Contributed equally
Sterol regulatory element binding protein-1c (SREBP-1c), which serves an essential role in the process of fat synthesis, is a key adjustment factor that regulates the dynamic balance of lipid metabolism. SREBP-1c activates the transcription of multiple genes encoding for enzymes involved in the synthesis of triglycerides (TG) and fatty acids (FA) and accelerates lipid synthesis. Previous analysis indicated that long non-coding RNA HCV regulated 1 (lncHR1) participates in lipid metabolism
Sterol regulatory element binding protein-1c (SREBP-1c) is one of SREBP family member and regulate the expression of lipogenic genes (
SREBP-1c is also a target of insulin, which activates transcription of the gene encoding SREBP1 by partially increasing the activity of liver X receptor α (LXRα) (
Long noncoding RNA (lncRNA) are transcribed RNA molecules that lack an open reading frame and are longer than 200 nucleotides (
Long non-coding RNA lncHR1 (HCV regulated 1, termed lncHR1), was first reported as upregulated in Huh7 cell infected by hepatitis C virus (HCV). As a new long non-coding RNA, lncHR1 exhibited obviously regulatory functions via SREBP-1c, the accumulation of TG and lipid droplets in cells, and in transgenic mouse model (
In this study, we initially studied the molecular mechanisms behind the regulation of SREBP-1c by lncHR1. The results showed that lncHR1 may affect the phosphorylation of the PDK1/AKT/FoxO1 signaling pathway, subsequently regulating SREBP-1c protein levels in hepatocellular carcinoma lines. Thus, our study offered a new information regarding lncRNA regulation of SREBP-1c through the AKT/FoxO1 signaling pathway and provided a practical and efficient platform for studying the function of lncRNA in lipid metabolism.
The Huh7 human hepatoma cell line was purchased from Apath, Inc. (Brooklyn, NY, USA) (
LncHR1 was cloned into the
Cultured cells were inoculated in 24 well plates with 10% FBS medium and incubated overnight at 37°C and 5% CO2 until the cells grew to 80% confluence. Before transfection, cell medium was replaced with fresh medium not containing with FBS for 1 h. DNA (1 µg) plasmids were diluted with serum-free DMEM medium and 2 µl of the transfection reagent D293, which was diluted with serum-free DMEM medium, and then mixed well. The diluted transfection reagents were added to the diluted plasmid by drop by drop, gently mixed and then kept at room temperature for 15 min. The suspension of the plasmid and the transfection reagent was added into the transfected cells evenly with gently agitation to ensure even distribution. The medium was replaced with fresh medium containing 10% FBS after 5–8 h of transfection. After 48 h of continuous culture, cells were collected and then used for experiments.
Huh7 cells were inoculated in the 24 well plates with the sterilized round glass slices and then carried out routine cell processing. Pretreated cells were fixed with 3% paraformaldehyde for 10 min at room temperature and then were permeabilized with 0.4% Triton X-100 for 10 min at room temperature. Pretreated cells were stained for 1 h with freshly diluted 0.5 mM oil red O dissolved in 60% isopropanol. Stained cells were washed with 50% ethyl alcohol twice for 5 min and then was washed with PBS twice for 5 min. When washing the second times, PBS with 4′,6-diamidino-2-phenylindole (DAPI) was added to stain the nuclei. Last, at least 5 random fields were observed under a Leica TCS-SL confocal microscope. The intensity was quantified with Image J software (NIH).
Treated cells were collected and washed twice with PBS. For cell lysate preparation, the monolayer of plates cells was lysed with lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 50 mM NaF, 1 mM Na3VO4, 5 mM β-glycerophosphate, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). The lysate was clarified by centrifuging at 14,000 × g for 20 min. Samples were mixed with 2X SDS loading buffer, boiled, and loaded onto a 10–12% polyacrylamide gel. After electrophoresis, the proteins were transferred onto a polyvinylidene difluoride membrane (Pall Corporation, East Hills, NY, USA). The resulting blots were blocked with 5% bovine serum albumin (BSA) for phosphoprotein antibodies, or 10% skim milk for non-phosphoprotein antibodies for 1 h and incubated with the selected primary antibody overnight at 4°C. The secondary antibody used in the immunoblot was a 1:2,000 or 1:5,000 dilution of HRP-linked anti-IgG. ECL reagent (Amersham Biosciences, England, UK) was used as the substrate for detection, and the membrane was exposed to an x-ray film for visualization. The density of the bands was quantified using the NIH image software and were normalized to the density of the control band.
