Pairs of HCC tissues and peritumoral normal tissues were gathered from 48 hepatocellular carcinoma patients in Shandong Provincial Hospital affiliated to Shandong University during January 2016 to December 2018. Specimens was immediately frozen in liquid nitrogen and then stored at -80℃ after surgery. We obtained the written informed consent and the Ethics Committees of Shandong Provincial Hospital affiliated to Shandong University approved for this study.
We purchased HCC cells HuH-7 and a normal hepatocyte cell L-02 from American Type Culture Collection (ATCC; Rockville, MD, USA). All the cells were incubated in DMEM medium (Invitrogen, Carlsbad, CA, USA) with 10% FBS (Sigma-Aldrich, Louis, MO, USA) at 37℃ in a humidified chamber with 5 % CO2.
The specific plasmids of miR-577 mimic or miR-577 inhibitor as well as their negative control were designed and synthetized from Gene-Pharma (Shanghai, China). The transfection was carried out using HuH-7 cells that were incubated in 6-well plate. The Lipofectamine 2000 Reagent (Invitrogen, USA) diluted using Opti-MEM/Reduced serum medium (Thermo Scientific, Shanghai, China) was used to perform the transfection. Geneticin (G418; Thermo Scientific, Shanghai, China) was used to select the stable transfection cells, while we harvest the transient transfection cells after transfected 48 h.
Quantitative real-time PCR
TRIzol Reagent (Invitrogen) and miRNeasy Mini Kit (Qiagen, Hilden, Germany) were employed to extract total mRNAs and miRNAs from tissues or cells. Omniscript Reverse Transcription Kit (Qiagen) and TaqMan miRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) were used to synthesize the first cDNA chain; followed QuantiTect SYBR Green PCR Kit (Qiagen) and miRNA-specific TaqMan MiRNA Assay Kit (Applied Biosystems) were conducted to carry out the qPCR in a Quantitect SYBR green PCR system (Qiagen). The relative levels of mRNA and miRNA were derived using 2-ΔΔCt method, the GAPDH and U6 small nuclear RNA utilized as normalization. The primers used for RT-qPCR were as follows: miR-577 forward 5'-TGCGGTAGATAAAATATTGG-3', reverse 5'-GTGCAGGGTCCGAGGT-3'; U6 forward 5'-GCTTCGGCAGCACATATACTAAAAT-3', reverse 5'-CGCTTCACGAATTTGCGTGTCAT-3'; CXCL5 forward 5'-AGCTGCGTTGCGTTTGTTTAC-3', reverse 5'-TGGCGAACACTTGCAGATTAC; GAPDH forward 5'-AAGGTGAAGGTCGGAGTCAA-3', reverse 5'-AATGAAGGGGTCATTGATGG-3'.
Western blot analysis
The total proteins were lysed by RIPA Lysis Buffer (Sigma, USA) containing 10% PMSF (Sigma). The SDS-PAGE was applied to separate the protein and then the blots were electro-transferred to PVDF membranes (Millipore, USA). After being blocked by 5% fat-free milk at ream temperature for 1 h, the membranes were incubated with primary antibodies. The primary antibodies were against CXCL5 (1:1000; Abcam, Cambridge, USA), E-cadherin (1:1000; Abcam), N-cadherin (1:1000; Abcam), Vimentin (1:1000; Abcam), c-Myc (1:1000, Abcam ), TRAF6 (1:1000, Abcam). Next, the blots were incubated by secondary anti-rabbit HRP-conjugated antibody (Cell Signaling). The protein signals were captured using Enhanced Chemiluminescence Detection Kit (ECL, Pharmacia Biotech, Arlington, USA).
The HuH-7 cells were plated into 96-well plates and cultivated for 24h, 48h, 72h and 96h. We added 20 μl of MTT (5 mg/ml, Sigma) into each well and followed cultured for 6 h. Next, we discarded the supernatant and added 100 μl of DMSO (Sigma) to each well. After agitating for 10 min, the absorbance at a wavelength of 570 nm was evaluated using an ELISA reader (Bio-Rad, Hercules, CA, USA).
The transwell insert (8 μm membrane, Corning, Cambridge, MA) were placed in 24-well plate to evaluate the cell invasive ability. The HuH-7 cells were suspended by FBS free RPMI-1640 medium and we added 200 μl in the upper chamber, whereas the lower chamber was filled with 500 μl medium containing 15% FBS, which acted as inducer. After the cells were incubated for 24 h at 37℃, the non-invasive cells, which still on the upper surface, were removed by cotton swabs. We fixed and then stained the invasive cells using 4% paraformaldehyde and 10% crystal violet respectively; and followed counted the cells under a microscope (Olympus Corporation, Tokyo, Japan).
miRNA targets prediction and dual-luciferase reporter assay
TargetScan was conducted to peform the prediction of target genes of miR-577 and we discovered that CXCL5 was one of potential target gene. We mutated the binding sequences from UUUAUCU to AAAUAGA to confirm miR-577 binding to CXCL5 in HCC cells. Followed, we inserted the wild type and the mutational 3'-UTR of CXCL5 into the dual luciferase reporter vectors, which were designated as WT or MUT. We utilized Lipofectamine 2000 Reagent (Invitrogen, USA) to co-transfect miR-577 mimic and WT or MUT vector into HuH-7 cells. Finally, the luciferase activity was measured using dual luciferase reporter assay system (Promega, USA).
All the statistical analysis was performed to use SPSS 16.0 software (IBM, Armonk, NY, USA) and the data were presented as mean ± SD. Student’s t test was performed to compare the differences between two groups, besides, one-way ANOVA was utilized to compare the differences between three or more groups. The association between miR-577 expression and the overall survival for HCC patients were assessed by Kaplan-Meier curve and log-rank test. P< 0.05 was considered to be statistical significant.