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MiRNA-185, MiRNA-96 and MiRNA-223 Repress Selective HDL-Cholesterol 1
Uptake through Posttranscriptional Inhibition of Scavenger Receptor Class BI 2
in Hepatic Cells 3
Li Wang*, Xiao-Jian Jia*, Hua-Jun Jiang, Yu Du, Fan Yang, Shu-Yi Si and Bin Hong# 4
From the Key Laboratory of Biotechnology of Antibiotics of Ministry of Health, Institute of 5
Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, 6
Beijing, China 100050. 7
8
Running Title: MiRNAs Repress HDL-C Uptake through Hepatic SR-BI 9
10
* These authors equally contributed to this study. 11
# To whom correspondence should be addressed. 12
13
word count for the Materials and Methods: 1318 14
word count for the introduction, Results, and Discussion: 3970 15
16
Bin Hong, Ph.D., Prof., Institute of Medicinal Biotechnology, Chinese Academy of Medical 17
Sciences & Peking Union Medical College, No. 1 Tiantan Xili, Beijing 100050, China. Email: 18
[email protected] , [email protected] ; Tel: +86 10 63028003; Fax: +86 10 19
63017302. 20
21
Copyright © 2013, American Society for Microbiology. All Rights Reserved.Mol. Cell. Biol. doi:10.1128/MCB.01580-12 MCB Accepts, published online ahead of print on 4 March 2013
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Abstract 22
Hepatic scavenger receptor class B type I (SR-BI) plays an important role in selective high-density 23
lipoprotein cholesterol (HDL-C) uptake, which is a pivotal step of reverse cholesterol transport. In 24
this study, the potential involvement of microRNAs (miRNAs) in posttranscriptional regulation of 25
hepatic SR-BI and selective HDL-C uptake was investigated. The level of SR-BI expression was 26
repressed by miR-185, miR-96 and miR-223, while the uptake of DiI-HDL was decreased by 27
31.9% (p<0.001), 23.9% (p<0.05) and 15.4% (p<0.05) in HepG2 cells, respectively. The inhibition 28
of these miRNAs by their anti-miRNAs had opposite effects in these hepatic cells. The critical 29
effect of miR-185 was further validated by the loss of regulation in constructs with mutated 30
miR-185 target sites. In addition, these miRNAs directly targeted the 3'UTR of SR-BI with a 31
coordinated effect. Interestingly, the decrease of miR-96 and miR-185 coincided with the increase 32
of SR-BI in the liver of ApoE KO mice on a high fat diet. These data suggest that miR-185, 33
miR-96 and miR-223 may repress selective HDL-C uptake through the inhibition of SR-BI in 34
human hepatic cells, implicating a novel mode of regulation of hepatic SR-BI and an important 35
role of miRNAs in modulating cholesterol metabolism. 36
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Introduction 37
Atherosclerotic cardiovascular disease is one of the leading causes of morbidity and mortality in 38
industrialized and developing nations. Numerous long-term clinical trials have demonstrated an 39
inverse relationship between plasma high-density lipoprotein cholesterol (HDL-C) levels and 40
atherosclerosis independent of low-density lipoprotein cholesterol (LDL-C), the latter of which is 41
a well-established major risk factor for atherosclerosis (1). The protective effect of HDL may 42
mainly be attributed to its role in reverse cholesterol transport (RCT), whereby peripheral 43
(extrahepatic) cholesterol is transported back to the liver for excretion into the bile and ultimately 44
the feces (2,3). Lipoprotein receptors play critical roles in cholesterol homeostasis both 45
physiologically and pathologically, as exemplified by endocytosis of LDL-C via the LDL receptor 46
(4). The selective uptake of HDL-C into the liver, a pivotal step of RCT, is mediated by scavenger 47
receptor class B type I (SR-BI or SCARB1) (5,6). 48
SR-BI is a single-chain plasma membrane glycoprotein structurally related to CD36; the human 49
