The HCV core region DNA (genotypes 3a and 1b) was synthesized according to the sequnce published in NCBI (GenBank, genotype 3a: AM263171.1, genotype 1b: EU155369.2) by Beijing AuGCT DNA-SYN Biotechnology Co., Ltd. (Beijing, China). The core region DNA was cloned into the PCMV5 vector. The HA-tagged HCV core region was cloned into the pcDNA3.1(+) vector using appropriate restriction sites. Direct sequencing of the expression vectors was carried out by AuGCT. Sequences were aligned in the NCBI BLAST server.

Human hepatoma Huh-7 cells were cultured at 37°C in a 5% CO₂ atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd., China). pcDNA3.1(+) plasmids containing the HA-tagged HCV genotype 3a or 1b core-encoding regions were transfected into Huh-7 cells with Lipofectamine 2000 (Invitrogen) as previously described (9). Briefly, cells were incubated with Opti-MEM medium (Invitrogen) under standard conditions for the first 6 h after transfection and then the medium was changed to DMEM containing 10% fetal calf serum without antibiotics. Twenty-four hours after transfection, the cells were cultured in the pressure selection medium containing 800 μg/ml G418 (Invitrogen). After 10–12 days, the neomycin-resistant cell colonies were isolated and maintained in the culture medium containing 400 μg/ml G418.

Total-RNA was purified from Huh-7 cells expressing the HCV core using the miRNeasy mini kit (Qiagen, Valencia, CA, USA). The quality of the total-RNA was confirmed by the Agilent 2100 Bioanalyzer and RNA LabChip^® kits (Agilent Technologies Inc., Santa Clara, CA, USA). Total-RNA (100 ng) was dephosphorylated with calf intestine alkaline phosphatase (GE Healthcare Europe GmbH), denatured with dimethyl sulfoxide, and labeled with pCp-Cy3 using T4 RNA ligase (GE Healthcare Europe GmbH). The labeled RNAs were hybridized to Agilent human miRNA microarrays for 20 h at 55°C with rotation. After hybridization and washing, the arrays were scanned with an Agilent micro-array scanner using high dynamic range settings as specified by the manufacturer. The Agilent Feature Extraction software was used to extract the data.

Quantitative stem-loop real time PCR (qRT-PCR) was carried out to detect and quantify miRNAs whose alteration was more than 3-fold between the HCV core 3a and 1b treatment groups according to the microarray profiling results. Primers for qRT-PCR were synthesized by AuGCT. All used primers were listed in Table I. A cDNA synthesis was carried out with the One Step PrimeScript^® miRNA cDNA Synthesis kit (Takara, Dalian, China) according to the manufacturer’s protocols. A quantitative PCR was performed using SYBR^® Premix Ex Taq™ II (Takara). The miRNA expression level was quantified using the Bio-Rad MyiQ™ Detection system (Bio-Rad, Hercules, CA, USA). The reactions were incubated in 96-well optical plates at 95°C for 10 sec followed by 40 cycles of 5 sec at 95°C and 20 sec at 60°C. Expression analysis was performed in triplicate for each sample. The small nuclear RNA U5 was used as the normalization control.

We applied different computational methods to maximally analyze functions and pathways targeted by differentially expressed miRNAs. Firstly, an open access platform for miRNA database miRBase (http://www.mirbase.org) was performed to generate the targets of significantly altered miRNAs. In this webserver, we obtained the website links for validated and predicted targets while entering an miRNA accession number. For validated targets, the results from miRTarBase and TarBase were combined. For predicted targets, the microRNA.org server, PicTar and TargetScan was adopted and the predicted targets with mirSVR score ≤-0.1 was adopted in the microRNA.org server (10). Only the common target gene predicted by two different servers was listed in the final target gene table. Thereafter, the databases of GO and PANTHER were searched for functional annotation and pathway analysis. To exclude these categories identified by chance alone, the enrichment analysis was further executed using the free access program DAVID (11), where its significance was measured by the Fisher’s exact test.

Cells were fixed with 10% formaldehyde for 1 h, permeabilized with 0.01% digitonin for 30 min, and treated with 3% BSA before immuno labeling for HA. Images were obtained with an Olympus fluorescence microscope. Lipid droplets (LDs) were stained with BODIPY 493/503 (Invitrogen). Hoechst 33258 (Invitrogen) were used to stain the cell nucleus. Contrast and brightness of micrographs were adjusted by Adobe Photoshop CS for data presentation.

The cell monolayer was washed with ice-cold PBS and scraped in 1 ml PBS. Lipids were extracted by the Folch method from 1-well of a 6-mutiwell dishes (12). The total lower phase was dried down, resuspended in isopropanol, and assayed with a triglyceride kit (InTec Products, Inc., Xiamen, China). Proteins were measured by a kit (Bio-Rad, Hercules, CA, USA).

Cells were collected for Annexin V/PI staining according to the procedures described by BD Biosciences. In brief, Huh-7 cells were washed twice with cold PBS and then resuspended in 1X binding buffer at a concentration of 1×10⁶ cells/ml. Subsequently, 100 μl of the solution was transferred into a 5 ml culture tube, and 5 μl Annexin V and 2 μl PI was added. The cells were gently mixed then incubated for 15 min at room temperature in the dark. Finally, 400 μl of 1X binding buffer was added to each tube, followed within 30 min by flow cytometry (FCM) analysis.

