Pro-inflammation NF-κB signaling triggers a positive feedback via enhancing cholesterol accumulation in liver cancer cells
- Mingyan He†1,
- Wenhui Zhang†2,
- Yinying Dong†1,
- Lishun Wang3,
- Tingting Fang1,
- Wenqing Tang1,
- Bei Lv1,
- Guanglang Chen3,
- Biwei Yang1,
- Peixin Huang1 and
- Jinglin Xia1, 3Email author
© The Author(s). 2017
Received: 7 October 2016
Accepted: 4 January 2017
Published: 18 January 2017
Hepatocellular carcinoma (HCC) develops in a complex microenvironment characterized by chronic inflammation. In recent years, cholesterol metabolic abnormalities have been implicated the importance in cancer cell physiology. This study was designed to investigate the relationship between inflammation and cholesterol accumulation in HCC cells.
Human HCC cells HepG2 and Huh7 were cultured and stimulated with lipopolysaccharide (LPS) for 24 h. The changes of HCC cells related to cholesterol metabolism including intracellular cholesterol concentrations, cholesterol uptake, and the expression of cholesterol-related genes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), LDL receptor (LDLR), sterol regulatory element-binding transcription factor 2 (SREBF2), and proprotein convertase subtilisin/kexin 9 (PCSK9) were comparatively analyzed. Simultaneously, the effects of nuclear factor-kappa B (NF-κB) signaling pathway on cholesterol metabolism were clarified by knocking-down of nuclear factor kappa-B kinase subunit alpha (IKKα) and TGF-beta-activated kinase 1 and MAP3K7-binding protein 3 (TAB3) via RNAi and microRNA (miR)-195. Subsequently, the roles of cholesterol accumulation in LPS induced pro-inflammatory effects were further investigated.
Pro-inflammatory factor LPS significantly increased intracellular cholesterol accumulation by upregulating the expression of HMGCR, LDLR, and SREBF2, while downregulating the expression of PCSK9. These effects were revealed to depend on NF-κB signaling pathway by knocking-down and overexpression of IKKα and TAB3. Additionally, miR-195, a regulator directly targeting IKKα and TAB3, blocked the effects of cholesterol accumulation, further supporting the critical role of pro-inflammation NF-κB signaling in regulating cholesterol accumulation. Intriguingly, the accumulation of cholesterol conversely exerted an augmented pro-inflammation effects by further activating NF-κB signaling pathway.
These results indicated that pro-inflammation effects of NF-κB signaling could be augmented by a positive feedback via enhancing the cholesterol accumulation in liver cancer cells.
KeywordsLipopolysaccharide Nuclear factor-kappa B Cholesterol accumulation microRNA-195 Hepatocellular carcinoma
Metabolic reprogramming in the uncontrolled proliferation of cancer cells has been thought to play important roles in cancer biology . Recently, many studies have demonstrated that cholesterol accumulate in a series of human cancers, including breast , colon , prostate , HCC , and others . In addition, enzymes involved in cholesterol metabolism have been reported abnormal expression in cancer tissues [7, 8]. For instance, cholesterol acyltransferase (ACAT)2, is found to be induced and promotes esterification of excess oxysterols for secretion to avoid cytotoxicity in a subset of hepatocellular carcinomas (HCCs) for tumor growth , suggestive of a specific cholesterol metabolic pathway in HCCs. On the one hand, cholesterol is needed for the synthesis of membranes, signaling molecules, lipid raft formation, and other factors to support the rapid growth of tumor cells . On the other hand, cholesterol oxidized products, namely oxysterols, exhibit inhibitory activities in cell growth and promote cell apoptosis and dampen antitumor responses [10–12]. However, mechanism and pathological significance underlying the aberrant cholesterol metabolism are still elusive.
Epidemiological studies have shown that 80% of HCCs develop in fibrotic or cirrhotic livers as a consequence of chronic liver injury [13, 14]. The NF-κB pathway, the key link of inflammatory responses, plays an important role in HCC promotion by increasing proliferation and preventing apoptosis . In the inactive state, NF-κB transcription factors are complexed in the cytoplasm, either with members of the inhibitor of κB (IκB) family (in the canonical pathway) or with the NF-κB precursor p100 (in the non-canonical pathway). Lipopolysaccharide (LPS), a component of the gram-negative bacterial wall, could activate NF-κB pathway and stimulate inflammatory responses. Previous studies have demonstrated that LPS/NF-κB signaling pathway promotes atherosclerotic progression and macrophage foam cell formation by increasing intracellular cholesterol accumulation [16, 17]. These reports inspired us to hypothesize whether there is a crosstalk between NF-κB signaling and cholesterol metabolism in cancer cells.
