CircRNA_0000392 promotes colorectal cancer progression through the miR-193a-5p/PIK3R3/AKT axis

Forty pairs of CRC tissues and adjacent normal tissues were collected from patients who were diagnosed with CRC at the Longhua Hospital affiliated with Shanghai University of Traditional Chinese Medicine (Shanghai, China). Tumor and normal adjacent tissue samples were obtained during surgical treatment at the Department of General Surgery. The samples were isolated, immediately snap frozen in liquid nitrogen and stored at − 80 °C before use. All patients signed informed consent forms prior to surgery and did not receive preoperative chemotherapy or radiotherapy. This study was approved by the Ethics Committee of Longhua Hospital.

Total RNA was isolated from the tissue samples using TRIzol reagent (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. Then, we assessed the RNA integrity and DNA contamination by using electrophoresis on a denaturing agarose gel. After confirming that the RNA was intact and pure, we used the Ribo-Zero rRNA Removal Kit (Illumina, San Diego, CA, USA) and the CircRNA Enrichment Kit (Cloud-seq, USA) to remove the rRNA and enrich the circRNAs, respectively. The RNA-seq libraries were constructed using pretreated RNAs with the TruSeq Stranded Total RNA Library Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. The libraries were denatured as single-stranded DNA molecules, captured on Illumina flow cells, amplified in situ as clusters and finally sequenced for 150 cycles on an Illumina HiSeq™ 4000 Sequencer (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Paired-end reads were harvested from an Illumina HiSeq™ 4000 sequencer and were quality controlled by Q30. The reads were aligned to the reference genome/transcriptome with STAR software, and circRNAs were detected and annotated with DCC software. The circBase database and circ2Trait disease database were used to annotate the identified circRNAs. The differentially expressed circRNAs between the two groups were identified using T test statistical methods.

CircRNA-miRNA interactions were predicted by popular target prediction software, including Circular RNA Interactome and RegRNA. Specific predictions for the target genes of miRNAs were based on the miRanda, miRDB, miRWalk, RNA22 and TargetScan databases. All circRNA-miRNA-mRNA networks were constructed using Cytoscape software.

Human CRC cell lines (HT29, HCT116, SW480, SW837, SW48, SW620 and RKO) were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). A normal human colon mucosal epithelial cell line (NCM460) and the 293 T cell line was obtained and preserved in our lab. HT29 and HCT116 cells were cultured in McCoy’s 5A (Gibco, Carlsbad, CA, USA), while SW480, SW620, SW48, SW837 and 293 T cells were cultured in DMEM (Gibco, Carlsbad, CA, USA). NCM460 cells were cultured in M3:10 media (INCELL, San Antonio, TX), and RKO cells were cultured with MEM (Gibco, Carlsbad, CA, USA). All culture media contained 10% fetal bovine serum and 1% penicillin. All these cell lines were maintained in a humidified atmosphere of 5% CO₂ at 37 °C.

Anti-PIK3R3 antibody (ab97862, 1:1000 dilution for immunoblotting and 1:200 for IHC) was purchased from Abcam. Anti-AKT1 antibody (#2938), anti-phospho-Akt (Ser473) antibody (#4058), anti-mTOR antibody (#2972), and anti-phospho-mTOR (Ser2448) antibody (#2971) were obtained from Cell Signaling Technology, and all antibodies were diluted 1:1000 for immunoblotting. Anti-actin (sc-1616, 1:5000 dilution), HRP-conjugated anti-mouse IgG (sc-2055, 1:5000 dilution) and HRP-conjugated anti-rabbit IgG (sc-2054, 1:5000 dilution) were purchased from Santa Cruz. Actinomycin D and crystal violet were purchased from Sigma-Aldrich (St Louis, MO, USA). RNase R was purchased from Epicentre Technologies (Madison, WI, USA).

Total RNA was extracted by using TRIzol reagent (Life Technologies, Carlsbad, CA) and then reverse-transcribed into cDNA using the SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). cDNA was used for qPCR performed with the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and gene-specific primers, and the results were normalized using β-actin or U6 as a control. PCR primers are listed in Additional file 1: Table S1.

