miRNA target site prediction for the VEGF-A gene was performed using the starBase database (http://starbase.sysu.edu.cn) (17). StarBase links to five commonly used miRNA target prediction programs including TargetScan (www.targetscan.org), picTar (www.mdc-berlin.de/en/research/research_teams/systems_biology_of_gene_regulatory_elements/projects/pictar), RNA22 (http://cbcsrv.watson.ibm.com/rna22.html), PITA (http://genie.weizmann.ac.il/pubs/mir07/mir07_data.html) and miRanda (http://www.microrna.org/microrna/home.do), which provide a comprehensive search for intersections among the putative miRNAs predicted by the programs and target gene VEGF-A.

Specific pathogen-free eight-week-old male C57BL/C mice were purchased from the Experimental Animal Medicine Centre of Sichuan University (Chengdu, China). The mice were housed at room temperature and 55% humidity and given access to food and water ad libitum throughout the experiment. After quarantine for one week, 12 mice, with a mean weight of 18.2±2.7 g, were subjected to 70% PHx using the procedure described previously (18) under chloral hydrate (50 mg/kg, i.p.) anesthesia. Following surgery, the rats were further divided into three groups (12, 24 and 48 h following PHx, n=4/group). At the indicated times, four mice were randomly sacrificed and the remnant regenerating liver tissues were collected and stored at −80°C. All experiments were approved by the Committee of the Experimental Use of Animals, Sichuan University, and performed in accordance with institutional animal care instructions approved by the Ethics Committee for Animals, Sichuan University.

Primary hepatocytes were obtained from mouse liver using in situ two-step collagenase liver perfusion (19) with minor modifications. The mean viability of hepatocytes was 90%, as determined by trypan blue exclusion. The isolated hepatocytes were suspended in DMEM (Hyclone, Logan, UT, USA) supplemented with 10% fetal calf serum (Invitrogen, Carlsbad, CA, USA), 1 mM insulin and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA) and seeded in 6-well culture plates precoated with rat tail tendon collagen type I (Shengyou Biotech, Co., Ltd., Hangzhou, China) at 1×10⁶ cells/ml. Subsequently, the primary hepatocytes were maintained for 4 h at 37°C in a 95% O₂ and 5% CO₂ atmosphere to allow for stabilization. Four hours later, the unattached cells were removed.

Hypoxia induction was achieved using DMEM medium containing HIF-1α-stabilizing agent CoCl₂ (Sigma-Aldrich) at a final concentration of 100 μM (20). In order to evaluate the gene expression levels, the cells were divided into three groups. In group A, the hepatocytes were cultured without CoCl₂ and used as a control. In groups B and C, hepatocytes were cultured with CoCl₂ for 12 and 48 h, respectively.

Hepatocytes were seeded in 96-well plates at a density of 5×10³ cells/well and transfected with micrOFF™ miR-150 inhibitor or a negative control (RiboBio Biotech., Co., Ltd., Guangzhou, China) at a final concentration of 100 nM using Lipofectamine™ 2000 (Invitrogen), according to the manufacturer's instructions. Total RNA and protein were extracted 24 and 48 h after transfection for quantitative PCR (qPCR) and western blot analysis.

Total RNA was extracted from prepared samples using TRIzol reagent (Invitrogen), according to the manufacturer's instructions. The quality and quantity of each RNA sample were measured spectrophotometrically using a NanoDrop 1000 (Thermo Scientific, Waltham, MA, USA) and the integrity of the RNA was assessed by 1% ethidium bromide agarose gel electrophoresis (Invitrogen). Equal amounts of 1 μg RNA were reverse transcribed into cDNA using the miRcute miRNA first-strand cDNA synthesis kit (Tiangen Biotech Co., Ltd., Beijing, China), according to the manufacturer's instructions. Subsequently, qPCR was performed using the miRcute miRNA qPCR detection kit (Tiangen). The 20 μl qPCR mixture consisted of 10 μl 2X miRcute miRNA premix, 2 μl forward primer (2 μM), 2 μl reverse primer (2 μM), 1 μl cDNA and 5 μl RNase-free ddH₂O. The primers used are listed in Table I. PCR amplification was performed using a 1000-Series Thermal Cyclers real-time PCR detection system (Bio-Rad, Hercules, CA, USA) under the following conditions: 94°C for 2 min followed by 40 cycles of 94°C for 20 sec, 62°C for 30 sec and 72°C for 30 sec. The production of specific products was confirmed using melting curve analysis (65–95°C) at the end of the amplification cycle. All experiments were performed in triplicate for each miRNA to obtain Ct values, and a non-template control was included on the same plate. A small stably expressed housekeeping RNA molecule, 5s rRNA (Rn5s), was included as an internal control to normalize gene expression levels and its primer product was purchased from Tiangen Biotech Co., Ltd. Data were analyzed using Bio-Rad CFX Manager software (Bio-Rad) and the relative expression (fold difference) of candidate genes (miR126–5p, 134, 150, 185, 29a, 29b and 29c) was calculated using the 2^−ΔΔCt method.

