Background
Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease commonly seen in over-weight or obese people. Almost 90% of obese people have been diagnosed with diseases associated with fatty liver [
1]. As the obesity epidemic grows, the incidence of NAFLD is accordingly increasing [
2]. Besides, NAFLD has become the leading cause for liver cancer [
3]. Therefore, efficiently treating NAFLD will be beneficial to prevent hepatocellular carcinoma (HCC). However, limited investigations on the mechanisms underlying the development of NAFLD have been reported [
4].
Long non-coding RNAs (lncRNAs) may act as potential markers for prognosis and progression of liver diseases and furthermore as direct targets for therapeutic purposes. Several lncRNAs have been proved to be associated with liver diseases [
5]. LncRNA activated in renal cell cancer (RCC) with Sunitinib Resistance (lncARSR), which is located on chromosome 9q82, was found to be up-regulated in sunitinib resistance of RCC [
6]. According to a prior study [
7], lncARSR is also potentially involved with hepatic steatosis. However, the role of lncARSR in NAFLD is less clear.
Yes-associated protein (YAP) is an effective transcriptional co-activator in the Hippo pathway, which is associated with the regulation of organ size through mediating cell proliferation, cell cycle and apoptosis. A higher level of YAP has frequently been detected in various types of human cancers [
8,
9]. Moreover, the AKT pathway was demonstrated to be activated by YAP [
10]. Further investigation highlighted that the Hippo pathway interacts with the AKT pathway by influencing insulin receptor substrate 2 (IRS2) expression in NAFLD, which affects the development of NAFLD and even HCC [
11].
In this study, we investigated the effects of lncARSR/YAP1 on NAFLD, and finally identified that lncARSR promoted NAFLD through YAP1 and the IRS2/AKT pathway, thus providing a novel insight into NAFLD treatment.
Materials and methods
Ethical statement
All the animal experiments complied with the standard ethical guidelines prescribed in Guide for the Care and Use of Laboratory Animals by National Institutes of Health. All efforts were made to avoid unnecessary distress to the animals.
Mouse model
A total of 60 C57BL/6 male mice (aged 6 weeks) were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China). The mice were fed with normal diet (carbohydrate accounted for 62.3% of total calories; fat 12.5%; protein 24.3%) or high fat diet (HFD) (carbohydrate accounted for 32.6% of total calories; fat 51.0%; protein 16.4%) (NAFLD mice). After 4 weeks, mice were euthanized by intraperitoneal injection of tripled 3% pentobarbital sodium (P3761, Sigma-Aldrich, St. Louis, MO, USA). The livers were separated for further analyses.
Cell culture
The human HCC cells (HepG2) (
http://www.cellbank.org.cn/) were cultured overnight in four-well chamber slides with Dulbecco’s modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS). Then, the cells were cultured with DMEM supplemented with 1% w/v fatty acid-free bovine serum albumin (BSA) and 0.5 mM oleate. Meanwhile, HepG2 cells were infected with lentivirus of short hairpin (sh)-lncARSR, over-expression (oe)-lncARSR, sh-YAP1, oe-YAP1 or YAP1S127D (a phosphorylated mimic form of YAP1) singly or in combination. After 24 h of infection, lipid accumulation in HepG2 cells was determined by Nile Red Staining. The level of intracellular triglyceride (TG) was measured following the protocols of detection kits (Applygen, Beijing, China).
RNA isolation and quantitation
Total RNA was extracted from cells and tissues with the Trizol kit (Thermo Fisher Scientific, Waltham, MA, USA) and then reversely transcribed into cDNA using the PrimeScript RT Reagent Kit (TaKaRa, Tokyo, Japan). Fluorescence quantitative polymerase chain reaction (qPCR) was subsequently carried out referring to the operation instruction provided by SYBR
® Premix Ex Taq™ II kit (Tli RNaseH Plus, TaKaRa) on Thermal Cycler Dice Real Time System (TP800, TaKaRa). The primers were synthesized by Guangzhou Ribobio Science & Technology Co., Ltd. (Guangzhou, China) (Table
1). Gene expression was measured by the 2
−ΔΔCt method with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal reference.