SW620 and RKO cells were treated with 3 U/mg RNase R (Epicentre, WI, USA) or 2 mg/L actinomycin D (Sigma, USA) and then cultured at 37 °C. The cells were harvested at the indicated time points, and the stability of circRNA_0000392 and YAF2 mRNA was detected by quantitative real-time PCR (qRT-PCR) assay.

SW620 and RKO cells were seeded in dishes and cultured until 70–80% confluence. Then, the cells were fixed at room temperature with 4% paraformaldehyde and treated with protease K. Then, the cells were overlaid with FITC-labeled circRNA_0000392 probe (Gefanbio, China) at 65 °C for 48 h. The signals of the probe were detected by a Fluorescent In Situ Hybridization Kit (Gefanbio, China) according to the manufacturer’s protocol. Nuclei were counterstained with DAPI.

The sequences of circRNA_0000392 and the PIK3R3 3′ UTR and their corresponding mutant versions without miR-193a-5p binding sites were synthesized and subcloned into the luciferase reporter vector pmirGLO (Promega, Madison, WI, USA), and the resulting constructs were named circRNA_0000392 -WT, circRNA_0000392-Mut, PIK3R3 3′ UTR-WT and PIK3R3 3′ UTR-Mut, respectively. The plasmids were validated by sequencing and then cotransfected with the miRNA mimics or inhibitor or the corresponding negative controls. The relative luciferase activity was measured using a Dual Luciferase Assay Kit (Promega, Madison, WI, USA).

A Transwell chamber (Corning, Kennebunk, ME, USA) was used for the migration assays, and a transwell chamber precoated with Matrigel was used for the invasion assays. According to the protocol, single-cell suspensions were added to the upper chambers and incubated for 24 h. Then, the cells were washed, fixed, and stained with crystal violet. Based on the crystal violet staining data, we calculated the migration and invasion rates by counting the cells in at least five random fields.

RIP assays were performed in SW620 and RKO cells. A total of 1 × 10⁷ cells were completely lysed by RNA lysis buffer and then incubated with RIP immunoprecipitation buffer containing magnetic beads conjugated with human anti-Argonaute2 (AGO2) antibody (Millipore, USA) or negative control mouse IgG (Millipore, USA). Proteinase K was added to the RIP sample and incubated at 55 °C for 30 min. Then, immunoprecipitated RNA was isolated and analyzed by qRT-PCR to quantify the enrichment of circRNA_0000392.

Biotin-labeled circRNA_0000392 probe or oligo probe (GenePharma, China) were synthesized. SW620 and RKO cells were lysed with lysis buffer and incubated with specific circRNA_0000392 probes. Then, SW620 and RKO cells were lysed with lysis buffer and incubated with probe-coated beads at 4 °C overnight. The beads were washed, the RNA complexes were extracted with TRIzol (Life Technologies, Carlsbad, CA) and detected by qRT-PCR.

Detection of the expression level of PIK3R3 by immunohistochemistry was performed on 5-μm thick paraffin sections of patient tissue samples. Briefly, the sections were deparaffinized and rehydrated followed by antigen retrieval using 0.01 M sodium citrate buffer (pH 6.0) at a boiling temperature for 10 min. Then, the sections were incubated with 3% hydrogen peroxide for 10 min, 5% bovine serum albumin for 1 h and primary antibodies at 4 °C overnight. The sections were incubated with secondary antibodies after washing three times with PBS. Finally, the DAB system was used to visualize the signal, and hematoxylin was used to stain the nucleus. The immunostaining images were captured using an Olympus FSX100 microscope (Olympus, Japan).

BALB/c nude mice (male, 3- to 4-week-old) were injected subcutaneously with 5 × 10⁶ SW620 cells. Tumor volumes were measured with a caliper every 3 days and calculated from the length (a) and the width (b) by using the following formula: volume (mm³) = ab²/2. Thirty days after injection, the animals were sacrificed, and the excised tumor tissues were removed to further assess tumor weight and pathological staining.

The cell transfection, western blot, cell proliferation, and apoptosis assays are described as the Supplementary Methods in Additional file 2.

