Human HCC tissue samples
From August 2016 to September 2018, 40 paired HCC tissues as well as adjoining normal tissues were harvested from diagnosed patients in our hospital. All included patients had been diagnosed, and they didn’t receive treatment of radiotherapy or chemotherapy before. All included subjects inscribed an informed consent and the investigation got approval from Ethics Committee of Affiliated Hospital of Youjiang Medical University for Nationalities. We maintained these tissues in liquid nitrogen.
Five HCC cell lines (HB611, HHCC, H-97, HuH-7, Li-7), normal liver cell line (LO2) and human umbilical vein endothelial cells (HUVECs) were acquired from Shang Hai Ze Ye Biotechnology Co., Ltd. (Shang Hai, China) and American Type Culture Collection (Manassas, VA, USA). We verified the authenticity of each cell line with short tandem repeat analysis. We preserved all cell lines in Dulbecco's modified Eagle's medium (DMEM; Gibco, USA), with 1% penicillin-streptomycin and 10% fetal bovine sera (FBS; Gibco, USA), and stored them in a 5% CO2 humidified incubator at 37℃.
QRT-PCR (Quantitative reverse transcriptase-polymerase chain reaction)
For total RNA was extraction we used RNeasy Mini Kit (Qiagen, Germany) and for reversed transcription Quantitect Reverse Transcription Kit (Qiagen, Germany) was used, during which we refered to the manufacturer’s instructions. We performed amplification on a Lightcyle 480 platform (Roche, Germany) for each sample in triplicate. According to GAPDH levels, we normalized relative expression levels. Here followed primer sequences:
MiR-455-3p, 5’-ACACTCCAGCTGGGCACAUAUACGGGUAC-3’ (F),
Western blot analysis
We prepared Protein lysates with RIPA buffer (Thermo Scientific Inc., Waltham, MA, USA) which contains 1% protease inhibitor and performed western blotting in triplicate experiments. We subjected protein lysates to electrophoresis on a 4% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and electrotransferred proteins to polyvinylidene fluoride membranes (Millipore, USA). With 5% non-fat dry milk in TBS, we incubated membranes and probed them with anti-VEGFC and anti-VEGFR-2 (sc-1881, sc-6251 respectively; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-pAKT, anti-AKT and anti-β-actin (#4056, #9272, #4970, respectively; Cell Signaling Technology, USA) in TBST (0.1% Tween 20 in TBS). For detection of immunoreactive proteins, we used Horseradish peroxidase-conjugated anti-rabbit (or mouse) IgG (Cell Signaling Technology, USA) by chemiluminescence (Pierce® ECL kit)
Cell proliferation assay
Via the Cell Counting Kit-8 assay (CCK-8, Dojindo, Japan), we referred to the manufacturer’s protocol and performed cell proliferation assays. We seeded a sum of 1×103 cells per well and cultured them for 1, 2, 3, 4 and 5 days. We also added ten microlitres of the CCK-8 solution to each well, and incubated sample at 37℃ for 4 h. We read absorbance at 450 nm on a microplate reader (MultiSkan Spectrum).
For EDU assay, we used EDU labeled solution (KeyGen Biotech, Nanjing, China) for cell culture for 2 h. As recommended by the manufacturer we performed EdU staining, after 15 min of cell fixation which was in 4% paraformaldehyde.
For colony-formation assays, with trypsin–collagenase we treated transfected HCC cells for digesting them into a single cell suspension, and we seeded them at a rate of 400 cells/well into 12-well plates before culturing them at 37℃ for 14 days in a 5% CO2 incubator. We then use PBS to wash the cells twice and for 15 min stained them with 2% crystal violet, then at room temperature we dried the plate. We counted the clone number which were formed in the plate.
We performed all experiments thrice, consequently calculated the average of the results.
We seeded cells in six-well plates and cultured them. After they reached confluence, we scratched confluent monolayer cells and washed them with PBS for three times for clearing suspension cells and cell debris. We added fresh serum-free medium and under normal conditions allowed the cells to close the wound for 24h. At the same position of the wound, with a computer-assisted microscope (Nikon) we took photographs.