The nuclear-cytosol fractionation experiments were performed using a Nuclear-Cytosol Extraction kit (Applygen Technologies, Inc., Beijing, China). Western blotting was performed to quantify protein according to standard protocols published in the literature. Cells were mounted on glass slides and the immunofluorescence assay was performed as previously described (
Intracellular TG were measured with a TG Assay kit from Applygen Technologies, Inc., according to the manufacturer's instructions. TG values are expressed as µM (at the cellular level).
Bar graphs depict means ± standard deviation of at least three independent experiments. A Student's t-test was used to analyze differences between means for two independent samples, and P<0.05 was considered to indicate a statistically significant difference.
It has been reported that lncHR1 regulated SREBP-1c levels in Huh7 cells and transgenic mice fed a high fat diet (
The cell model of OA-induced TG accumulation was used. The results showed that, in Huh7 cells, OA treatment for 24 h obviously increased SREBP-1c protein (
In the model of the TG accumulation, overexpression of lncHR1 inhibited SREBP-1c protein expression, suppressed the phosphorylation of AKT (Thr308) and total AKT protein levels. β-actin was used as a loading control (
To study how lncHR1 regulates the phosphorylation of AKT, an activator (IGF-1) and inhibitor (LY294002) of the PI3K/AKT pathway were used in this study. As shown in
The major biological function of phosphatase and tensin homolog (PTEN) relies on its phosphatase activity and PTEN exerts tumor suppressive functions by suppressing the PI3K pathway (
3-phosphoinositide-dependent protein kinase 1 (PDK1) is downstream of PI3K and activated PDK1 can stimulates the phosphorylation of Threonine 308 in the central catalytic domain (
A study by Kamei (
Phosphorylation of FoxO1 (Ser256) (p-FoxO1) by AKT occurs in the cytoplasm. Non-phosphorylated FoxO1 located in the nucleus inhibits SREBP-1c expression. Therefore, Western blotting and immunofluorescence assays were used to measure the lncHR1-regulated translocation of FoxO1. First, we separated the nuclear protein fraction for each treatment and then quantified FoxO1 accumulation. The results indicated that a greater amount of FoxO1 accumulated in the nucleus when lncHR1 was overexpressed (
Above all, these results consolidated our findings that lncHR1 suppressed SREBP-1c levels through decreased the phosphorylation level of PDK1/AKT/FoxO1 and increased expression of intranuclear FoxO1. According to these results, lncHR1 may inhibit the expression of SREBP-1c through modulation of the PDK1/AKT/FoxO1 pathway in Huh7 cells. Further studies will investigate how lncHR1 regulate the phosphorylation of PDK1.
In this study, we found that lncHR1 inhibited SREBP-1c and TG accumulation when the phosphorylation level of the PDK1/AKT/FoxO1 pathway was reduced through increased lncHR1. Based on the results of previous experiments (
SREBPs are key transcriptional factors that control lipogenesis and lipid uptake (
The long non-coding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) showed significantly higher expression in advanced gastric cancer tissues, and the study results indicated that UCA1 regulates PI3K-AKT-mTOR signaling proteins
In particular, SREBP-1c is the chief factor regulating the transcription of genes involved in fat synthesis, and can be up-regulated by LXRα (
Activation of the PI3K/AKT signaling pathway results in AKT-dependent phosphorylation of FoxO1, reducing FoxO1 nuclear translocation and inhibiting its transcriptional function. PI3K/AKT signaling upregulates glucose uptake and glycolysis and controls the metabolic flux from glucose and glutamine to de novo lipid synthesis (
SREBP-1c may be activated by the PI3K/AKT oncogenic signaling pathway either PI3K/AKT/GSK3-β/SREBP-1c (
It is well established that the major biological function of PTEN relies on its phosphatase activity. PTEN dephosphorylates PIP3 to PIP2, thereby inhibiting the PI3K signaling pathway (
There are several limitations in this study. i) The detailed molecular mechanism for the effect of lncHR1 on the phosphorylation of PDK1 has not been elucidated. ii) The phosphorylation of AKT is regulated by mTOR, but whether lncHR1 is involved in the mTOR pathway was not verified. Therefore, these areas will be the next focus of our research.
