Cell lines and culture
HB cell lines including HepG2, HuH-7, HuH-1, HuH-6 and normal epithelial cell line (THLE-3) were selected for this investigation. HepG2 and THLE-3 cell lines were both obtained from ATCC (Manassas, VA, USA) while HuH-7, HuH-1, HuH-6 cell lines were purchased from Huatuo Biological Technology Co., Ltd. (Shenzhen, China). Among them, HepG2 and THLE-3 cell lines were cultured in Eagle’s Minimum Essential Medium (EMEM) and BEGM respectively. HuH-7 and HuH-6 cells were incubated in Dulbecco’s Modified Eagle’s medium (DMEM) while HuH-1 cell line was cultured in RPMI-1640 Medium. All mediums contained 10% fetal bovine serum (FBS) and incubated at 37 °C with 5% CO2.
Transfection of cells
The knockdown of MIR205HG in HB cells was implemented by transfection with sh-MIR205HG#1/2/3 plasmids acquired from Ribobio (Guangzhou, China). MiR-514a-5p mimics/inhibitor, miR-205-5p mimics/inhibitor and their respective negative controls were commercially obtained from GenePharma (Shanghai, China). Furthermore, pcDNA3.1 vectors were obtained from Invitrogen to overexpress MAPK9. Lipofectamine 2000 was used to transfect HB cells as per the instruction of manufacturer.
RNA extraction and quantitative real-time PCR (RT-qPCR)
The total RNA was subjected to extraction from cells using Trizol reagent. PrimeScript™ RT Master Mix (TaKaRa, Shiga, Japan) was used to obtain cDNA through reverse transcribing two mircrograms of total RNA. SYBR Premix Ex TaqTM II (Takara Bio, Tokyo, Japan) was applied to perform RT-qPCR. GAPDH and U6 were utilized as internal references. The 2−ΔΔCt method was used to evaluate gene expression. The experiment was implemented in triplicate.
Colony formation assay
In brief, cultured cells were inoculated into 6-well plates. After 14 days of incubation, cells were subjected to fixation using ethanol, followed by the staining with crystal violet. Finally, the stained cell colonies were observed and counted. The experiment was independently conducted in triplicate.
5-ethynyl-20-deoxyuridine (EdU) staining assay
Cells at logarithmic growth stage were taken and seeded in 96-well plates with 4 × 103 ~ 1 × 105 cells per well. Subsequently, the cells were cultured to the normal growth stage. A total of 50 μM EdU culture medium was prepared via the dilution of EdU solution (reagent A) by 1:1000. The cells were subjected to fixation, followed by the staining using Apollo. The assay was independently conducted in triplicate.
Wound healing assay
A total of 3 × 103 cells were seeded into 6-well plates and cultured in serum-free medium for 24 h at 37 °C. When cells reached 80% confluence, a straight scratch wound was made. Then cells were cultured for another 24 h, and scratches were monitored and photographed at 0 and 24 h. An inverted microscope (DMi1, Leica, Wetzlar, Germany) at 100 × magnification was used to monitor the wound healing. The assay was independently conducted in triplicate.
Transwell assay
The transfected HB cells were harvested and put into the upper chamber of Transwell inserts. Afterwards, 10% FBS was added to the lower chamber. The chambers coated with Matrigel (BD Biosciences, San Diego, CA, USA) were used for invasion assay, while without Matrigel for migration assay. After 24 h, migrated or invaded cells were finally visualized using optical microscope (Olympus) after being stained by crystal violet. The assay was independently conducted in triplicate.
Subcellular fractionation
Cytoplasmic and nuclear RNA were isolated with the employment of PARIS™ Kit (Ambion, Austin, TX, USA) in line with the manufacturer’s protocols. Cells were washed in PBS. Afterwards, cell samples were treated with cell fractionation buffer and disruption buffer. GAPDH and U6 were seen as the cytoplasmic control and the nuclear control respectively. The isolated RNA in the nucleus and the cytoplasm was measured by PCR analysis. The assay was independently conducted in triplicate.
Fluorescent in situ hybridization (FISH)
RNA FISH probe (Ribobio) targeting MIR205HG was designed and utilized as per the guide of provider. After hybridization using FISH probe, cells were counterstained by DAPI solution, followed by the detection using fluorescence microscope (Olympus). The experiment was independently performed in triplicate.
RNA pull down assay
RNA pull down assay was implemented by use of Pierce Magnetic RNA–Protein Pull-Down Kit (Thermo Fisher Scientific, Waltham, MA, USA). The cell extracts from HepG2 and HuH-6 cells were used for incubation with biotinylated probes of miR-514a-5p-WT/Mut or miR-205-5p-WT/Mut. After adding magnetic beads, relative RNA enrichment was assessed by RT-qPCR. The experiment was independently performed in triplicate.
RNA-binding protein immunoprecipitation (RIP)
Z-Magna RIP™ RNA-binding Protein Immunoprecipitation kit (Millipore Corporation, USA) was adopted to perform RIP assay. Anti-Ago2 (Abcam) antibody as well as anti-IgG (Abcam) antibody were used for immunoprecipitation with cell lysates. Finally, the RNA complexes were subjected to extraction for RT-qPCR analysis. The experimental procedure was independently carried out in triplicate.
Luciferase reporter assay
MIR205HG or MAPK9 sequence with the wild-type (Wt) and mutant type (Mut) miR-514a-5p binding sites was inserted into pmirGLO dual-luciferase vector to form pmirGLO-MIR205HG Wt/Mut and pmirGLO-MAPK9 3’-UTR-Wt/Mut respectively. Later, miR-514a-5p mimics or control mimics were co-transfected with the reporter construct into HB cells. Similarly, MIR205HG sequence of Wt and Mut of miR-205-5p was also utilized to pmirGLO-MIR205HG Wt/Mut and then was co-transfected with NC mimics and miR-205-5p mimics into HB cells. After 48 h, luciferase activity was measured via the employment of the Dual Luciferase Reporter Assay Kit (Promega, Madison, WI, USA). The assay was independently conducted in triplicate.
Western blot analysis
Separated protein samples were subjected to transference to PVDF membranes (Millipore, Bedford, MA, USA). After being blocked with skimmed milk, the membranes were subjected to incubation with the following primary antibodies against MAPK9, ERK, p-ERK, JNK, p-JNK, P38, p-P38, p-PI3K, PI3K, p-AKT, AKT and GAPDH. Subsequently, the blots were subjected to incubation with secondary antibody. At last, Chemiluminescence system (GE Healthcare, Chicago, USA) was applied for the quantification of proteins. The assay was independently conducted in triplicate.
In vivo study
Ten male BALB/c nude mice (4–5 weeks old) were purchased from the Beijing Chrles River Experimental Animal Technology Co., LTD. A total of 1 × 107 HepG2 cells stably transfected with sh-NC or sh-MIR205HG#1 were independently injected subcutaneously into the right back of each nude mouse. Seven days after the injection, tumor volume was recorded every 3 days. Twenty-eight days after the injection, all the mice were sacrificed for tumor weighing.
Statistical analysis
Experimental data were subjected to analysis by use of SPSS 22.0 statistical software package. All data were presented as mean ± standard deviation (SD). For differences comparison between two groups or more, Student’s t-test or ANOVA was adopted. All the experiments were independently implemented in triplicate. Differences were considered to be statistically significant when P < 0.05.