homologue of SR-BI (hSR-BI) is also known as CLA-1 (CD36 and Lysosomal integral membrane 50
protein-II Analogous-1), which is mapped to chromosome 12 (7). As a high-affinity HDL receptor, 51
SR-BI is most highly expressed in the liver and steroidogenic tissues, where it delivers HDL-C for 52
excretion and steroid hormone synthesis (5). Abundant data from SR-BI gene manipulated mice 53
have definitely established that hepatic SR-BI is a critical regulator of overall HDL-C metabolism 54
and presents anti-atherogenic activity in vivo (8). The SR-BI expression level in the liver directly 55
modulates HDL metabolism, which is delicately regulated at the transcriptional level. For example, 56
SR-BI is under the control of different nuclear receptors and transcription factors, including 57
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peroxisome proliferator-activated receptors (PPAR) (9), liver X receptor (LXR) (10), liver receptor 58
homolog-1 (LRH-1) (11), and sterol regulatory element-binding proteins (SREBPs) (12). Some 59
posttranscriptional regulation mechanisms have also been reported, best exemplified by the 60
alternative splicing of SR-BI pre-mRNA (13) and the interaction of SR-BI with its tissue-specific 61
adaptor PDZK1 (14). 62
MicroRNAs (miRNAs) are a class of highly conserved, single-stranded, non-coding small 63
RNAs (approximately 22 nucleotides in length) that regulate gene expression on the 64
posttranscriptional level by promoting the degradation or inhibiting the translation of its target 65
mRNA (15). These short non-coding RNAs can be transcribed from their own promoter or 66
encoded in the intron of other genes (15,16). Regardless, most original pri-miRNA is processed by 67
the ribonuclease Drosha cotranscriptionally, resulting in a pre-miRNA, which is then exported to 68
the cytoplasm for further cleavage by ribonuclease Dicer to generate the mature miRNA (15,16). 69
In this study, a significant increase of SR-BI expression were observed by silencing the miRNA 70
processing enzymes Drosha and Dicer with siRNA, implying that miRNAs are involved in the 71
complex mechanism to ensure the appropriate SR-BI gene expression regulation. Therefore, we 72
further investigated the contribution of miRNAs in the posttranscriptional regulation of SR-BI and 73
their potential involvement in the modulation of HDL-mediated selective uptake. Three miRNAs 74
(miR-185, miR-96 and miR-223) were identified as regulators to effectively repress hSR-BI 75
expression and HDL mediated cholesterol selective uptake in hepatic cells by directly targeting the 76
binding sites within the hSR-BI 3’-untranslated region (3’UTR), suggesting the importance of the 77
miRNAs in the modulation of cholesterol metabolism. 78
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Materials and methods 79
Cell culture. Human hepatoma HepG2 cells were cultured in Eagle minimal essential medium 80
(MEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, v/v, 81
Gibco, Grand Island, NY, USA), sodium pyruvate, and non-essential amino acids (Invitrogen). 82
Human hepatocellular carcinoma Bel-7402 cells, a human normal hepatic immortal cell line 83
HL-7702 and acute monocytic leukemia THP-1 cells were grown in RPMI-1640 medium 84
(Invitrogen) containing 10% FBS (v/v). For differentiation to macrophages, THP-1 cells were 85
cultured at a density of 200,000 cells per cm2 and stimulated with 160 nM 86
phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Chinese 87
hamster ovary (CHO) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, high 88
glucose, Invitrogen)/F-12K (1:1) containing 10% FBS (v/v). The cells were maintained at 37 °C in 89
a humidified 5% CO2 incubator. 90
Animal. Experiments were performed in ApoE knock-out (KO) mice purchased from the 91
Department of Laboratory Animal Science of the Peking University Health Science Center 92
