Human colorectal cancer and adjacent non-tumorous tissue samples for qRT-PCR analysis were obtained from a total of 82 patients who underwent surgical resection in the Department of General Surgery of Nanfang Hospital affiliated to Southern Medical University. Twenty colorectal cancer samples were randomly selected for immunohistochemistry (IHC) detection and analysis. All the samples were gathered with informed consent according to the Institutional Review Board of Ethical Committee–approved protocol.
Cell culture and treatment
The human monocyte cell line THP-1 and CRC cell lines (SW480, HCT116, LoVo, and RKO) were obtained from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). Lentiviruses carrying full-length CPEB3 or short hairpin RNA (shRNA_CPEB3) sequences targeting against human CPEB3 mRNA and matched negative controls were constructed by the Shanghai Institute of Biochemistry and Cell Biology. SW480, HCT116, LoVo and RKO cells were transfected with the indicated lentivirus overnight, then 2 μg/mL puromycin was added after 72 h of transfection to obtain stably transfected CRC cells. For macrophage generation, THP-1 cells were treated with 100 ng/mL phorbol- 12-myristate-13-acetate (PAM) (Beyotime, Shanghai, China) for 12 h to differentiate into adhered macrophages. To obtain TAM supernatants, CRC cells were seeded in 0.4-μm pore inserts, then transferred to a 6-well plate seeded with THP-1 macrophages in advance and co-cultured for another 24 h. For co-culture experiments, stably transfected CRC cells were co-cultured with THP-1 macrophages for another 24 h.
Five-week-old BALB/c male mice were purchased from the Experimental Animal Center of Southern Medical University (Guangzhou, China) and sheltered under specific pathogen-free conditions. For tumor formation in mice, mice were randomly assigned to four groups (five mice per group): HCT116-CPEB3 group, LoVo-shCPEB3 group, and matched negative control groups. HCT116-Ctrl/CPEB3 (5 × 106) and LoVo-shCtrl/shCPEB3 (5 × 106) were subcutaneously injected into the right back portion of male BALB/c mice at five weeks of age. Tumor nodules were examined every five days and the volume was evaluated using the following formula: tumor volume = (width2 × length)/2. Mice were sacrificed after a period of 30 days and examined for the growth of subcutaneous tumors. For liver metastasis assay, LoVo-shCtrl/shCPEB3 (5 × 106) were injected into the spleen of nude mice, then 5mg/kg IL-6R inhibitor (tocilizumab) was injected intraperitoneally weekly. After 30 days, mice injected with CRC cells were sacrificed and livers were removed for examination. All animal care and handling procedures were performed in accordance with the NIH's Guide for the Care and Use of Laboratory Animals.
Cell proliferation and colony formation assay
Stably transfected CRC cells were treated with supernatants from TAMs (co-cultured with CRC cells), IL-6 (20 ng/mL, Peprotech, Rocky Hill, NJ, USA), tocilizumab (5 μg/mL, Selleck Chemicals, Houston, TX, USA) and neutralizing antibodies to IL-6 (anti-IL-6; R&D Systems, Minneapolis, MN, USA). The proliferation rate of these stably transfected CRC cells was assessed at 24, 48, 72 and 96 h using the Cell Counting Kit-8 (CCK-8; Beyotime). Each time-point was assessed in replicates of three wells. For the colony formation assay, the stable cell lines (400 cells/well) were seeded in 6 well plates. After 2 weeks, the cells were fixed in 4% paraformaldehyde and stained with crystal violet for 30 min at room temperature. Colonies consisting of >50 cells were counted.
Matrigel invasion assay
The Matrigel invasion assays were carried out in 24 well plates with 8 μm polycarbonate nucleopore filters (Corning, Tewksbury, MA, USA). The membrane for the invasion assay was covered with 100 μL BD Matrigel (diluted 1:4 with serum-free medium) in advance. The stable cells lines treated with or without tocilizumab (5 ug/mL, Selleck Chemicals) were seeded in the upper chambers; the lower chambers were filled with medium containing 10% FBS along with or without 20 ng/mL IL-6. In addition, the lower chambers were also filled with TAM cells supernatants along with or without neutralizing antibodies to IL-6 (anti-IL-6; R&D Systems). After a 48 h incubation, the cells adhering to the lower filter surface were counted.
THP-1 macrophages were co-cultured with stably transfected CRC cells along with or without neutralizing antibodies to CCL2 (anti-CCL2; R&D Systems). Then these macrophages were processed into single cell suspensions, incubated with antibodies (BV421 Mouse anti-Human CD68, BB515 Mouse anti-Human CD86, PE Mouse anti-Human CD163, all from BD (BD Biosciences, San Jose, CA, USA) for 1 h at 4 ℃. Mouse macrophages were then stained with CD206-APC (mouse), CD86-FITC (mouse), F4/80-PE (mouse) antibodies (eBiosciences, San Diego, CA, USA), respectively. Macrophages of nude mice subcutaneous tumor were separated and obtained using Percoll (Sigma-Aldrich, St. Louis, MO, USA) following the instruction. Flow cytometry was performed using a FACS Calibur flow cytometer (BD Biosciences). Flow cytometric analysis was performed on FlowJo software (FlowJo, Ashland, OR, USA).
