Ethics statement
All procedures for animal handling were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Yunnan University of Chinese Medicine (Permit Number: R-062021G049).
Preparation of cbFeD aqueous extract
The dry cbFeD was purchased from Yunnan International Pty, Ltd. (Yunnan, China). 1 kg of dry cbFeD was soaked in cold water for 30 min, decocting twice with 1:6 w/v distilled water for 1 h. Filtration was then performed to the appropriate concentrations, the first decoction comprised in 1:10 w/v distilled water for 90 min and second comprised 1:8 w/v distilled water for 60 min. A final quantity of 450 g dried powder was obtained by spray drying at room temperature, which was then sealed and stored in the dark at 4˚C. The cbFeD powder was dissolved in normal saline for gavage. The drug containing serum solutions were collected from mice following exposure to the following treatments (once per day for 1 week): Treatment with normal saline by gastrogavage (n = 8; normal control group); 5 g/kg of cbFeD by gastrogavage (n = 8; cbFeD treat group). The mice were euthanized at day 30, and the colon tissue samples were obtained and stored at 80˚C.
Establishment of CRC mice model and immunohistochemistry (HE)
The Kunming mice (average age is 8 weeks, average live weight is 22 g) were provided by Kunming Medical University, and housed in consistent and standard environmental conditions with laboratory. Based on our criteria, experimental samples were made up of three groups of mice, including CRC group induced by DMH (Dimethylhydrazine, TCI Company, Japan) (CaD) and the control group (C). We set out to compare miRs changes among three groups with a high variation in CRC development. The chosen mice were slaughtered according to guidelines for the ethical use and treatment of animals in experiments in China. Colon tissue were separated and stored in liquid nitrogen until analyzed. The total RNA from colon was extracted by using TRIzol. The quality of miRs was assessed by the 2100 Bioanalyzer (Agilent, USA).
The CRC mice tissues were embedded in paraffin and HE staining was carried out on the 5-µm-thick sections. The sections were dewaxed with xylene for 5 min and repeated for 3 times. Then rinse with anhydrous ethanol for 5 min and repeat for 3 times. Then, 95%, 80%, 75% ethanol and distilled water were used for gradient elution for 2min each time. The sections were stained with hematoxylin drops for 5min, and rinsed with tap water for 2min. After 30s of differentiation with 1% hydrochloric acid ethanol, the water was soaked in antiblue treatment for 15min. The sections were stained with Kay red dye drops for 2min, and rinsed with tap water for 3-5s, dehydration were carried with 70% ethanol, 80% ethanol, 95% ethanol, 100% ethanol (2 times), xylene (3 times) for 1min. Finally, adding neutral resin for seal.
Small RNA library construction and sequencing
After extraction of the total RNA, the RNA molecules with a size range of 18-30nt were enriched by PAGE. Small RNA libraries were then constructed using a NEBNest multiplex small RNA library prep set (NEB E7300, Illumina) according to the manufacturer’s protocol. The small RNA libraries were then sequenced on an Illumina HiSeq TM 2500 by Gene Denovo Biotechnology Co. (Guangzhou, China).
Alignment and identification of miRs
Reads obtained from the sequencing machines included dirty reads containing adapters or low quality bases which would affect the following assembly and analysis. Thus, to get clean tags, raw reads were further filtered. After filtering, all of the clean tags were aligned with small RNAs in GeneBank database and Rfam database (11.0) to identify and remove other RNA. All of the clean tags were also aligned with reference genome and then searched against miRBase database (Release 21) to identify known mouse miR. Total miRNA consists of exist miRNA, known miRNA and novel miRNA, based on their expression in each sample, the miRNA expression level was calculated and normalized to transcripts per million.
Bioinformatic analysis of miRs data and target generation
Based on the sequences of the exist miRs, known miRs and novel miRs, the candidate target genes were predicted using three softwares RNAhybrid (v2.1.2) + svm_light (v6.01), Miranda (v3.3a) and TargetScan (Version:7.0). Gene Ontology classification, enrichment and KEGG analysis was performed base on the genes.
