Normal liver cell line LO2 (BNCC100012) and HCC cell lines HepG2 (BNCC102171) and Bel-7402 (BNCC338237) were got from BNCC cell library. Cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (Gibco, Rockville, MD, USA) in an incubator (Thermo Fisher Scientific, Waltham, USA) at 37 °C and 5% CO2. Cells in logarithmic growth phase were chosen for experiments.
Cell groups and administration
HCC cell lines were taken for experiment. The experiment was classified to three groups: (1) blank control; (2) miR-29a mimic group: transfected miR-29a mimic into cells; (3) mimic-NC: add miR-29a mimic negative control reagent. miR-29a mimic (mir10000802-1-5) and miR-29a mimic control (mir01101-1-5) were purchased from Ribobio( Guangzhou, China).
After 0.25% trypsin digestion and passage, 2 × 105 cells at the logarithmic stage were put in 6-well plates. After 24 h, the cell growth was watched by microscope. When the cell density reached 30–50%, the HCC cells were transfected. Add 5µL miR-29a mimic and miR-29a mimic control into 250µL serum-free medium as liquid A. Add 5µL LipofectamineTM2000 into 250µL serum-free medium as liquid B. Liquid A and B were mixed at 25 °C for 15 min. After 24 h, replaced the mixture with a fresh medium containing 10% FBS. miR-29a expression was evaluated by RT-qPCR.
Total RNA isolation kit (A27828, MagMAX™ mirVana™ Total RNA Isolation Kit, Thermo Fisher Scientific, Waltham, USA) was used to extract total RNA from human normal liver cells, HCC cells and transfected HCC cells according to the instructions. RNAs reversely transcribed into cDNAs in reaction system using miR-29a primer (mirq0000802-2-1, Ribobio, Guangzhou, China). qPCR was subsequently performed using SYBR Green Qpcr Master Mix (MedChemExpress) and 2 µl cDNA as a template. The PCR reaction was performed according to the following conditions: every cycle was 10 min at 95 °C, 15 s at 95 °C and 1 min at 60 °C and 40 cycles were performed. With U6 and GAPDH genes (mqp-0202, Ribobio, Guangzhou, China) as internal reference, the relative expressions of miR-29a and VEGF were conducted by 2−ΔΔCt. Primers as follows:
miR-29a: F: 5'- TGTCTCGAGCAAGGGACGCCGTGGAAGA − 3', R: 5'- TGTGTCGACCCGCACACCGATATGGTT − 3'; U6: F: 5'-GCTTCGGCAGCACATATACTAAAAT-3', R: 5'-CGCTTCACGAATTTGCGTGTCAT-3'; VEGF: F: 5'-CAACATCACCATGCAGATTATGC-3', R: 5'-CCCACAGGGATTTTCTTGTCTT-3'; GAPDH: F: 5'-GAAGGTGAAGGTCGGAGTC-3', R:5'-GAAGATGGTGATGGGATTTC-3'.
Cells at the logarithmic stage were digested with trypsin. The concentration was 1 × 105/ml, and then put in 96-well plates with 100 µL per well and 4 parallels. Then the cells were cultured at 37 °C with 5% CO2. 10 µL CCK-8 solution was added into each well at 24 h, 48 h, 72 h and 96 h respectively. After 4 h, the absorbance value (OD) was detected at 450 nm.
Clone Formation Assay
The cells in each group at logarithmic growth stage were digested and prepared to single cell suspension. About 2 mL cell suspension was respectively taken from each group and put in a 6-well culture plate with 500 cells per well. The cells were dispersed evenly by shaking the culture plate and cultured at 37 °C and 5% CO2. When there were clones visible to the naked eye, the medium was discarded. Cells were washed by phosphate buffered solution (PBS) for 2 times and fixed by methanol at 25 °C for 15 min. Each well was added with 1 mL Giemsa Stain solution(G1015, Solarbio, Beijing, China) and stained for 30 min. After washed with ultra-pure water, the record was imaged by a camera. The clone formation number was directly counted by naked eye.