(Beijing, China). All experimental procedures involving animals were approved by the 93
Institutional Laboratory Animal Care and Use Committee of Peking University Health Science 94
Center (Peking, China). Eight-week-old male ApoE KO mice were fed a high fat diet (HFD) 95
containing 0.15% cholesterol and 20% lard or fed a regular rodent chow as a control. After 8 96
weeks of feeding, the animals were sacrificed; cholesterol was then measuremed, and the tissues 97
were collected for further analyses. 98
3'UTR luciferase reporter assays. Firefly luciferase coding sequence was amplified from the 99
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pGL3-Basic vector (Promega, Madison, WI, USA) and inserted upstream of the multiple cloning 100
site in the pcDNA3.1 vector (Invitrogen) at the Hind III and Xba I sites, resulting a 101
pcDNA-luciferase reporter vector (pc-luc). The entire 3'UTR of human SR-BI (U1) was generated 102
by reverse transcription-polymerase chain reaction (RT-PCR) from total RNA extracted from 103
HepG2 cells and fused downstream of the luciferase coding sequence in the pc-luc vector at the 104
Xba I site. Using the U1 fragment as a template, other luciferase reporter constructs with truncated 105
SR-BI 3'UTR, U2-U6, were obtained by PCR. The deletions of the predicted seed regions of 106
miR-185 and miR-96 within human SR-BI 3'UTR were generated by overlap extension PCR and 107
then cloned into the pc-luc vector. All of the constructs were confirmed by sequencing. Each 108
purified reporter plasmid (0.2 μg) was transfected into HepG2 cells of 70% confluence using 109
Lipofectamine 2000 (Invitrogen) for 4 h before introduction of miRNA mimics, anti-miRNAs or 110
negative control duplexes (ctl-miR) for another 48 h. The cells were cotransfected with 0.02 μg 111
Renilla luciferase control reporter vector pRL-TK (Promega) to normalize the transfection 112
efficiency. The luciferase activity was measured using the Dual-Glo Luciferase Assay System 113
(Promega) and detected by a Victor X5 multilabel plate reader (PerkinElmer, Waltham, MA, USA). 114
Firefly luciferase activity was normalized to the corresponding Renilla luciferase activity and 115
plotted as a percentage of the control (ctl-miR). 116
hSR-BI expression in CHO cells. The full-length human SR-BI cDNA was amplified by RT-PCR 117
from total RNA extracted from HepG2 cells and inserted into the multiple cloning sites of the 118
pcDNA3.1 vector (Invitrogen) at Hind III and Kpn I, generating the recombinant SR-BI expression 119
plasmid pc-SR-BI. The entire 3'UTR of human SR-BI (U1 fragment) or with mutated miR-185 120
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target sites (DEL1+2 fragment) was then cloned downstream of the SR-BI coding sequence in the 121
pc-SR-BI at the Xba I site, generating pc-WT and pc-185m, respectively. These constructs were 122
transfected into CHO cells using Lipofectamine 2000 (Invitrogen). 123
Transfection with small RNAs. Transfection of miRNA mimics, anti-miRNAs and small 124
interfering RNAs (siRNA) was carried out using the Lipofectamine RNAiMAX reagent 125
(Invitrogen) according to the manufacturer’s instructions. The miRNA mimics (double-stranded 126
RNA oligonucleotides), anti-miRNA (2'-O-methyl antisense oligonucleotides against the target 127
miRNAs) and negative control duplexes were obtained from Qiagen (Hilden, Germany) and 128
applied at a final concentration of 100 nM. Pre-designed siRNAs specifically targeting the mRNA 129
of human Dicer, Drosha and SR-BI and the scrambled negative control siRNA were obtained from 130
Invitrogen and used at a final concentration of 100 nM. After the incubation of the small RNAs for 131
24-72 h, the cells were harvested for further analyses. 132
