Participants and liver tissue samples
A total of 25 patients with HBV-associated liver cancer (Liver cancer group, N = 25, 13 males and 12 females, 49.3 ± 1.1 years old) and 25 patients with HBV-associated cirrhosis (Cirrhosis group, N = 25, 12 males and 13 females, 48.6 ± 1.3 years old) were screened from our hospital between September 2015 and August 2018. A total of 25 healthy participants were enrolled as the Control group (Control group, N = 25, 14 males and 11 females, 48.8 ± 1.2 years old). All participants were diagnosed as HBV-associated cirrhosis or liver cancer at the first time, and no other diseases and tumor metastasis were observed. No significant differences were observed on the age and gender among these three groups. Liver tissues were collected from participants undergoing cancer resection or outpatient liver biopsy. This study was approved by the ethics committee of our hospital, and informed consents were obtained from all participants.
Human normal liver cell line HL-7702 cells, liver cancer cell line HepG2 cells, and HBV-associated liver cancer cell line HepG2.2.15 cells were purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). HL-7702 and HepG2 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS), and HepG2.2.15 cells were cultured in RPMI1640 medium containing 10% FBS and 380 μg/mL G418 (Sigma, Dorset, UK). All cells were maintained in a 5% CO2 incubator at 37° C and 95% humidity.
MiRNA-210 inhibitor, miRNA-210 inhibitor negative control (miRNA-210-NC), siRNA1-EGR3 (si1-EGR3), siRNA2-EGR3 (si2-EGR3), and siRNA negative control (si-NC) were purchased from Shanghai GenePharma Co., Ltd (Shanghai, China). HepG2 and HepG2.2.15 cells were digested with 0.25% trypsin and seeded into 24-well plate at a density of 1.3 × 105 cells/well. When reaching 90% confluence, cells were transfected with the above agents using Lipfectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Cells without transfection were considered as the Blank group. After 48 h of transfection, cells were used for further assays.
After the transfection, cells were cultured for 7 days and collected every day. The collected cells were seeded into 96-well plates at a density of 1 × 104 cells/well, and then incubated with MTT solution (20 µL, 5 mg/mL) for 4 h at 37°C. Dimethyl sulfoxide (DMSO, 150 μL) was used to dissolve the MTT formazan crystals. The absorbance at 590 nm (A590) was measured by a microplate reader (Thermo Fisher Scientific).
Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining
After the transfection, cells were cultured for 24 h, and seeded into 24-well plates at a density of 1 × 105 cells/well. After stained with Annexin V- FITC and PI for 15 min under darkness, the apoptosis rate was detected by a Flow Cytometry (BD, San Jose, CA, USA).
Quantitative Real-Time PCR (qRT-PCR)
Total RNA was extracted from specific tissues and cells using TRIZOL reagent (Thermo Fisher Scientific). RNA was reverse transcribed into cDNA on a Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA) using a RevertAidTM H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). qRT-PCR was performed on a Rotor-Gene 3000 Real-time PCR instrument (Corbett Research, Sydney, Australia). The PCR program of miRNA-210 was 95°C for 10 min, 40 cycles at 95°C for 15 s and 60°C for 60 s. The PCR program of EGR3 was 95°C for 10 min, 35 cycles at 94°C for 30 s, 53°C for 45 s and 72°C for 45 s. U6 was used as an internal reference for miRNA-210, and GAPDH was used as an internal reference for EGR3. The primers sequences were shown as follows: miRNA-210-F: 5'-GTGCAGGGTCCGAGGT-3', miRNA-210-R: 5'-TATCTGTGCGTGTGACAGCGGCT-3'; U6-F: 5'-CTCGCTTCGGCAGCAC-3', U6-R: 5'-AACGCTTCACGAATTTGCG-3'; EGR3-F: 5'-TACAATCAGATGGCTACAGAGAAT-3', EGR3-R: 5'-TTCCCAAGTAGGTCACGGTC-3'; GAPDH-F: 5'-TCGGAGTCAACGGATTTGGTC-3', GAPDH-R: 5'-GCCATGGGTGGAATCATATTGG-3'. Data was calculated in accordance with the 2-∆∆Ct method.
Total proteins were extracted from specific tissues and cells using RIPA lysis buffer, and then quantified using a Bradford protein assay kit (Beyotime, Shanghai, China). The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membrane. After blocked with 5% skim milk for 2 h, the membrane was incubated with Mouse anti-human EGR3 monoclonal antibody (1:1000, Abcam, UK) for 12 h at 4°C. The membrane was then washed with TBST for 3 times, and subsequently incubated with horseradish peroxidase-labeled goat anti-mouse IgG (l:2000, Zhongshan Jinqiao, Beijing, China) for 1 h at 25°C. The protein bands were visualized using an ECL kit, and the gray value was quantified by a gel imaging analysis system. GAPDH was used as an internal reference for EGR3.
The targets of miRNA-210 were predicted using TargetScan 7.1 (http://www.targetscan.org/vert_71/). A total of 4046 transcripts containing 5853 sites were predicted. A target gene EGR3 (ENST00000317216.2) was selected due to its anti-tumor role on liver cancer 16.
Dual luciferase reporter gene (DLR) assay
The regulatory relationship between EGR3 and miRNA-210 was identified by DLR assay. The sequences of EGR3 wild type (wild) and EGR3 mutation (mutation) were synthesized according to the predicted binding site. The fragments were then inserted to the luciferase reporter vector pGL3-promoter (GenePharma). HepG2 cells were co-transfected with the plasmids carrying wild/mutation and miRNA-210 mimic/miRNA-210 mimic negative control (NC) (GenePharma), and grouped as mimic + wild, mimic + mutation, NC + wild, and NC + mutation group. The fluorescence was detected by a Microplate Reader (Thermo Fisher Scientific). The relative fluorescence unit was calculated as the ratio of Fireny Luciferase and Renilla Luciferase.
All experiments were performed in triplicate. Statistical analysis was performed by SPSS version 17.0 (SPSS Inc., Chicago, IL). Data were expressed as mean ± standard deviation (SD). Differences among multi-groups were analyzed by one-way ANOVA followed by Tukey's test. A P-value of less than 0.05 was considered significantly different.