Cell culture and culture conditions
The human CRC (HCT116, RKO, HT29, HCT8, SW620, SW480, and LOVO), 293T and normal colon epithelial (FHC) cell lines were purchased from the Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences (Shanghai, China). HCT116 p53+/+ and HCT116 p53−/− cells were obtained from American Type Culture Collection (ATCC, Maryland, USA). All these cell lines were maintained in DMEM medium supplemented with 10% FBS at standard culture conditions (5% CO2, 95% humidity and 37 °C).
Patients and tissue samples
108 paired CRC and adjacent normal tissues were collected from Shanghai General Hospital between 2013 and 2014, and were paraffin embedded for the tissue microarray (TMA) construction (the final TMA contained 106 CRC tissues and 106 adjacent normal tissues). Meanwhile, sixty-three pairs of human CRC fresh tissues and adjacent normal tissues were collected from Shanghai General Hospital after radical surgical resection between 2015 and 2017. After resection, the tissues were transported in liquid nitrogen and stored at -80°C refrigerator for RNA and protein extraction. No patients had received chemotherapy and radiotherapy before surgery. The detailed clinicopathological feature was confirmed by at least two pathologists according to the American Joint Committee on Cance (AJCC). Our research was approved by the Ethics Committee for Clinical Research of Shanghai General Hospital.
RNA extraction, gDNA extraction, and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from human CRC cells lines and frozen tissues with RNAiso Plus reagent (Takara, Japan) according to the manufacturer’s protocol. Genomic DNA (gDNA) was extracted from tissues using Fast Pure Cell/Tissue DNA Isolation Mini Kit (Vazyme, DC102) in accordance with the manufacturer’s protocol. Cytoplasmic and nuclear RNAs were separated using PARIS™ Kit (Invitrogen, USA) following the manufacturer’s protocol. For circRNA and mRNA, reverse transcription was conducted using the PrimeScript RT Master Mix (Takara, Japan). For miRNA, reverse transcription was conducted using PrimeScript RT Reagent Kit (Takara, Japan) with corresponding stem-loop primers. qRT-PCR was conducted by using SYBR Green Master Mix (Yeasen, China) in line with the manufacturer’s protocol on Roche real-time PCR instrument (Roche Applied Science, USA). Human GAPDH was selected as internal control for circRNAs and mRNAs. U6 was selected as internal control for miRNAs. All the primers were listed in Additional file 1: Table S1. The relative RNA expression levels were determined by the 2-ΔΔCT method. Each qRT-PCR experiment was carried out in triplicate.
Transfection, oligonucleotides and plasmids
For lentivirus transfections, short hairpin RNA (shRNA) plasmid targeting human C2CD4A and the control plasmid (sh-Ctrl) were inserted into a lentiviral vector (Genechem Shanghai, China). The sequences to knockdown human C2CD4A were showed in Additional file 2: Table S2. The human C2CD4A gene was cloned into pLVX plasmids (Genechem, Shanghai, China) to construct the C2CD4A overexpression vector. Then, the indicated cells with lentiviral transduction were selected with puromycin (Beyotime Biotechnology, China) for two weeks. The efficiency of C2CD4A knockdown and overexpression were verified by qRT-PCR and western blotting. Meanwhile, to regulate circSLC6A6, miR-1265, and MDM2 expression, oligonucleotides and plasmids were constructed. The siRNAs targeting circSLC6A6 and MDM2 were provided by GenePharma (Shanghai, China). The relevant oligonucleotides sequences were presented in Additional file 2: Table S2. Additionally, full length of circSLC6A6 was successfully cloned into the lentiviral plasmid pEX-3 (GenePharma, Shanghai, China) to establish the cell line with stable expression of circSLC6A6. The mimics, inhibitor and controls for miR-1265 were purchased from GenePharma (Shanghai, China). Cells transfection were performed using Lipofectamine 3000 (Invitrogen).