Table 1
Primer sequences for RT-qPCR
lncARSR (Humo) | F: 5′-TGGATGGGCAAGGCAAGGTC-3′ |
R: 5′-AAGTTGGGCACGGAAGCAGG-3′ |
lncARSR (Mmu) | F: 5′-TTTGAAATGCTCTTTGAGGGAT-3′ |
F: 5′-TGCAGGTTGTCTGAAGTTGGA-3′ |
IRS2 (Humo) | F: 5′-CAAGAGCCCTGGCGAGTACA-3′ |
R: 5′-CCGCGGATGCCAGTAGTG-3′ |
IRS2 (Mmu) | F: 5′-ATATTGCTGAAGAGCTTGGCG-3′ |
R: 5′-TGTATGCGGTGCTCCGGGAAG-3′ |
GAPDH (Humo) | F: 5′-GGTCTCCTCTGACTTCAACA-3′ |
R: 5′-GTGAGGGTCTCTCTCTTCCT-3′ |
GAPDH (Mmu) | F: 5′-GTTGTCTCCTGCGACTTCA-3′ |
R: 5′-GCCCCTCCTGTTATTATGG-3′ |
Western blot analysis
Sufficiently ground liver tissues or HepG2 cells were lysed with radioimmunoprecipitation assay lysis buffer (C0481, Sigma-Aldrich), and total proteins were isolated. The proteins were then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto a polyvinylidene fluoride membrane, which was blocked by 5% skimmed milk for 1 h. The membrane was incubated at 4 °C overnight with the diluted primary antibodies against YAP1 (1:1000, ab205270, rabbit), phosphorylated YAP1 (1:10,000, ab76252, rabbit), IRS2 (1:1000, ab134101, rabbit), AKT (1:10,000, ab179463, rabbit), phosphorylated AKT (1:5000, ab81283, rabbit), fatty acid synthase (Fasn, ab99359, 1:2000, rabbit), stearoyl-coenzyme A desaturase 1 (Scd1, ab19862, 1:1000, mouse), glycerol-3-phosphate acyltransferase (GPAT, ab69990, 1:5000, rabbit) and GAPDH (1:1000, ab8245, rabbit). All the above antibodies were purchased from Abcam Inc. (Cambridge, UK). Next, the membrane was incubated with horseradish peroxidase conjugated goat anti-rabbit immunoglobulin G (TransGen Biotech, Beijing, China), and developed with enhanced chemiluminescence solution (BaomanBio, Shanghai, China). The relative expression was described as the ratio of gray value of the target band to that of GAPDH band, which was analyzed by the image analysis software Image J.
Nile red staining
The lipid accumulation of HepG2 cells was examined by staining with the lipophilic dye Nile Red (Sigma-Aldrich). In brief, cells were fixed with 4% paraformaldehyde for 10 min and incubated with Nile Red solution at a final concentration of 1 mg/L in phosphate buffer saline (PBS) for 20 min at 37 °C. Then the cells were mounted with Prolong® Gold antifade reagent containing 4′-6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA) and examined by a fluorescent microscope.
Histological analysis
Sections of liver were embedded in Tissue-Tek OCT Compound and frozen with carbon dioxide ice. Then, sections were stained with Oil-red-O/60% isopropyl alcohol solution (Thermo Fisher Scientific). After being rinsed with 60% isopropyl alcohol and distilled water, the sections were counterstained by hematoxylin for 4 min, and then observed under a Zeiss Axioplan 2 upright microscope (Carl Zeiss, Jena, Germany).
RNA immunoprecipitation (RIP) and RNA pull-down
RIP and RNA pull-down were conducted following the methods as previously described [
12]. EZ-Magna RIP RNA-Binding Protein Immunoprecipitation Kit (EMD Millipore, Billerica, MA, USA) was used for RIP, and Rneasy Mini Kit (Qiagen, Hamburg, Germany) was utilized to purify RNA in RNA pull-down.