Using a Transwell chamber (8-µm pore size; Corning, USA), we performed cell invasion experiments. In the upper chamber we seeded HCC cells at a density of 105 cells per well, in which we also added DMEM containing 1% FBS. We precoated the upper chamber with Matrigel (Corning, USA, dilution ratio: 1:6) while in the lower chamber there was 600μL DMEM which contains 10% FBS. We fixed cells with 4% methanol, which were migrated or invaded to the lower surface of membrane, after incubation at 37℃ for 24 hours then we strained them with crystal violet. We selected 5 random microscopic fields and counted the number of cells in each field. Each experiment was carried out three times.
Generation of stable cell lines expressing miR-455-3p
From LO2 cell line genomic DNA, a DNA fragment which contains the miR-455-3p was amplified then into the pcDNA-copGFP vector (System Biosciences, USA) we cloned it. Name LV-miR-455-3p was given to the lentivirus vector expressing miR-455-3p. Via Lipofectamine2000 (Invitrogen, CA, USA) Lentiviral packaging plasmids and LV-miR-NC (as a negative control) were co-transfected following the manufacturer’s instruction in 293FT packaging cells. To infect HCC cells and HUVECs, we collected, filtered and used the lentivirus in the supernatant at the time forty-eight hours after transfection. Using qRT-PCR, we obtained stable clones and confirmed the expression of mature miR-455-3p after two weeks of antibiotic selection.
Tumor xenograft treatment model
We got approval of animal experimental procedures from the Institutional Animal Care and Use Committee of Affiliated Hospital of Youjiang Medical University for Nationalities. Of each BALB/c nude mouse (5–6 weeks of age, 20–22 g) we injected a suspension of HuH-7 cells (1×107) into the right flank subcutaneously. From the Laboratory Animal Center (Shanghai, China), we purchased all mice and maintained them under specific pathogen-free conditions in laminar flow cabinets. On day 7 post-implantation, when tumors formed, we randomly divided mice into two treatment groups: one is Lv-miR-455-3p group which was treated with lentivirus encoding miR-455-3p; another is Lv-NC which was treated with negative control lentivirus. After treatment with calipers two major axes, we measured tumor volumes every 7 days, and we calculated with the formula: V = 0.5×L (length) ×W2 (width). With all the experiments approval from Affiliated Hospital of Youjiang Medical University for Nationalities, we cared all animals with humane care referring to the criteria by the National Academy of Sciences and the National Institutes of Health, which outlines in the “Guide for the Care and Use of Laboratory Animals”.
Dual-luciferase reporter gene assay
We cloned the 3’-UTR regions of human VEGFC mRNA, which is downstream of the luciferase reporter gene. We first amplified it by PCR and then transferred it into the pGL3-basic vector (Promega, Madison, WI, USA). The construct as Wt (wild-type) and named it VEGFC Wt. Using PCR and the site-directed mutagenesis kit (Takara), which acted as template, the mutated 3’-UTR was generated. Into the luciferase reporter we inserted the mutated sequence and named it VEGFC Mut. Then the Wt or Mut of VEGFC, firefly luciferase reporter and mock LV-miR-455-3p vector or miR-NC were co-transfected into HHCC and HuH-7 cells. 48 h after, we harvested cells and treated them with the Dual Luciferase Assay kit l.
In vitro angiogenesis assays
By tube formation assay, we determined the in vitro angiogenic activity of HUVECs, for a significant angiogenic property of HUVECs is capillary tube formation on Matrigel. After transfection, we serum-starved HUVECs in endothelial basal medium (EBM; Clonetics, CA, USA) with 0.2% BSA for 24 h by incubation. We harvested HUVECs and in a 12-well plate seeded 8×104 cells, which is coated with Matrigel basement membrane matrix (BD Biosciences, USA), after serum starvation. With a computer-assisted microscope (Nikon), we observed tube formation after 8 h of incubation. We defined tube formation as a tube-like structure with a length four times its width. We selected 10 random microscopic fields and took images of tube morphology at 100 magnification per sample. Using LAS software (Leica), we measured the number of tubes.
Using SPSS statistical software (16.0 for Windows), we performed statistical analyses. We displayed experimental data as means ± standard deviation (SD). Through Student’s t-test and one-way analysis of variance (ANOVA), we analysed the differences between groups. We performed all statistical tests two sided. P < 0.05 were considered significant statistically. We performed all experiments three times or more.