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MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression
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MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression Nan Zhang a, JiaYan Lei a, Han Lei a,n, Xiongzhong Ruan b,d, Qing Liu c, Yaxi Chen b, Wei Huang a a
Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China Centre for Lipid Research, Key Laboratory of Metabolism on Lipid and Glucose, Chongqing Medical University, Chongqing, China c Centre for Clinical Research, The First Affiliated Hospital, Chongqing Medical University, Chongqing, China d John Moorhead Research Laboratory, Centre for Nephrology, University College London Medical School, Royal Free Campus, London, United Kingdom b
art ic l e i nf o
a b s t r a c t
Article history: Received 24 December 2014 Received in revised form 24 May 2015 Accepted 27 May 2015
Background: MicroRNAs play key roles in regulating cholesterol homeostasis. Here, we investigated the role of microRNA-101 (miR-101) in regulating ATP-binding cassette transporter A1 (ABCA1) expression and cholesterol efflux under non-inflammatory and inflammatory conditions in human THP-1-derived macrophages and HepG2 hepatoblastoma cells. Methods: The cell lines were transfected with one of four lentiviral vectors: miR-101, miR-101 control, anti-miR-101, or anti-miR-101 control. A luciferase reporter assay was used to examine miR-101 binding to the 3’ untranslated region (UTR) of ABCA1. Western blotting was conducted to assess ABCA1 protein expression. Cells were loaded with BODIPY-cholesterol and stained with oil red O to assess cholesterol efflux. Results: The luciferase activity assay revealed that wild-type miR-101 binding at site 2 significantly repressed ABCA1 3’ UTR activity, suggesting that miR-101 directly targets the ABCA1 mRNA at site 2. In both cell lines, Western blotting revealed that miR-101 expression negatively regulates ABCA1 protein expression and significantly suppresses cholesterol efflux to ApoA1 under both low-density lipoprotein (LDL) and non-LDL conditions, which was confirmed by pronounced lipid inclusions visible by oil red O staining. In HepG2 cells, both IL-6 and TNF-α treatments produced significant miR-101 overexpression; however, in THP-1-derived macrophages, only IL-6 treatment produced significant miR-101 overexpression. Anti-mir-101 transfection under both IL-6 and TNF-α treatment conditions led to ABCA1 upregulation, indicating that miR-101 expression represses ABCA1 expression under inflammatory conditions. Conclusions: miR-101 promotes intracellular cholesterol retention under inflammatory conditions through suppressing ABCA1 expression and suggests that the miR-101-ABCA1 axis may play an intermediary role in the development of NAFLD and vascular atherosclerosis. & 2015 Elsevier Inc. All rights reserved.
Keywords: microRNA miRNA miR-101 ATP-binding cassette transporter A1 ABCA1 Cholesterol Atherosclerosis Non-alcoholic fatty liver disease NAFLD Inflammation IL-6 TNF-α
1. Introduction Excessive intracellular cholesterol accumulation is a key process underlying the development of both non-alcoholic fatty liver disease (NAFLD) and vascular atherosclerosis [1,2]. Under inflammatory conditions, both hepatocytes and vascular macrophages increase their intake of cholesterol, which can eventually lead to the formation of pathological lesions in the liver parenchyma and vascular wall, respectively [1,2]. In vitro, inflammatory cytokines have been shown to promote intake of lown Correspondence to: Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, 1 Youyi Road, Yuzhong, Chongqing 400016, China. E-mail address: [email protected] (H. Lei).
density lipoprotein (LDL) in HepG2 (human hepatoblastoma) cells, primary liver hepatocytes, THP-1-derived macrophages, and vascular smooth muscle cells [1–4]. Therefore, systemic inflammatory conditions contribute to a maladaptive cellular environment that facilitates cholesterol accumulation that, if unchecked, can lead to NAFLD, atherosclerosis, and more serious downstream conditions such as liver cirrhosis and coronary artery disease (CAD) [5,6]. Therefore, developing a better understanding of the mechanism (s) underlying cholesterol accumulation on a cellular level under inflammatory conditions is crucial. Cholesterol homeostatic equilibrium on a cellular basis is tightly regulated through a balance of intracellular cholesterol biosynthesis, cholesterol intake from the extracellular environment, and cellular cholesterol efflux [2]. A protein named adenosine triphosphate (ATP)-binding membrane cassette transporter
http://dx.doi.org/10.1016/j.yexcr.2015.05.023 0014-4827/& 2015 Elsevier Inc. All rights reserved.
Please cite this article as: N. Zhang, et al., MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.023i
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A1 (ABCA1, ABC1) was initially identified by Langmann et al. to be upregulated in the presence of acetylated LDL (AcLDL), which was reversed by LDL depletion through addition of high-density lipoprotein (HDL) [7]. Specifically, under conditions of intracellular cholesterol overload, ABCA1 is upregulated and mediates the cellular efflux of cholesterol and phospholipids to extracellular apolipoprotein A-I (apoA-I) [2,8,9]. Clinically, mutations in the ABCA1 gene produce Tangier disease – a rare, autosomal recessive disorder characterized by extremely low plasma HDL cholesterol levels, cholesterol accumulation in multiple tissues, peripheral neuropathy, and accelerated atherosclerosis [10]. Moreover, the interaction of apolipoproteins with ABCA1 is known to activate signaling pathways (e.g., JAK2/STAT3, protein kinase A, and Rho family G protein CDC42) that regulate ABCA1-mediated cholesterol efflux, making ABCA1 both a lipid exporter and a signaling receptor [11]. Notably, extracellular inflammatory conditions have been shown to inhibit ABCA1 expression, thereby decreasing ABCA1mediated cholesterol efflux [2]. For example, LXR/RXR agonists that inhibit inflammatory cytokine production have been shown to induce ABCA1 expression, making these agonists potential therapeutic agents for mobilizing cholesterol from tissues under inflammatory conditions [11]. However, the precise mechanism (s) by which ABCA1 expression is regulated on a cellular level under inflammatory conditions remain unclear. Recent studies have shown that several microRNAs (miRNAs) – small, non-coding RNAs that negatively regulate target mRNAs by binding to complementary sites on their 3’ untranslated regions (UTRs) – such as miR-26, miR-33, miR-122, miR-370, and miR-758 play key roles in regulating cholesterol homeostasis [12]. Dysregulation of another human miRNA in particular – hsamiR-101 – has been previously associated with NAFLD [13]. Moreover, miR-101 has been shown to inhibit autophagy, a process known to regulate the availability of free cholesterol for cellular efflux [14,15]. However, miR-101’s role in regulating cellular cholesterol homeostasis remains unclear. Therefore, in this study, we investigated the role of miR-101 in regulating ABCA1 expression and cholesterol efflux under non-inflammatory and inflammatory conditions in human THP-1-derived macrophages and human HepG2 hepatoblastoma cells.
2. Materials and methods 2.1. Cell lines and reagents The human monocyte cell line THP-1 and the human hepatoblastoma cell line HepG2 were obtained from American Type Culture Collection (ATCC, no. TIB-202 and no. HB-8065, respectively). THP-1 was cultured in RPMI 1640 medium containing 10% (v/v) fetal calf serum, 2 mmol/l glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. THP-1 was fully differentiated into macrophages after being triggered with 160 nmol/l phorbol-12myristate-13-acetate (PMA) for 72 h, and the differentiated THP-1 macrophages were washed extensively with phosphate-buffered saline (PBS) prior to use. LDL was isolated from fresh plasma of healthy human volunteers by sequential density gradient ultracentrifugation as described previously [16]. Written informed consents were obtained from all volunteers prior to sampling. Recombinant human interleukin (IL)-6 and tumor necrosis factor (TNF)-α were obtained from SinoBio (Shanghai, China) and PeproTech Asia (Rocky Hill, NJ, USA), respectively.