RNA-fluorescence in situ hybridization (FISH)
The expression and distribution of lncARSR and YAP1 in HCC cells were determined by FISH. Cells were cultured in a 24-well plate with 5 × 103 cells/well, and penetrated with PBS containing 0.5% Triton X-100. The cells were then blocked with pre-hybridization solution at 37 °C and hybridized with lncARSR probe at 37 °C overnight in dark. Next, the cells were washed with FISH solution at 42 °C and stained with DAPI. The FISH signals were detected using the tyramide signal amplification system (PerkinElmer Corporation, Norwalk, CT, USA) and analyzed with a fluorescence microscope (IX70, Olympus Medical Systems Co., Tokyo, Japan).
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay
Cell proliferation was examined by a MTT cell proliferation kit (Cell Biolabs Inc, San Diego, CA, USA) following the manufacturer’s instruction.
HepG2 cells (0.5 × 106) were suspended in 8 mL of 0.4% top agar (Sigma-Aldrich), cultured in a 6-cm petri dish in 2 × DMEM supplemented with 20% FBS and wrapped with 3.5 mL of 0.7% bottom agar. After 14 days, the number of cell colonies was counted in three randomly selected regions from each plate.
Transwell invasion assay
The in vitro transwell invasion assay was conducted in 24-well plates using transwell chambers (with 8-μM diameter; Corning Incorporated, Corning, NY, USA). Transwell chambers pre-coated with Matrigel were pre-supplemented with 600 μL DMEM containing 20% FBS at 37 °C for 1 h. The transwell basolateral chamber was also supplemented with DMEM containing 20% FBS. After 48 h of transfection, HepG2 cells were resuspended with DMEM containing 10% FBS. Cells were cultured in the apical chamber at 37 °C with 5% CO2 for 24 h, and then the cells in intimal microporous film were scrubbed off using a cotton swab. Cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The stained cells were observed under an inverted microscope and quantified.
Flow cytometry
HepG2 cells 24 h before transfection were seeded into a 6-well plate. After 48 h transfection, cells were fixed with 70% ethanol. Then, cells were resuspended in PBS and incubated with RNase (100 μg/mL) and propidium iodide (60 μg/mL; Sigma-Aldrich). Cells were subsequently sorted by the FACSCalibur System (BD Biosciences, San Jose, CA, USA) and the cell cycles were analyzed by CellQuest software. Proliferation index (PI) was calculated as PI = (S + G2/M)/G1, in which S, G2/M and G1 referred to the percentage of cells in S, G2/M and G1 phase, respectively.
Xenograft tumor assay
HepG2 cells were placed into a 6-well plate and transfected with shRNA against NC or shRNA targeting lncARSR. After 24 h, 5 × 105 cells resuspended in 0.1 × PBS were hypodermically injected into the right side of the back of athymic nude mice (n = 15). Tumors were observed and measured every 3 days, and the tumor volume (cm3) was calculated as d2 × D/2, in which d was the shortest diameter and D was the longest diameter. When the diameter of tumors reached 1.5 cm, the mice were euthanized, and tumors were removed and weighed.
Statistical analysis
The data were processed using SPSS 21.0 statistical software (IBM Corp., Armonk, New York, USA). Then data distribution was tested for normality and homogeneity of variance. Data were expressed as mean ± standard deviation. If departure from normality and variance was not observed, unpaired t-test was used for analysis between 2 experimental groups, while one-way analysis of variance (ANOVA) was employed for comparison among multiple groups. Repeated measures ANOVA were utilized to compare data among multiple groups at different time points. Pairwise comparison within group was examined by post hoc test. If departures from normality or variance were found, rank-sum test was conducted. p < 0.05 was considered statistically significant.