Treated hepatocytes were harvested at the indicated times and total RNA was extracted using TRIzol reagent. RNA was reverse transcribed using the PrimeScript^® RT reagent kit with gDNA Eraser (Takara, Dalian, China). cDNA was amplified by qPCR with a 1000-Series Thermal Cyclers real-time PCR detection system (Bio-Rad) and SYBR^® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara), according to the manufacturer's instructions. PCR was performed in a 10 μl reaction mixture containing 5 μl SYBR premix, 1 μl forward primer and reverse primer, 1 μl cDNA template and 3 μl RNase-free ddH₂O. The housekeeping gene β-actin mRNA normalized the levels of cDNA. Primers for PCR are listed in Table I. The PCR conditions were as follows: HIF-1α, 95°C for 3 min followed by 45 cycles of amplification at 95°C for 5 sec and 57°C for 15 sec; VEGF-A, 95°C for 3 min followed by 40 cycles of amplification at 95°C for 10 sec, 56°C for 10 sec and 72°C for 10 sec; and β-actin, 95°C for 3 min followed by 40 cycles of amplification at 95°C for 5 sec and 59°C for 10 sec. All the samples were amplified in triplicate and each experiment was repeated three times.

Liver tissues, or cultured hepatocytes, were homogenized and lysed in cell lysis buffer (Beyotime institute of Biotechnology, Nantong, China) containing phenylmethanesulfonyl fluoride (PMSF; Beyotime) for 30 min on ice. The lysates were centrifuged for 10 min at 10,612 × g at 4°C. Protein concentrations were determined using the BCA protein assay kit (Beyotime) with BSA as a standard. Equal amounts of protein were loaded onto a 10% SDS-polyacrylamide gel and separated intermittently for 1 h at 100 V on an electrophoresis system (Bio-Rad). Proteins were then transferred to a nitrocellulose membrane (Pall, Port Washington, NY, USA) for 2.5 h at 0.35 mA. Membranes were blocked for 1 h with a blocking buffer (Beyotime) and subsequently incubated overnight at 4°C with a 1:100 dilution of primary rabbit anti-HIF-1α or anti-VEGF polyclonal antibody (Boster Bioengineering Co., Ltd., Wuhan, China). A mouse anti-GAPDH polyclonal antibody (Boster) was used as a loading control to normalize protein levels. After washing four times with TBS-T for 15 min, the immunoblots were incubated for 1 h with a 1:5,000 dilution of secondary horseradish peroxidase-conjugated goat anti-rabbit IgG (Boster), followed by detection with enhanced chemiluminescence (Beyotime) according to the manufacturer's instructions. Protein levels were visualized on the gel imaging and analysis system and quantified using the Quantity One 4.5 software (Bio-Rad). The gray ratio value between the target protein and GAPDH was used to calculate the relative expression level of target protein for the subsequent statistical analysis.

All data are expressed as the mean ± SD. Statistical analysis was performed using one-way ANOVA and a Newman-Keuls test between groups using Statistical Package for the Social Science (SPSS) for Windows (SPSS, Inc., Chicago, IL, USA). Graphs were created with Origin 7.5 (OriginLab data analysis and graphing software, Northampton, MA, USA). P<0.05 was considered to indicate a statistically significant difference.

