Patient population and clinical data
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.
RNA sequencing, identification and quantification of human circRNAs
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.
Analyses of circRNA-miRNA-mRNA interactions in CRC
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.
Cell culture
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 293T 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 293T 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.
Antibodies and reagents
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).
RNA extraction and qRT-PCR
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: Supplementary Table 1.
CircRNA RNase R resistance analysis and actinomycin D assay
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.
Fluorescence in situ hybridization (FISH)
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.
Luciferase reporter assay
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).
Transwell migration and Matrigel invasion assays
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.
RNA immunoprecipitation (RIP)
RIP assays were performed in SW620 and RKO cells. A total of 1 x 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.
RNA pull-down
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.
Immunohistochemistry
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).
Xenograft tumor model
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.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software Inc., CA, USA). Student’s t-test and one-way ANOVA were used to compare differences between groups as appropriate. The correlation between groups was analyzed by Pearson correlation. ROC curve analysis was performed to evaluate the diagnostic value. Data are presented as the mean ± standard deviation (SD), and p < 0.05 was considered statistically significant.
Additional methods
The cell transfection, western blot, cell proliferation, and apoptosis assays are described as the Supplementary Methods in Additional file 2.