Taken together, these findings indicated that lncHR1 may induce a decrease in the phosphorylation of the PDK1/AKT/FoxO1 pathway and suppressed SREBP-1c protein levels. However, the precise role of lncHR1 in the phosphorylation of PDK1 remains to be elucidated, and further investigations is needed.
The authors would like to thank Dr Wei Yang of the MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College for providing technical and material support.
This work was supported by the Collective grants from the Programs for Science and Technology Development of Henan (grant nos. 172102310499 and 182102310240), Open Program of Henan Key Laboratory of Biological Psychiatry (grant no. ZDSYS2016007) and Dr. scientific research start-up fund (grant no. XYBSKYZZ201605).
The analyzed data sets analyzed during the study are available from the corresponding author on reasonable request.
DL and ZY are responsible for the study concept and design. LG and BD performed part of the cell biology experiment. ML, TY and FY performed the molecular biology experiment. DL and ZY were involved in the data analysis and manuscript drafting.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
long non-coding RNA
sterol regulatory element binding protein-1c
oleic acids
liver X receptor α
SREBP cleavage-activating protein
4′,6-diamidino-2-phenylindole
fatty acid
fatty acid synthase
triglyceride
acetyl-CoA carboxylase α
reverse transcription-quantitative polymerase chain reaction
The model of OA induces hepatic cell triglyceride accumulation in Huh7 cells. (A and B) In Huh7 cells, OA treatment for 24 h significantly increased the level of SREBP-1c protein and the expression of SREBP-1c mRNA compared with control cells. (C) OA treatment increased the volume of TG in Huh7 cells. (D) Lipid droplets were significantly increased compared with untreated controls (Scale bar=30 µM). (E) The volume of intracellular lipid droplets was also increased as determined through the quantification column. *P<0.05 and **P<0.01. OA, oleic acid; BSA, bovine serum albumin; SREBP-1c, sterol regulatory element binding protein-1c; TG, triglyceride.
LncHR1 suppresses the AKT/SREBP-1c pathway in the cellular model of Steatosis. (A) In the cellular model of the steatosis, overexpressed lncHR1 inhibited SREBP-1c protein levels and the phosphorylation of AKT (Thr308) level was suppressed. Total protein AKT and β-actin used as control. (B) Knockdown of lncHR1 elevated SREBP-1c protein levels and activated the phosphorylation of AKT (Thr308) in the steatosis cells. lncHR1, long non-coding RNA HCV regulated 1; SREBP-1c, sterol regulatory element binding protein-1c; OA, oleic acid.
LncHR1 affects the p-PDK1/AKT/FoxO1/SREBP-1c pathway. (A) IGF-1, an activator of PI3K/AKT, simultaneously increased SREBP-1c levels and the phosphorylation level of AKT (Thr308) and FoxO1 (Ser256). This elevated phosphorylation was significantly reversed by overexpression of lncHR1. IGF-1 induction of the SREBP-1c protein by was also reversed following lncHR1 overexpression. Total AKT and β-actin were used as controls. (B) LY294002, an inhibitor of PI3K/AKT, suppressed AKT (Thr308) and FoxO1 (Ser256) phosphorylation compared to AKT and β-actin. LncHR1 knockdown rescued LY2940002 inhibition of AKT and FoxO1 phosphorylation, as well as SREBP-1c protein levels. (C) The phosphorylation level of PDK1 at Ser241 site was suppressed by lncHR1 overexpression. β-actin was used as a loading control. (D) LncHR1 knockdown activated the phosphorylation level of PDK1 (Ser241). β-actin was used as a loading control. lncHR1, long non-coding RNA HCV regulated 1; SREBP-1c, sterol regulatory element binding protein-1c; FoxO1, forkhead Box O1; PDK1, 3-phosphoinositide-dependent protein kinase 1.
LncHR1 regulated the distribution of FoxO1 inside and outside of the nucleus. LncHR1 overexpression or knockdown plasmids were transfected into Huh7 cells. After 48 h post-transfection, the nuclear protein fraction was separated for western blotting. In the nucleus, FoxO1 was increased by lncHR1 overexpression (A) and was reduced after lncHR1 knockdown. (B) Immunofluorescence staining showed that FoxO1 was increased in the nucleus by lncHR1 overexpression. (C) The accumulation of FoxO1 in the nucleus was reduced by knockdown lncHR1 (Scale bar=20 µM). lncHR1, long non-coding RNA HCV regulated 1; FoxO1, forkhead Box O1.