Since this study investigated the effect of miRNA profile change in the proliferating hepatocytes upon VEGF-A gene expression and aimed to identify an miRNA that was associated with the VEGF-A gene with relative specificity, we first analyzed target site intersections between mouse miRNAs and the VEGF-A gene using a prediction assay of miRNA target genes. Initially, in silico analysis predicted 22 putative miRNAs based on three reference sequences of the mouse VEGF-A gene (GenBank accession numbers: NM_009505, NM_001025257 and NM_001025250) using five bioinformatics algorithms (Table II). The animal miRNA target genes were roughly estimated since there was incomplete base pairing on binding sites between the miRNA and mRNA target sequences (21). In order to make full use of all the algorithm programs with different characteristics and to improve the accuracy rate of target prediction, we selected miRNAs that were simultaneously predicted by two or more of the five bioinformatics algorithms as putative regulators of VEGF-A. In total, 7 of the 22 putative miRNAs were selected for further screening in order to identify a specific miRNA with close correlation to the VEGF-A gene.

Since PHx is considered to be the most potent reproducible stimulus for hepatocyte proliferation (22), we analyzed the liver tissues following PHx to explore changes in the expression of miRNAs in hepatocytes of the regenerating liver. We investigated which miRNA was regulated upon hypoxia in order to modulate the expression of VEGF-A in the seven miRNAs predicted to target VEGF-A. We measured the miRNA expression levels according to the variation tendency of VEGF-A expression in the early phase of LR. Alterations in the expression profiles of the miRNAs were examined using an miRNA microarray assay. However, the significantly altered miRNAs observed in the microarray results were selected for further validation by qPCR, as the microarray detects changes in gene expression without the need for high specificity and the microarray product of different sources may have inconsistent results. Thus, we directly detected substantial levels of seven miRNAs by qPCR instead of miRNA microarray assay and observed changes in their expression at three time-points (12, 24 and 48 h) once the total RNA was extracted from prepared liver samples. We identified that six of the miRNAs, with the exception of miR-29b, were downregulated >2-fold between 12 and 48 h (Fig. 1A). Among them, miR-150 was downregulated ~19-fold from 12–48 h and demonstrated the most marked downwards trend. The direct binding site of miR-150 to the VEGF target sequence is shown in Fig. 1B.

miRNAs with significant alterations in their expression levels, as determined by the microarray results, were generally considered to correlate closely with different physiological or pathological phenomena. We infer that the increased production of VEGF in the early stage of LR is due to a global downregulation in the production of certain miRNAs. Of all the miRNAs predicted to target VEGF-A, miR-150 was selected for further investigation as it was the most downregulated. In view of an interaction between miR-150 and VEGF-A mRNA with the highest degree of correlation, we evaluated the role of miR-150-mediated regulation of VEGF-A gene expression. The miR-150 inhibitors and a negative control were transfected into the hepatocytes for 24 and 48 h. As a chemosynthetic and modified inhibitor, the micrOFF miRNA inhibitor specifically bound the mature target miRNA and suppressed its expression. The efficacy of downregulating the expression of the miR-150 gene and upregulating the expression of the VEGF-A gene was examined by qPCR and western blot analysis. Fig. 2 demonstrates that the miR-150 inhibitors effectively suppressed the expression of miR-150 2.4-fold 48 h after transfection and promoted the corresponding transcription and translation of the VEGF-A gene. The mRNA and protein levels of VEGF-A increased 3.1-fold and 2.6-fold, respectively, 48 h after transfection with the miR-150 inhibitors compared with the control group. The differences were not statistically significant at 24 h when compared with the control group (Fig. 3).

In order to evaluate the hypoxic conditions in the early phase of LR, we selected the HIF-1α protein as a detection index to indicate the degree of hypoxia. Intraoperatively excised liver tissues served as the normal control (0 h). Western blot analysis demonstrated that HIF-1α protein expression levels were increased 12 h after PHx and continued increasing until the highest expression level was achieved 24 h after PHx with an ~4-fold increase relative to the control group (Fig. 4).

To determine whether miR-150 was regulated by hypoxia, primary hepatocytes were isolated and hypoxia was induced with CoCl₂. As shown in Fig. 5, the expression of HIF-1α mRNA was low in the normoxic state, but increased at 12 h (2.7-fold vs. control) and increased further at 48 h (4-fold vs. control) following the administration of CoCl₂. Moreover, the expression of miR-150 declined and VEGF-A mRNA increased 48 h after hypoxia with differences of 4.2-fold and 3.1-fold, respectively, relative to the normal control. However, the expression of miR-150 and VEGF-A mRNA was not significantly different from the control 12 h after hypoxia.