miR-30c affects the pathogenesis of ulcerative colitis by regulating target gene VIP | Scientific Reports – Nature.com

Active ulcerative colitis patients and normal healthy control individuals

The colon tissue samples of 4 active ulcerative colitis patients and 4 normal healthy controls were used to analyze miR-30c and VIP expression levels in this study, which were provided by the First Affiliated Hospital of Bengbu Medical College. The patients were included into this study according to the score of Sutherland Index (to describe the disease activity)37 and the modified Baron score (to represent an endoscopic classification)38. This study was approved by the Ethics Committee of Bengbu Medical College (No.2023LK401) and was conducted in accordance with the ethical guidelines of the Declaration of Helsinki. All participants in this study were over the age of 18, and informed consent was obtained from all subjects. We are committed to protecting the privacy and personal information of our participants throughout the research process and ensuring that all procedures performed in the study adhere to the applicable ethical standards.

The miR-30c gene knockout mice (miR-30c-/-, referred to as KO) used in this study were utilized by using CRISPR/cas9-mediated genome engineering technology (Shanghai Model Organisms Center, China). The target sequences of two sgRNAs were 5'-AAGTGTCCATGACAGTGTCA-3' and 5'-ACTAGACTTAGATGCTCTGC-3', which targeted the transcript of mmu-mir-30c-1 (ENSMUST00000083556.3). miR-30c KO mice were further validated by qRT-PCR test. The mice were fed and watered liberally under specific pathogen free (SPF) conditions, standardized temperature conditions (2122C) and illumination (12h light/12h dark). All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of Bengbu Medical College (No.2022LDK140). And methods were carried out in accordance with relevant guidelines and regulations. This study was carried out in compliance with the ARRIVE guidelines.

To induce experimental colitis, it was established by providing the mice with 2.5% (wt/vol) DSS (MP Biomedicals, Santa Ana, CA, USA; molecular weight of 36,00050,000Da; CAS Number: 9011-18-1) dissolved in drinking water for 7days. There were 4 groups (6 mice per group): WT and miR-30c KO mice untreated group (normal water without DSS); WT and miR-30c KO mice DSS treated group (water containing 2.5% DSS). Daily weight, diarrhea, and bleeding scores were recorded. The weight loss percentage was scored as: grade 0, none; grade 1, 1 to 5%; grade 2, 5 to 10%; grade 3, 10 to 15%; grade 4,>15%. Fecal bleeding was scored by Occult blood test kit (O-toluidine method, Yuanye Bio, China) and observation as grade 0, no bleeding and tested negative; grade 1, tested weakly positive; grade 2, some bleeding with tested positive; grade 3, gross bleeding; grade 4, blood filling the whole colon. The score for stool consistency was: grade 0, normal stool; grade 1, slightly loose stool; grade 2, loose stools; grade 3, watery stool; grade 4, severe diarrhea (according to Cooper and colleagues, with modifications). Disease Activity Index DAI=(Decreased Body Mass Score+Blood in Stool Score+Fecal Characteristics Score).

The plasma were obtained by orbital blood sampling and the colons were taken and cleaned with PBS (phosphate buffered saline) solution. The levels of inflammatory factors (IL-1, IL-6, IL-10, IL-12, IL-23, and TNF-) in plasma and colonic lavage fluid were measured using ELISA (Enzyme-Linked Immunosorbent Assay) kits (Jingmei Bio, China) according to the manufacturer's instructions.

After the fresh mice colon tissues were rolled up and fixed with 10% formaldehyde, the tissues were dehydrated, transparent, and were dipped in wax to make paraffin-embedded tissues. 10m-sections of tissues were taken for deparaffinization and rehydration. Slides were blocked with arborvitae after hematoxylin and eosin (H&E) staining, Alcian blue and Periodic acid Schiff (AB-PAS) staining. They were observed by microscopy (Olympus, Japan) and evaluated using Image J software. The sums of tissue damage scoring and inflammatory cell infiltration scoring were used for statistical analysis, both of which were scored on the following scale: grade 0, no colon mucosal damage, and few scattered inflammatory cells in the lamina propria of the colonic mucosa; grade1, colonic epithelial cells are damaged,and massive invasion of inflammatory cells into the lamina propria of the colonic mucosa; grade 2, colonic mucosal damage or with focal ulcers, and widespread inflammatory cells with extension into the colonic submucosa; grade 3, damage to the colonic mucosa that spreads to deeper colonic wall structures,and massive invasion of inflammatory cells into the colonic submucosa. The goblet cells were also counted on AB-PAS staining results.

Total RNA was isolated using the MiFure Cell/Tissue miRMA Kit (Vazyme, China). miR-30c expression was reverse transcribed and detected according to All-in-One miRNA qRT-PCR Detection Kit 2.0 (GeneCopoeia, China) and U6 was used as endogenous controls. The expression levels of VIP, IL-1, IL-6, IL-10, IL-12, IL-23, and TNF- was measured using GAPDH as the reference gene by qRT-PCR (Applied Biosystems 7500, USA). Relative gene expression level was calculated by 2Ct relative quantitative method. The primer sequences were listed in the Supplement Table S1.

The 293T cell line (KeyGEN BioTECH, China) was cultured in DMEM (GIBCO, USA). 10% fetal bovine serum (FBS) (ExCell Biology, USA) in the incubator at 37C and 5% CO2. The VIP 3'-UTR sequence and the mutated VIP 3'-UTR sequence were designed and synthesized in order to verify the targeting relationship between miR-30c and VIP. The synthesized fragments of the two target genes were cloned into pmirGLO dual luciferase reporter gene vectors to construct the VIP 3'-UTR dual luciferase reporter gene wild-type vector (pmirGLO-VIP) and its mutant vector (pmirGLO-mut-VIP), respectively. Recombinant vectors were identified by PCR electrophoresis and gene sequencing. The two recombinant vector plasmids were co-transfected with miR-30c mimics (mimics) or miR-30c Negative Control (NC) in 293T cells using the transfection reagent lipofectamineTM2000, respectively. The activity of luciferase was detected using the Dual-Luciferase Assay System Kit (Promega, Madison, USA) by Multi-function Enzyme Labeler (Molecular Devices M3, USA).

The terminal colon proteins of mice were extracted by RIPA Lysis Buffer kit (Beyotime, China). The extracted proteins were quantified by BCA Protein Assay Kit (Epizyme, China), and the protein expression was analyzed by Western blotting assay. Membranes were incubated overnight at 4C with the primary antibodies VIP (DF6627, Affinity Biosciences, China) and GAPDH (AF7021, Affinity Biosciences, China) mixed by primary antibody diluent (Epizyme, China) and incubated with Goat Anti-Rabbit IgG (H+L) HRP (S0001, Affinity Biosciences, China) subsequently. Immunodetected proteins were visualized using YosiSuper West Pico PLUS Chemiluminescent Substrate Kit (Yoshi Bio, China). The protein expression was quantified using the ImageJ software (version 1.53s; ImageJ can be downloaded from https://imagej.net/ij/download.html).

The slides were incubated with diluted primary antibody at 4C overnight followed by secondary antibody incubation. Staining was performed as described in the operating manual of DAB Horseradish Peroxidase Color Development Kit (Solarbio, China). The antibodies were the same as those used in the WB analysis. And the average optical density values of positive signals were analyzed using the ImageJ software.

Data were presented as meansstandard deviation (SD) using R software version 4.0.3. The experimental data were analyzed using an unpaired two-tailed t-test. P values<0.05 were considered statistically significant.

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miR-30c affects the pathogenesis of ulcerative colitis by regulating target gene VIP | Scientific Reports - Nature.com