IHC staining was performed on 5-µm sections of paraffin-embedded tissue samples to detect the protein expression levels of CD68, CD86, CD163, CPEB3, E-cadherin, vimentin and Ki67. In brief, the slides were incubated in anti-CD68(1:500, Servicebio, Wuhan, China), anti-CD86 (1:100, BOSTER, Wuhan, China), anti-CD163 (1:500, BOSTER), anti-CPEB3 (1:200, Abcam, Cambridge, MA, USA), anti-E-cadherin (1:1000, Proteintech, Chicago, IL, USA), vimentin (1:1000, Proteintech) and Ki67 (1:1000, Proteintech) antibodies at 4℃ overnight. All slides were independently evaluated by two observers. The score for CPEB3, E-cadherin and vimentin staining was based on the integrated staining intensity and the proportion of positive cells. IHC staining of CD68, CD86, CD163 and Ki67 was calculated by the positive cell numbers in the per high field. All the percentages/numbers of positive cells were expressed as the average of six randomly selected microscopic fields.
Western blot analysis
Protein extracts were probed with antibodies against human CPEB3 (1:500, Abcam), phospho-STAT3(Tyr705) (1:1000, Cell Signaling Technology, Danvers, MA, USA), STAT3 (1:1,000, Cell Signaling Technology), IL-6R (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-6ST (1:500, Santa Cruz Biotechnology), phospho-JAK1(Y1022/1023) (1:1000, ABclonal Technology, Wuhan, China), JAK1 (1:1,000, ABclonal Technology), ZEB2 (1:1,000, Proteintech), E-cadherin (1:1,000, Proteintech), N-cadherin (1:1,000, Proteintech), vimentin (1:1000, Proteintech), slug (1:1000, Proteintech), snail1 (1:1000, Proteintech), and GAPDH (1:1,000, Proteintech). Peroxidase-conjugated anti-mouse (1:5000, Proteintech) or rabbit antibody (1:2000, Proteintech) was used as a secondary antibody and the antigen-antibody reaction was visualized by an enhanced chemiluminescence assay (Millipore, Bedford, MA, USA).
The levels of cytokines in cell culture supernatants were measured using IFN-γ, IL-1β, IL-1RA, IL-4, IL-6, IL-10, IL-12p70, IL23, IP10, TNF-α and CCL2 Human ProCartaPlex™ simplex kit and Human Basic kit (eBioscience). Briefly, 50 μL samples or standard recombinant protein dilution were added to a mixture of capture beads coated with related monoclonal antibodies to a group of cytokines, washed beads were further incubated with biotin-labeled anti-human cytokine antibodies for 1 h at room temperature followed by incubation with streptavidin–phycoerythrin for 30 min. Samples were analyzed using Luminex 200™ (Luminex Corporation, Austin, TX, USA).
Enzyme-linked immunosorbent assay (ELISA)
The cytokine of IL-6 in cells supernatants was estimated by ELISA, using a commercial kit (MultiSciences, Hangzhou, China), according to the manufacturer’s instructions. Positive controls were supplied in the kit.
RNA isolation, reverse transcription (RT) and real-time PCR
Total RNA from tissues and cultured cell lines was isolated using the Trizol reagent (TaKaRa, Osaka, Japan) according to the manufacturer’s instruction, and cDNA was synthesized using random primers and the TaKaRa PrimeScript RT regent kit. qRT-PCR reactions were performed in triplicate using the SYBR Green method on a Light Cycler 480 Real Time PCR System (Roche Diagnostics, Mannheim, Germany). The PCR primers are listed in Supplementary Table S1.
RNA immunoprecipitation (RIP)
RNA immunoprecipitation of CPEB3 targeting RNAs was performed in SW480 and LoVo cells. Briefly, SW480 and LoVo cells were lysed in a polysome lysis buffer according to the Magna RIP RNA-Binding Protein Immunoprecipitation Kit guidelines (Millipore). RNA was extracted for RT-PCR and qRT-PCR detection.
Luciferase reporter assay
SW480-Ctrl, SW480-CPEB3, HCT116-Ctrl and HCT116-CPEB3 cells of 80% confluence were transfected with indicated plasmids using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). Wild-type and mutated forms of the IL6R-3' UTR were subcloned into a pmir-GLO vector were co-transfected per well of a 24-well plate. Cell extracts were prepared at 36 h after transfection. The luciferase activity was measured with a Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA).
All statistical analyses were performed using Graph-Pad Prism software (version 6.0, GraphPad Software, San Diego, CA, USA). Pearson’s correlation analysis was performed to assess the relationship between CD86, CD163 expression and CPEB3 expression in patients with CRC. Groups of discrete variables were compared using the Student's t test or nonparametric ANOVA. All experiments for cell cultures were performed independently at least three times and in triplicates each time. In all in vitro experiments, data represented at least three independent experiments and are expressed as means ± SEM. In in vivo experiments, data are expressed as the mean ± SEM. P-values < 0.05 were considered statistically significant (in all figures: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns = not significant).