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Following partial hepatectomy (PH), the complex process of liver regeneration is initiated, which encompasses the synchronized induction of hepatocyte proliferation. Hepatocyte proliferation can be regulated by multiple stimuli, including long non-coding RNAs (lncRNAs) and Wnt/β-catenin signaling, although the underlying mechanism of lncRNA/Wnt in liver regeneration remains unclear. In the present study, a liver regeneration-associated functional lncRNA was identified, and its function was delineated
Liver regeneration occurs after events that could lead to the loss of liver mass, including surgery, trauma, infection and liver transplantation (
lncRNAs are non-coding transcripts (>200 nucleotides) which have been widely demonstrated as vital regulators of numerous cellular responses, developmental processes and disease (
Small nucleolar RNA host gene 12 (SNHG12) is a newly identified lncRNA that is associated with various malignancies, and is considered to be a useful tumor biomarker (
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 (
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 (
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 (
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 (
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 (
Mouse primary hepatocytes were isolated and treated with HGF.
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.
The results of the BrdU ELISA assays also indicated that hepatocyte proliferation was increased after SNHG12-overexpression, and suppressed by SNHG12-knockdown (
Previous studies have demonstrated the Wnt/β-catenin pathway as the key factor in triggering liver regeneration after PH (
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 the present study, the role of SNHG12-associated hepatocyte proliferation and liver regeneration after 2/3 PH was verified. The data demonstrated that: i) SNHG12 expression was significantly increased following 2/3 PH; ii) SNHG12 enhanced hepatocyte proliferation
It is acknowledged that different cytokines, growth factors and miRNAs regulate the genes responsible for cellular proliferation during liver regeneration (
Previous studies have widely reported that the Wnt/β-catenin pathway plays a primary role in organogenesis, homeostasis and various human diseases (
Finally, the use of the Wnt inhibitor IWR-1 revealed that the promotion of hepatocyte proliferation was partially attenuated, further confirming that SNHG12 may exert its regenerative role by activating Wnt signaling. Subsequent
Collectively, the results of the present study demonstrated the potential regulatory role of SNHG12 in liver regeneration.
Not applicable.
The present study was supported by grants from the National Natural Science Foundation of China (grant no. 81770613) and the Science and Technology Commission of Shanghai Municipality (grant no. 19411967000).
All data generated or analyzed during this study are included in this published article.
YaZ and ZQ participated in the design of the main research ideas and manuscript correction. YiZ edited the manuscript and conducted the experiments. BL and XJ performed statistical evaluation of the final data and agreed to publish the paper. All authors read and approved the final manuscript.
The present study was approved by the Institutional Animal Ethics Committee of the Second Military Medical University.
Not applicable.
The authors declare that they have no competing of interests.
SNHG12 expression is significantly upregulated after 2/3 PH. (A) Heatmap of RT-qPCR data for the expression of 13 liver cancer-related SNHGs (SNHG1, SNHG2, SNHG3, SNHG5, SNHG6, SNHG7, SNHG8, SNHG12, SNHG14, SNHG15, SNHG16, SNHG18, and SNHG20). NCTC 1469 cells were treated with HGF (20 ng/µl), and then total RNA was extracted and used to perform RT-qPCR analysis. (B) RT-qPCR analysis of SNHG12 expression level in primary mouse hepatocytes (2×105 cells/well) after treatment with different concentrations of HGF (5, 10, 20, 30 and 40 ng/µl). (C) RT-qPCR analysis of SNHG12 expression in liver tissues (n=5) at different time-points after 2/3 PH. *P<0.05 and **P<0.01. SNHG12, small nucleolar RNA host gene 12; PH, partial hepatectomy; RT-qPCR, reverse transcription-quantitative PCR; HGF, hepatocyte growth factor.
SNHG12 promotes hepatocyte proliferation
SNHG12 promotes liver regeneration
SNHG12 activates Wnt/β-catenin signaling in hepatocytes. Activation of canonical Wnt/β-catenin signaling was assessed in NCTC 1469 and BNL CL.2 cells using TOPflash/FOPflash after (A) SNHG12 overexpression and (B) SNHG12 knockdown. RLUs were calculated using ≥3 results. (C) Nuclear translocation of β-catenin was assessed using an immunofluorescence assay. Scale bar, 20 µm. Nuclear translocation of β-catenin induced by (D) SNHG12 overexpression or (E) SNHG12 knockdown was indicated using western blot analysis. Relative Axin2 mRNA expression was analyzed using RT-qPCR in BNL CL.2 cells after (F) SNHG12 overexpression and (G) SNHG12 knockdown. *P<0.05. SNHG12, small nucleolar RNA host gene 12; RLU, Relative Luciferase Unit; RT-qPCR, reverse transcription-quantitative PCR.
SNHG12 accelerates liver regeneration by activating Wnt signaling. CCK-8 analysis was carried out to assess (A) NCTC 1469 and (B) BNL CL.2 cell proliferation after SNHG12 overexpression in the presence or absence of 20 µM IWR-1 (an inhibitor of Wnt signaling). (C) Quantification of PCNA-positive cells at different time-points after 2/3 PH following SNHG12 overexpression in the presence or absence of 60 µM IWR-1. (D) The liver/body weight ratio of SNHG12-treated mice was assessed after 2/3 PH in the presence or absence of 60 µM IWR-1 (n=5). *P<0.05 vs. control. #P<0.05 vs. SNHG12. SNHG12, small nucleolar RNA host gene 12; PH, partial hepatectomy; CCK-8, Cell Counting Kit-8; PCNA, proliferating cell nuclear antigen.