Construction of Sec1−/− mice and Sec1-siRNA intestinal epithelial cells
Knockout of sec1 (gene ID: 56546) in C57BL/6 mice were carried out by Cyagen Biosciences (Guangzhou, China) using CRISPR/Cas9 gene editing technology that has been published elsewhere (16). The exon 4 of Sec1 located on the mouse chromosome 7 was selected as the target site for gRNA target sequences (Table 1). The genotype of Sec1−/− mice was verified using polymerase chain reactions (PCR), using the following primers F: 5’-AGGTGACAGAAAGATTCAGAGGTAC-3’, R:5’-GAGTGAGTGTGAGTGTGCTAGAAAC-3’. Undifferentiated colon carcinoma cell lines CT26.WT and CMT93 (Xiamen Immocell Biotechnology Co. Ltd) were used to construct Sec1-siRNA intestinal epithelial cells (IEC). The mouse Sec1 sequence was from the NCBI gene library and used as the target of suppression following the design principle of siRNA (si-m-Sec1: GGACACTGTTTACCTGGCT, NC: TTCTCCGAACGTGTCACGT). Cells were maintained in the Dulbecco’s modified Eagle’s medium (DMEM, Gibco, NY) supplemented with 10% fetal calf serum (Excell Bio, Shanghai, China) and 1% penicillin-streptomycin solution. This study was approved by the Experimental Animal Ethics Committee of Wenzhou Medical University (Approval number: wydw-2016-0215). All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals.
Table 1
Genes | Sequence |
gRNA1 | TCACAGTGCTCCAGCGACTCAGG |
gRNA2 | CCATGACAGTAAACACGTTGGGG |
Mouse model of colitis
Colitis was induced in C57BL/6 wildtype (WT) and Sec1−/− mice with 3% (w/v) dextran sulphate sodium (DSS) (Colitis grade, 36–50 kDa, MP Biomedicals, Canada) pre-dissolved in drinking water and given ad libitum (days 1–5) as described (17). Body weight, rectal bleeding, diarrhoea and mental status of the animals were monitored daily. As for the mental status, sign of alertness/sleeping were monitored, including being dull or depressed, presenting little response to handling, and being unconscious. Mice were euthanized on day 7 and the peripheral blood and colonic tissues were collected for further analyses.
Histopathological analysis and fluorescence microscopy
Colonic tissues were fixed with 10% formalin and embedded in paraffin. Sections of 5 µm in thickness were stained with hematoxylin and eosin (H&E) for pathological analysis. For immunofluorescence microscopy, paraffin-embedded tissue sections were incubated with FUT3 antibody (Affinity), DR5 antibody (Invitrogen), CY3 Conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) secondary antibody (Boster Biological Technology), and DAPI (Beyotime Biotechnology, China). Sections were examined using an Olympus digital sight BX53 fluorescence microscope. The apoptosis of colonic epithelial cells was assessed using TUNEL BrightRed Apoptosis Detection Kit (A113-03, Vazyme), following the manufacturer's instruction.
Flow cytometry
The numbers of Th17 and T reg cells in the spleen and colon of WT and Sec1−/− mice with induced colitis were analyzed by flow cytometry. The lamina propria mononuclear cells (LPMCs) and spleen lymphocytes were isolated using a monocyte separator (LTS1092PK, Tianjin Haoyang Biological Manufacture, China) and resuspended in phosphate buffered saline (PBS). LPMCs and spleen lymphocytes were counted and stimulated using PMA/Ionomycin (Yeasen, Shanghai, China) for 1 h, followed by harvesting, fixation using IC fixation buffer (eBioscience, USA) and permeabilization using 1× permeabilization buffer (eBioscience, USA). Cells were then incubated with the following antibodies for 30 min on ice: CD4 antibody (eBioscience, USA), CD25 antibody (eBioscience, USA), Foxp3 antibody (eBioscience, USA), IL-17A antibody (eBioscience, USA), and examined using a flow cytometer (CytoFLEX, Beckman). The numbers of CD4/IL-17A/Th17 and CD4/CD25/FoxP3/T reg cells were calculated using CtyExpert 2.3.