The HCC cells were trypsinized and were collected by centrifugation at 999 x g for 5 min at 4˚C after transfection 48 h. Cells were subsequently thoroughly washed twice with pre-cooled sterile PBS at 4˚C. We chose single staining with PI for cell cycle dection. After washing, the cells were fixed overnight with 70% ethanol and precooled at 4 °C. After PBS washing, 100 µL RnaseA (Solarbio, Shanghai, China) was added to resuspend at 37 °C for 30 min. Then 400 µL propidium iodide (PI, Solarbio, Shanghai, China) was added and avoided light for 30 min. The cell cycle was detected by flow cytometer (Beckman Coulter, Brea, CA, USA). We chose AnnexinV-FITC apoptosis assay kit (BD Biosicences, San Diego, US) for apoptosis detection. After PBS washing, 300 µL 1 × binding buffer was added to the cells for suspension. Then 5 µL AnnexinV-TIFC was added. After mixed and incubated in dark for 15 min, 5 µL PI was added and mixed. CellQuest software (Version, 5.1, BD Biosicences, San Diego, USA) was used to analyze the data. The Q1 represented necrotic cells, the Q2 presented late apopotic cells, the Q3 represented living cells, the Q4 represented early apoptotic cells. The apoptosis rate = Q2 + Q4.
Total proteins were extracted with a total protein extraction kit (BC3640-50T, Solarbio, Beijing, China) according to the instruction. Protein concentration was measured by the BCA Protein Assay Kit (23225, Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, Waltham, USA). Samples (40 µg) from each group were isolated by 10% SDS-PAGE electrophoresis (Mini-Protean-3, Bio-Rad, Hercules, CA, USA) and then transferred to PVDF membrane (Millipore, Massachusetts, USA). 5% skim milk was used to seal for 1 h. The membranes were incubated overnight at 4˚C with the following primary antibodies diluted in 5% bovine serum albumin (BSA): rabbit anti-human Bcl-2 antibody (1:1000, ab32124, Abcam), rabbit anti-human Bax antibody (1:1000, ab32503, Abcam), rabbit anti-human CDK4 antibody (1:1000, ab108357, Abcam), rabbit anti-human Cyclin D1 antibody (1:1000, ab134175, Abcam), rabbit anti-human Rb antibody (1:1000, ab181616, Abcam), rabbit anti-human VEGF antibody (1:1000,AV202, Beyotime), rabbit anti-human p53 (1:1000,ab32389༌Abcam),rabbit anti-human MCL1 (1: 1000༌ab32087༌Abcam) and rabbit anti-human beta-actin antibody (1:1000, ab8227, Abcam). After overnight at 4 °C, TBST(TBS, 1 ml/L tween-20) washed the membrane three times with 10 min each time. Then the membrane was incubated with goat anti-rabbit IgG HRP (1:2000, ab6721, Abcam) at room temperature for 2 h. Again, TBST washed the membrane three times with 10 min each time. ECL chemiluminescence was used for detection. ImageJ software (version 6;National Institutes of Health) was used for grayscale scanning and quantification.
Dual Luciferase assay
Wild-type (WT) and mutant (MUT) 3 ‘UTRs of VEGF were amplified in pGL3/ luciferase vector (Promega, Madison, WI, USA) and cloned into the downstream of luciferase gene. According to the instruction, luciferase activity of cells was detected after transfection with the dual luciferase reporting system (Promega) 48 h.
To further verify the effect of miR-29a on HCC cells via targeting VEGF, HCC cells were randomly divided into 6 groups: normal control group (Control), miR-29a mimic group (miR-29 mimic), VEGF siRNA group (VEGF siRNA), VEGF siRNA control group (si-NC), miR-29a mimic + VEGF mimic-NC group (miR-29a mimic + mi-NC), miR-29a mimic + VEGF mimic group (miR-29a mimic + VEGF mimic). These reagents were purchased from Ribobio( Guangzhou, China). Similarly, the above indexes were analyzed repeatedly.
SPSS 19.0 (IBM Corp.) was used to analyze the statistical data. The data was expressed as mean ± SD. Statistical differences between two groups were analyzed by t test. Data analysis about multiple groups was performed by ANOVA followed by LSD and Turkey test for multiple comparisons.. P < 0.05 was considered statistically significant.