Prior to animal experimentation, the present study was approved by the Institutional Animal Ethics Committee of the Second Military Medical University. Male BALB/c mice (6–8 weeks of age) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were maintained in sterile conditions with free access to food and water, under a 12-h light/dark cycle. 2/3 PH was performed according to previously described methods (17), where the left and median liver lobes were removed under isoflurane anesthesia (2% v/v). Euthanasia was carried out by CO₂ inhalation for 5 min. Humane endpoints included weight loss of >20%, dehydration, severe lameness, ruffled fur, and hunched appearance. None of mice exhibited the humane endpoint criteria. The liver tissues were collected and the liver/body weight ratio was calculated on days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 after 2/3 PH.

Mouse primary hepatocytes were isolated from the livers of 2/3 PH model mice and control mice. Liver tissues were fragmented using ophthalmic scissors, and the resulting tissue pieces were washed with PBS containing with 100 U/ml of penicillin and 100 mg/ml of streptomycin. The liver tissues were then digested (0.25% trypsin; 0.04% EDTA), filtrated through a 4 µm cell strainer and centrifuged for 5 min at 350 × g at 4°C. Then, mouse primary hepatocytes (5–6×10⁵/ml) were suspended and cultured in high glucose DMEM (Gibco; Thermo Fisher Scientific, Inc.) with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO₂, with medium replaced every 2 days. Cells were subcultured for 3–4 days until confluence reached to 70–80%. Mouse primary hepatocytes were treated with different concentrations of HGF (5, 10, 20, 30, and 40 ng/µl).

The NCTC 1469 and BNL CL.2 mouse hepatocyte cell lines were purchased from the American Type Culture Collection, and cultured in DMEM containing 10% fetal bovine serum at 37°C with (5% CO₂). NCTC 1469 cells were treated with HGF (20 ng/µl) or the Wnt inhibitor IWR-1 (60 µM) at 37°C for 24 h. Recombinant lentivirus (Lv)-SNHG12 and the scrambled control (Lv-NC) were constructed by Shanghai GeneChem Co., Ltd. The cells were transfected using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) by exposure to diluted viral supernatant containing polybrene for 48 h, as previously described (18). SNHG12 small interfering (si)RNA was purchased from ZHBY Biotech Co. Ltd. and scramble siRNA was used as the control.

Total RNA from tissues and cells was extracted using a TRIzol^® kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Reverse transcription (RT) was carried out using a PrimeScript RT reagent Kit (Takara Bio, Inc., Tokyo, Japan). The RT system of 10 µl was carried out according to the manufacturer's instructions. The RT conditions were 37°C for 15 min and 85°C for 5 sec. mRNA and lncRNA expression levels were determined using the SYBR Green Supermix (Invitrogen; Thermo Fisher Scientific, Inc.) on an Applied Biosystems 7300 real-time PCR system. Thermocycling conditions were 95°C for 10 min, following by 35 cycles at 95°C for 10 sec, 58°C for 15 sec and 72°C for 20 sec, and a final 72°C for 20 min. β-actin was used as the internal control. All qPCR experiments were performed at ≥3 times, and the primer sequences were as follows: SNHG12 forward, 5′-CATCAAGACTGAGAAAAAGCACACC-3′ and reverse, 5′-TACCTTAAAGCACAGCTCCAGAAAC-3′; Axin2 forward, 5′-GATGTTGGAGAGTGAGCGGCAG-3′ and reverse, 5′-TGTTGGGTGGGGTAAGGGGAG-3′; β-actin forward, 5′-CAACTGGGACGACATGGAG-3′ and reverse, 5′-TAGCACAGCCTGGATAGCAAC-3′.

The total protein from liver tissues and hepatocytes was extracted and homogenized in RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.). The concentrations of the extracted nuclear and cytoplasmic fractions were quantified using the bicinchoninic acid assay method. A total of 50 µg protein per sample was separated by SDS-PAGE (10%) and then transferred to a PVDF membrane, prior to blocking with 5% non-fat milk in 1X TBST, overnight at 4°C. The membrane was then incubated with anti-β-catenin (1:1,000; product no. ab32572), anti-β-actin (1:5,000; product no. ab8227), anti-histone 3 (1:2,000; product no. ab62642; all from Abcam) primary antibodies for 2 h at room temperature. After washing three times in 1X TBST, the membranes were incubated with the corresponding HRP-conjugated secondary antibody (1:5,000; cat. no. A6154-1 ml; Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. The membranes were washed once more, exposed to ECL (Sangon Biotech Co., Ltd.), and then photographed by ChemiDoc™ XRS+ Imaging system (Bio-Rad Laboratories, Inc.). The protein expression levels were quantified using ImageJ software version 1.46 (National Institutes of Health). Histone 3 and β-actin were used as internal controls.