2.2. Lentiviral transfection Two lentiviral vectors, GV259 (Con-miR) and GV159 (Con-antimiR), were constructed as controls for their corresponding miR101 and anti-miR-101 lentiviral vectors, respectively (Shanghai Ji Kai Gene Chemical Technology Co., Ltd., China). Then, HepG2 cells and THP-1 macrophages at 50–70% confluence were transfected with either miR-101, Con-miR, anti-miR-101, or Con-anti-miR (1 108 TU/ml lentivirus) using the manufacturers’ protocols (Shanghai Ji Kai Gene Chemical Technology Co., Ltd., China). Cells were transfected for 48 h and treated with or without LDL (150 mg/ml) and with either IL-6 (20 ng/ml) or TNF-α (25 ng/ml) for an additional 24 h [17,18]. Then, Western blotting was conducted to assess ABCA1 protein expression with β-actin used as an internal control. 2.3. Western blotting As previously described with minor modifications [19], identical amounts of protein from extracts or nuclear extracts of cultured THP-1 macrophages and HepG2 cells (with or without lentivirus infection) were denatured and then subjected to 6% or 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE; Bio-Rad Laboratories, UK). Electrophoretic transfer to nitrocellulose was performed at 85 V with 250 mA for 3 h. The membrane was then blocked with 5% skimmed milk for 2 h at room temperature and probed with rabbit anti-ABCA1 antibody (1:500 dilution; Abcam, USA) and mouse anti-human β-actin polyclonal antibody (1:2000 dilution; Abcam, USA) in 5% skimmed milk in PBS with 1% Tween at 4 °C overnight. After three 5-min washes in PBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1:4000 dilution; ZSGB-BIO, Beijing, China) at room temperature for 1 h. After rinsing, the membranes were subjected to enhanced chemiluminescence (Amersham Biosciences, USA). With β-actin was used as loading control, the protein band intensities were analyzed by an imaging analysis system to assess relative protein expression. 2.4. 3’ UTR luciferase reporter assays cDNA fragments corresponding to the entire 3’ UTR of human ABCA1 were amplified by reverse transcription-polymerase chain reaction (RT-PCR) from total RNA extracted from HepG2 cells with XhoI and NotI linkers (Jiang Su Beyotime Co. Ltd., China) The PCR products were directionally cloned downstream of the Renilla luciferase open reading frame (ORF) in a pc-DNA3.1 vector (Promega) that also contained a constitutively-expressed firefly luciferase gene used to normalize transfections. Site-directed mutations in the seed region of the predicted miR-101 sites within the 3’ UTR of human ABCA1 were generated using Multisite-Quickchange (Stratagene) according to the manufacturer's protocol. All constructs were confirmed by sequencing. HepG2 cells were plated onto 96-well plates and co-transfected with 0.2 mg of the indicated 3’ UTR luciferase reporter vectors and the miR-101 vector. Luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega). Renilla luciferase activity was normalized to the corresponding firefly luciferase activity and plotted as a percentage of the control (Con-miR). All experiments were independently performed in triplicate. 2.5. Quantitative real-time polymerase chain reaction (RT-PCR) As previously described with minor modifications [20], total RNA was isolated from THP-1 macrophages and HepG2 cells using a RNAiso kit (Takara, Dalian, China) according to the
Please cite this article as: N. Zhang, et al., MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.023i
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manufacturer's protocol. Total RNA (1 mg) was used as a reverse transcription template using a PrimeScriptH RT reagent kit (Takara, Dalian, China). RT-PCR was performed using a Sequence Detection System (CFX96TM Real-Time PCR Detection System, BioRad, USA) with Power SYBR Green PCR Master Mix (Takara, Dalian, China). U6 was used as a reference gene. U6, miR-101, and miR33a-5P were analyzed by Bulge-Loop miRNA RT-PCR. The U6, miR101, and miR-33a-5P primers were purchased from Guangzhou RiboBio Co., Ltd. (MQP-0202, miRQ0000099, and miRQ0000091). The 22DDCt method was applied to obtain the relative expression of the genes. The amplification efficiencies of the target and reference were within the 95–100% range. Controls (H2O or samples) that were not reverse-transcribed were negative for both target and reference genes. 2.6. Cholesterol efflux assays As previously described with minor modifications [21,22], we examined apoA-I-mediated cholesterol efflux using boron dipyrromethene difluoride linked to sterol carbon-24 (BODIPY-cholesterol). THP-1-derived macrophages and HepG2 cells (with or without lentivirus) were plated onto 96-well plates and cultured in serum-free medium containing 0.1 ml labeling media (consisting of 0.025 mM BODIPY-cholesterol (Avanti Polar Lipids), 10 mM methyl-b-cyclodextrin (Sigma-Aldrich, USA), and 0.1 mM egg phosphatidylcholine (Avanti Polar Lipids, USA) in MEM-HEPES (Sangon, China)) for 1 h. The cells were washed twice with MEMHEPES and then cultured in serum-free medium containing treatment factors for 18 h. Next, the cells were cultured in serumfree medium containing treatment factors and 10 mg/ml apoA-I (Sigma-Aldrich, USA) for 4 h. Single layer cells were dissolved in 0.1 N NaOH overnight. After centrifugation at 10,000 rpm for 10 min, the supernatant was collected, and the fluorescence intensity value that represented total cholesterol efflux was recorded using a BioTek microplate reader (excitation 482 nm, emission 515 nm). The fluorescence intensity value of the liquid supernatant of cells cultured in serum-free medium without treatment factors and 10 mg/ml apoA-I was applied to represent background cholesterol efflux. Intracellular cholesterol levels were assessed by extraction of intracellular lipids by a chloroform/methanol (2:1) mixture followed by vacuum drying. Total cholesterol (TC) contents were measured by an enzymatic assay normalized by total cellular protein determined by Lowry assay. The concentration of cholesterol ester (CE) was calculated from TC. The apoA-I-mediated cholesterol efflux was calculated as follows: apoA-I-mediated cholesterol efflux¼ (apoA-I-mediated cholesterol efflux/[intracellular cholesterol þTC efflux]) 100%. 2.7. Oil red O staining As previously described with minor modifications [2], THP-1 macrophages and HepG2 cells (with or without lentivirus) were incubated in either serum-free experimental medium or experimental medium. After 24 h of incubation, the cells were washed two times in PBS, fixed for 30 min with 5% formalin solution in PBS, stained with oil red O for 30 min, and counterstained with hematoxylin for another 5 min. Cells were examined by light microscopy (Axio Imager 2, Zeiss, Germany) through six randomlyselected high-power fields (HPF) (4006). Semi-quantitative analysis of oil red O staining was performed using Image-J software. 2.8. Statistical analysis The results were expressed as means 7SEMs. Statgraphics Plus v5.0 (Statistical Graphics, USA) was used for all statistical analyses.
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Statistical comparisons between groups were performed by Student’s t-test or analysis of variance (ANOVA), and post hoc multiple comparisons were performed using the Student–Newman– Keuls test.
