shanghai Biotechnology, Shanghai, China). The labeled cRNA targets were then hybridized with the slides. After hybridization, slides were scanned on the Agilent Microarray Scanner (Agilent Technologies, Santa Clara, CA, USA). Data were extracted using Feature Extraction software, v10.7 (Agilent Technologies). Raw data were normalized by the quantile algorithm of the limma package in the R program. Microarray experiments were performed at Shanghai Biotechnology following the protocol of Agilent Technologies. Ratios between CRC patients and normal control (NC) subjects were calculated. Genes with a fold change of at least 2 were selected for further analysis. The selected parent genes of circRNA were grouped into functional categories based on the Gene Ontology database (http://geneontology.org/), and functional pathways (Kyoto Encyclopedia of Genes and Genomes) were also analyzed using the online enrichment analysis tool of Shanghai Biotechnology.

TCGA Dataset Analysis

The data and the corresponding clinical information of patients were collected from the TCGA database (http://cancergenome.nih.gov/). We used the edgeR package of the R packages to perform the difference analysis (http://www.bioconductor.org/packages/release/bioc/html/edgeR.html) and used the pheatmap package of the R packages to perform the cluster analysis (https://cran.r-project.org/web/packages/pheatmap/index.html). The Sva R package was used to remove the batch effect. Genes with adjusted p values < 0.05 and absolute fold changes (FCs) > 1.5 were considered differentially expressed genes. Kaplan-Meier survival curves were drawn to analyze the relationships between genes and overall survival in the survival package. The corresponding statistical analysis and graphics were performed in R software (R v.3.3.2).

Cell Lines and Culture Conditions

The human CRC cell lines (HCT116, DLD1, SW480, RKO, and HT-29) were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). The human colonic epithelial cell line HCoEpiC and the 293T cell line were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI 1640 or DMEM (GIBCO, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (10% FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin (GIBCO) in humidified air at 37°C with 5% CO2.

Quantitative Real-Time PCR Analyses

Total RNA from cells and tissues was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA). Total RNA from plasma was extracted using TRIzol LS Reagent (Invitrogen, Carlsbad, CA, USA). The RNA extraction was performed according to the manufacturer’s instructions. We designed specific divergent primers spanning the back-splice junction site of circIFT80. For circRNA and mRNA, PrimeScript RT Reagent Kit (TaKaRa, Dalian, China) was used to generate cDNA, the TB GreenPremix Ex Taq II (TaKaRa, Dalian, China) was used to perform real-time PCR, and 18S rRNA was utilized as internal control. For miRNA, the miRcute Plus miRNA First-Strand cDNA Kit (TIANGEN, Beijing, China) was used to generate cDNA, the miRcute Plus miRNA qPCR Kit (SYBR Green) was used to perform real-time PCR, and U6 small nuclear RNA (snRNA) was utilized as internal control. Real-time PCR was performed using an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The 2−ΔΔCt method was used to calculate the relative expression of RNAs. The primers used in this study were synthesized by Sangon Biotech (Shanghai, China).

Cell Transfection

The siRNAs targeting the back-splice junction sequences of circ-IFT80 and the respective negative-control oligonucleotides were synthesized by GenePharma (Shanghai, China). A pcDNA 3.1 circRNA mini-vector was used to ectopically express circ-IFT80 levels in CRC cells. The miRNA mimics, inhibitor, and siRNAs were obtained from GenePharma (Shanghai, China). We used Lipofectamine 3000 for transfection following the protocols of the manufacturer.

RNase R Treatment

For RNase R treatment, total RNA (2 μg) was incubated for 30 min at 37°C with or without 3 U/μg of RNase R (Epicenter Technologies, Madison, WI, USA). After treatment with RNase R, qRT-PCR was performed to determine the expression levels of circIFT80 and IFT80 mRNA.

CCK8 Assay

Cell proliferation assay was performed using a CCK8 kit (Dojindo Laboratories, Kumamoto, Japan) following the manufacturer’s instructions. Approximately 1 × 103 transfected cells in 100 μL were incubated in 96-well plates. At 0, 24, 48, 72, and 96 h, cells in each well were treated with the CCK8 reagent (10 μL) and incubated at 37°C for 2 h. The optical density (OD) at 450 nm was read using a Varioskan Flash Spectral Scanning Multimode Reader (Thermo Fisher Scientific, Waltham, MA, USA). Cells in each group were tested for 5 replicates.

Colony Formation Assay

For colony formation assay, 24 h after transfection, cells were seeded into 6-well plates and then incubated for about 2 weeks. Then, the colonies were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 15 min at room temperature. The cell colonies were counted and photographed.

Apoptosis Analysis

Cell apoptosis was analyzed using the Annexin V-FITC Apoptosis Detection Kit (KeyGen Biotech, Nanjing, China) according to the manufacturer’s instructions. 48 h after transfection, cells were stained with fluorescein isothiocyanate (FITC) and propidium iodide. Then, flow cytometry was performed using the BD FACSCanto II (BD Biosciences), and the data were analyzed using BD FACSDiva software and FCS Express 5 software (De Novo Software, Los Angeles, CA, USA).