Cell culture, miR transfection and cbFeD incubating
The MC38 cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS (cat. no. 10099158; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 µg/ml of streptomycin and 100 U/ml of penicillin in a humidified atmosphere of 5% CO2 at 37˚C. The logarithmically growing cells were transfected with miR-488-3p mimics and its corresponding control RNA (mimics NC). The sequence of miR-488-3p mimics: 5’-UUGAAAGGCUGUUUCUUGGUC-3’. For transfection, 1.5×105 cells were cultured in 6 well plates 1 day for 24 h. And then, the MC38 cells were transfected with of miR-488-3p mimics or mimics NC (10 nmol/L) using INTERFER in siRNA Transfection Reagent (Polyplus Transfections, Illkirch, France). For cbFeD incubating, 1.5×105 cells were cultured in 6 well plates 1 day for 24 h. And then, the MC38 cells were incubated with 20µg/mL cbFeD for 48h. According protocol, cells were harvested after 48h, and total RNA was extracted using TRIzol regent (Invitrogen, CA) and determined by Nanodrop 2000.
Quantitative real-time PCR analysis
Total RNA was isolated from MC38 cells and converted in cDNA using AMV reverse transcriptase (Promega Corp, WI), and then transcript copies were calculated relative to those of the housekeeping gene GAPDH by qPCR that was performed using TaqMan MicroRNA Assays with the 7300 PCR Assay System (30 cycles, denaturation at 94℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s with final extension at 72℃ for 10 min). The primers were designed with Primer 5.0 and synthesized by BGI (Beijing Genomics Institute) Co., Ltd. The primer sequences are: PTEN Forward 5’-TTGTTAGCCTCTTCATGTGTGC-3’, Reverse 5’- TGGTAGCCAAACGGAACTTCA-3’;GAPDH Forward 5’-GAGTCAACGGATTTGGTCGT-3’, Reverse 5’-TTGATTTTGGAGGGATCTCG-3’. The relative expression of genes was calculated using the 2−ΔΔCt method and the standard deviation was calculated based on three biological replicates.
Western blot analysis and antibodies
The cells were harvested using protein extraction solution (Intron Biotechnology, Sungnam, Korea), and incubated for 30 min at 4˚C. Following removal of cell debris, the supernatants were collected by centrifuging at 13,000 x g for 15 min at 4˚C and the protein concentrations were determined using a Bio-Rad protein assay reagent, according to the manufacturer's protocol. PTEN (1:500; cat. no. ab137337) level was detected on western blot. 30 µg proteins were separated by 10% SDS-PAGE at 110V of 2hrs. Gel was transferred to PVDF membranes (BioRad, USA). Ponceau-S stain was used to evaluate the transfer efficiency. The strips were blocked with 5% nonfat dry milk powder in 0.1% TBST for 1 hr at RT and then incubated with primary antibodies in 2% BSA/TBST overnight at 4°C or 2 h at room temperature. The membranes were washed with 0.1% TBST buffer for 1 hr followed by probing with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:1000 dilution in PBS) (Sigma-Aldrich, USA). The membranes were then washed 0.1% TBST for 1 hr and bound antibody was detected by enhanced chemi-luminescence detection reagents (GE Biosciences, USA) according to manufacturer's instructions. Relative expression was calculated and normalized to the values of β-actin (Santa Cruz, Texas, USA) control.
Cell counting kit assay, flow cytometry apoptosis analysis
Cell counting kit-8 (CCK-8; Dojindo, Tokyo, Japan) was applied to assess cell growth. After 48 hours of transfection, 1×103 cells were incubated in each well of the 96-well plates. Then, each well was added with 10 microliters of CCK-8 solution mixed with a non-serum medium at days 1, 2, 3, and 4. After another incubation of 2 hours, the absorbance was examined at 450 nm using a microplate reader.
For analyzing cell apoptosis, cells were seeded in 6-well plates with the destiny of about 1×l04 each well. After 48 hours of transfection and cbFeD incubating, cells were collected, washed 3 times using cold PBS, and re-suspended with binding buffer at a concentration of 1×106/mL. Next, the cells were stained using fluorescein isothiocyanate (BioVision, San Francisco, CA) and PE (Beyotime). The signal was captured with the use of a FACS Calibur flow cytometer (BD Biosciences) and subjected to the analysis on FACS Diva software (BD Biosciences).
Statistical analysis
Each treatment was performed in triplicate. All data were compared via analysis of variance and multiple testing using the R-package (R v3.02). Differences were declared significant at P < 0.05 and P < 0.01.