Immunohistochemistry (IHC)
The slides were incubated with primary antibodies including P53 (1:100, Abcam, USA), Cleaved-caspase3 (1:400; CST, USA), P21 (1:50, CST, USA), Bax (1:400, CST, USA), Ki-67 (1:500, Abcam, USA) at 4°C overnight, respectively, and then incubated with HRP labeled secondary antibody for 1 h at room temperature. The details about IHC staining score were shown in our previously published research [23].
Nucleic acid electrophoresis and RNase R treatment
Nucleic acid electrophoresis and RNase R treatment were performed as previously described [19, 24] .
Western blotting analysis
All cells and tissues were lysed with RIPA lysis buffer (New Cell & Molecular Biotech Co, China). Total protein concentration was measured by BCA Protein Assay Kit (Beyotime Biotechnology, China). Protein lysates (50μg) were separated in different concentrations of sodium dodecyl sulfate-polyacrylamide gels electrophoresis (SDS-PAGE) and then transferred to PVDF membranes (Millipore, Billerica, USA). The membranes were subsequently blocked in 5% non-fat milk at room temperature for 2h and then incubated at 4 °C overnight with the primary antibodies: anti-C2CD4A (1:500; Sigma-Aldrich, USA. The applications not including IHC staining), anti-Flag (1:5000; Sigma-Aldrich, USA), anti-His (1:1000; CST, USA), anti-HA (1:1000, CST, USA), anti-GFP (1:1000; Abcam, USA), anti-MDM2 (1:1000; CST, USA), anti-P53 (1:1000; Abcam, USA), anti-P21 (1:1000; CST, USA), anti-Ki67 (1:5000; Abcam, USA), anti-Bax (1:1000; CST, USA), anti-Cleaved-caspase3 (1:1000; CST, USA), anti-GAPDH (1:1000; CST, USA). Then, the membranes were incubated with appropriate secondary antibodies (1:000; CST, USA) for 2h. After membranes being washed three times, the brands were detected using ECL chemiluminescent reagent (Millipore, MA, USA). GAPDH was used as a loading control.
Cell proliferation assay
For the cell counting kit-8 (CCK-8) (Dojindo, Japan) assay, the transduced CRC cells were seeded in 96-well microplates with 1000 cells per well in humidified air containing 5% CO2 at 37˚C. OD values at 450 nm were measured every 24 hours by Gen5 microplate reader (BioTek). For the 5-Ethynyl-2’-deoxyuridine (EdU) cell growth assay, the transduced CRC cells were seeded into 96-well microplates and cultured for 24h. Then, the cells were incubated with 50 μM EdU (RiboBio, Guangzhou, China) for 2h, then, cell nuclei were stained with Hoechst 33342 for 30 min. The percentage of EdU-positive cells was presented by: (EdU positive cells/Hoechst positive cells) ×100%. Colony formation assays were performed to evaluate the cloning capability of CRC cells, the transduced CRC cells were seeded into 6-well plates (2000 cells/well) and incubated at 37 °C to facilitate colony formation. Two weeks later, the cells were fixed in methyl alcohol for 30min and then stained with 1% crystal violet for 20min. All experiments were performed in triplicate.
Apoptosis analysis
The cell apoptosis assay was conducted by using the AnnexinV-PE/7-AAD Apoptosis Detection Kit (MultiSciences Biotech Co, Ltd) in accordance with the manufacturer’s protocol [23]. The apoptotic cells rate were detected and analyzed by a flow cytometrer (BD Biosciences, San Jose, CA, USA). All experiments were performed in triplicate.
The cancer genome atlas (TCGA) database, GEPIA dataset and Oncomine database
The CRC data was downloaded from TCGA database (https://cancergenome.nih.gov).