Cell Counting Kit (CCK)-8 reagent (Biotech Well) was used to determine the level of cellular proliferation, per the manufacturer's protocol. Briefly, NCTC 1469 or BNL CL.2 cells were seeded into 96-well plates at a density of 3×10⁵/ml, and transfected with the aforementioned constructs for 48 h. CCK-8 reagent was then added to each well and the cells were incubated for ~30 min at 37°C. The absorbance was measured by a microplate reader (Bio-Rad Laboratories, Inc.) at a wavelength of 450 nm. The number of cells was calculated in reference to a standard curve obtained under the same conditions.

NCTC 1469 or BNL CL.2 cells were seeded at 3×10³ cells/well in 96-well plates. After 2 days of culture, proliferation was assessed using Cell Proliferation ELISA, BrdU (colorimetric) kit (product no. 11647229001; Roche), per the manufacturer's protocol. Briefly, BrdU-labeling solution (100 µM) was added into each well and incubated for 4 h at 37°C. The absorbance was measured at 450 nm by a microplate reader (Infinite 200; Tecan Group).

The liver specimens were fixed using formalin (10%) and embedded in paraffin. Serial sections of 5-µm thickness were sliced from the paraffin embedded blocks, and used for subsequent experiments. The sections were incubated overnight at 4°C with an anti-proliferating cell nuclear antigen (PCNA) primary antibody (1:500; product no. ab29; Abcam), then washed thrice prior to subsequent incubation with an HRP-conjugated goat anti-rabbit antibody (1:200; product no. 5571; Cell Signaling Technology, Inc.). The sections were then stained with DAB (Sangon Biotech Co., Ltd.) for 5 min at 37°C to observe the expression of PCNA.

NCTC 1469 or BNL CL.2 cells (5×10⁴/well) were seeded into 24-well plates. TOP-FLASH or FOP-FLASH constructs and Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) were used to transfect the cells and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega Corporation) as previously described (19). Luciferase activity was normalized to that in cells transfected with TOPFlash/FOPFlash.

Statistical analysis was conducted using SPSS software version 16 (SPSS, Inc.). All data are presented as the mean ± SD (standard deviation) from three or more separate experiments. Statistical significances between two groups were analyzed using one-way ANOVA followed by the Scheffé test (Figs. 2C-F, 3 and 5), or two-tailed Student's t-test (Figs. 1, 2A and B, 4 and Fig. S1). P<0.05 was considered to indicate a statistically significant difference.

lncRNAs play crucial roles in the regulation of omnigenous cellular activities, which may provide unique functions and improve the regenerative ability of the liver. Recently, the role of SNHGs in tumor progression was verified (20–22), but their role in liver regeneration remains unexplored. In the present study, 13 liver cancer-related SNHGs (SNHG1, SNHG2, SNHG3, SNHG5, SNHG6, SNHG7, SNHG8, SNHG12, SNHG14, SNHG15, SNHG16, SNHG18 and SNHG20) were assayed using RT-qPCR. Since HGF is a key factor in liver regeneration (23,24), the expression of these SNHGs was assessed in NCTC 1469 cells following treatment with HGF. Among the 13 SNHGs, the expression level of SNHG2 was decreased, and that of SNHG6, 12 and 20 was significantly increased after HGF treatment (Fig. 1A). SNHG12 was selected for further analysis, since its level of upregulation was the most significant.

Mouse primary hepatocytes were isolated and treated with HGF. Fig. 1B revealed that in primary hepatocytes, the expression level of SNHG12 was upregulated in a dose-dependent manner following HGF treatment. Therefore, 2/3 PH was performed and the SNHG12 level was assessed once more. The SNHG12 expression was also significantly increased at various time-points after 2/3 PH (Fig. 1C); 2/3 PH in mice resulted in enhanced SNHG12 levels that were detectable at 6 h, that peaked between 24 h, and that had returned to almost normal levels by 72 h. Thus, the time-dependent alterations in SNHG12 expression indicated that SNHG12 may potentially be involved in liver regeneration.

