Tissue samples
HCC tissues and paracancerous tissues were collected from 36 patients who received hepatectomy in Jiamusi Central Hospital from May 2012 to April 2014. All subjects did not received radiotherapy or chemotherapy before undergoing tumor resection. The research was endorsed by the Ethics Committee of Jiamusi Central Hospital and written informed consent was acquired from each subject.
Acquisition of GEO data
GEO database is a public database that provides access to gene expression datasets. CircRNA microarray datasets of HCC were searched using the following keywords “circRNA” and “liver cancer”, and GSE94508 and GSE97332 were obtained. The corresponding raw data were then downloaded and analyzed with GEO2R online analysis tool. The circRNAs with P < 0.05 and │log2(Fold Change)│ > 1 in each data set were regarded the differentially expressed circRNAs.
Cell culture and transfection
Human HCC cell lines (Huh7, Hep3B, HCCLM3, and MHCC97-L) and human normal liver epithelial cell line (THLE-3) were procured from American Type Culture Collection (ATCC, Manassas, VA, USA) and Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA) and 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Invitrogen, Carlsbad, CA, USA) at 37 °C with 5% CO2. Circ_0064288 overexpression plasmid pcDNA3.1-circ_0064288 (circ_0064288), empty vector (NC), circ_0064288 siRNA (si-circ_0064288#1 and si-circ_ circ_0064288#2), scramble siRNA (si-NC), miR-335-5p mimics/miR-335-5p inhibitors and negative controls (mimics NC/inhibitors NC) were procured from Invitrogen (Carlsbad, CA, USA). Transient transfection was performed with Lipofectamine® 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s direction .
Quantitative real-time polymerase chain reaction (qRT-PCR)
Homogenized HCC tissue and cells were collected, and total RNA was extracted using TRIzol Reagent (Yeasen Biotech, Shanghai, China). cDNA synthesis was implemented using the TaqMan MiRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) for miR-335-5p and employing a PrimeScript RT Master Mix Kit (TaKaRa, Dalian, China) for ROCK1 and circ_0064288, respectively. Then with cDNA as the template, qRT-PCR was implemented to determine the relative expressions of ROCK1 and circ_0064288 with the SYBR® Premix Ex Taq™ II kit (TaKaRa, Dalian, China). Meanwhile, a stem-loop primer SYBR Green qRT-PCR kit (Synbio Tech, Suzhou, China) was used for qRT-PCR to evaluate miR-335-5p expression. With U6 and GAPDH as internal references, the relative expressions of circ_0064288, miR-335-5p and ROCK1 were calculated by 2−ΔΔCt method. The primer sequences are listed in Table 1. The cytoplasm and nuclear fractions of the HCC cells were separated using a PARIS™ Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions, and next, the RNA in cytoplasm and nuclei was respectively extracted. Then, the expression of circ_0064288 in the cytoplasm and nucleus was measured by qRT-PCR. GAPDH and U6 were used as cytoplasm and nuclear controls, respectively.
CCK-8 experiment
Transfected HCC cells were planted in 96-well plates (2 × 103 cells/well) containing 100 μL of medium/well. 10 μL of CCK-8 solution (Beyotime, Shanghai, China) was supplemented to each well at the indicated time points (0 h, 24 h, 48 h, 72 h, 96 h). Subsequently, the cells were incubated at 37 °C for 2 h and the absorbance at 450 nm was measured with a microplate reader.
Transwell experiment
Transwell chambers (Corning Life Sciences, Corning, NY, USA) were utilized to detect the migration of HCC cells. The cells of each group in the logarithmic growth period were taken, trypsinized with 0.25% trypsin and re-suspended in serum-free medium, and the cell concentration was modulated to 1 × 105 cell/ml. In the top compartment, 200 μL of cell suspension was supplemented, and 500 μL of medium containing 10% FBS was added into the bottom compartment, and subsequently, the cells were cultured for 24 h. Next, methanol-fixed migrated cells were stained with 0.1% crystal violet. Five fields of view were randomly selected under a microscope, and the cells were counted, and the average value was calculated.
Wound healing experiment
HCC cells were planted in 6-well plates (1 × 104 cells/well) and cultured, and when the cells were spread all over the plates, the tips of 200 μL sterile pipette tubes were used to make scratches in the middle. The cells were then gently rinsed 3 times with serum-free medium, and then cultured with serum-free medium. Photographs were taken at 0 h and 24 h after scratch formation and the width of the scratch was recorded.
Western blot
Transfected HCC cells were lysed using RIPA lysis buffer (Beyotime, Shanghai, China), the supernatant of the lysate was harvested to collect the total protein. A BCA protein assay kit (Beyotime, Haimen, China) was applied to determine protein concentration. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane. 5% skim milk was adopted to block the membrane for 1 h at room temperature. Subsequently, anti-ROCK1 antibody (1:1000, ab134181, Cambridge, UK) and anti-GAPDH antibody (1:2000, ab8245, Cambridge, UK) were applied to incubate the PVDF membrane at 4 °C overnight. The next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000, Beyotime, Shanghai, China) for 50 min at room temperature. The protein bands were developed using an Amersham Imager 600 (GEHealthcare, Chicago, IL, USA) with an electrochemiluminescence kit (Biosharp, Hefei, China).
Luciferase reporter experiment
StarBase database was utilized to predict the binding sites between miR-335-5p and circ_0064288 / ROCK1 3’UTR, respectively, and the binding fragments of circ_0064288 and ROCK1 3’UTR were amplified by PCR. The amplification products were inserted into the PGL3-promoter plasmid vector (Promega, Madison, WI, USA) to construct the circ_0064288 and ROCK1 wild-type (WT) plasmids; the binding fragment was mutated using gene mutation technology to construct the circ_0064288 and ROCK1 mutant type (MUT) plasmid. The recombinant plasmids were co-transfected with miR-335-5p mimic (or mimic NC) respectively in HEK-293 T cells. After 48 h, the cells were collected. Fluorescence values were measured using a dual-luciferase reporter gene assay system (Promega, Madison, WI, USA).
Statistical analysis
All of the experiments were performed at least in triplicate, and at least repeated for 3 times. All data were expressed as “mean ± SD”, and GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, US) was employed for graphing and statistical analysis. Kolmogorov-Smirnov test was used to examine the normality and equal variance of the data. Student’s t-test was utilized to make the comparison between the two groups. One-way ANOVA and Dunnett’s post-hoc multiple comparisons were employed for comparisons among three or more groups. For data that were skewed distributed, comparisons between two groups were performed by Wilcoxon signed-rank test. Chi-square test was employed to elucidate the correlation between LINC00518 expression and pathological parameters. Kaplan-Meier survival curve and log-rank test were adopted to compare patient’s prognosis in different groups. Gene expression correlations were analyzed by Pearson correlation coefficient. P < 0.05signified statistical significance.