Real-time RT-PCR. HepG2, Bel-7402, HL-7702 cells or PMA-induced THP-1 cells were plated 133
in 24-well culture dishes (200,000 cells/well) and transfected with either miRNA mimics or 134
anti-miRNAs, as well as their control miRNAs, for 48 or 72 h. For assessment of the mRNA level, 135
total RNAs were isolated from the cultured cells by the SV Total RNA Isolation System (Promega) 136
and quantified by Nanodrop spectrophotometry (Thermo Scientific, Wilmington, DE, USA). RNA 137
was reverse transcribed using the SuperScript III First-Strand Synthesis System (Invitrogen) 138
following the manufacturer's instructions. Specific real-time PCR primers were used to quantify 139
the mRNA levels. Real-time PCR was performed in the CFX96 Real-time System (Bio-Rad, 140
California, USA) using a SYBR Green Master Kit (Roche, Mannheim, Germany) as described 141
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previously (17). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the 142
normalization control. For the quantitative analyses of the miRNAs, RNeasy Mini Kit (Qiagen) 143
was used to purify small RNAs from the cultured cells and tissue samples with the inclusion of a 144
DNase I (Qiagen) treatment step. Reverse transcription was performed by the miScript II RT Kit 145
(Qiagen). miRNA expression levels in the sorted samples was assessed by real-time PCR using the 146
miScript SYBR Green PCR Kit (Qiagen). U6 small nuclear RNA (RNU6) was served as the 147
endogenous control for the miRNA expression levels. 148
Western blot. The cells were treated as described above for transcript analysis. Whole-cell protein 149
lysates were prepared using RIPA buffer (25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1% sodium 150
deoxycholate, 0.1% SDS, pH 7.6) containing complete an EDTA-free protease inhibitor cocktail 151
(Roche). The protein extracts (50 μg) were subjected to 10% SDS-polyacrylamide gel 152
electrophoresis and transferred to a 0.45-μm polyvinylidene difluoride (PVDF) membrane 153
(Millipore, Bedford, MA). The membranes were probed with antibodies to hSR-BI (1:2,000; BD 154
Biosciences, San Jose, CA, USA) and β-actin (1:1,000; Cell Signaling Technology Inc., Beverly, 155
Massachusetts, USA). The proteins were visualized using the appropriate horseradish 156
peroxidase-conjugated secondary antibodies (1:1,000, BD Biosciences) and chemiluminescence 157
reagents (Thermo Scientific). 158
Analysis of cell surface expression by flow cytometry. The cells were transfected with miRNA 159
mimics, anti-miRNAs or control miRNAs for 72 h, as described above. The cells were detached 160
from the plates by trypsinization (0.25% trypsin with 0.02% EDTA) for 2 min before PBS 161
containing 10% FBS was immediately added to stop the trypsin action. The cells were then 162
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washed with PBS and incubated with the primary mouse anti-human SR-BI monoclonal antibody 163
(1:1,000, BD Biosciences) for 50 min at 4 °C and, subsequently, with the secondary 164
FITC-conjugated rabbit anti-mouse antibody (1:2,000, BD Biosciences) for 50 min at 4 °C. For 165
flow cytometry analysis, the cells were collected by centrifugation (800× g, 3 min, 4 °C), and the 166
obtained pellet was resuspended in PBS buffer. A total of 10,000 cells were counted, and FITC 167
fluorescence was analyzed using an Accuri C6 flow-cytometer (BD Biosciences). 168
Assays for the cellular uptake of DiI-HDL. Different cells were transfected with either miRNA 169
mimics, anti-miRNAs, or their control miRNAs as described above, followed by incubation with 2 170
μg/ml 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI)-labeled HDL 171
(Biomedical Technologies, Stoughton, MA, USA) for 4 h at 37 °C. After the cell harvest and the 172
washes, fluorescence measurements were performed with the flow cytometer as described 173