To obtain the expression profiles of circRNAs and identify differentially expressed circRNAs in CRC patients, secondary sequencing was used to profile circRNA expression in paired CRC tissues and adjacent normal tissues (ANT) from 6patients with CRC. First, the scatter plot showed the differences in circRNA expression between the tumor tissues and adjacent normal tissues (Fig. 1a). Then, under the cutoff criteria of fold change > 2.0 and P < 0.05, the significant differentially expressed circRNAs between the two groups are presented in a volcano plot (Fig. 1b) and hierarchical cluster (Fig. 1c). In total, 66,855 circRNAs were detected in the tissue samples by sequencing, including 19 significantly upregulated and 1668 significantly downregulated circRNAs (Fig. 1d and Additional files 3 and 4). GO and KEGG analyses of the host genes of differentially expressed circRNAs are shown in Additional file 1: Fig. S1 and S2. Based on their genomic origin, circRNAs were classified as intronic, exonic, intergenic, antisense and sense overlapping circRNAs and are presented as a pie chart showing the percentage of significantly differentially expressed circRNAs in each category (Fig. 1e). We then identified the circRNAs that can function as miRNA sponges from the significantly differentially expressed circRNAs and constructed a network map using Cytoscape software (Additional file 1: Fig. S3). We overlapped the top 10 circRNAs that were significantly upregulated or downregulated (Additional file 1: Table S2) with circRNAs that function as miRNA sponges. From the overlap, we first selected 4 circRNAs and verified the circRNA-seq results in 40 paired CRC tissues and adjacent normal tissues (ANT) by qRT-PCR. As shown in Fig. 1f, circRNA_0000392 expression was significantly upregulated in CRC tissues, which was consistent with the sequencing data. Of the two, circRNA_0000392 had an approximately 25-fold change in sequencing and an approximately 13-fold change in 16 patients, indicating a more significant difference. Therefore, in this study, we will focus on the role of circRNA_0000392 in the tumorigenesis and progression of colorectal cancer.

To further confirm our results, we increased the number of tissue samples to 40 pairs and detected the expression level of circRNA_0000392 by qRT-PCR. The results showed that in 29 of the 40 pairs of CRC tissues and adjacent normal tissues, the expression level of circRNA_0000392 was higher in tumor tissues than in adjacent normal tissues and was significantly increased in the overall statistics of the 40 pairs (Fig. 2a and b). Then, ROC curve analysis was performed to assess the diagnostic value of circRNA_0000392 in CRC. The results showed that circRNA_0000392 could discriminate CRC from adjacent normal tissues with an AUC of 0.713 (95% CI: 0.598–0.827, P < 0.01) (Fig. 2c). The associations between the circRNA_0000392 expression level and clinical parameters of the CRC patients are listed in Table 1. There was no significant difference in the expression level of circRNA_0000392 between the two groups according to age, gender, tumor size or location. However, the expression of circRNA_0000392 was significantly correlated with pathological stage (P = 0.0123), lymph node metastasis (P = 0.0055) and distant metastasis (P = 0.0084) (Fig. 2d–g, and Additional file 1: Fig. S4A-B). The circRNA_0000392 expression level was also measured in the normal human colon mucosal epithelial cell line NCM460 and in 7 CRC cell lines, namely, HT29, HCT116, SW480, SW837, SW48, SW620, and RKO. circRNA_0000392 expression was markedly upregulated in human CRC cell lines compared with normal colon mucosal epithelial cell lines (Fig. 2h). These results indicate that circRNA_0000392 expression was elevated in CRC and may be involved in the tumorigenesis and development of CRC.

Before we delved into the specific role of circRNA_0000392 in CRC, we first investigated the characteristics of circRNA_0000392. The genomic locus of circRNA_0000392 is shown in Fig. 3a, and the spliced mature sequence length of circRNA_0000392 is 326 bp. Further, SW620 and RKO cells were treated with RNase R exonuclease and actinomycin D to verify the authenticity of circRNA_0000392. CircRNA_0000392 was resistant to RNase R (Fig. 3b) and actinomycin D (Fig. 3c and Additional file 1: Fig. S4C), whereas YAF2 mRNA was significantly reduced after RNase R and actinomycin D treatment. RNA fluorescence in situ hybridization (FISH) assays demonstrated that circRNA_0000392 was mainly localized in the cytoplasm (Fig. 3d). These data showed that the circRNA_0000392 species was indeed circular.