3. Results 3.1. ABCA1 expression negatively regulated by miR-101 Western blotting of ABCA1 expression in THP-1-derived macrophages revealed that miR-101 expression negatively regulates ABCA1 protein expression under both LDL and non-LDL conditions (Fig. 1A) with LDL treatment more pronouncedly upregulating ABCA1 expression in the absence of miR-101 (Fig. 1B). Western blotting of ABCA1 expression in HepG2 cells revealed that LDL treatment significantly raised ABCA1 expression, and miR-101 expression negatively regulates ABCA1 expression under LDL conditions (Fig. 1C and D). 3.2. miR-101 targets the 3’ UTR of the ABCA1 mRNA Sequences of the four hsa-miR-101's binding sites on the human ABCA1 3’ UTR are detailed in Fig. 2A. The luciferase activity assay revealed that wild-type (WT) miR-101 binding at site 2 significantly repressed ABCA1 3’ UTR activity (Po 0.05; Fig. 2B), suggesting that miR-101 directly targets site 2 on the ABCA1 mRNA. The luciferase activity assay also revealed that wild-type (WT) Con-miR binding at site 4 significantly increased ABCA1 3’ UTR activity (Po 0.05; Fig. 2B). We attribute this phenomenon to some molecular effect of the Con-MiR on the WT site 4 that served to derepress ABCA1 3’ UTR activity. 3.3. miR-101 suppresses cholesterol efflux Total cholesterol efflux from HepG2 cells was evaluated six hours after incubation with ApoA1. We found that miR-101 significantly suppresses cholesterol efflux to ApoA-1 under both LDL and non-LDL conditions (Po 0.05; Fig. 3A and B). A similar pattern of findings was observed in THP-1-derived macrophages (P o0.05; Fig. 3E and F). In the presence of ApoA-1, miR-101 expression negatively regulates ABCA1 protein expression in HepG2 cells under both LDL and non-LDL conditions (P o0.05; Fig. 3C and D). A similar pattern of findings was observed in THP-1-derived macrophages (P o0.05; Fig. 3G and H). miR-101-transfected THP-1derived macrophages and HepG2 cells displayed pronounced lipid inclusions (Fig. 4A and B), significantly increased Oil red O staining (P o0.05; Fig. 4C and D), and significantly increased intracellular cholesterol content (P o0.05; Fig. 4E and F) relative to Con-miRtransfected counterparts, while anti-miR-101-transfected counterparts displayed the opposite effects. 3.4. Inflammatory conditions increase miR-101 expression and repress ABCA1 expression In HepG2 cells, both IL-6 and TNF-α treatments produced significant miR-101 overexpression (P o0.05; Fig. 5A). However, in THP-1-derived macrophages, only IL-6 treatment produced significant miR-101 overexpression (P o0.05; Fig. 5B). Using miR33a-5P as a previously established positive control under inflammatory conditions [2], both IL-6 and TNF-α treatments produced significant miR-33a-5P overexpression in both cell lines as expected (Po 0.05; Fig. 5C and D). In HepG2 cells, anti-mir-101 transfection under both IL-6 and TNF-α treatment conditions led to significant ABCA1 upregulation, indicating that miR-101 expression represses ABCA1 expression under inflammatory
Please cite this article as: N. Zhang, et al., MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.023i
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Fig. 1. ATP-binding cassette transporter A1 (ABCA1) expression negatively regulated by microRNA (miR)-101. Western blotting of ABCA1 and β-actin expression induced with low-density lipoprotein (LDL) in (A) THP-1-derived macrophages infected with Con-miR or miR-101, (B) THP-1-derived macrophages infected with Con-anti-miR or antimiR-101, (C) HepG2 cells infected with Con-miR or miR-101, and (D) HepG2 cells infected with Con-anti-miR or anti-miR-101. nPo 0.05 (comparison versus Con-miR or Conanti-miR), △Po 0.05 (comparison versus non-LDL or LDL).
conditions (Po0.05; Fig. 5G). A similar pattern of findings were shown in anti-mir-101-transfected THP-1-derived macrophages (P o0.05; Fig. 5H). Accordingly, miR-101 transfection under IL-6 treatment conditions in both cell lines led to significant intracellular cholesterol retention over either miR-101 transfection alone or Il-6 treatment conditions alone, indicating that miR-101 expression increases intracellular cholesterol content under inflammatory conditions (P o0.05; Figs. 5E, F and 6).
4. Discussion Here, we investigated the role of miR-101 in regulating ABCA1 expression and cholesterol efflux under non-inflammatory and inflammatory conditions in human THP-1-derived macrophages and human HepG2 hepatoblastoma cells. First, we found that miR101 directly targeting the ABCA1 3’ UTR in both cell lines (Fig. 2A and B). Next, we found that miR-101 suppresses cholesterol efflux to ApoA-1 under both LDL and non-LDL conditions (Fig. 3A, B, E and F) and negatively regulates ABCA1 expression (Fig. 3C, D, G and H). More specifically, this negative regulation of ABCA1
expression by miR-101 was found to suppress cholesterol efflux, which led to pronounced lipid inclusions (Fig. 4A and B), increased Oil red O staining (Fig. 4C and D), and increased intracellular cholesterol retention (Fig. 4E and F) in both cell lines. Lastly, we discovered that inflammatory conditions produce miR-101 overexpression (Fig. 5A and B) and suppressed ABCA1 expression (Fig. 5G and H) in both cell lines. Accordingly, miR-101 transfection under IL-6 treatment conditions in both cell lines led to significant intracellular cholesterol retention over either miR-101 transfection alone or Il-6 treatment conditions alone (Fig. 5E and F). These combined findings reveal that miR-101 overexpression under inflammatory conditions promotes intracellular cholesterol retention through suppressing ABCA1 expression and suggests that the miR-101-ABCA1 axis may play an intermediary role in the development of NAFLD and vascular atherosclerosis. In addition to its role in regulating ABCA1 expression in human HepG2 hepatoblastoma cells, miR-101 has been previously associated with regulating several other proteins and pathways in liver cells and macrophages. For example, miR-101 has been identified as a tumor suppressor in hepatocellular carcinoma (HCC) cells that represses several oncogenes (including STMN1, JUNB, and CXCR7)
Please cite this article as: N. Zhang, et al., MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.023i
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Fig. 2. MicroRNA (miR)-101 targets the 3’ untranslated region (UTR) of the ATP-binding cassette transporter A1 (ABCA1) mRNA. (A) Sequence alignment of the human hsamiR-101 mature sequence with the four binding sites of the human ABCA1 3’ UTR. Relative positions of the four binding sites are indicated. (B) Activity of the luciferase reporter construct infected with Con-miR or miR-101 along with wild-type (WT) or site-directed mutations (SDM) in the miR-101 target sites of the ABCA1 3’ UTR. Luciferase activity was normalized with Renilla. Relative luciferase activity expressed as means 7 SEMs from six independent experiments. nPo 0.05 (comparison versus other site 2 groups), #Po 0.05 (comparison versus other site 4 groups). WT: wild-type.