Apoptosis and cell cycle analysis
CT26.WT or CMT93 cells were seeded in 6-well plates, at a density of approximately 2×105 cells per well. After transfection with SiRNA using the lipofectamineTM 2000 transfection reagent (Invitrogen, USA) for 24 h, cells were harvested and washed with PBS, resuspended in 5 µL AnnexinV-APC/7-AAD and incubated in the dark for 20 mins. For cell cycle analysis, SiRNA-treated CT26.WT and CMT93 cells were resuspended in 500 µL propidium iodide (PI) solution for 30 mins. Cell apoptosis and cycle were analyzed using the CytoFLEX flow cytometer (Beckman). For cell viability assays, CT26.WT and CMT93 cells were seeded in 6-well plates at a density of 5×103 cells per well. After receiving treatments for 24 h, cell viability was assessed using a Cell Counting Kit (CCK-8; E-CK-A362, Elabscience) per the manufacturer’s instructions.
Western blot
Western blot
Proteins were extracted from colonic tissues and cell lines using RIPA Lysis Buffer (Beyotime Biotechnology, Shanghai, China) and PMSF non-specific protease inhibitor solution (Aladdin, Shanghai, China), following the manufacturers’ instructions. The protein concentrations were determined using the BCA method (Beyotime Biotechnology, Shanghai, China). Protein extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to Immobilon-P polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, USA). Membranes were probed with the following primary antibodies: GAPDH antibody (Goodhere Biology, Hangzhou, China), FUT3 antibody (Affinity), DR5 antibody (Invitrogen) and detected using secondary HRP-coupled antibodies (Goodhere Biology, Hangzhou, China). This experiment was carried out in three biological repeats.
Quantitative RT-PCR (q RT-PCR)
Total RNA was extracted with Trizol and reverse transcribed into cDNA with ReverTra Ace qPCR RT Kit (Toyobo, Japan). qPCR was carried out using 2×SYBR Green qPCR Mix (With ROX) (Sparkjade, China) and a fluorescence quantitative PCR instrument (Bio-rad, USA). The relative gene expression was calculated using the 2−ΔΔCT method (18). The primers used for qPCR were listed in Table 2.
Table 2
Sequences of the primers for Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR).
Genes | | Sequence |
GAPDH | Forward: | 5′-ATGGGTGTGAACCACGAGA-3′ |
Reverse: | 5′-CAGGGATGATGTTCTGGGCA-3′ |
FUT2 | Forward: | 5′-CACCATCAGAGTCAAAGGCC-3′ |
Reverse: | 5′-TAACGCTCCTCCATCCAGTC-3′ |
DR5 | Forward: | 5′-TACGGTGTGTCGATGCAAAC-3′ |
Reverse: | 5′-GCTTATGCCAAGATGCCCAA-3′ |
IL-6 | Forward: | 5′-AGACTTCCATCCAGTTGCCT-3′ |
Reverse: | 5′-CATTTCCACGATTTCCCAGAGA-3′ |
IL-1β | Forward: | 5′-TCAGGCAGGCAGTATCACTC-3′ |
Reverse: | 5′-AGCTCATATGGGTCCGACAG-3′ |
TNF-α | Forward: | 5′-CGTCAGCCGATTTGCTATCT-3′ |
Reverse: | 5′-CGGACTCCGCAAAGTCTAAG-3′ |
The enzyme-linked immunosorbent assay (ELISA)
The cell supernatants from CT26.WT and CMT93 cells, as well as the serum samples of mice with induced colitis were collected. Cytokine levels, including that for IL-1β, IL-6, and TNF-α were measured using ELISA kits (Elabscience Biotechnology, Wuhan, China) according to the manufacturer's instructions.
Statistical analysis
Statistical analysis was performed using IBM SPSS ® Statistics version 22 (IBM Corporation, USA). Normality of data was examined and confirmed with the Kolmogorov-Smirnov test. Variables between groups were analyzed by Student's t test and one-way ANOVA. Results were presented as median ± standard error of the means. Spearman or Pearson correlation was used for correlation analysis. p < 0.05 was considered to be statistically significant.