In the present study, we investigated the relationship between inflammation and cholesterol metabolism, and found that pro-inflammatory factor Lipopolysaccharide (LPS) increased intracellular cholesterol accumulation through NF-κB pathway in HCC cells. Cholesterol accumulation conversely promoted LPS/NF-κB pathway and inflammatory responses.
Human HCC cells HepG2 and Huh7 were purchased from Cell Bank of the institute of Biochemistry and Cell Biology, China Academy of Sciences, Shanghai, China. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (Gibco, BRL, USA), 10ug/mL streptomycin sulfate and 100ug/mL penicillin G. All cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2.
LDL was purchased from AngYu Biotechnologies (Shanghai, China). It is isolated from blood bank produced human plasma. It is purified via ultracentrifugation to homogeneity determined by agarose gel electrophoresis. Each lot is analyzed on agarose gel electrophoresis for migration versus LDL.
Measurement of intracellular cholesterol
Total cellular cholesterol measurement was determined with a detection kit from Applygen (Peking, China) according to the manufacturer’s protocol. Intracellular lipids were extracted using a chloroform-methanol (2:1) mix and dried under vacuum. Total cholesterol contents were measured by an enzymatic assay. The value was normalized against total protein concentration from each sample, as determined with Bicinchoninic Acid assay (Beyotime biotechnology, China).
The low density lipoprotein labeled with 1,1′-Dioctadecyl-3,3,3′,3′-tetramethyl- indocarbocyanideperchlorate (DIL-LDL) (Biomedical Technologies Inc., MA, USA) uptake represented the ability of cell cholesterol uptake. HCC cells were treated with different addressment, then changed to serum-free medium and incubated with 10ug/ml DIL-LDL at 37 °C for 5 h. The cells were detached by trypsin, washed and suspended by PBS and analyzed by flow cytometry using FL2 emission filter (FACScan, BD Biosciences, San Jose, CA, USA). Data were analyzed with FlowJo software.
Total RNA isolation and quantitative real-time quantitative PCR
Primers for PCR used in this study
Forward primer (5′ → 3′)
Reverse primer (5′ → 3′)
HCC cells were lysed in cell lysis buffer containing 1nM PMSF for 30 min at 4 °C. Lysates were collected by centrifugation at 12,000 rpm for 30 min at 4 °C. Proteins from cell lysates were separated on the SDS-PAGE and transferred onto PVDF membrane (Immobion-P Transfer Membrane, Millipore Corp., Billerica, MA, USA). The membrane was blocked with TBST containing 5% non-fat dry milk for 1 h and further incubated overnight at 4 °C with primary antibodies against PCSK9, LDLR, HMGCR, SREBF2 (Abcam, Cambridge, MA, USA), NF-κB p65, phospho-NF-κB p65, IKKα, TAB3 (Cell Signaling Technology, USA) and β-actin (Santa Cruz, CA, USA). After that, the membrane was incubated with horseradish peroxidase (HRP)-linked secondary antibodies (Santa Cruz Biotechnology, USA) for 2 h at room temperature. All protein bands were visualized using an electrochemiluminescence kit (Thermo, USA). Intensity of each protein band was quantified by Quantity One 4.6.2 software (Bio Rad).
MiRNA inhibitor or mimic transfection
For transfections in 12-well plates, the HCC cells were seeded at a density of 1 × 105 cells/well and incubated overnight and then transfected with 50 nM miR-195 mimic or 100 nM miR-195 inhibitor (GenePharma, Shanghai, China) using lipo2000 Transfection Agent (Invitrogen, USA) according to the manufacturer’s instructions. The corresponding negative sequence of mimic or inhibitors (GenePharma, Shanghai, China) were transfected with the same concentration as controls. At 24 h after the transfection, cells were harvested or further incubated with LPS (1000 ng/mL) for the following experiments.
The sequences of siRNA used in this study
Sense Primer Sequence (5′ → 3′)
Anti-sense Primer Sequence (3′ → 5′)
HCC cells were stimulated with LPS (1000 ng/ml) for 24 h in serum-free medium in the presence or absence of LDL (200ug/ml). The concentrations of IL-8 and TNF-α in culture supernatants were measured with ELISA kits (R&D Systems, USA) according to the manufacturer’s instruction. Absorbance was recorded at 450 nm using a microplate reader.
Data analysis was performed using SPSS16.0 statistical software (SPSS Inc, Chicago, IL). Student’s t-tests were used to assess significant differences among study groups. P < 0.05 was considered statistically significant. All experiments have been performed at least three times.