and further increases expression of endogenous miR-101 through inhibiting PRC2 activation [23]. In addition, miR-101 has been identified as an anti-fibrotic miRNA with miR-101 expression significantly blunting pro-fibrotic TGF-β signaling in hepatic stem cells (HSCs) and hepatocytes [24]. In macrophages, miR-101 has been identified as a pro-apoptotic, pro-inflammatory miRNA that represses anti-apoptotic mitogen-activated protein kinase phosphatase-1 (MKP-1) expression as well as, in response to pro-inflammatory lipopolysaccharide (LPS), increases supernatant levels of TNF-α, nitric oxide (NO), IL-1β, and IL-6 [25,26]. Based on these findings, future studies on miR-101's regulation of lipid retention in liver cells and macrophages should control for changes in these other proteins, as these proteins may also influence ABCA1
expression or other lipid retention pathways. Aside from miR-101, several other miRNAs have been identified as negative regulators of ABCA1 expression in hepatocytes. For example, miR-758 also negatively regulates the expression of ABCA1 in hepatocytes, thereby attenuating cholesterol efflux to apoA-1 [27]. Moreover, in a mouse model, delivery of miR-144 oligonucleotides has been shown to suppress hepatic ABCA1 expression and plasma high-density lipoprotein (HDL) levels, while miR-144 silencing increases ABCA1 expression and plasma HDL levels [28]. Additionally, in HepG2 cells, miR-128-2 and miR-145 have been shown to significantly decrease ABCA1 expression and cholesterol efflux with this suppressive effect being rescued by their respective anti-miRs [29,30]. As all the foregoing miRNAs
Please cite this article as: N. Zhang, et al., MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.023i
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Fig. 3. MicroRNA (miR)-101 suppresses cholesterol efflux by HepG2 and THP-1-derived macrophages. Total cholesterol efflux from HepG2 were evaluated six hours after incubation with apolipoprotein A1 (ApoA-1) in the presence of (A) Con-miR-101 or miR-101 or (B) Con-anti-miR-101 or anti-miR-101. In the presence of ApoA-1, transfection of low-density lipoprotein (LDL)-treated HepG2 cells using (C) Con-miR or miR-101 or (D) Con-anti-miR or anti-miR-101 modifies ATP-binding cassette transporter A1 (ABCA1) expression. Total cholesterol efflux from THP-1-derived macrophages were evaluated six hours after incubation with ApoA-1 in the presence of (E) Con-miR-101 or miR-101 and (F) Con-anti-miR-101 or anti-miR-101. Transfection of LDL-treated THP-1-derived macrophages using (G) Con-miR or miR-101 or (H) Con-anti-miR or anti-miR101 modifies ABCA1 expression. Data expressed as means 7 SEMs of six independent experiments. nPo 0.05 (comparison versus Con-miR or Con-anti-miR), △Po 0.05 (comparison versus LDL).
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Fig. 4. Oil red O staining of THP-1-derived macrophages. (A) Representative images of THP-1-derived macrophages examined for lipid inclusions by oil red O staining (400 ). (B) Representative images of HepG2 cell examined for lipid inclusions by Oil red O staining (400 ). (C) miR-101 cells displayed higher Oil red O staining relative to THP-1-derived macrophages with Con-miR and anti-miR-101. (D) miR-101 cells displayed higher oil red O staining relative to HepG2 cell with Con-miR and anti-miR-101. (E) Effects of miR-101 and anti-miR-101 on intracellular cholesterol content in THP-1-derived macrophages. (F) Effects of miR-101 and anti-miR-101 on intracellular cholesterol content in HepG2 cells. Data expressed as means 7SEMs of six independent experiments. nPo 0.05 (comparison versus Con-miR or Con-anti-miR), #Po 0.05 (comparison versus miR-101).
have been shown to negatively regulate ABCA1 expression in hepatocytes, miRNA antisense oligonucleotide (ASO)-based therapeutics targeting NAFLD should endeavor to simultaneously target multiple miRNAs in order maximize therapeutic efficacy. Several other miRNAs have been identified as negative regulators of ABCA1 expression in THP-1-derived macrophages. For example, in THP-1-derived macrophages, a miR-144-3p mimic has been shown to inhibit cholesterol efflux via suppressing ABCA1, resulting in decreased HDL-C circulation and accelerated atherosclerosis in apoE / mice [31]. Moreover, miR-33a also downregulates ABCA1 expression in THP-1-derived macrophages, resulting in decreases in circulating HDL-C levels [32]. In THP-1derived macrophages, miR-27a/b has also been shown to affect the efflux, influx, esterification, and hydrolysis of cholesterol through regulating ABCA1, apoA-1, lipoprotein lipase (LPL), CD36, and ACAT1 expression [33]. In PPARγ-activated THP-1-derived macrophages, miR-613 has been shown to suppress LXRα and ABCA1 expression, thereby inhibiting cholesterol efflux [34]. In THP-1derived macrophages, miR-19b has also been shown to suppress ApoA-I-mediated ABCA1-dependent cholesterol efflux; more notably, treatment with inhibitory miR-19b ASO was shown to reverse the suppression of ABCA1 expression as well as reversing the following indicators of atherosclerotic progression: decreases in
plasma HDL levels, increases in plasma low-density lipoprotein (LDL) levels, increases in aortic plaque size and lipid content, and reductions in collagen content [35]. As all the foregoing miRNAs have been shown to negatively regulate ABCA1 expression in THP1-derived macrophages, miRNA ASO-based therapeutics targeting vascular atherosclerosis should endeavor to simultaneously target multiple miRNAs in order maximize therapeutic efficacy. There are several limitations to this study. First, although this study clearly demonstrates that mir-101 negatively regulates ABCA1 expression and cholesterol efflux through directly targeting the ABCA1 3’ UTR (with a more pronounced effect under inflammatory conditions), we did not conduct animal model experiments to assess the in vivo effects of miR-101 on plasma HDL and LDL levels, arterial and hepatic plaque size, arterial and hepatic lipid content, and arterial and hepatic collagen content. Second, although we determined that IL-6 and TNF-α treatment produced miR-101 overexpression and suppressed ABCA1 expression in both THP-1-derived macrophages and HepG2 hepatoblastoma cells, we did not examine the signal transduction mechanism(s) underlying this phenomenon, which may be different in these two cell lines. Third, we did not examine miR-101’s effects upon the expression of other proteins that may have also influenced ABCA1 expression or other lipid retention pathways.
Please cite this article as: N. Zhang, et al., MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.023i
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Fig. 5. Inflammatory conditions increase microRNA (miR)-101 expression and repress ATP-binding cassette transporter A1 (ABCA1) expression. miR-101 overexpression in (A) HepG2 cells and (B) THP-1-derived macrophages treated with either TNF-α or IL-6. miR-33-5p control overexpression in (C) HepG2 cells and (D) THP-1-derived macrophages treated with either TNF-α or IL-6. Effects of IL-6 and miR-101 on intracellular cholesterol content in (E) HepG2 cells and (F) THP-1-derived macrophages under nonLDL and LDL conditions. ABCA1 expression in (G) HepG2 cells and (H) THP-1-derived macrophages transfected with anti-miR-101 and either TNF-α or IL-6. Data expressed as means 7 SEMs of six independent experiments.nPo 0.05 (comparison versus control), #Po 0.05 (comparison versus IL-6 þmiR-101), ^Po 0.05 (comparison versus IL-6), & P o0.05 (comparison versus TNF-α).
Future studies should aim at addressing these limitations. In conclusion, this study reveals that miR-101 promotes intracellular cholesterol retention under inflammatory conditions through suppressing ABCA1 expression. These findings suggest that the miR-101-ABCA1 axis may play an intermediary role in the development of NAFLD and vascular atherosclerosis.
Author contributions Conceived and designed the study: NZ, XZR, and HL. Performed the experiments: NZ,YXCand JYL. Analyzed the data: YXC and QL. Drafted the manuscript: NZ and WH. Modified the manuscript: XZR.
Please cite this article as: N. Zhang, et al., MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.023i
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Fig. 6. Confirmation of lentiviral transfection by fluorescence microscopy. Light and fluorescence microscopy of green fluorescent protein (GFP) expression in (A) THP-1 cells (pre-macrophage) infected with lentivirus, (B) THP-1-derived macrophages infected with lentivirus, and (C) HepG2 cells infected with lentivirus. All images captured at 100 magnification.
Conflicts of interest None.
