2.1. Sample collection
A total of 121 pairs of HCC tissues and para-carcinoma tissues, 33 control liver tissues (liver tissues from patients with liver hemangioma) and 31 cirrhosis tissues were collected from patients who underwent surgery from May 2015 to May 2019, in the Chengdu First People's Hospital and Sichuan Cancer Hospital. Tissue samples were immediately soaked in RNAlater@ RNA Stabilization Solution (Invitrogen, CA, USA) after surgery, then stored at −80 °C until use. All patients were confirmed by pathological diagnosis of liver biopsy. Clinical features of patients with HCC are shown in Table 1. All the healthy controls were with no hepatitis, hepatic diseases and abnormal biochemical results of liver function tests. Patients who had an additional history of solid organ tumors, underwent radiotherapy, chemotherapy or targeted therapy were excluded.
2.2 RNA extraction and qRT-PCR analysis
Samples of patient tissues and cell lines were lysed using TRIzol Reagent (Invitrogen, CA, USA). Total RNA was isolated and reverse transcribed into cDNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Japan). QRT-PCR was performed using iTaq Universal SYBR Green supermix (Bio-Rad, Hercules, CA, USA) on a CFX96 system (Bio-Rad, CA, USA). The relative mRNA expression was analyzed by the comparative threshold cycle method followed by normalization to GAPDH expression. Relative mRNA expression was calculated by the 2-ΔΔ Ct method. The primer sequences used in this paper were shown in Supplementary table1.
2.3 Cell culture and transfection
Human HCC cell lines (Huh7, LM9 and 97L) were cultured in DMEM (Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA), 100 μg/ml streptomycin and 100 U/ml penicillin. The cells were maintained at 37 °C in a humidified incubator with 5% (v/v) CO2. HCC cell lines were authenticated by short tandem repeat (STR) profiling with ABI 3130 sequencing system (ABI, USA). The open reading frame (OFR) of circC3 was amplified from 97L cells and subcloned into pcDNA 3.1. Transfection was performed with FuGENE® HD Transfection Reagent (Promega, USA) according to the manufacture's protocol.
2.4 Cell proliferation assay
Cell proliferation assay was performed using CCK8 (Dojindo, Japan) according to the manufacturer's instructions. At 24 h after transfection, the cells were seeded into 96-well plates with 1000 cells per well and cultured for 0, 24, 48, 72 or 96 h, respectively. About 10 μl CCK8 reagent was added to the wells and incubated for 2 h at 37 °C. The absorbance was measured at 450 nm. Each experiment was replicated three times.
2.5 Apoptosis detection
At 24 h after transfection, the level of apoptosis was detected with Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology, Shanghai, China) on a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA) and Tunel assay (Roche, Cat. No: 11 684 817 910) according to the manufacturer's protocol. Fluorescent images were captured using a Zeiss fluorescence microscope at magnification of ×200. Apoptosis rate was calculated according to the formulas: number of TUNEL positive cells/number total cells ×%.
2.6 Western blotting
HCC cells were lysed by RIPA lysis buffer (Wu han Goodbio technology CO., LTD Hubei, China). The concentration of total protein was determined by BCA protein concentration assay kit (Wu han Goodbio technology CO., LTD Hubei, China). Then sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred, soaked and incubated with anti-GLUT1 and anti-LDHA (1:1000; Wuhan proteintech group Hubei, China), anti-GAPHD (1:3000; Wuhan Goodbio technology CO., LTD Hubei, China) antibodies at 4 ° C overnight. The proteins were detected using a luminescence method (ECL Western Blotting detection with IgG antibodies respectively 1:5000 dilution). The GAPDH was used as the loading control. Quantification of protein bands was carried by using Quality One software.
2.7 Statistical analysis
Data were presented as the mean±standard deviation (SD). Differences within multiple groups were tested by analysis of One-way analysis of variance (ANOVA) followed by the Bonferroni correction for post hoc t test. Differences between two groups were tested by Student’s t test. The steps of ROC curve analysis were as follows: (1) Calculate the predicted probabilities. The Binary Logistic process of SPSS was used for logistic regression, then obtained the logistic regression equation, and a new variable containing the prediction probability of each individual was generated in the working data table of SPSS. (2) ROC curve analysis. The ROC curve was performed using GraphPad, the prediction probability was used as the test variable. The AUC, 95% CI, P value, sensitivity and specificity could obtain from the ROC curve. Statistical significance was set at P < 0.05.