Expression data of mRNA and clinical information of CRC patients were obtained from the public TCGA and GEO databases. Besides, the level of HTSeq-FPKM was converted into Transcripts Per Million (TPM) for the further analysis. Boxplots were used to evaluate ENO3 expression in normal and CRC tissues from TCGA and GEO datasets. Receiver operating characteristic curves (ROC) and the area under the curve (AUC) were performed to assess the diagnostic value of ENO3 in CRC patients. Kaplan–Meier method was used to explore the association between ENO3 expression level and OS or PFS of CRC patients.
Cell or tissue total proteins were extracted with RIPA lysis buffer (Beyotime, China). Total proteins were loaded on 10% SDS-PAGE gel and electrophoretically transferred to PVDF membranes. After blocked with 5% BSA solutions, membranes were incubated using then ENO3 antibody (1:1000, Proteintech, USA) at 4℃ overnight. β-actin (1:2000, Abways, China) was used as an endogenous control. Afterwards, horseradish peroxidase (HRP)-conjugated secondary antibodies were incubated for 2h at room temperature. Specific immune complexes were exposed with ECL Western Blotting Substrate (NCM Biotech, China), and detected using ChemiDoc™ Touch Imaging System (Bio-Rad, USA) and GE Amersham Imager 600 (USA).
RNA isolation and qRT-PCR
Tissues and cells total RNAs were extracted with TRIzol Reagent (Invitrogen, China) and cDNA was reversed by using a Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed with SYBR Green Kit (Vazyme, China) using Roche LightCyclerTM 480 according to the manufacturer’s protocols. The data was analyzed by 2-ΔΔCT method. β-actin was used as the endogenous control. Sequence of all primers used was provided in Supplementary Table 1.
Cell culture and transfection
Human CRC cell lines (LoVo, HCT116, RKO, HT29) and HEK293T were purchased from American Type Culture Collection (ATCC, USA), and routinely maintained at 37°C in a 5% CO2 incubator with recommended culture medium.
The ENO3 over-expression plasmid and shRNA were designed and synthesized by Tsingke Biotechnology Co., Ltd. Detail sequence of shRNAs was presented in Supplementary Table 2. Lenti-viruses were generated by co-transfecting the pLKO.1- copGFP-puro vectors, del8.9 (Beyotime, China) and vesicular stomatitis virus-G (Beyotime, China) into HEK293T cells. The viral supernatants were harvested and enriched via centrifugation. PolyJet (Thermo Scientific, USA) was used for plasmid transfection. The transfection efficiency of viral infection was examined by western blot and qRT-PCR.
Cell proliferation assay
For cell viability assays, 3000 cells per well were seeded in 96-well plates. At 6h of culture, as well as 24-, 48-, 72- and 96h after that, cells were measured using CCK-8 assay reagents (Meilun Biotechnology, China) and the cell growth curves were then obtained.
For plate colony formation assay, 800 cells per well were plated into 6-well plates and maintained for 2 weeks at 37℃ in 5% CO2. At the end point, colonies were stained with 0.05% crystal violet (Sigma, USA) for 30 half an hour after fixation by 4% PFA for 20 minutes. The result was determined by counting the stained colonies number and detecting the 570-nm absorbance of the stained colonies eluent.
Cell migration and wound healing assay
Cell migration assay was conducted using transwell chambers (Corning, USA). Briefly, 20 × 104 cells in 100 μl of serum-free medium were plated into the upper chamber with 700 μl of 10% FBS medium added to the lower chamber. After 24 hours, cells migrated to other side of the membrane were fixed by 4% PFA and stained with 0.05% crystal violet, and finally photographed under the optical microscope.
For wound-healing assay, same amount of CRC cells was seeded into 6-well plates. Then a pipette tip to draw gaps on the cell monolayers. After this, we used serum-free DMEM to allow cells to migrate. The cells that migrated into these gaps were observed at 0-, 12- and 24h using an inverted microscope (Leica, DMI1, China).
Immunohistochemical (IHC) analysis
Tissue samples were fixed in 4% PFA and embedded in paraffin. Antigen retrieval was enforced in citrate buffer (pH 6.5) for 20 min. Tissue slices were incubated overnight at 4℃ with anti-ENO3 antibody (1:1000, Proteintech, USA), followed by secondary antibody incubation using Immunohistochemical secondary antibody kit (ZSGB-Bio, China). Antigen-antibody complexes were then determined with a DAB kit (ZSGB-Bio, China). Slices were lightly counterstained with hematoxylin to visualize nuclei. The data analysis was performed as previously described .
Collected cell samples were added Trizol reagent and then handed over to a biotechnology company (LC-Bio Technology, China) for the subsequent transcriptome sequencing. The sequencing results were obtained as the FPKM (fragment per kilobase of exons per million reads). The DEGs were selected with p<0.05 and fold change>2 by R software (package DESeq2), and then the differentially expressed genes was further used for GO enrichment analysis.
Detection of Lactate production and ATP
Lactate of culture medium was detected by the Lactate Assay kit (Solarbio, China). ATP of cell were assessed by a luciferase-based ATP assay kit (Beyotime, China). Lactate and ATP level were normalized to protein concentration, which was determined using BCA Kit (FDbio Science, China).
Graphpad Prism 8.0 software was used for statistical analysis. Data from experiments performed in triplicates were expressed as the mean ± SD. Two-tailed t-test or two-way ANOVA was used to evaluate the statistical differences of groups. p<0.05 was considered to be statistically significant.