Role of NF-κB signaling in cholesterol accumulation
IKKα and TAB3 regulates LPS-induced cholesterol accumulation
MiR-195 regulates cholesterol accumulation and cholesterol-related gene expression induced by LPS
Cholesterol increases LPS induced pro-inflammatory effects
MicroRNAs (miRNAs) are a class of small non-coding RNAs, which repress translation or induce cleavage of target mRNAs by base pairing with their 3′ untranslated regions. MiR-195 is a member of the miR-15/16/195/424/497 family . It has been reported that miR-195 suppresses cancer development and is downregulated in multiple types of cancer, such as prostate [25, 26], lung , osteosarcoma , HCCs , and so forth. Genome-wide screening suggests that miR-195 mediates NF-κB activity by directly targeting the expression of IKKα TAB3 in HCCs . Here, we demonstrated that not only miR-195 but also its targeted genes TAB3 and IKKα mediate LPS/NF-κB-induced cholesterol accumulation and cholesterol-related gene expression, providing further evidence that NF-κB pathway play an important role in HCC intracellular cholesterol accumulation.
PCSK9, the ninth member of the proteinase K subfamily of subtilases, plays an important role in post-transcriptional degradation of LDLR, which decreases intracellular cholesterol uptake [30–32]. Decreased PCSK9 and increased LDLR expression have been demonstrated in HCC tissues, supporting a constant cholesterol supply in the HCC microenvironment . HMGCR is the rate-limiting enzyme in de novo synthesis of cholesterol in vivo. Recent studies have reported that HMGCR is upregulated in several types of cancer including gastric , ovarian  and breast cancers , suggesting that HMGCR plays an oncogenic role. SREBF2 is a membrane-bound transcription factor that regulates cholesterol homeostasis in cells. It has been demonstrated that PCSK9, LDLR, and HMGCR expression are co-regulated by SREBF2 [36–38]. When cholesterol levels fall, SREBF2 is activated to up-regulate the expression of genes responsible for cholesterol synthesis, such as HMGCR, and for cholesterol uptake, such as LDLR. In this study, the expression of PCSK9, LDLR, HMGCR, and SREBF2 were investigated in HCC cells after stimulation with LPS. We found that LPS significantly inhibited the expression of PCSK9 and increased LDLR, HMGCR, and SREBF2 expression, suggesting that LPS may increase native LDL cholesterol uptake via LDLR and promote de novo cholesterol synthesis via HMGCR.
There are growing evidences that cholesterol, as an important molecule, impacts upon cancer cell physiology, however, the concrete role of cholesterol in cancer progression remains elusive and controversial. Analyses of the cancer Genome Atlas (TCGA) database revealed a correlation between increased activity of the cholesterol synthesis pathway and decreased survival in patients with sarcoma, acute myeloid leukemia and melanoma [39, 40], supporting the concept that cholesterol promotes carcinogenesis. However, some epidemiological studies have reported objective observation that poor prognosis in HCC patients were linked to decreased serum cholesterol [41, 42]. In this study, we have suggested that cholesterol further activated the NF-κB signaling pathway and promotes the expression of NF-κB target genes, indicating the pro-inflammatory effects of cholesterol in HCC cells.
In summary, we have experimentally demonstrated that LPS/NF-κB signaling pathway triggers an increase in intracellular cholesterol levels by promoting the expression of HMGCR and LDLR in HCC cells. Cholesterol accumulation conversely promotes LPS/NF-κB induced pro-inflammatory effects. MiR-195, as a regulator of NF-κB pathway, inhibited cholesterol accumulation by decreasing the expression of TAB3 and IKKα. These data provide us with a better understanding of the relationship between LPS/NF-κB pathway and cholesterol abnormalities in cancer cells.
Low density lipoprotein labeled with 1,1′-Dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanideperchlorate
Nuclear factor kappa-B kinase subunit alpha
Nuclear factor-kappa B
Proprotein convertase subtilisin/kexin 9
Sterol regulatory element-binding transcription factor 2
TGF-beta-activated kinase 1 and MAP3K7-binding protein 3
This study was sponsored by grants from the National Natural Science Foundation of China (Nos. 81272732 and 81572395), the Shanghai Leading Talent Projects (No. 048, 2013), the Shanghai Leading Academic Discipline Project (Project Number: B115), and the Shanghai Science and Technology Commission (Project Number: 14XD1401100).
Availability of data and materials
JLX and LSW conceived and designed the study. MYH, WHZ, YYD performed the experiments. TTF, WQT, BL, GLC, BWY, PXH analyzed the data. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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