Patients and samples
A total of 392 paraffin sections of CRC tissues and adjacent paired non-cancerous tissues were collected to design a tissue array chip from the Department of Gastrointestinal Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University. All patients with CRC underwent surgery at the Department of Gastrointestinal Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University between January 2014 and January 2016. The study was approved by the Research Ethics Committee of Renji Hospital and carried out in accordance with ethical standards as formulated in the Helsinki Declaration. Informed consents were provided by all patients.
Cell culture
All cell lines used in this study were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin.
Small-interfering RNA (siRNA) transfection
The siRNAs for GLUT1 were purchased from GenePharma (Shanghai GenePharma Co., Ltd., Shanghai, China). Sequences are shown in Table S1 and experimental method was performed as previously described(16).
Lentivirus transfection
Full-length human BRIX1 cDNA was transfected into CRC cell lines using a lentivirus to generate Lentivirus-BRIX1 (BRIX1 overexpression). Lentivirus-NC was used as the negative control (Vector). In addition, one short-hairpin RNA (shRNA) sequence against BRIX1 was transfected into CRC cell lines to generate shRNA-BRIX1, while shNC was used as the negative control. Sequences are shown in Table S1.
RNA isolation and RT-qPCR
Trizol was used to extract RNA and total RNA was reverse transcribed to cDNA by PrimeScriptTM (TAKARA). We used 18S RNA as an internal control. The sequences of the primers used are shown in Table S1. The relative expression of the target gene was calculated by the −△△Ct method.
Western blot analysis
Total protein was extracted using radioimmunoprecipitation assay (RIPA) buffer supplemented with 1% protease inhibitors and phosphatase inhibitors. Protein concentration was measured with the bicinchoninic acid (BCA) assay. Western blot analysis was performed as previously described(16). BRIX1 (sc-373680, Santa Cruz), GLUT1 (ab115730, Abcam), β-catenin (ab32572, Abcam), Ki67 (Proteintech Group, Inc.) primary antibodies were used. Horseradish peroxidase (HRP)-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) and HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) were obtained from Proteintech Group, Inc (Jackson).
Immunohistochemistry (IHC)
All tissues were paraffin-embedded and cut into 4-m thick sections. All sections were dewaxed with xylene and hydrated with alcohol. Sodium citrate was used for antigen retrieval and 0.3% hydrogen peroxide (H2O2) was used to block endogenous peroxidase. After blocking non-specific sites with bovine serum albumin (BSA), all the sections were incubated with an appropriate primary antibody and secondary antibody. We used 3,3-diaminobenzidine (DAB) kit for visualization and hematoxylin was used to stain nuclei. All the sections were dehydrated with alcohol and sealed with neutral resin. IHC staining score was calculated based on pixel intensity; staining was scored as follows as per the staining intensity: no staining, 1; weak staining, 2; moderate staining, 3; and strong staining, 4.
Seahorse analyses
The Seahorse XF96 Flux Analyzer (Seahorse Bioscience, Agilent) was used to carry out the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in the CRC cell lines. Briefly, all CRC cells used in this paper, including HCT-116, SW480 and HT29 cells, were seeded into an XF96-well plate. The media were replaced with assay media 1 hr before the assay. For ECAR assay (Seahorse Cat.#103020-100), 10 mM glucose, 1 μM oligomycin, and 50 mM 2-deoxyglucose (2-DG) were added to the wells. For the OCR test (Seahorse Cat.#103015-100), 1 μM oligomycin, 1 μM FCCP, 0.5 μM rotenone, and 0.5 μM actinycin A were added to the wells at a special time point. Both measurements were normalized by total protein quantitation. The above experiments were performed in triplicate and repeated twice.
Glucose and lactate measurement
The Amplex® Red Glucose/Glucose Oxidase Assay Kit (Invitrogen, Cat.#A22189) was used to measure the glucose uptake. Glucose consumption was calculated by the net content of the original glucose concentration deduced the measured glucose concentration in the medium. The Lactate Assay Kit (BioVision, Cat.#ABIN411683) was used to measure lactate production. Total proteins were used for the normalization of the results obtained above. These experiments were performed in triplicate and repeated twice.
CCK8 assay
1×103 cells were seeded into 96-well plates. Adding detection reagent (10μl CCK8 reagent+ 90μl DMEM) to each plate and incubating at 37℃for 1h. Measure the absorbance at 450nm with a microplate reader. Each experiment was carried out independently in triplicate.
Polysome fractionation and RNA isolation
cellular extracts were centrifuged at 10,000 rpm for 5 min at 4°C and the supernatant was carefully isolated and loaded onto 10-50% sucrose gradients containing 0.1 mg/ml heparin and 2mM DTT and centrifuged at 37,000 rpm for 2.5 h at 4°C (SW40 rotor). The sucrose gradient was subsequently fractionated with a gradient fractionation system (ISCO) connected to a UV detector to monitoring absorbance at 252nm. RNA was isolated from polysomal fractions using the PureLink RNA Mini Kit (Invitrogen).
Animal model
For the generation of an orthotopic model of CRC, all nude mice were anesthetized with 0.5% pentobarbital. After opening the abdominal cavity, 1x106 SW480luc cells/null mouse were injected into the ileocecum. Tumor growth was monitored by animal CT imaging. After 4 weeks, the mice were sacrificed and the tumor tissues were excised and weighed. All tissues were fixed with 4% paraformaldehyde. All animal experiments were approved by the Research Ethics Committee of Renji Hospital and adhered to the local or national requirements for the care and use of laboratory animals.
Statistical analysis
Measurement data are presented as the mean±standard deviation (SD). SPSS 20.0 (Chicago, IL, USA) and GraphPad Prism 7 software were used to conduct the statistical analyses. The correlation of BRIX1 expression with categorical clinical variables in patients with CRC was evaluated using chi-square analysis or Student’s t-test. Measurement data, such as age and tumor size, were evaluated using Student’s t-test, while categorical variables and ranked data, such as gender, T stage, lymph node invasion, and distant metastasis, were analyzed using the chi-square test. Spearman’s rank correlation was used for the analysis of two-way ordered categorical data. Survival curves were generated using the Kaplan–Meier method and analyzed by the log-rank test. Statistical significance was accepted at p < 0.05.