There are 461 colon cancer samples with available data in TCGA database, including 458 RNA-seq samples, of which 41 pairs with paired sample data and pathological information. There are 171 rectal cancer samples with available data in TCGA database, of which RNA-seq samples have 166; there are 9 pairs with paired sample data and pathological information. Our expression profile analysis is based on these 50 paired samples with RNA-seq data, paired sample data and pathological information. In the above 50 pairs of CRC tissues and adjacent tissues, after filtering and screening, significantly differentially expressed genes were found. Gene Expression Profiling Interactive Analysis (GEPIA) dataset (http://gepia.cancer-pku.cn/detail.php) were used to determine the expression of C2CD4A in CRC tissues and normal tissues. Oncomine database (https://www.oncomine.org) was performed to detect the level of C2CD4A expression in CRC and normal adjacent tissues. The expression of C2CD4A mRNA was significantly higher in 45 colorectal adenocarcinoma (Skrzypczak Colorectal Statistics, 2010) and 36 colorectal carcinoma compared with normal colorectal mucosa tissues, respectively.
cDNA array analysis
cDNA array were performed as previously described to identify differentially expressed genes (DEGs) between three pairs RKO/sh-C2CD4A-1 and RKO/sh-Ctrl cells [25]. Then, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to analyze the DEGs.
Animal experiments
Four-week-old male BALB/c athymic nude mice were randomly divided into 5 groups (n=5). And injected subcutaneously into subscapular with 2.0×106 cells from the stable transfected RKO cell lines (shCtrl, sh-C2CD4A-1, sh-C2CD4A-2) to establish the CRC cancer xenograft model, respectively. Tumor size was measured every week to monitor tumor growth. Both of minimum (Width) and maximum (Length) diameters were measured for all tumors; the tumor volumes were calculated using the formula: 0.5×length×width2. All mice were sacrificed at 38 days, and tumors were surgically removed and weighed. After fixed in 4% paraformaldehyde, all tumors were embedded in paraffin and sliced into 4μm slides for IHC staining. All animal experiments were approved by the Institutional Animal Care of Shanghai General Hospital.
Luciferase reporter assay
Luciferase plasmids (pGL3-Firefly-Renilla containing circSLC6A6 sequence or Mutant sequence, pGL3-Firefly-Renilla containing C2CD4A 3’-UTR sequence or Mutant sequence) were purchased from GenePharma (Shanghai, China) and were co-transfected with miR-1265 mimics or inhibitor to indicated cells using LipofectamineTM 2000 reagent. In short, the transfected cells were lysed with 100μl of passive lysis buffer and the supernatant was collected after centrifuge. Cell samples (20μl) and 100μl of LARII were added into 96-well luminometer plates, followed by firefly luciferase activity detection. Then, 100μl of Stop & GloR reagent was added into 96-well luminometer plates to detect Renilla luciferase activity. The ratio of firefly luciferase/Renilla luciferase activity was used to detect the effects of miR-1265 on luciferase reporter plasmids. All experiments were independently performed in triplicate.
Fluorescence in situ hybridization (FISH)
FISH assay was performed to locate circSLC6A6 using a Cy3-labeled probe and to locate miR-1265 using a FAM-labeled probe in HCT8 and RKO cells, respectively. The Cy3-labeled circSLC6A6 probe and FAM-labeled miR-1265 probe were designed and synthesized by GenePharma (Shanghai, China). Cells were seeded in 35-mm glass bottom dishes with 10-mm microwells. After washing with PBS and being fixed with anhydrous ethanol, the cells were treated with 100μl of 0.1% Triton-100 at room temperature for 15 min. The cells were hybridized with 5µl of probe in hybridization buffer (10% dextran sulfate, 40% formamide, 4×saline-sodium citrate (SSC), 1×Denhardt's solution, 1000 mg/ml sheared salmon sperm DNA, 10 mM DDT, 1000 mg/ml yeast transfer RNA) at 37°C overnight. Then, cells were continuously washed and dyed with 100μl of DAPI for 20 min. Then, confocal laser scanning microscopy was used to observe the staining. Simultaneously, FISH assay was also performed in TMA, which contained 106 paired CRC samples by Cy3-labeled miR-1265 (Boster, Shanghai). The staining scores were evaluated by two independent pathologists to avoid bias as previously described in our published study [24]. Finally, the samples scores were divided into two groups: the high expression group (4-8) and the low expression group (0-3). The sequences of circSLC6A6 and miR-1265 probe for FISH and TMA were listed in Additional file 1: TableS3.