previously (18). For visualization, the cells were additionally stained with 174
4',6-diamidino-2-phenylindole (DAPI) and photographed at 360 nm/460 nm (DAPI) and 480 175
nm/600 nm (DiI), by IN Cell Analyzer 1000 (GE Healthcare, Piscataway, NJ) after washing with 176
PBS twice. 177
Statistical analyses and bioinformatics. The results presented in the manuscript are derived from 178
at least three independent experiments. The data are presented as the mean ± SEM, and statistical 179
significance was evaluated by Student’s t-test. Multiple comparisons were made using ANOVA 180
followed by a post hoc Newman-Keuls test. Values of P < 0.05 were considered statistically 181
significant. MicroRNA.org (http://www.microrna.org), TargetScan (http://www.targetscan.org) and 182
miRBase (http://www.mirbase.org) databases were used for in silico computational target 183
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predictions and miRNA sequence analyses. 184
Results 185
hSR-BI expression in hepatic cells is modulated by miRNAs. miRNAs posttranscriptionally 186
regulate gene expression by binding to the 3'UTR of their target mRNAs. The 3'UTR of the human 187
SR-BI gene is 959-bp-long, which raises the potential possibility of it being regulated by miRNAs. 188
RNA interference-mediated knockdown of the components of the miRNA biogenesis pathway was 189
applied to detect the involvement of miRNAs in SR-BI gene regulation. The transfection of 190
HepG2 cells with siRNAs directed against Drosha or Dicer (the inhibitory efficiency equaled to 191
64% or 62%, respectively), resulted in a marked increase of SR-BI mRNA and protein level (Fig. 192
1A). Therefore, we hypothesized that the 3'UTR of the SR-BI mRNA is susceptible to being 193
targeted by miRNAs in either a direct or an indirect manner. 194
To identify the potential miRNA candidates that target the SR-BI 3’UTR, both TargetScan 195
(http://www.targetscan.org) and miRanda (http://www.microrna.org), two bioinformatic tools, 196
were applied for miRNA target prediction. The former tool revealed 45 predicted miRNAs that 197
target the SR-BI 3'UTR, whereas the latter predicted 20 conserved miRNAs with good mir-SVR 198
scores. To assess whether these potential miRNAs regulated SR-BI expression, the mRNA level of 199
SR-BI was detected by RT-PCR after transfection with some of the predicted miRNAs in HepG2 200
cells. Interestingly, several effective miRNAs were found and among these, and miR-185, miR-96 201
and miR-223 had the most pronounced inhibitory effect on SR-BI mRNA level. 202
miR-185, miR-96 and miR-223 repress hSR-BI expression in hepatic cells by targeting its 203
3'UTR. The transfection of HepG2 cells with 100 nM miR-185, miR-96 or miR-223 significantly 204
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decreased SR-BI at both the mRNA (Fig. 1B) and the protein level (Fig. 1C). Similar results were 205
also observed when the cell surface SR-BI protein level was measured by flow cytometry (Fig. 1D 206
and Supplemental Figure 1D). To further determine whether these miRNAs are specifically 207
involved in regulating SR-BI expression, antisense oligonucleotides directed against miR-185, 208
miR-96 and miR-223 were applied. The results showed that inhibition of endogenous miRNAs by 209
anti-miR-185, anti-miR-96 or anti-miR-223 significantly increased the expression of SR-BI in 210
HepG2 cells at 72 h (Fig. 1B-1D), further confirming that miR-185, miR-96 and miR-223 may be 211
involved in regulating SR-BI expression. The results were similar in other two human hepatic cell 212
lines Bel-7402 and HL-7702 (Supplemental Figure 1A, 1B and 1C), indicating that the modulating 213
effect of these miRNAs is not due to cell line specificity. 214
The predicted binding sites for the 3 miRNAs are located in different sections of the hSR-BI 215