To investigate the role of SNHG12 in hepatocyte proliferation, a CCK-8 assay was performed using normal mouse liver cell lines (NCTC 1469 cells and BNL CL.2 cells) following SNHG12-overexpression or -knockdown. Fig. 2A and B revealed that SNHG12 expression was significantly increased in NCTC 1469 cells after treatment with Lv-SNHG12, and decreased after treatment with SNHG12-specific siRNA (siSNHG12). Furthermore, NCTC 1469 hepatocyte proliferation was significantly increased by the overexpression of SNHG12 (Fig. 2C), which also promoted BNL CL.2 cell proliferation (Fig. 2D), whereas SNHG12-knockdown decreased NCTC 1469 and BNL CL.2 cell proliferation (Fig. 2C and D).

The results of the BrdU ELISA assays also indicated that hepatocyte proliferation was increased after SNHG12-overexpression, and suppressed by SNHG12-knockdown (Fig. 2E and F). Subsequently, to assess hepatocyte proliferation in vivo, the expression level of PCNA was investigated by IHC in mouse-model tissues. At 36 h post-2/3 PH, SNHG12-treated mice exhibited a higher number of PCNA-positive nuclei in the hepatocytes (Fig. 3A and B). Furthermore, the role of SNHG12 in regulating liver regeneration was assayed. As revealed in Fig. 3C, after 2/3 PH the liver/body weight ratio of mice overexpressing SNHG12 was significantly greater than that of the control mice. Conversely, SNHG12-inhibition suppressed hepatocyte proliferation in vivo and reduced the liver/body weight ratio (Fig. 3A-C). These data indicated that SNHG12 upregulation contributed to hepatocyte proliferation in vitro and in vivo.

Previous studies have demonstrated the Wnt/β-catenin pathway as the key factor in triggering liver regeneration after PH (25). Furthermore, lncRNAs can control liver regeneration by regulating Wnt/β-catenin signalling (10). lncRNA SNHG12 was revealed to promote cellular proliferation and metastasis by activating the Wnt/β-catenin pathway in papillary thyroid cancer (13). Therefore, the present study sought to investigate whether SNHG12 regulated Wnt/β-catenin in hepatocytes following PH. A TOPflash/FOPflash assay was first carried out to verify the role of SNHG12 in Wnt regulation. Fig. 4A and B indicated that the relative luciferase activity of BNL CL.2 and NCTC 1469 cells was significantly increased following SNHG12 overexpression, whereas SNHG12 knockdown suppressed the luciferase activity, compared with the control. When Wnt signaling is activated, β-catenin is dephosphorylated and translocates into the nucleus, where it promotes the expression of downstream target genes (26,27). In the present study, the effects of the SNHG12-induced nuclear translocation of β-catenin were also investigated. SNHG12-induced nuclear translocation was further investigated using immunofluorescence (Fig. 4C). When SNHG12 was overexpressed in NCTC 1469 cells, β-catenin nuclear translocation was markedly increased (Fig. 4D), whereas SNHG12-knockdown inhibited the nuclear translocation of β-catenin (Fig. 4E). Axin-2, a downstream target of Wnt/β-catenin signaling, was also enhanced in BNL CL.2 cells after SNHG12 overexpression, yet suppressed following SNHG12 knockdown, compared with the control (Fig. 4F and G). These results demonstrated that SNHG12 is a regulator of Wnt/β-catenin signaling.

To investigate the role of the SNHG12/Wnt pathway in the regulation of liver regeneration following 2/3 PH, SNHG12 was overexpressed and Wnt was concurrently inhibited using a specific inhibitor (IWR-1); hepatocyte proliferation and liver regeneration were then assessed in vitro and in vivo. As revealed in Fig. 5A and B, the cell proliferation of mouse hepatocytes was significantly increased after SNHG12 overexpression, whereas IWR-1-induced Wnt inhibition attenuated SNHG12-associated cellular proliferation. As anticipated, SNHG12 overexpression was revealed in vivo, as indicated by increased nuclear translocation of β-catenin (Fig. S1). The effect of SNHG12 overexpression on hepatocyte proliferation and liver regeneration was thus investigated in vivo. After 2/3 PH, the SNHG12-treated mice exhibited a larger number of PCNA-positive hepatocyte nuclei than those in the control group (Fig. 5C). However, Wnt inhibition partially suppressed SNHG12-associated hepatocyte proliferation in vivo. Furthermore, the liver/body weight ratio of SNHG12-treated mice was significantly increased. Conversely, Wnt inhibition attenuated SNHG12-induced liver regeneration after 2/3 PH (Fig. 5D). These results demonstrated that PH-induced upregulation of SNHG12 accelerated hepatocyte proliferation and liver regeneration by activating Wnt/β-catenin signaling.