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MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression Nan Zhang a, JiaYan Lei a, Han Lei a,n, Xiongzhong Ruan b,d, Qing Liu c, Yaxi Chen b, Wei Huang a a
Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China Centre for Lipid Research, Key Laboratory of Metabolism on Lipid and Glucose, Chongqing Medical University, Chongqing, China c Centre for Clinical Research, The First Affiliated Hospital, Chongqing Medical University, Chongqing, China d John Moorhead Research Laboratory, Centre for Nephrology, University College London Medical School, Royal Free Campus, London, United Kingdom b
art ic l e i nf o
a b s t r a c t
Article history: Received 24 December 2014 Received in revised form 24 May 2015 Accepted 27 May 2015
Background: MicroRNAs play key roles in regulating cholesterol homeostasis. Here, we investigated the role of microRNA-101 (miR-101) in regulating ATP-binding cassette transporter A1 (ABCA1) expression and cholesterol efflux under non-inflammatory and inflammatory conditions in human THP-1-derived macrophages and HepG2 hepatoblastoma cells. Methods: The cell lines were transfected with one of four lentiviral vectors: miR-101, miR-101 control, anti-miR-101, or anti-miR-101 control. A luciferase reporter assay was used to examine miR-101 binding to the 3’ untranslated region (UTR) of ABCA1. Western blotting was conducted to assess ABCA1 protein expression. Cells were loaded with BODIPY-cholesterol and stained with oil red O to assess cholesterol efflux. Results: The luciferase activity assay revealed that wild-type miR-101 binding at site 2 significantly repressed ABCA1 3’ UTR activity, suggesting that miR-101 directly targets the ABCA1 mRNA at site 2. In both cell lines, Western blotting revealed that miR-101 expression negatively regulates ABCA1 protein expression and significantly suppresses cholesterol efflux to ApoA1 under both low-density lipoprotein (LDL) and non-LDL conditions, which was confirmed by pronounced lipid inclusions visible by oil red O staining. In HepG2 cells, both IL-6 and TNF-α treatments produced significant miR-101 overexpression; however, in THP-1-derived macrophages, only IL-6 treatment produced significant miR-101 overexpression. Anti-mir-101 transfection under both IL-6 and TNF-α treatment conditions led to ABCA1 upregulation, indicating that miR-101 expression represses ABCA1 expression under inflammatory conditions. Conclusions: miR-101 promotes intracellular cholesterol retention under inflammatory conditions through suppressing ABCA1 expression and suggests that the miR-101-ABCA1 axis may play an intermediary role in the development of NAFLD and vascular atherosclerosis. & 2015 Elsevier Inc. All rights reserved.
Keywords: microRNA miRNA miR-101 ATP-binding cassette transporter A1 ABCA1 Cholesterol Atherosclerosis Non-alcoholic fatty liver disease NAFLD Inflammation IL-6 TNF-α
1. Introduction Excessive intracellular cholesterol accumulation is a key process underlying the development of both non-alcoholic fatty liver disease (NAFLD) and vascular atherosclerosis [1,2]. Under inflammatory conditions, both hepatocytes and vascular macrophages increase their intake of cholesterol, which can eventually lead to the formation of pathological lesions in the liver parenchyma and vascular wall, respectively [1,2]. In vitro, inflammatory cytokines have been shown to promote intake of lown Correspondence to: Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, 1 Youyi Road, Yuzhong, Chongqing 400016, China. E-mail address: [email protected] (H. Lei).
density lipoprotein (LDL) in HepG2 (human hepatoblastoma) cells, primary liver hepatocytes, THP-1-derived macrophages, and vascular smooth muscle cells [1–4]. Therefore, systemic inflammatory conditions contribute to a maladaptive cellular environment that facilitates cholesterol accumulation that, if unchecked, can lead to NAFLD, atherosclerosis, and more serious downstream conditions such as liver cirrhosis and coronary artery disease (CAD) [5,6]. Therefore, developing a better understanding of the mechanism (s) underlying cholesterol accumulation on a cellular level under inflammatory conditions is crucial. Cholesterol homeostatic equilibrium on a cellular basis is tightly regulated through a balance of intracellular cholesterol biosynthesis, cholesterol intake from the extracellular environment, and cellular cholesterol efflux [2]. A protein named adenosine triphosphate (ATP)-binding membrane cassette transporter
http://dx.doi.org/10.1016/j.yexcr.2015.05.023 0014-4827/& 2015 Elsevier Inc. All rights reserved.
Please cite this article as: N. Zhang, et al., MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.023i
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A1 (ABCA1, ABC1) was initially identified by Langmann et al. to be upregulated in the presence of acetylated LDL (AcLDL), which was reversed by LDL depletion through addition of high-density lipoprotein (HDL) [7]. Specifically, under conditions of intracellular cholesterol overload, ABCA1 is upregulated and mediates the cellular efflux of cholesterol and phospholipids to extracellular apolipoprotein A-I (apoA-I) [2,8,9]. Clinically, mutations in the ABCA1 gene produce Tangier disease – a rare, autosomal recessive disorder characterized by extremely low plasma HDL cholesterol levels, cholesterol accumulation in multiple tissues, peripheral neuropathy, and accelerated atherosclerosis [10]. Moreover, the interaction of apolipoproteins with ABCA1 is known to activate signaling pathways (e.g., JAK2/STAT3, protein kinase A, and Rho family G protein CDC42) that regulate ABCA1-mediated cholesterol efflux, making ABCA1 both a lipid exporter and a signaling receptor [11]. Notably, extracellular inflammatory conditions have been shown to inhibit ABCA1 expression, thereby decreasing ABCA1mediated cholesterol efflux [2]. For example, LXR/RXR agonists that inhibit inflammatory cytokine production have been shown to induce ABCA1 expression, making these agonists potential therapeutic agents for mobilizing cholesterol from tissues under inflammatory conditions [11]. However, the precise mechanism (s) by which ABCA1 expression is regulated on a cellular level under inflammatory conditions remain unclear. Recent studies have shown that several microRNAs (miRNAs) – small, non-coding RNAs that negatively regulate target mRNAs by binding to complementary sites on their 3’ untranslated regions (UTRs) – such as miR-26, miR-33, miR-122, miR-370, and miR-758 play key roles in regulating cholesterol homeostasis [12]. Dysregulation of another human miRNA in particular – hsamiR-101 – has been previously associated with NAFLD [13]. Moreover, miR-101 has been shown to inhibit autophagy, a process known to regulate the availability of free cholesterol for cellular efflux [14,15]. However, miR-101’s role in regulating cellular cholesterol homeostasis remains unclear. Therefore, in this study, we investigated the role of miR-101 in regulating ABCA1 expression and cholesterol efflux under non-inflammatory and inflammatory conditions in human THP-1-derived macrophages and human HepG2 hepatoblastoma cells.
2. Materials and methods 2.1. Cell lines and reagents The human monocyte cell line THP-1 and the human hepatoblastoma cell line HepG2 were obtained from American Type Culture Collection (ATCC, no. TIB-202 and no. HB-8065, respectively). THP-1 was cultured in RPMI 1640 medium containing 10% (v/v) fetal calf serum, 2 mmol/l glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. THP-1 was fully differentiated into macrophages after being triggered with 160 nmol/l phorbol-12myristate-13-acetate (PMA) for 72 h, and the differentiated THP-1 macrophages were washed extensively with phosphate-buffered saline (PBS) prior to use. LDL was isolated from fresh plasma of healthy human volunteers by sequential density gradient ultracentrifugation as described previously [16]. Written informed consents were obtained from all volunteers prior to sampling. Recombinant human interleukin (IL)-6 and tumor necrosis factor (TNF)-α were obtained from SinoBio (Shanghai, China) and PeproTech Asia (Rocky Hill, NJ, USA), respectively.