Cell Migration and Invasion Assays

Cell migration and invasion assays were conducted using Transwell permeable supports, with polycarbonate (PC) membrane (Corning, Corning, NY, USA), which was coated with (for invasion assays) or without (for migration assays) Matrigel (Corning, Corning, NY, USA) according to the manufacturer’s instructions. 24 h after transfection, cells in 200 μL serum-free medium were seeded into the upper compartment, with 700 μL culture medium containing 20% FBS in the lower compartment. After 24 h incubation, the cells located on the upper surfaces of the Transwell chamber were erased with cotton swabs, and the cells located on the lower surfaces were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 15 min at room temperature. The stained cells were photographed and counted in five randomly selected fields under a Leica DM4000B microscope (Leica, Wetzlar, Germany).

Xenografts in Mice

Approximately 1 × 106 cells were injected subcutaneously into the right neck of male BALB/C nude mice (age, 4–6 weeks; weight, 18–22 g; five mice per group) purchased from Slaccas (Slaccas Laboratory Animal, Shanghai, China). The length and width of tumor xenografts were measured weekly by Vernier calipers, and the tumor volume was calculated using the following formula: volume (mm3) = 0.5 × width2 × length. Six weeks after injection, the mice were killed by cervical dislocation. The animal studies were conducted in accordance with the ethical guidelines for animal experiments and approved by the Animal Care and Use Committee of the First Affiliated Hospital of Huzhou University.

Immunohistochemistry

For each patient sample, three 5-μm paraffin sections were prepared; one for H&E staining and the other two for immunohistochemical staining. PBS, instead of primary antibodies, was used for negative control, and the breast cancer tissue was used for positive control. Sections were dewaxed using xylene, followed by hydration with ethanol solutions and the addition of EDTA for antigen retrieval. Later, sections were blocked with normal goat serum for 30 min to eliminate non-specific binding. Sections were incubated with anti-human HOXB7 polyclonal antibody (1:1,000; Abcam, Cambridge, MA, USA). Sections were then incubated with biotin-labeled secondary antibodies for 30 min at room temperature, followed by staining with diaminobenzidine (DAB). Finally, the sections were counterstained with hematoxylin. The result of staining was determined by two doctors who did not know the clinical condition of the patients. The proportions of positive cells of 0%, 1%–5%, 6%–25%, 26%–75%, and 76%–100% were assigned with scores of 0, 1, 2, 3, and 4, respectively. Scores of 0–2 were considered as negative expression, and scores of 3–4 were considered as positive expression.

Isolating RNAs from Nucleus and Cytoplasmic Fractions

The nuclear and cytoplasmic fractions were isolated using the PARIS Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Briefly, cells were collected and lysed with cell fractionation buffer, followed by centrifugation to separate the nuclear and cytoplasmic fractions. The supernatant containing the cytoplasmic fraction was collected and transferred to a fresh RNase-free tube. The nuclear pellet was lysed with cell disruption buffer. The cytoplasmic fraction and nuclear lysate were mixed with 2× lysis/binding solution and then mixed with 100% ethanol. The sample mixture was drawn through a filter cartridge, followed by washing with wash solution. The RNAs of nuclear and cytoplasmic fractions were eluted with elution solution. U6 snRNA and 18S rRNA were used as positive controls for nuclear and cytoplasmic fractions, respectively.

Luciferase Reporter Assay

Cells (5 × 103) were seeded into 96-well plates and co-transfected with corresponding plasmids and miRNA mimics or inhibitors using the Lipofectamine 2000 transfection reagent. Luciferase activity was measured using the dual-luciferase reporter assay system (Promega, Madison, WI, USA) after 48 h of incubation according to the manufacturer’s instructions. Independent experiments were performed in triplicate. Relative luciferase activity was normalized to the Renilla luciferase internal control.

RIP Assay

RNA immunoprecipitation (RIP) assay was performed using an EZ-Magna RIP Kit (Millipore, Billerica, MA, USA) in accordance with the manufacturer’s instructions. Cells were lysed at 70%–80% confluence in RIP lysis buffer and then incubated with magnetic beads conjugated with human anti-Ago2 antibody (Millipore) and normal mouse IgG control (Millipore) in RIP buffer. The RNAs in the immunoprecipitates were isolated with TRIzol reagent and analyzed by qRT-PCR.

Statistical Analysis

Results are presented expressed as mean ± SD. A Student’s t test was performed to measure the difference between two groups, and differences between more than two groups were assessed using one-way ANOVA. p < 0.05 was considered significant.

Ethical Approval and Consent to Participate

The study was approved by the First Affiliated Hospital of Huzhou University. The informed content was obtained from all participants. The research has been carried out in accordance with the World Medical Association Declaration of Helsinki.

Availability of Data and Materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request, but no information infringing on the privacy of the participants will be given.

Author Contributions

W.F. and G.C. performed primers’ design and experiments. H.G. and Yongchun Wang contributed the flow cytometry assay and animal experiments. G.Z. collected and classified the human tissue samples. T.X. contributed to RT-PCR and qRT-PCR. Yao Wang analyzed the data. G.C. wrote the paper. All authors read and approved the final manuscript.

Conflicts of Interest

The authors declare no competing interests.