mRNA 3'UTR according to the bioinformatic miRNA target prediction (Fig. 2A), and there are 2 216
computationally predicted miR-185 binding sites (site 1 and site 2, Fig. 2A). Sequence alignment 217
revealed that the 4 sites for the 3 miRNAs are conserved among different mammals (Fig. 2B). As 218
for rodents, the predicted target site for miR-96 is conserved in the mouse (Mmu) SR-BI 3'UTR, 219
while there is no predicted target site for miR-223 in the SR-BI 3’UTR of mouse, rat (Rno) and 220
guinea pig (Cpo). For miR-185, site 2, but not site 1, is conserved in guinea pig. In addition, 221
within a stretch of phylogenetically conserved sequence of the mouse and rat SR-BI 3’ UTR, there 222
is a G:U wobble at the second nucleotide of the miR-185 target site 1, which is a 223
thermodynamically favorable base pair in RNA secondary structure. To assess the effects of the 3 224
miRNAs on the SR-BI mRNA 3'UTR, the luciferase reporter assay was conducted. The results 225
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showed that miR-185, miR-96 and miR-223 markedly reduced the activity of the full-length 226
SR-BI mRNA 3'UTR reporter construct (U1, Fig. 2C). According to the 4 predicted target binding 227
sites for these 3 miRNAs, luciferase reporter constructs with different 5'-end truncated SR-BI 228
3'UTR were constructed (U2-U6) and applied to evaluate the effects of the 3 miRNAs on their 229
target sites in HepG2 cells. The lack of a binding site for miR-185 (site 1, U2 vs U1; site 2, U3 vs 230
U2), miR-96 (U4 vs U3) or miR-223 (U6 vs U5) markedly diminished each miRNA-induced 231
repression of SR-BI mRNA 3'UTR activity (Fig. 2C), implying that all the 3 miRNAs are involved 232
in SR-BI posttranscriptional regulation. It was further confirmed that direct removal of the seed 233
region of the binding sites for miR-185 (DEL1, 84 bp to 89 bp and/or DEL2, 274 bp to 281 bp) or 234
miR-96 (461 bp to 468 bp) abolished the miR-185- or miR-96-induced downregulation and 235
anti-miR-185- or anti-miR-96-induced upregulation (Fig. 2D and Supplemental Figure 2). Taken 236
together, these results demonstrate that miR-185, miR-96 and miR-223 may regulate SR-BI 237
posttranscriptionally by binding to their target sites located at the SR-BI 3’UTR. 238
miR-185, miR-96 and miR-223 regulate selective HDL-C uptake in hepatic cells. As the HDL 239
receptor, the main ability of SR-BI is to mediate selective HDL-C uptake in the liver, the final 240
phase of RCT, which is critical to the antiatherosclerotic role of HDL. To assess the inhibitory 241
effect of the 3 miRNAs on the selective uptake of lipids from HDL, fluorescence-labeled DiI-HDL 242
uptake by HepG2 cells was measured. HepG2 cells were treated with miR-185, miR-96 or 243
miR-223 mimics or their anti-miRNAs for 72 h, followed by an additional 4 h incubation with 244
DiI-HDL. All 3 miRNAs markedly attenuated DiI-HDL uptake into HepG2 cells (Fig. 3A) in 245
accordance with the decreased cellular and cell surface SR-BI levels (Fig. 1C and 1D). Notably, 246
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antagonism of endogenous miR-185 or miR-96 significantly increased the uptake of DiI-HDL into 247
HepG2 cells (Fig. 3A) along with the increased expression of SR-BI (Fig. 1C and 1D). Similar 248
results were obtained in Bel-7402 and HL-7702 cells with miR-185/96 and anti-miR-185/96 249
treatment (Supplemental Figure 1B and 1C). Fluorescence visualization by IN Cell Analyzer 1000 250
(GE Healthcare) showed that lipid accumulation was detected in HepG2 cells after incubation of 2 251
μg/mL DiI-HDL for 4 h (Fig. 3Bg vs 3Bc). Compared with control miRNA (ctl-miR)-transfected 252
cells, the fluorescence was clearly decreased in miR-185, miR-96 or miR-223 transfected cells 253
(Fig. 3Bk, 3Bo, 3Bs vs 3Bg). 254