2.2. Lentiviral transfection Two lentiviral vectors, GV259 (Con-miR) and GV159 (Con-antimiR), were constructed as controls for their corresponding miR101 and anti-miR-101 lentiviral vectors, respectively (Shanghai Ji Kai Gene Chemical Technology Co., Ltd., China). Then, HepG2 cells and THP-1 macrophages at 50–70% confluence were transfected with either miR-101, Con-miR, anti-miR-101, or Con-anti-miR (1 108 TU/ml lentivirus) using the manufacturers’ protocols (Shanghai Ji Kai Gene Chemical Technology Co., Ltd., China). Cells were transfected for 48 h and treated with or without LDL (150 mg/ml) and with either IL-6 (20 ng/ml) or TNF-α (25 ng/ml) for an additional 24 h [17,18]. Then, Western blotting was conducted to assess ABCA1 protein expression with β-actin used as an internal control. 2.3. Western blotting As previously described with minor modifications [19], identical amounts of protein from extracts or nuclear extracts of cultured THP-1 macrophages and HepG2 cells (with or without lentivirus infection) were denatured and then subjected to 6% or 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE; Bio-Rad Laboratories, UK). Electrophoretic transfer to nitrocellulose was performed at 85 V with 250 mA for 3 h. The membrane was then blocked with 5% skimmed milk for 2 h at room temperature and probed with rabbit anti-ABCA1 antibody (1:500 dilution; Abcam, USA) and mouse anti-human β-actin polyclonal antibody (1:2000 dilution; Abcam, USA) in 5% skimmed milk in PBS with 1% Tween at 4 °C overnight. After three 5-min washes in PBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1:4000 dilution; ZSGB-BIO, Beijing, China) at room temperature for 1 h. After rinsing, the membranes were subjected to enhanced chemiluminescence (Amersham Biosciences, USA). With β-actin was used as loading control, the protein band intensities were analyzed by an imaging analysis system to assess relative protein expression. 2.4. 3’ UTR luciferase reporter assays cDNA fragments corresponding to the entire 3’ UTR of human ABCA1 were amplified by reverse transcription-polymerase chain reaction (RT-PCR) from total RNA extracted from HepG2 cells with XhoI and NotI linkers (Jiang Su Beyotime Co. Ltd., China) The PCR products were directionally cloned downstream of the Renilla luciferase open reading frame (ORF) in a pc-DNA3.1 vector (Promega) that also contained a constitutively-expressed firefly luciferase gene used to normalize transfections. Site-directed mutations in the seed region of the predicted miR-101 sites within the 3’ UTR of human ABCA1 were generated using Multisite-Quickchange (Stratagene) according to the manufacturer's protocol. All constructs were confirmed by sequencing. HepG2 cells were plated onto 96-well plates and co-transfected with 0.2 mg of the indicated 3’ UTR luciferase reporter vectors and the miR-101 vector. Luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega). Renilla luciferase activity was normalized to the corresponding firefly luciferase activity and plotted as a percentage of the control (Con-miR). All experiments were independently performed in triplicate. 2.5. Quantitative real-time polymerase chain reaction (RT-PCR) As previously described with minor modifications [20], total RNA was isolated from THP-1 macrophages and HepG2 cells using a RNAiso kit (Takara, Dalian, China) according to the
Please cite this article as: N. Zhang, et al., MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.023i
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manufacturer's protocol. Total RNA (1 mg) was used as a reverse transcription template using a PrimeScriptH RT reagent kit (Takara, Dalian, China). RT-PCR was performed using a Sequence Detection System (CFX96TM Real-Time PCR Detection System, BioRad, USA) with Power SYBR Green PCR Master Mix (Takara, Dalian, China). U6 was used as a reference gene. U6, miR-101, and miR33a-5P were analyzed by Bulge-Loop miRNA RT-PCR. The U6, miR101, and miR-33a-5P primers were purchased from Guangzhou RiboBio Co., Ltd. (MQP-0202, miRQ0000099, and miRQ0000091). The 22DDCt method was applied to obtain the relative expression of the genes. The amplification efficiencies of the target and reference were within the 95–100% range. Controls (H2O or samples) that were not reverse-transcribed were negative for both target and reference genes. 2.6. Cholesterol efflux assays As previously described with minor modifications [21,22], we examined apoA-I-mediated cholesterol efflux using boron dipyrromethene difluoride linked to sterol carbon-24 (BODIPY-cholesterol). THP-1-derived macrophages and HepG2 cells (with or without lentivirus) were plated onto 96-well plates and cultured in serum-free medium containing 0.1 ml labeling media (consisting of 0.025 mM BODIPY-cholesterol (Avanti Polar Lipids), 10 mM methyl-b-cyclodextrin (Sigma-Aldrich, USA), and 0.1 mM egg phosphatidylcholine (Avanti Polar Lipids, USA) in MEM-HEPES (Sangon, China)) for 1 h. The cells were washed twice with MEMHEPES and then cultured in serum-free medium containing treatment factors for 18 h. Next, the cells were cultured in serumfree medium containing treatment factors and 10 mg/ml apoA-I (Sigma-Aldrich, USA) for 4 h. Single layer cells were dissolved in 0.1 N NaOH overnight. After centrifugation at 10,000 rpm for 10 min, the supernatant was collected, and the fluorescence intensity value that represented total cholesterol efflux was recorded using a BioTek microplate reader (excitation 482 nm, emission 515 nm). The fluorescence intensity value of the liquid supernatant of cells cultured in serum-free medium without treatment factors and 10 mg/ml apoA-I was applied to represent background cholesterol efflux. Intracellular cholesterol levels were assessed by extraction of intracellular lipids by a chloroform/methanol (2:1) mixture followed by vacuum drying. Total cholesterol (TC) contents were measured by an enzymatic assay normalized by total cellular protein determined by Lowry assay. The concentration of cholesterol ester (CE) was calculated from TC. The apoA-I-mediated cholesterol efflux was calculated as follows: apoA-I-mediated cholesterol efflux¼ (apoA-I-mediated cholesterol efflux/[intracellular cholesterol þTC efflux]) 100%. 2.7. Oil red O staining As previously described with minor modifications [2], THP-1 macrophages and HepG2 cells (with or without lentivirus) were incubated in either serum-free experimental medium or experimental medium. After 24 h of incubation, the cells were washed two times in PBS, fixed for 30 min with 5% formalin solution in PBS, stained with oil red O for 30 min, and counterstained with hematoxylin for another 5 min. Cells were examined by light microscopy (Axio Imager 2, Zeiss, Germany) through six randomlyselected high-power fields (HPF) (4006). Semi-quantitative analysis of oil red O staining was performed using Image-J software. 2.8. Statistical analysis The results were expressed as means 7SEMs. Statgraphics Plus v5.0 (Statistical Graphics, USA) was used for all statistical analyses.
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Statistical comparisons between groups were performed by Student’s t-test or analysis of variance (ANOVA), and post hoc multiple comparisons were performed using the Student–Newman– Keuls test.