Discussion
NAFLD, which is characterized by lipid accumulation of the liver, is prevalent in around 25% of adults, especially in diabetic patients [
14]. Normally, NAFLD is frequently related to obesity and HFD, and originates from the abnormal accumulation of TG in livers, which may finally develop to liver cancer or cirrhosis [
15]. However, the pathogenesis of NAFLD still needs to be further explored. In a previous study, over-expressing lncARSR was reported to accelerate the accumulation of liver fat in vivo and in vitro, which suggested that lncARSR may participate in NAFLD and may function as a novel therapeutic target for NAFLD [
7]. Based on these facts, this study was designed to explore the effects of lncARSR on NAFLD. The collected evidences derived from this study demonstrated that lncARSR silencing could alleviate NAFLD by inactivating IRS2/AKT pathway via YAP1.
Initially, increased lncARSR expression was found in oleate-treated HepG2 cells and HFD-fed mice, suggesting that lncARSR may be associated with the progression of NAFLD. LncARSR, as one of the up-regulated lncRNAs in NAFLD, was proved to promote liver cancer stem cell expansion and HCC differentiation [
16]. Similarly, lncARSR was upregulated in HCC cells and promoted doxorubicin resistance of HCC cells by activation of the PI3K/AKT pathway [
17]. The present study also found that lncARSR could specifically bind to YAP1 and promote YAP1 nuclear translocation through inhibiting its phosphorylation. The transcriptional activator YAP acts as a key regulator in the Hippo pathway [
18]. In liver disease, YAP has also been reported to be increased with the increased degree of liver impairment [
19]. Moreover, another study has reported that LATS2 modulated phosphorylation of YAP1 and regulated YAP1 in NAFLD [
20]. Consistently, Qu et al. have also confirmed that lncARSR interacted with YAP and promoted YAP import into nucleus [
12].
Furthermore, another important finding in this study was that lncARSR activated the IRS2/AKT pathway to elevate lipid accumulation in vivo and in vitro, accompanied by increased expression of Fasn, Scd1 and GPA, and accelerate NAFLD progression through inhibition of YAP1 phosphorylation. A previous study [
21] revealed that the IRS2/PI3K/AKT pathway was activated in the liver of NAFLD models. LncARSR regulating AKT-related pathway was also exhibited in another study [
17], in which lncARSR activates the PI3K/AKT pathway by promoting PTEN expression. Furthermore, Jeong et al. reported that Hippo-YAP/TAZ connecting with activation of IRS2/AKT plays a role in the development of NAFLD [
22]. The previous study showed NAFLD was characterized by dysregulation of lipid metabolism in the liver, and less-differentiated HepG2 cells could suppress lipid accumulation [
23]. Likewise, another study also explained that in the setting of NAFLD, lipid accumulation gives rise to liver damage and disease fibrosis [
24]. Fasn and Scd1 play critical roles in hepatic fatty acid synthesis [
25]. The treatment with GPA was identified to be associated with hepatic steatosis and lipid accumulation [
26]. Reduction of Fasn and Scd1 expression was observed after alleviation of oleate-induced NAFLD [
27]. Moreover, a study showed that depletion of lncARSR suppressed hepatic lipid accumulation in vivo and in vitro by decreasing Fasn and Scd1 expression via inactivation of the PI3K/AKT/mTOR pathway [
7], which was in line with our results. Additionally, it is documented that the pathogenesis of NAFLD correlates to metabolic disorders such as lipid accumulation and insulin resistance [
28]. Meanwhile, another study also showed promotion of insulin resistance and repression of hepatocellular glucose uptake in HepG2 cells treated with 0.5 mM oleate [
29]. More importantly, the IRS2/AKT pathway was critical for liver insulin signaling to regulate insulin resistance in muscle and liver of diabetic rats [
30]. However, although IRS2/AKT may also play a role in insulin resistance, it is not enough to resist the role of IRS2/AKT activation in promoting NAFLD and HCC. Therefore, the effect of lncARSR/YAP1/IRS2/AKT axis on NAFLD by regulating insulin resistance needs further elaboration.
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