The Mann-Whitney U test or the Student’s t-test were used to determine statistical significance by the SPSS 12.0 software.

First, we obtained the cDNA fragments of encoding HCV different genotype core proteins and then constructed HCV genotypes 3a and 1b core recombinants. Both of the recombinants were transfected into the Huh-7 cells and G418-resistant stable clones were selected and expanded under G418 selecting for 10–12 days. The total-RNA and protein were extracted from the G418-resistant clones. Different genotype core mRNA and protein expressions were identified by real-time PCR and western blot analyses. As shown in Fig. 1A and B, the core mRNA and protein were detected in the extracts from the Huh-7 cells transfected with HCV core 3a and 1b recombinants respectively, but not in the extract from Huh-7 cells transfected with a blank vector. Furthermore, HA-tagged 3a and 1b core proteins were also detected by indirect immunofluorescence only in Huh-7 cells stably transfected by HCV core recombinant (Fig. 1C). These results suggest that these two recombinants could express core protein effectively in the Huh-7 cells. Thus, we established independent Huh-7 cell lines that constitutively expressed core proteins of HCV genotypes 3a and 1b.

For microarray analysis, we used the commercial Agilent human miRNA (8x60K) v16 array set to examine the miRNA expression profiles of transfected Huh-7 cells with the HCV genotypes 3a and 1b core protein. miRNAs showing at least a 3-fold change in expression were considered for further investigation. In contrast to genotype 1b, there were 8 miRNAs upregulated and 19 miRNAs downregulated in Huh-7 cells stably expressing the core of the genotype 3a (Table II). To confirm the microarray results, qRT-PCR was performed on the above 27 miRNAs. U5 was used as control. The primers for the miRNAs are listed in Table I. After statistical analysis, 5 miRNAs (miR-16-2^*, miR-423-3p, miR-30a^*, miR-663 and miR-92b) and 11 miRNAs (miR-224, miR-629^*, miR-542-3p, miR-132, miR-455-5p, hsa-miR-192^*, miR-34b^*, miR-95, miR-885-5p and miR-664) were determined to be respectively upregulated and downregulated (Table III).

The analysis of miRNA target genes and pathways were based on the data from the qRT-PCR result. We first generated a list of all validated target genes to those dysregulated miRNAs (5 miRNAs upregulated and 11 miRNAs downregulated) from an open access platform for the miRNA database miRBase. The result showed a total of 29 and 188 target genes, respectively. Then, a list of all predicted target genes to those dysregulated miRNAs, including 714 and 1160 genes respectively, was also generated by analyzing the targets from three miRNA target prediction web servers TargetScan, PicTar and the microRNA.org server. Thereafter, a functional annotation and over-representation analysis of the GO biological process (GO BP) terms for those genes were performed using the DAVID program. In this study, a complete list of GO BP terms contained 451 and 495 categories for upregulated and downregulated miRNAs. Table IV lists the top 15 categories of those upregulated and downregulated miRNAs. They mainly include regulation of cellular metabolic processes, regulation of cellular biosynthetic processes, and regulation of gene expression. A similar approach was applied to the enrichment analysis of PANTHER pathway for those genes. A total of 4 and 13 pathways (Table V) were enriched for upregulated and down-regulated miRNAs with 3 overlaps, including TGF-β signaling pathway, p53 pathway and integrin signaling pathway.

By GO and PANTHER analyses, dysregulated miRNAs between cells expressing HCV genotype 3a and 1b core are mainly cellular metabolism and apoptosis associated. Several trials were carried out to confirm the functions regulated by HCV core proteins and related miRNAs. Firstly, cellular lipid storage was detected by LDs staining and TAG measuring. As shown in Fig. 2A, the number and size of lipid droplets were increased in cells transfected with HCV core recombinants and cells transfected with genotype 3a had more and larger lipid droplets than those transfected with genotype 1b. Furthermore, TAG measurement was performed. As shown in Fig. 2B, TAG was gradually increased from blank vector to HCV genotype 1b and 3a groups (TAG content, vector, 55.9±1.7; HCV 1b, 94.5±3.6; HCV 3a, 159.5±7.3 μg/mg protein).

Afterwards, cellular apoptosis was assessed by Hoechst 33258 staining and FCM assays. According to the Hoechst 33258 staining of cell nuclei, the percentage of apoptotic cells of the core 1b group was greater than that in the core 3a group, and the lowest in the blank vector group (Fig. 3A). To determine the apoptotic rate of cells accurately, quantitative analysis of apoptosis by FCM of AV/PI dual staining was performed. As shown in Fig. 3B and C, among the blank vector and HCV 3a and 1b groups, the apoptotic rate of cells was gradually increased (apoptotic rate, vector, 8.30±0.36%; HCV 3a, 16.43±0.57%; HCV 1b, 21.47±0.60%). So, HCV core 3a and 1b could affect cellular lipid metabolism and apoptosis differently in Huh-7 cells.