3. Results 3.1. ABCA1 expression negatively regulated by miR-101 Western blotting of ABCA1 expression in THP-1-derived macrophages revealed that miR-101 expression negatively regulates ABCA1 protein expression under both LDL and non-LDL conditions (Fig. 1A) with LDL treatment more pronouncedly upregulating ABCA1 expression in the absence of miR-101 (Fig. 1B). Western blotting of ABCA1 expression in HepG2 cells revealed that LDL treatment significantly raised ABCA1 expression, and miR-101 expression negatively regulates ABCA1 expression under LDL conditions (Fig. 1C and D). 3.2. miR-101 targets the 3’ UTR of the ABCA1 mRNA Sequences of the four hsa-miR-101's binding sites on the human ABCA1 3’ UTR are detailed in Fig. 2A. The luciferase activity assay revealed that wild-type (WT) miR-101 binding at site 2 significantly repressed ABCA1 3’ UTR activity (Po 0.05; Fig. 2B), suggesting that miR-101 directly targets site 2 on the ABCA1 mRNA. The luciferase activity assay also revealed that wild-type (WT) Con-miR binding at site 4 significantly increased ABCA1 3’ UTR activity (Po 0.05; Fig. 2B). We attribute this phenomenon to some molecular effect of the Con-MiR on the WT site 4 that served to derepress ABCA1 3’ UTR activity. 3.3. miR-101 suppresses cholesterol efflux Total cholesterol efflux from HepG2 cells was evaluated six hours after incubation with ApoA1. We found that miR-101 significantly suppresses cholesterol efflux to ApoA-1 under both LDL and non-LDL conditions (Po 0.05; Fig. 3A and B). A similar pattern of findings was observed in THP-1-derived macrophages (P o0.05; Fig. 3E and F). In the presence of ApoA-1, miR-101 expression negatively regulates ABCA1 protein expression in HepG2 cells under both LDL and non-LDL conditions (P o0.05; Fig. 3C and D). A similar pattern of findings was observed in THP-1-derived macrophages (P o0.05; Fig. 3G and H). miR-101-transfected THP-1derived macrophages and HepG2 cells displayed pronounced lipid inclusions (Fig. 4A and B), significantly increased Oil red O staining (P o0.05; Fig. 4C and D), and significantly increased intracellular cholesterol content (P o0.05; Fig. 4E and F) relative to Con-miRtransfected counterparts, while anti-miR-101-transfected counterparts displayed the opposite effects. 3.4. Inflammatory conditions increase miR-101 expression and repress ABCA1 expression In HepG2 cells, both IL-6 and TNF-α treatments produced significant miR-101 overexpression (P o0.05; Fig. 5A). However, in THP-1-derived macrophages, only IL-6 treatment produced significant miR-101 overexpression (P o0.05; Fig. 5B). Using miR33a-5P as a previously established positive control under inflammatory conditions [2], both IL-6 and TNF-α treatments produced significant miR-33a-5P overexpression in both cell lines as expected (Po 0.05; Fig. 5C and D). In HepG2 cells, anti-mir-101 transfection under both IL-6 and TNF-α treatment conditions led to significant ABCA1 upregulation, indicating that miR-101 expression represses ABCA1 expression under inflammatory
Please cite this article as: N. Zhang, et al., MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.023i
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Fig. 1. ATP-binding cassette transporter A1 (ABCA1) expression negatively regulated by microRNA (miR)-101. Western blotting of ABCA1 and β-actin expression induced with low-density lipoprotein (LDL) in (A) THP-1-derived macrophages infected with Con-miR or miR-101, (B) THP-1-derived macrophages infected with Con-anti-miR or antimiR-101, (C) HepG2 cells infected with Con-miR or miR-101, and (D) HepG2 cells infected with Con-anti-miR or anti-miR-101. nPo 0.05 (comparison versus Con-miR or Conanti-miR), △Po 0.05 (comparison versus non-LDL or LDL).
conditions (Po0.05; Fig. 5G). A similar pattern of findings were shown in anti-mir-101-transfected THP-1-derived macrophages (P o0.05; Fig. 5H). Accordingly, miR-101 transfection under IL-6 treatment conditions in both cell lines led to significant intracellular cholesterol retention over either miR-101 transfection alone or Il-6 treatment conditions alone, indicating that miR-101 expression increases intracellular cholesterol content under inflammatory conditions (P o0.05; Figs. 5E, F and 6).
4. Discussion Here, we investigated the role of miR-101 in regulating ABCA1 expression and cholesterol efflux under non-inflammatory and inflammatory conditions in human THP-1-derived macrophages and human HepG2 hepatoblastoma cells. First, we found that miR101 directly targeting the ABCA1 3’ UTR in both cell lines (Fig. 2A and B). Next, we found that miR-101 suppresses cholesterol efflux to ApoA-1 under both LDL and non-LDL conditions (Fig. 3A, B, E and F) and negatively regulates ABCA1 expression (Fig. 3C, D, G and H). More specifically, this negative regulation of ABCA1
expression by miR-101 was found to suppress cholesterol efflux, which led to pronounced lipid inclusions (Fig. 4A and B), increased Oil red O staining (Fig. 4C and D), and increased intracellular cholesterol retention (Fig. 4E and F) in both cell lines. Lastly, we discovered that inflammatory conditions produce miR-101 overexpression (Fig. 5A and B) and suppressed ABCA1 expression (Fig. 5G and H) in both cell lines. Accordingly, miR-101 transfection under IL-6 treatment conditions in both cell lines led to significant intracellular cholesterol retention over either miR-101 transfection alone or Il-6 treatment conditions alone (Fig. 5E and F). These combined findings reveal that miR-101 overexpression under inflammatory conditions promotes intracellular cholesterol retention through suppressing ABCA1 expression and suggests that the miR-101-ABCA1 axis may play an intermediary role in the development of NAFLD and vascular atherosclerosis. In addition to its role in regulating ABCA1 expression in human HepG2 hepatoblastoma cells, miR-101 has been previously associated with regulating several other proteins and pathways in liver cells and macrophages. For example, miR-101 has been identified as a tumor suppressor in hepatocellular carcinoma (HCC) cells that represses several oncogenes (including STMN1, JUNB, and CXCR7)
Please cite this article as: N. Zhang, et al., MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.023i
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Fig. 2. MicroRNA (miR)-101 targets the 3’ untranslated region (UTR) of the ATP-binding cassette transporter A1 (ABCA1) mRNA. (A) Sequence alignment of the human hsamiR-101 mature sequence with the four binding sites of the human ABCA1 3’ UTR. Relative positions of the four binding sites are indicated. (B) Activity of the luciferase reporter construct infected with Con-miR or miR-101 along with wild-type (WT) or site-directed mutations (SDM) in the miR-101 target sites of the ABCA1 3’ UTR. Luciferase activity was normalized with Renilla. Relative luciferase activity expressed as means 7 SEMs from six independent experiments. nPo 0.05 (comparison versus other site 2 groups), #Po 0.05 (comparison versus other site 4 groups). WT: wild-type.
