From January 2015 to March 2015, 134 continuous CRC specimens and 20 matching adjacent non-tumor specimens that obtained from CRC patients at the Zhongshan Hospital, Fudan University were collected. None of the patients received any preoperative chemotherapy or radiation. All tissue samples were pathologically diagnosed. The study was authorized by the Ethic Committee of the Zhongshan Hospital, Fudan University. Written informed consent was obtained from every patient.
Collection of TCGA public data and identification of significantly differential expression miRNA
We collected gene expression profiles of 551 COADREAD samples from TCGA cohort (http://gdac.broadinstitute.org/). Data including mRNA expression level and miRNA expression level. All the data have been standardized. For miRNAs differential analysis, we calculated the mean expression level of these miRNAs to divide them into high and low expression groups.
The R package DESeq2 (version 1.22.2) were used to calculate the differential expressed t statistics for microarray and RNA sequencing data. We used univariate Cox proportional hazards model to examine the associations between gene expression and overall survival. MiRNAs with P values less than 0.05 were considered to be statistically significant and included in consensus survival analysis.
Total 134 CRC specimens were stained with the CDK6 antibody (ab124821, Abcam) and HK2 antibody (2867T, Cell Signaling Technology). The 20 matched adjacent non-tumor specimens were stained with CDK6 antibody (ab124821, Abcam). IHC scores were conducted according to the ratio and intensity of positive-staining areas. The staining areas were scored as follow: 0-25%, score 1; 25-50%, score 2; 50-75%, score 3; and beyond 75%, score 4. The signal intensity was scored on a scale of 0-3: 0-negative, 1-weak, 2-moderate and 3-strong. Two experienced pathologists scored the IHC staining independently and the final IHC score was the score average from both pathologists.
Cell lines and culture
Human CRC cell lines (HCT116 (RRID: CVCL_0291) and SW480 (RRID: CVCL_0546)) and normal human colon epithelial cell line NCM460 (RRID: CVCL_0460) were obtained from National Collection of Authenticated Cell Cultures (Shanghai, China). All human cell lines have been authenticated using STR profiling within the last three years and all experiments were performed with mycoplasma-free cells. These cell lines were maintained in Dulbecco’s Modified Eagle Medium (Logan Utah, HyClone, USA) with 10% fetal bovine serum (FBS; Gibco), 1% penicillin (10 U/mL) and 1% streptomycin (10 μg/mL) in an incubator with the environment of 37 °C and 5% CO2.
Quantitative real-time polymerase chain reaction (qRT-PCR)
RNA was isolated with Trizol reagent. The purity and the concentration of different RNA samples were measured using NanoDrop ND-1000 (Thermo Scientific, USA). Reverse transcription reaction was performed using miRcute miRNA cDNA synthesis Kit (kr211, Tiangen, China) for miRNAs and SuperScript IV VILO cDNA synthesis kit (11756050, Thermo Scientific, USA) for mRNAs. Quantitative RT-PCR (qRT-PCR) was performed using SYBR® Premix Ex Taq™ (Takara) containing mRQ 3’ Primer on an ABI 7500 platform (Applied Biosystems, Carlsbad, CA, USA). The relative quantities of miR-500a-3p in cells was normalized to U6. Beta actin was used as an internal control for mRNA detection. The primer sequences for miRNA and mRNA detection are listed in supplementary Table 3.
Lentivirus and miRNA transfection
The lentivirus overexpressing human miR-500a-3p and CDK6 was purchased from Genomeditech (Shanghai, China). miR-500a-3p mimics were synthesized by Genomeditech (Shanghai, China). The sequences of miR-500a-3p mimic were 5’-AUGCACCUGGGCAAGGAUUCUG-3’. The miRNA oligonucleotides were transfected using Lipofectamine® RNAiMAX™ (50 nmol/L; Invitrogen, CA, USA).
Cell viability assay
After 72 h lentivirus infection or 24 h miRNA transfection, HCT-116 (2,500/well) and SW480 (2000/well) cells were planted into 96-well culture plates. Cell viability was measured at 24, 48, 72 and 96h post seeding, by using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions.
HCT-116 (1500/well) and SW480 (2000/well) cells were seeded in six-well plates after 72 h lentivirus infection. The culture medium was changed every 3 days. After incubating for 2 weeks, colonies were fixed with 4% paraformaldehyde for 15 min and stained with 5% Giemsa for 20 min. The colonies containing at least 50 cells were scored.
Cell cycle analysis
Seventy-two hours after infection, cells were harvested, washed with phosphate-buffered saline (PBS), and fixed in 70% ethanol at 4°C overnight. After fixation, cells were washed with PBS before suspension in RNase A/propidium iodide solutions (100 mg/mL RNase A and 5 μg/mL propidium iodide). Cells were incubated at room temperature for an hour. Stained cells were analyzed by a FACScan flow cytometer (BD Biosciences, Mountain View, CA, USA).
Cell apoptosis analysis
Cell apoptosis was assessed by annexin V/propidium iodide (BD Biosciences, San Jose, CA, USA). Cells were harvested, washed with PBS, and resuspended in 1× binding buffer at a concentration of 1×106 cells/mL. A total of 100 μL solution was transferred to a new tube and added with 5 μL of APC Annexin V and 5 μL of propidium iodide. Cells were incubated at room temperature for 15 min in the dark and then analyzed by a FACScan flow cytometer.
