Animal assays

CRC is a life-threatening disease that can be a complication of inflammatory bowel diseases or develop spontaneously, and AOM/DSS and ApcMin/+ mice models were used in our research. C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animal, China. ApcMin/+ mice were purchased from Nanjing Biomedical Research Institute of Nanjing University, China. All mice were maintained in ventilated cages with 12-h light/dark cycles, constant temperature and humidity, enriched water, and ad libitum feeding under SPF conditions. All animal experiments used in this study were approved by the Institutional Animal Care and Use Committee of Zhejiang University.

For inflammation-related carcinogenesis model, C57BL/6 male mice (8 weeks old) were given one single intraperitoneal injection of carcinogen AOM (Sigma-Aldrich, USA) at 10 mg/kg body weight, followed by five successive days of 2% DSS (Sigma-Aldrich, USA) in the drinking water, and then given regular drinking water for 2 weeks. This cycle was then repeated twice. Meanwhile, the three groups were given the following treatments separately: common feed + PBS, common feed + 5% HFCS (45% glucose + 55% fructose), 20% RS (Hi-maize® 260, a commercial type 2 RS supplementation produced from naturally modified high amylose corn [26], Ingredion, USA) + 5% HFCS. PBS and HFCS were administrated by gavage every day (400 μl), while 20% RS was fed freely and mixed with their daily feed. During the modeling process, the disease severity of mice in each group was evaluated by the DAI score, which included the index of weight, stool characteristics, and degree of blood in the stool of mice. After 2 months, mice were killed and the colons were surgically excised for further analysis.

For spontaneous adenoma model, C57BL/6 J ApcMin/+ male mice (3–5 weeks old) were randomly assigned to three groups. The three groups were given different treatments separately, consistent with the grouping in AOM/DSS mice model. At the indicated time intervals, colon and small intestine tissues were harvested after fasting.

In addition, for the subcutaneous tumor model, C57BL/6 mice (4–6 weeks old) were randomly divided into three groups. Drinking water was supplemented with antibiotics cocktail (0.2 g/L ampicillin, neomycin, and metronidazole, and 0.1 g/L vancomycin) for the whole duration of the experiment to deplete the gut microbiota as previously reported [27]. Dietary treatment with either RS or control diet was continuously given to mice through the entire experiment. 5 × 106 MC38 cells were injected subcutaneously into the right flank of C57BL/6 mice (100 μl per mouse). After 5 days of implantation, the tumor volume was monitored every 2 days and calculated as follows: Volume = 0.5 × L × W2, where L is the longest diameter and W is the shortest diameter. At the end of the time, mice were killed and the subcutaneous tumors were surgically excised for further analysis.

Ki67 staining

Colon tumor tissues were fixed in 4% buffered formalin immediately after dissection of mice. The fixed tissues were then dehydrated in ethanol, embedded with paraffin and sectioned at 5 μm. IHC staining analysis of Ki67 was performed as previously described [28]. Tissue microarrays were scanned with a digital slide scanner (Pannoramic MIDI, 3D HISTECH) after staining and processed with Pannoramic viewer software. The intensity of staining in cells was automatically calculated by Quant center software. H-score was acquired according to the formula: H-score = (percentage of weak intensity area × 1) + (percentage of moderate intensity area × 2) + (percentage of strong intensity area × 3).

16S rRNA sequencing of the microbial community

Genomic DNAs were extracted from mice colon tissues by QIAamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. 16S rRNA sequencing and bioinformatics analysis were performed by Majorbio BioPharm Technology Company in China. Raw fastq files were demultiplexed and quality filtered by Trimmomatic and merged by FLASH (Fast Length Adjustment of Short Reads to Improve Genome Assemblies). Samples were identified by barcodes and primers, then sequences were dereplicated and discarded. OTUs were clustered with 97% similarity cutoff using UPARSE (version 7.1 http://drive5.com/uparse/). We used Shannon index to measure species richness (α-diversity) of the gut microbiome. β-Diversity of the gut microbiome was calculated using the UniFrac distance between samples and visualized using the principal coordinate analysis (PCoA) (http://www.majorbio.com/).

Targeted metabolomics of SCFAs

2.5 g metaphosphoric acid was dissolved in 100 ml deionized water, and then 0.6464 g crotonic acid was added to prepare a crotonic acid/metaphosphoric acid solution. The fermentation broth and crotonic acid/metaphosphoric acid solution were evenly mixed and stored at – 40 °C for 24 h. After acidification, samples were centrifuged to separate the supernatants from the precipitate (13,000 r/min, 4 °C) and filtered via a 0.22 μm hydrophilic micron membrane. Then, 150 μl filtered solution was used for gas chromatography. The column temperature heating conditions were: column temperature: 80 °C for 1 min, increased to 190 °C (10 °C per minute), and maintained for 0.5 min; then increased to 240 °C (40 °C per minute) and maintained for 5 min; FID detector: 240 °C; gasification chamber: 240 °C; carrier gas: nitrogen flow rate 20 ml per minute, hydrogen flow rate 40 ml per minute, air flow rate 400 ml per minute. The obtained data were recorded.