and further increases expression of endogenous miR-101 through inhibiting PRC2 activation [23]. In addition, miR-101 has been identified as an anti-fibrotic miRNA with miR-101 expression significantly blunting pro-fibrotic TGF-β signaling in hepatic stem cells (HSCs) and hepatocytes [24]. In macrophages, miR-101 has been identified as a pro-apoptotic, pro-inflammatory miRNA that represses anti-apoptotic mitogen-activated protein kinase phosphatase-1 (MKP-1) expression as well as, in response to pro-inflammatory lipopolysaccharide (LPS), increases supernatant levels of TNF-α, nitric oxide (NO), IL-1β, and IL-6 [25,26]. Based on these findings, future studies on miR-101's regulation of lipid retention in liver cells and macrophages should control for changes in these other proteins, as these proteins may also influence ABCA1
expression or other lipid retention pathways. Aside from miR-101, several other miRNAs have been identified as negative regulators of ABCA1 expression in hepatocytes. For example, miR-758 also negatively regulates the expression of ABCA1 in hepatocytes, thereby attenuating cholesterol efflux to apoA-1 [27]. Moreover, in a mouse model, delivery of miR-144 oligonucleotides has been shown to suppress hepatic ABCA1 expression and plasma high-density lipoprotein (HDL) levels, while miR-144 silencing increases ABCA1 expression and plasma HDL levels [28]. Additionally, in HepG2 cells, miR-128-2 and miR-145 have been shown to significantly decrease ABCA1 expression and cholesterol efflux with this suppressive effect being rescued by their respective anti-miRs [29,30]. As all the foregoing miRNAs
Please cite this article as: N. Zhang, et al., MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.023i
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Fig. 3. MicroRNA (miR)-101 suppresses cholesterol efflux by HepG2 and THP-1-derived macrophages. Total cholesterol efflux from HepG2 were evaluated six hours after incubation with apolipoprotein A1 (ApoA-1) in the presence of (A) Con-miR-101 or miR-101 or (B) Con-anti-miR-101 or anti-miR-101. In the presence of ApoA-1, transfection of low-density lipoprotein (LDL)-treated HepG2 cells using (C) Con-miR or miR-101 or (D) Con-anti-miR or anti-miR-101 modifies ATP-binding cassette transporter A1 (ABCA1) expression. Total cholesterol efflux from THP-1-derived macrophages were evaluated six hours after incubation with ApoA-1 in the presence of (E) Con-miR-101 or miR-101 and (F) Con-anti-miR-101 or anti-miR-101. Transfection of LDL-treated THP-1-derived macrophages using (G) Con-miR or miR-101 or (H) Con-anti-miR or anti-miR101 modifies ABCA1 expression. Data expressed as means 7 SEMs of six independent experiments. nPo 0.05 (comparison versus Con-miR or Con-anti-miR), △Po 0.05 (comparison versus LDL).
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Fig. 4. Oil red O staining of THP-1-derived macrophages. (A) Representative images of THP-1-derived macrophages examined for lipid inclusions by oil red O staining (400 ). (B) Representative images of HepG2 cell examined for lipid inclusions by Oil red O staining (400 ). (C) miR-101 cells displayed higher Oil red O staining relative to THP-1-derived macrophages with Con-miR and anti-miR-101. (D) miR-101 cells displayed higher oil red O staining relative to HepG2 cell with Con-miR and anti-miR-101. (E) Effects of miR-101 and anti-miR-101 on intracellular cholesterol content in THP-1-derived macrophages. (F) Effects of miR-101 and anti-miR-101 on intracellular cholesterol content in HepG2 cells. Data expressed as means 7SEMs of six independent experiments. nPo 0.05 (comparison versus Con-miR or Con-anti-miR), #Po 0.05 (comparison versus miR-101).
have been shown to negatively regulate ABCA1 expression in hepatocytes, miRNA antisense oligonucleotide (ASO)-based therapeutics targeting NAFLD should endeavor to simultaneously target multiple miRNAs in order maximize therapeutic efficacy. Several other miRNAs have been identified as negative regulators of ABCA1 expression in THP-1-derived macrophages. For example, in THP-1-derived macrophages, a miR-144-3p mimic has been shown to inhibit cholesterol efflux via suppressing ABCA1, resulting in decreased HDL-C circulation and accelerated atherosclerosis in apoE / mice [31]. Moreover, miR-33a also downregulates ABCA1 expression in THP-1-derived macrophages, resulting in decreases in circulating HDL-C levels [32]. In THP-1derived macrophages, miR-27a/b has also been shown to affect the efflux, influx, esterification, and hydrolysis of cholesterol through regulating ABCA1, apoA-1, lipoprotein lipase (LPL), CD36, and ACAT1 expression [33]. In PPARγ-activated THP-1-derived macrophages, miR-613 has been shown to suppress LXRα and ABCA1 expression, thereby inhibiting cholesterol efflux [34]. In THP-1derived macrophages, miR-19b has also been shown to suppress ApoA-I-mediated ABCA1-dependent cholesterol efflux; more notably, treatment with inhibitory miR-19b ASO was shown to reverse the suppression of ABCA1 expression as well as reversing the following indicators of atherosclerotic progression: decreases in
plasma HDL levels, increases in plasma low-density lipoprotein (LDL) levels, increases in aortic plaque size and lipid content, and reductions in collagen content [35]. As all the foregoing miRNAs have been shown to negatively regulate ABCA1 expression in THP1-derived macrophages, miRNA ASO-based therapeutics targeting vascular atherosclerosis should endeavor to simultaneously target multiple miRNAs in order maximize therapeutic efficacy. There are several limitations to this study. First, although this study clearly demonstrates that mir-101 negatively regulates ABCA1 expression and cholesterol efflux through directly targeting the ABCA1 3’ UTR (with a more pronounced effect under inflammatory conditions), we did not conduct animal model experiments to assess the in vivo effects of miR-101 on plasma HDL and LDL levels, arterial and hepatic plaque size, arterial and hepatic lipid content, and arterial and hepatic collagen content. Second, although we determined that IL-6 and TNF-α treatment produced miR-101 overexpression and suppressed ABCA1 expression in both THP-1-derived macrophages and HepG2 hepatoblastoma cells, we did not examine the signal transduction mechanism(s) underlying this phenomenon, which may be different in these two cell lines. Third, we did not examine miR-101’s effects upon the expression of other proteins that may have also influenced ABCA1 expression or other lipid retention pathways.
Please cite this article as: N. Zhang, et al., MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.023i
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Fig. 5. Inflammatory conditions increase microRNA (miR)-101 expression and repress ATP-binding cassette transporter A1 (ABCA1) expression. miR-101 overexpression in (A) HepG2 cells and (B) THP-1-derived macrophages treated with either TNF-α or IL-6. miR-33-5p control overexpression in (C) HepG2 cells and (D) THP-1-derived macrophages treated with either TNF-α or IL-6. Effects of IL-6 and miR-101 on intracellular cholesterol content in (E) HepG2 cells and (F) THP-1-derived macrophages under nonLDL and LDL conditions. ABCA1 expression in (G) HepG2 cells and (H) THP-1-derived macrophages transfected with anti-miR-101 and either TNF-α or IL-6. Data expressed as means 7 SEMs of six independent experiments.nPo 0.05 (comparison versus control), #Po 0.05 (comparison versus IL-6 þmiR-101), ^Po 0.05 (comparison versus IL-6), & P o0.05 (comparison versus TNF-α).
Future studies should aim at addressing these limitations. In conclusion, this study reveals that miR-101 promotes intracellular cholesterol retention under inflammatory conditions through suppressing ABCA1 expression. These findings suggest that the miR-101-ABCA1 axis may play an intermediary role in the development of NAFLD and vascular atherosclerosis.
Author contributions Conceived and designed the study: NZ, XZR, and HL. Performed the experiments: NZ,YXCand JYL. Analyzed the data: YXC and QL. Drafted the manuscript: NZ and WH. Modified the manuscript: XZR.
Please cite this article as: N. Zhang, et al., MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.023i
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Fig. 6. Confirmation of lentiviral transfection by fluorescence microscopy. Light and fluorescence microscopy of green fluorescent protein (GFP) expression in (A) THP-1 cells (pre-macrophage) infected with lentivirus, (B) THP-1-derived macrophages infected with lentivirus, and (C) HepG2 cells infected with lentivirus. All images captured at 100 magnification.
Conflicts of interest None.
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Please cite this article as: N. Zhang, et al., MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.023i