Patients and specimens
This study was carried out in accordance to the principles of the Declaration of Helsinki and approved by the Medical Ethics Committee in Ningbo University Medical College. All clinical HCC tissues and matched adjacent normal tissues were obtained from Yinzhou Hospital between 2015 and 2017. Written informed consent was obtained from all the participants prior to enrollment. All patients recruited in this study were diagnosed with HCC based on histopathological evaluation, and were not received any chemotherapy or radiotherapy before surgical operation. All collected tissues were immediately stored at liquid nitrogen until further analysis. TNM staging of HCC samples were performed according to 7th edition AJCC/UICC TNM staging systems. Clinicopathological characteristics analyses of all the specimens were provided in Tables 1.
Cell lines and culture
The human HCC cell lines (7721 and Huh7) were obtained from Chinese Academy of Sciences, the normal hepatocyte cell line (L02) was presented by Department of General Surgery of Changhai Hospital. Cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco) containing 10% fetal bovine serum (Gibco) in a humidified atmosphere of 5% CO2 at 37 °C. The nutrient-deficient induction was established by Earle’s Balanced Salt Solution (EBSS. Gibco).
RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from tissue and cell samples using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. Reverse transcription was performed using PrimeScript RT Reagent Kit (TaKaRa). The diluted cDNAs were amplified using SYBR Premix Ex Taq (TaKaRa). Three independent biological replicates were set at least for the cell experiments. β-actin was used as a loading control. The sequences of the primers were listed in supplemental Table S1.
Western blot analysis
Total cell lysates were subjected to 10% SDS-PAGE and the proteins were transferred to nitrocellulose filter membranes, followed by blocking for 1 hour in 5% non-fat dry milk. The membranes were incubated with primary antibodies (LC3, 1:1000 dilutions, ab51520 from Abcam. BECN1, 1:1000 dilutions, ab62557 from Abcam. HIF-1α, 1:500 dilutions, BM4083 from Boster. P-mTOR and mTOR, 1:1000 dilutions, ab32028 and ab2732 from Abcam. β-actin, 1:2000 dilutions, BM0627 from Boster) at 4 °C overnight, and then with secondary antibodies (HRP-conjugated anti-mouse and anti-rabbit secondary antibodies, 1:5000 dilutions, BA1051 and BM2006 from Boster) at room temperature for 1 hours. Proteins were visualized by ECL Plus Western Blotting Substrate (Thermo Scientific) on ChemiDoc MP system (Bio-Rad). β-actin was used as a gel loading control.
Small interfering RNA (siRNA) and transient transfection
SiRNA targeting HIF1A-AS1 (5’-GUCAAUUGGUUGAUCACCCG-3’, si-HIF1A-AS1) and scrambled control (5’-UUCUCCGAACGUGUCACGUTT-3’, si-NC) were designed and synthesized by Shanghai GenePharma company. When the confluence of cells reached to 70%–80%, siRNAs were transfected at a final concentration of 100 nmol/L with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The non-off target effects were confirmed by an additional siRNA targeted GAPDH (supplemental Figure S1). Knockdown efficiency of the siRNA was determined by qRT-PCR.
Cell viability analysis
The HCC cells vitality were identified by Cell Counting Kit‐8 assay (CCK-8. Beyotime). Briefly, HCC cells were seeded in a 96-well plate (6 wells per group) and incubated overnight, followed by siRNA transfection and nutrient-deficient induction for 24 h. After adding 10 μL CCK‐8 solution, the relative growth vitality was detected on a microplate reader (BioTek) according to the manual.
Cell apoptosis analysis
The cell apoptosis was determined using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining (BD Biosciences). After treatment for 48 h, cells were harvested and resuspended in 200 μL Annexin-binding buffer. Then, the cells were incubated with 10 μL Annexin V-FITC and 5 μL PI for 30 min in the dark. The stained cells were examined by a FACScan flow cytometer (BD Biosciences).
The statistical analysis was carried out by SPSS 22.0 software. The qualitative data was analyzed by Chi-Square test or Fisher’s exact test when necessary. The quantitative data were expressed as the means ± standard deviations, and analyzed by t-test for 2 groups, and one-way ANOVA test for multiple groups. The survival curves were analyzed by Kaplan-Meier test. A P value less than 0.05 was considered statistically significant.