1.1 Bioinformatic analysis
The miRNA (50 normal samples, 375 cancer samples) and mRNA (50 normal samples, 374 cancer samples) expression profiles of HCC were downloaded from The Cancer Genome Atlas (http://www.tcga.org/, TCGA) database. EdgeR package was employed to screendifferentially expressed genes (DEGs)and |logFC|>2, padj < 0.01were set as the threshold. Survival analysis of DEmiRNA was conducted based on the clinical information of samples to determine the target miRNA. Then miDIP(http://ophid.utoronto.ca/mirDIP/index.jsp#r) and starBase (http://starbase.sysu.edu.cn/) were utilized to predict the potential targets of miRNAand the Venn diagram was plotted with down-regulated DEmRNA to find the potential targeted genes.
1.2 Cell culture
Human HCC cell lines SMMC7721 (BNCC352197), HepG2 (BNCC338070) and human normal liver cell lines HL-7702 (BNCC351907) were purchased from BeNa Culture Collection (Shanghai, China).HL-7702 and SMMC7721 were cultured in 90% RPMI-1640 with 10% FBS.HepG2 was cultured in 90% DEEM-Hcontaining10%FBS.These cells were grown at 37 °C with 5% CO2in an incubator.
1.3 Cell transfection
MiR-18a-5p mimic (Mimic) and NC-mimic were obtained from RiboBio (Guangzhou, China), CPEB3 overexpression plasmid (oe-CPEB3) and corresponding negative control NC (oe-NC) were purchased from Genechem (Shanghai, China). Lipofectamine 2000 (Thermo Fisher Scientific, USA) was used to transfect miR-18a-5p mimic,NC-mimic,oe-CPEB3 and oe-NC vectors into SMMC7721 and HepG2 cell lines at a concentration of 50 nM according to the instructions.
1.4 qRT-PCR
Total RNA was extracted from cells using TRIzol reagent (Life Technologies, Grand Island, NY, USA) and the RNA concentration was measured by NanoDrop 2000 system (Thermo Fisher Scientific, Inc., Waltham, MA, USA).MiRNA was transcribed into cDNA using SYBR miRNA RT-PCR kit, and mRNA was transcribed into cDNA using PrimeScript RT Master Mix (Takara, Dalian, P.R. China). The expression levels of miR-18a-5p and CPEB3 were measured by Applied Biosystems 7500 real-time PCR instrument (Thermo Fisher Scientific, Inc.). The procedureswere as follows: denaturation at 95℃ for 5 min followed by 40 cycles of denaturation at 95℃ for 10 s, annealing at 60℃ for 20 s, extension at 72℃ for 20 s. GAPDH served as an internal reference for CPEB3 and U6 served as an internal reference for miR-18a-5p.The primer sequenceswere shown in Supplement table 1.
1.5 Western blotting
The cell lysates were extracted with RIPA lysate buffer (Beyotime, China) and the protein concentration was determined by BCA protein assay kit (Beyotime, China). After high-temperature denaturation, proteins were separated by SDS-PAGEand transferredonto PVDF membrane (PVDF, Millipore). The membrane wasblocked with 5% skim milk for 2 hours, and incubated with primary antibody overnight at 4℃. The primary antibodies were all rabbit polyclonal antibodies, including CPEB3 (NBP1-56919, 1:1000, Novus Biologicals, USA) and GAPDH (ab9485, 1:2500, Abcam, China).Thereafter, the membrane was incubated with secondary antibody IgG (ab6721; 1:5000; Abcam) for 2 hours at room temperature. Protein signals were detected using enhanced chemiluminescence (ECL) kits (GE Healthcare, Chicago, IL, USA).
1.6 MTT assay
MTT assay was used to detect cell proliferation. Cells were seeded in 96-well plates at a density of 5 × 103. After 1, 2, 3, 4 and 5 days of cells culture, the cell viability was determined using the MTT Cell Proliferation and Toxicity Assay Kit (Beyotime, China) according to the instructions. Cells were incubated at 37℃ for 4 h with 20 µl MTT solution, then incubated with 100 µl solution buffer at 37℃ overnights. The absorbance at 570 nm was read with a spectrophotometer (Molecular Devices, USA). All experiments were repeated three times.
1.7 Wound healing assay
Wound healing assay observed the migration ability of cells. Cells were seeded in a 6-well plate and when the cell coverage reached 80%, the 200 ul pipette tip was used to gently scrape the single layer through the center of the well.The wells were washed twice briefly in the medium to remove the isolated cells.Then fresh medium was added, the cells were regrown for 48 h, and the cell migration at 0 h and 48 h was observed and photographed microscopically.
1.8 Transwell
The cells were suspended in serum-free medium, and then seeded in the upper chamber of a 24-well plateTranswellinvasion chamber (8 µm pores, BD Biosciences, USA).After incubation at 37 ° C for 24 h, the cells on the upper side were cleared away with a cotton swab and the migratory cells on the lower side were stained with 0.2% crystal violet. The number of stained cells was then counted. Each experiment was repeated three times.
1.9 Dual luciferase assay
The 3’UTR of Wild-type (Wt) or mutant (Mut) CPEB3 was cloned into pmirGLO (Promega, WI, USA) vectors to construct CPEB3-Wt and CPEB3-Mut vectors, and then the plasmid vectors and miR-18a-5p mimic or NC-mimic were co-transfected into HepG2 cell lines with empty luciferase reporter vectors.After 24 hours of transfection, the activity of firefly luciferase and Renilla luciferase in the lysed cells was determined using the dual luciferase assay system (Promega, USA) according to the manufacturer's instructions.
1.10 Statistical analysis
Data were processed by SPSS 21 (IBM Corp., Armonk, NY, USA) and exhibited as Mean ± standard deviation (SD). Differences between two groups were compared using t-test, and differences among the groups were evaluated by one-way analysis of variance (ANOVA).P < 0.05 was considered statistically significant.