Targeted detection of glycolysis products

Targeted detection of glycolysis products and further analysis were performed by Metware Technology Company. Briefly, colon tumor tissues stored in a – 80 °C refrigerator were thawed and smashed, and then mixed with 70% methanol/water. After different speeds of centrifugation, the supernatant was transferred for further LC–MS analysis. Next, the sample extracts were analyzed using an LC–ESI–MS/MS system (UPLC, ExionLC AD, https://sciex.com.cn/; MS, QTRAP® 6500 + System, https://sciex.com/) and AB 6500 + QTRAP® LC–MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in both positive and negative ion modes and controlled by Analyst 1.6 software (AB Sciex). For differential metabolites selected, significantly regulated metabolites between groups were determined by VIP ≥ 1 and absolute Log2FC (fold change) ≥ 1.0. The VIP values were extracted from OPLS-DA result, which also contains score plots and permutation plots, and generated using R package MetaboAnalystR. The data was log transformed (log2) and mean centered before OPLS-DA.

Cell culture

Human CRC cell lines (LoVo, HCT116), human normal colonic epithelial cell line NCM460, and mouse CRC cell line MC38 were purchased from American Type Culture Collection (ATCC). HCT116 cultured in Maccoy 5A (Genom, China), LoVo cultured in F-12 K (Genom, China), and NCM460 and MC38 cultured in DMEM medium (GIBCO, China) were supplemented with 10% FBS (Sijiqing, China) at 37 °C in a humidified 5% CO2 atmosphere.

CCK-8 assay

For CCK-8 assay, HCT116, LoVo, or NCM460 cells with the indicated treatment were seeded at 2 × 103 cells per well in 96-well plates, and added with PBS, HFCS, or butyrate plus HFCS in the culture medium. Then CCK-8 assay was performed by the Cell Counting Kit-8 assay kit (Meilunbio, China) according to the manufacturer’s instructions. Briefly, after removing the medium, cells were incubated with CCK-8 for 2 h and the absorbance was determined at 450 nm by a Bio-Rad microplate reader (BioTek, Winooski, VT, USA) at 0, 24, 48, 72, and 96 h, respectively. Each test was repeated five times.

Lactic acid detection

PBS, HFCS, or butyrate plus HFCS was added to the culture medium of LoVo or HCT116 cells with the indicated treatment separately. After 48 h of treatment, the culture medium was collected for lactic acid detection by lactic acid assay kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions.

RNA extraction and quantitative RT-PCR

Total RNAs were extracted from CRC cell lines with the indicated treatments using TRIzol reagent (Invitrogen, USA), and cDNAs were reversed by HiScript® II Q RT SuperMix for qPCR (Vazyme, China) according to the manufacturer’s instructions. Quantitative RT-PCR analysis was performed in triplicate in a ROCHE LightCycler480 System (Rotor gene 6000 Software, Sydney, Australia). Each reaction was tested in triplicate in 10 μl reaction system containing ChamQ Universal SYBR qPCR Master Mix (Vazyme, China), primers and template cDNAs. The relative mRNA expression was calculated using the comparative cycle method (2−ΔΔCt). β-Actin served as the internal reference genes. The primers are listed in Supplementary Table 1.

Western blot analysis

Cell extracts were collected using RIPA buffer (Beyotime, China) containing protein phosphatase inhibitor (Solarbio, China) on ice and quantified by BCA Protein Assay Kit (Fdbio science, China). Total proteins were loaded on to 10% polyacrylamide-SDS gels, followed by the transfer of electrophoresed proteins onto the PVDF membranes. The membranes were blocked in 5% BSA (Fdbio science, China) for 2 h and then incubated with primary and secondary antibodies. Subsequently, the signals were detected using an ECL Kit (Fdbio science, China) by ChemiDoc Touch Imaging System (Bio-Rad, USA). The following antibodies were used: HK2 (22029-1-AP, Proteintech), β-actin (AC026, ABclonal), HRP goat anti-rabbit IgG (H + L) (AS014, ABclonal), HRP goat anti-mouse IgG (H + L) (AS003, ABclonal).

Cell transfection

The siRNAs were transfected into cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific, USA) in opti-MEM (Genom, China) according to the manufacturer’s instructions. The following sequences of siRNAs were used:

siNC: 5ʹ-UUCUCCGAACGUGUCACGUTT-3ʹ;

siHK2-1: 5ʹ-CCAAAGACAUCUCAGACAUUG-3ʹ;

siHK2-2: 5ʹ-CCAGAAGACAUUAGAGCAUCU-3ʹ.

Plasmid construction and transfection

Plasmid expressing HK2 (NM_000189.5:455-3208) was constructed by GenePharma (Shanghai, China). Transient plasmid transfection was carried out using FuGENE HD transfection reagent (Promega, USA) according to the manufacturer’s instructions.

Statistical analysis

Statistical analysis was performed using the GraphPad Prism 7.0 software. Data were analyzed with Student’s t test, Wilcoxon rank-sum test, or linear regression as shown in figure legends. P value less than 0.05 was considered statistically significant.