Protein half-life detection
HCT116 p53+/+ cells were respectively stably transfected with shC2CD4A-1, shC2CD4A-2 and pLVX-C2CD4A, and were treated with 10 mg/mL cycloheximide (CHX). Then, the treated cells were harvested after CHX treatment for 0, 20, 40, 60, 90 and 120 min, respectively. Equal amount of protein was extracted from the treated cells which was performed western blotting analysis with anti-C2CD4A or anti-P53 antibody. GAPDH was selected as an internal control to verify basal level expression in different groups.
Immunoprecipitation and Mass Spectrometry (IP/MS)
293T cells transfected with Flag-C2CD4A were lysed in RIPA buffer (20 mM Tris-HCl (pH 8), 137 mM NaCl, 0.5% Triton X-100, 2 mM EDTA) and protease inhibitor cocktail (Sangon Biotech, Shanghai, China). Cell lysates were incubated with Flag-M2 agarose beads (Sigma) overnight and eluted by Flag peptides (Sigma). The Flag peptide elution was resolved on SDS-PAGE and Coomassie blue stained. Lysates from 293T cells transfected with control Flag-vector were served as control. Protein bands specific to the Flag-C2CD4A transfection were digested with trypsin and analyzed by mass spectrometry for protein identification.
Co-immunoprecipitation assay (Co-IP) and ubiquitination assay
Protein extracts were immunoprecipitated with Protein A+G Agarose beads (Beyotime Biotechnology, Shanghai, China) using appropriate antibodies or control IgG (Sigma, USA). Then, the protein of immunocomplexes were subject to immunoblotting analysis with antibodies as indicated. For ubiquitination assay, 293T cells were co-transfected with Flag-C2CD4A, HA-P53 and His-ubiquitin. Alternatively, 293T cells were co-transfected with sh-C2CD4A, HA-P53 and His-ubiquitin. 24 hour later, 293T cells were treated with MG132 (10μM) for 8 hours to inhibit proteasomal degradation. The transfected cells were lysed in lysis buffer. The cell lysates were centrifuged. Then the supernatants were pulled-down by incubation with Ni-NTA beads, and the protein of immunocomplexes was immunoblotted using anti-HA antibody.
RNA immunoprecipitation (RIP)
RIP was performed by using Magna RIP™ RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA) according to manufacturer’s protocol. The AGO2-RIP experiments were conducted in RKO cells transiently overexpressing miR-1265 mimics or miR-NC. 48h later, approximately 1×107 RKO cells were lysed in complete RNA lysis buffer. Then, RKO cell lysates were incubated with RIP immunoprecipitation buffer containing magnetic beads conjugated with human anti-Argonaute2 (AGO2) antibody (Millipore, Billerica, MA, USA) or negative control IgG (Millipore, Billerica, MA, USA) at 4℃ overnight. Each sample was digested with Proteinase K and then immunoprecipitated RNA were analyzed by qRT-PCR to test the concentration of circSLC6A6. RIP assay was repeated in triplicate.
RNA Pull-down assay
The biotinylated circSLC6A6 probe and miR-1265 probe were designed and synthesized by GenePharma (Shanghai, China) and the sequences were listed in Additional file 1: TableS4. Briefly, cells were collected, lysed, and sonicated. Probe-coated beads were generated by coincubating the circSLC6A6 and miR-1265 probes with probes-M280 streptavidin dynabeads (Invitrogen, USA) at 25°C for 2h. The cell lysates were incubated with circSLC6A6 and miR-1265 probes or oligo probe at 4°C overnight. After washing with wash buffer, the RNA complexes bound to the beads were eluted and extracted with the RNAisoPlus (TaKaRa, Japan) and measured by qRT-PCR.
Statistical analysis
All quantitative data were enumerated by a χ2 or Fisher’s exact test. The associations were analyzed by Pearson’s test. Comparisons between different groups were analyzed using a paired or unpaired t-test. Survival curves were drawn by Kaplan-Meier method and analyzed by log-rank test. The SPSS 23.0 software was deployed for statistical analysis. P<0.05 was considered statistically significant in all tests.