Cell invasion assay
The cell invasion assay was performed in 24-well transwell chambers pre-coated with Matrigel (Corning, NY, USA). HCT-116 cells (105/well) in 200 μL serum-free medium were seeded into the upper chamber, and 600 μL complete medium (with 10% FBS) was filled into the lower chamber. Cells on the inner membrane were removed with a cotton swab after 24 h. The outer membrane was fixed in 4% formaldehyde (in PBS) and stained with 0.5% crystal violet. Cell numbers were counted and averaged in five random fields at a magnification of 100×.
Xenograft tumor assay
BALB/c nude mice (6 weeks old) were purchased from Shanghai SLAC Laboratory animal co. ltd (shanghai, China) and randomly divided into 2 groups (n = 7/6). SW480 (3×106) or HCT116 (4×106) cells that stably transfected with miR-NC or miR-500a-3p were injected at the back region of BALB/c mice subcutaneously. The volume of tumors was measured twice a week using a vernier caliper with the method of volume = (length × width2)/2. The mice were killed after inoculation for 30 d, and the tumors were weighed. Tumor tissues were subjected to
measure the expression of ki67. The procedures in this study were permitted by the Animal
Research Committee of the Zhongshan Hospital, Fudan University.
Luciferase reporter assay
The putative miR-500a-3p binding sequence and the matching mutant binding sequence in the 3’untranslated region (3’UTR) of CDK6 were amplified and inserted to pGL3 luciferase reporter vector (Genomeditech, shanghai, China). SW480 cells were transfected with miR-NC or miR-500a-3p and the above constructed reporter plasmids. After transfection for 48 h, the luciferase activities in different groups were determined by the dual-luciferase reporter assay system (Promega).
Glucose, lactate and ATP measurement
SW480 and HCT116 cells were cultured with FBS-free medium and culture medium was collected after 24h. Glucose Uptake Colorimetric Assay kit (Biovision, Milpitas, California, USA), Lactate Assay Kit (Biovision) and ATP Colorimetric Assay kit (Biovision) were utilized to detect the glucose consumption, the lactate production and ATP in CRC cells according to the manufacturer’s instructions. The glucose, lactate and ATP levels were normalized to total cell protein.
Extracellular acidification rate (ECAR) was detected using a Seahorse XF96 analyzer (Seahorse Biosciences, USA). SW480 cells (105/well) were seeded in a 96-well XF96 microplate (Seahorse Biosciences, USA). Before experiments, cell culture medium was replaced and cells were then incubated with assay medium for 1 h at 37 °C in a CO2-free incubator. ECAR was detected using sequential injection of 10mM glucose, 2mM oligomycin (Sigma-Aldrich) and 50mM 2-deoxyglucose (2-DG, Sigma-Aldrich). Each cycle of measurement involved mixing (3 min), waiting (2 min), and measuring (3 min) cycles.
The targeted metabolomics analyses were performed using an HPLC system (Agilent 1290, Agilent Technologies) and mass spectrometer (Agilent 5500, Agilent Technologies). A 10-cm dish of cultured tumor cells were collected, adding 1 ml acetonitrile / methanol water (v, 2:2:1) and storing at -80℃ after quick freezing in liquid nitrogen. Sample preparation processes were performed in accordance with the above method of parallel preparation of QC samples. MRM transitions representing the metabolites were simultaneously monitored, and the positive/negative polarity switching was used. Data analyses were performed as instructions by Shanghai Applied Protein Technology.
Whole protein extracts were lysed by radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific) according to the manufacturer’s protocol. At that time, 30 μg proteins were run on a 10% sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) gel at 100 V for 2 h and transferred to a polyvinylidene fluoride membrane at 80 V for 2 h. Membranes were blocked with 5% bovine serum albumin (BSA) in Tris buffered saline with Tween-20 (TBST) at room temperature for 1 h. They were then incubated overnight at 4°C with one of the following primary antibodies: anti-CDK6 (ab124821, Abcam) (1:1000), anti-GLUT1 (ab115730, Abcam) (1:1,000) and anti-HK2 (2867T, Cell Signaling Technology) (1:1,000) and anti-beta actin (1:1,000) from Santa Cruz Biotechnology (Dallas, Texas, USA). After washing with TBST, membranes were incubated with secondary antibodies for 1 h at room temperature and signals were developed using an enhanced chemiluminescence kit (Pierce, Waltham, MA, USA).
SPSS (version 22.0, SPSS Inc.) or GraphPad Prism software (version 7.0, USA) were used to analyze the data and generate the graphs. The differences between the miR-500a-3p expression and the clinical characteristics of CRC patients were analyzed using χ2 test. Survival curves were generated using the Kaplan-Meier method and log-rank tests. Univariate and multivariate Cox regression analyses were conducted to identify the independent factors. The correlation among the expression of miR-500a-3p, CDK6 and HK2 was analyzed using Spearman’s correlation test. Student’s t-test or the Mann–Whitney U test were used to calculate the P value between two groups. P < 0.05 was identified as statistically significant. R software 3.5.1 was applied in this study for the statistical analyses. All miRNAs expression data were normalized.