To explore the function of circRNA_0000392 in CRC cells, we first designed two siRNAs targeting the back-splice region. Then, loss-of-function assays were performed in SW620 and RKO cells with relatively high expression of circRNA_0000392. After transfection of the two siRNAs, the expression of circRNA_0000392 was significantly reduced by siRNA #1 in both cell lines (Fig. 4a). The WST-1 assay demonstrated that downregulation of circRNA_0000392 significantly inhibited the proliferation viability of SW620 and RKO cells (Fig. 4b and c). We further investigated whether circRNA_0000392 has an effect on the apoptosis of CRC cells by flow cytometry. Double staining with Annexin V and PI showed that circRNA_0000392 knockdown significantly enhanced cell apoptosis at 48 h post transfection with siRNA #1 or si-NC (Fig. 4d). Next, cell migration and invasion abilities after siRNA transfection were performed using Transwell assays with or without Matrigel. As a result, the cell migration and invasion abilities of SW620 and RKO cells were significantly inhibited after knocking down the expression of circRNA_0000392 (Fig. 4e and f). We further measured the protein expression level of the EMT markers. As shown in Additional file 1: Fig. S9, after the expression level of circRNA_0000392 was knocked down, the expression of E-Cadherin was significantly up-regulated and the expression of Vimentin was significantly decreased. These results indicated that circRNA_0000392 contributed to CRC cell proliferation and motility in vitro.

It is well known that acting as a miRNA sponge is one of the important mechanisms by which circRNAs exert their biological functions [10]. Given that we previously found by prediction software that circRNA_0000392 could function as a miRNA sponge, we mainly focused on its function as a miRNA sponge to further explore its underlying mechanism in CRC cell proliferation. First, the RIP assay was performed in SW620 and RKO cells. The results showed that circRNA_0000392 was enriched in AGO2 immunoprecipitates, confirming that circRNA_0000392 functions to adsorb miRNA (Fig. 5a). Then, circRNA-miRNA-mRNA interactions based on circRNA_0000392 were predicted by bioinformatics analysis using Cytoscape software (Fig. 5b). From the prediction results, we selected the top 5 candidate miRNAs to validate the specific interaction by RNA pull-down assay. The results showed that miR-193a-5p had a dramatic difference in the pull-down level by the circRNA_0000392 probe compared with the oligo probe in both SW620 and RKO cells (Fig. 5c and Additional file 1: Fig. S5A). To further confirm the interactions between circRNA_0000392 and miR-193a-5p, a dual-luciferase reporter assay was performed in 293 T cells. The circRNA_0000392-wt or circRNA_0000392-mut plasmid was constructed based on a luciferase reporter vector (Fig. 5d) and then cotransfected with miR-193a-5p mimic or NC in 293 T cells (Additional file 1: Fig. S5B-C). The dual-luciferase reporter assay results showed that miR-193a-5p mimics significantly reduced the luciferase activity of the circRNA_0000392-WT group but had no effect on the mutant group (Fig. 5e). The expression levels of miR-193a-5p in 40 pairs of CRC tumor tissues and adjacent normal tissues were measured by qRT-PCR. The results showed that the miR-193a-5p expression level in CRC tumor tissues was significantly reduced compared with that in adjacent normal tissues (Fig. 5f and Additional file 1: Fig. S6). Spearman correlation coefficient analysis revealed a negative correlation between miR-193a-5p and circRNA_0000392 expression in CRC tumor tissues (r = − 0.365, P = 0.021) (Fig. 5g). Overall, these results demonstrate that circRNA_0000392 acts as a sponge for miR-195-5p in CRC.

