This study used Huh 7.5.1 cells and OR-6 cells. Huh 7.5.1 cells were provided by Dr. Francis Chisari and the OR-6 cell line obtained from Dr Kato,33 harbored full-length genotype 1b HCV RNA and coexpressed Renilla luciferase. Huh 7.5.1 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). OR-6 cells were grown in DMEM supplemented with 10% FBS and 500 μg/ml of G418 (Promega, Madison, WI).
After the identification of highly significant target genes, the primers for Q-PCR were designed using Primer Express 3.0 (Applied Biosystems, USA). Total cellular and viral RNA was isolated post-infection using RNeasy Mini columns (QIAGEN), reverse transcribed by random priming with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Foster City, CA), and then quantitated by real-time PCR using the DyNAmo HS SYBR Green qPCR Kit (Finnzyme; Espoo, Finland). The primers for the genes are as follows: PHOSPHO2-KLHL23 forward 5'- GGAATAGTTGGGATGTGTTGCTT-3' and reverse 5'-GAGTGTGGAATAGATGGTCTCACAGA-3', TSNAX-DISC1 forward 5’-GGAAGATGCAGTTGAGAATGATGA-3’ and reverse 5’-TCTTGTTCCAGGTCTTCTAATCTTTG-3’, TRIM39 forward 5'- ACCACCACACCTTTTACCCC-3' and reverse 5'- TATGAGAGCGGTCTGTGACAT-3', RPP21 forward 5’- CTACACTGAGAGGACCATTGCG-3’ and reverse 5’- TGTTAGGCAGGTCTGTACGGT-3’, using GAPDH as normalization control.
Cell viability assay
Cell viability assay was performed as previously described.34 Briefly, hepatoma cells were seeded into 96-well white plates overnight prior to treatment. The cells were lysed to determine cell viability with CellTiter Glo (Promega Corporation, G7571), which is a bioluminescent assay for measuring cellular ATP level, an indicator of metabolically active cells.
Three-dimensional cell culture was performed as previously described, with minor modifications.34 NanoCulture system was used for 3D cell culture (organogenix). First, 100 ul of medium was added to each well. The plate was then centrifuged at 2000 g for 5 min to remove microbubbles, followed by incubation for 15-30 min at incubator. Each well was seeded with 50 ul of medium containing 5 × 103 cells. (If needed, transfection was conducted at this time point.) The plate was then kept on the bench at room temperature for 10-15 min until cells adhered to the bottom film. To avoid excessive evaporation of medium, phosphate buffered saline (PBS) was added to the gutter surrounding the plate. At 24 h, 50 μl of medium (including drug) was added, and colony formation was observed after 4-6 days. Pictures of the colonies formed were taken and cell viability was measured with 3D CellTiter Glo (Promega Corporation)
In vitro wound assays were performed using IBIDI Culture-Inserts (35 mm with high culture-insert coating). First, 1.5 × 105 transfected cells in 140 μl DMEM were seeded in the culture insert for 16 h. The confluent monolayers of cells were washed twice with PBS to remove residual cell debris. Subsequently, culture inserts were removed and wound healing as indication of cancer migration took 6-9 h. Migration distance was measured in triplicate.
Library preparation for transcriptome sequencing
A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using TruSeq stranded mRNAlibrary prep Kit(cat# RS-122-2101, Illumina, SanDiego, CA,USA)following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNAusing poly-T oligo-attached magnetic beads and fragmented by heating. The first strand cDNA was synthesized using SuperScript II Reverse Transcriptase. PCR amplification was performed using 2X PCR Master Mix. After adenylation of 3’ ends of DNA fragments, adaptors were ligated and the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). The final library quality was assessed on the Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips. The libraries were sequenced on an Illumina NextSeq 500 platform and 75bp paired-end reads were generated.
The bioinformatics analysis pipeline is followed from sequencing step. Low quality bases and sequencing adapters in raw data which generated from Illumina sequencer was removed using program Trimmomatic, then the reads were aligned to reference transcript sequences using Bowtie2. Then, gene expression level was calculated by RSEM with Maximum likelihood abundance estimates using the Expectation Maximization (EM) algorithm for its statistical model. Differentially expressed genes (DEGs) are calculated by EBSeq, and the functional analysis, include GO and KEGG analysis, were using clusterProfiler.
Establishment of gene knockdown cells (siRNA and transfection)
Chemically synthesized siRNA oligonucleotides targeting candidate genes were purchased from Qiagen-Xeragon (Germantown, MD) and designed using an siRNA design algorithm applying stringent homology analysis (https://www1.qiagen.com/Products/GeneSilencing/)CustomSiRna/CustomSiRna Order.aspx). Indicated siRNAs were transfected into cells using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA). The negative control siRNA was obtained from QIAGEN. All siRNAs used for gene knockdown were SMART pools. Gene knockdown was confirmed by Q-PCR.
Data were reported as mean ± standard error of the mean (SEM) from three independent experiments. Results of cell proliferation assay and migration assay were analyzed using non-parametric two-tailed Student’s t-test. Gene expression in the HCC patient dataset obtained from TCGA database (https://cancergenome.nih.gov/) was analyzed using analysis of variance (ANOVA) with Tukey’s post-hoc test. Different levels of candidate genes between the tumor and corresponding non-tumor part were evaluated using the Wilcoxon signed-rank test. The cutoff for each gene expression level was determined according to receiver operating characteristic (ROC) curve and was divided into high and low groups. Cumulative survival curves were estimated using the Kaplan-Meier method. Univariate and multivariate Cox proportional hazards models for overall and disease-free survival were used for crude and adjusted hazard ratios, respectively. A p value less than 0.05 (2-sided) was considered statistically significant.