According to our previous predictions, EPHA2, PIK3R3, EGFR, USP22 and DDX58 are the most likely potential target genes for miR-193a-5p. Then, we detected the mRNA expression levels of these genes after transfection of SW620 cells with the miR-193a-5p mimics or inhibitor. The results revealed that the EPHA2 and PIK3R3 expression levels were significantly downregulated by the miR-193a-5p mimic and that the PIK3R3 expression level was upregulated after transfection with the miR-193a-5p inhibitor (Fig. 6a and Additional file 1: Fig. S7A). The dual-luciferase reporter assay was performed to confirm the binding relationship between PIK3R3 and miR-193a-5p (Fig. 6b). The PIK3R3 3’UTR WT or mutant plasmid was cotransfected with the miR-193a-5p mimic in 293 T cells. The results showed that cotransfection of the PIK3R3 3’UTR WT plasmid and miR-193a-5p mimic significantly reduced the relative luciferase activity (Fig. 6c). Subsequently, we also tested whether miR-193a-5p affects the expression of PIK3R3. The qRT-PCR results revealed that the miR-193a-5p mimic could markedly reduce the expression level of PIK3R3, whereas the expression level of PIK3R3 was significantly upregulated by the miR-193a-5p inhibitor in both SW620 and RKO cell lines (Fig. 6d). PIK3R3 protein levels were significantly downregulated by intervention with the miR-193a-5p mimic (Fig. 6e). Then, we detected the expression levels of PIK3R3 in 40 pairs of CRC tumor tissues and adjacent normal tissues. The results showed that the PIK3R3 expression level in CRC tumor tissues was significantly increased compared with that in adjacent normal tissues and was negatively correlated with miR-193a-5p expression in CRC tissues (r = − 0.34, P = 0.032) (Fig. 6f and Additional file 1: Fig. S7B-D). To explore whether PIK3R3 expression levels could also be regulated by circRNA_0000392, we detected PIK3R3 expression after transfection with circRNA_0000392 siRNA. We found that knockdown of circRNA_0000392 significantly decreased the expression of PIK3R3 (Fig. 6g). Based on IHC staining of PIK3R3 in 40 CRC tissues, we found that PIK3R3 was positively correlated with circRNA_0000392 expression in CRC tissues (r = 0.385, P = 0.014) (Fig. 6h and i). Collectively, these results demonstrated that PIK3R3 was a target gene of miR-193a-5p and could be regulated by circRNA_0000392.

To investigate whether circRNA_0000392 plays its role in promoting tumor progression through the/miR-193a-5p/PIK3R3 axis, rescue experiments were performed by transcription of circRNA_0000392-knockdown cells with the miR-193a-5p inhibitor. The results of WST-1 and Transwell assays indicated that the inhibition of proliferation and invasion by circRNA_0000392 knockdown in SW620 and RKO cells could be rescued by the miR-193a-5p inhibitor (Fig. 7a-d). The miR-193a-5p inhibitor also rescued the effect of circRNA_0000392 knockdown on apoptosis (Additional file 1: Fig. S8). Then, we detected PIK3R3 mRNA expression by qRT-PCR and found that the reduction in PIK3R3 expression due to circRNA_0000392 siRNA could be alleviated by the miR-193a-5p inhibitor (Fig. 7e). In addition, the western blot assay revealed that knockdown of circRNA_0000392 decreased the PIK3R3 protein levels and AKT and mTOR phosphorylation levels, and the effects could be reversed by the miR-193a-5p inhibitor (Fig. 7f-i). Collectively, these results demonstrated that circRNA_0000392 could act as a regulator of miR-193a-5p to further affect the expression of PIK3R3 and play a regulatory role in CRC.

To explore the effects of circRNA_0000392 in vivo, circRNA_0000392-knockdown SW620 cells and negative control cells were subcutaneously injected into the backs of BALB/c nude mice. After 30 days of observation, the results showed that circRNA_0000392 knockdown reduced the volume and weight of SW620-derived tumors in vivo (Fig. 8a-c). Then, the expression levels of Ki-67 and PIK3R3 in the two groups of tumor tissues were evaluated by immunohistochemical staining. The results demonstrated that Ki-67 and PIK3R3 expression levels decreased in the tumor tissues in which the expression of circRNA_0000392 was knocked down (Fig. 8d - e). Therefore, inhibiting the expression of circRNA_0000392 significantly inhibited the growth of CRC in vivo (Fig. 8f).