To assess the role of SR-BI in the miRNA mediated repression of selective HDL-C uptake, HepG2 255
cells transfected with small interfering RNA against SR-BI were applied. The results showed that 256
the inhibitory effect of miR-185 on cell surface SR-BI levels and DiI-HDL uptake by HepG2 cells 257
was significantly attenuated after SR-BI silencing (Fig. 3C), suggesting that miR-185 regulates 258
DiI-HDL uptake via SR-BI. To further confirm this, an SR-BI expression vector containing its 259
entire 3'UTR (pc-WT) or with mutated miR-185 target sites (pc-185m) was constructed and 260
introduced into CHO cells, which exhibited relatively low background of DiI-HDL uptake (data 261
not shown). When the CHO cells transfected with the expression plasmids were subjected to 262
miRNA-185 mimics, the hSR-BI protein level and DiI-HDL uptake was inhibited in pc-WT 263
transfected CHO cells, but not in pc-185m transfected CHO cells (Fig. 3D). Taken together, it was 264
demonstrated that miR-185 blocks selective cholesterol uptake by repressing the HDL receptor 265
SR-BI expression. 266
Next, we evaluated whether the 3 miRNAs might function in a coordinated manner. The results 267
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showed that miR-185, miR-96 and miR-223 mimics decreased luciferase activity in HepG2 cells 268
in a dose-dependent manner and reached the maximal effect at 200 nM (Fig. 4A). Thus, coordinate 269
effects of miR-185, miR-96 and miR-223 were detected at 200 nM in the full-length SR-BI 3'UTR 270
luciferase reporter assay. The results showed that the combination of two or three miRNAs was 271
much more effective than that of each alone (Fig. 4B), implicating their coordination with each 272
other in SR-BI gene regulation. The coordinated effect of these miRNAs was further confirmed by 273
the selective HDL-C uptake in HepG2 cells (Fig. 4C). 274
miRNA expression and regulation by cholesterol in vivo. The functional significance of miR-96 275
was addressed by determining its in vivo expression in various ApoE KO mouse tissues, for its 276
predicted binding sites within mouse SR-BI 3'UTR. As shown in Fig. 5A, miR-96 is broadly 277
expressed in different tissues and particularly abundant in the kidney, liver and intestine compared 278
with other tissues. The expression of miR-185 was also detected in the different tissues (Fig. 5A), 279
although there is a G:U wobble at the seed region of target site for miR-185 in the mouse SR-BI 280
3'UTR. To check whether these miRNAs are modulated in cholesterol loaded physiological 281
conditions in vivo, their expression in the liver of ApoE KO mice fed either a normal chow or 282
high-fat diet (HFD) for 8 weeks was measured. As expected, the HFD treated ApoE KO mice 283
showed a notable increase in body weight, plasma total cholesterol and LDL cholesterol levels 284
compared with the normal fed ApoE KO mice (Fig. 5B). Interestingly, hepatic levels of miR-96 285
were significantly decreased in the HFD treated group, whereas the expression of SR-BI showed 286
an inverse effect (Fig. 5C), suggesting a possible involvement of miR-96 in the regulation of 287
SR-BI functioning in the delivery of excess cholesterol to the liver. The hepatic miR-185 was also 288
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decreased in the HFD treated group (Fig. 5C), implicating that miR-185 may be involved in 289
mouse SR-BI regulation in a direct or an indirect manner. 290
miR-185, miR-96 and miR-223 regulate SR-BI expression in macrophages. In addition to its 291
pivotal role in RCT in the liver, human SR-BI is expressed in cultured macrophages and 292
atherosclerotic lesions (19). Therefore, we further evaluated the expression level of the miRNAs 293
identified above in PMA-stimulated THP-1 cells, which display macrophage-like differentiation. 294
The level of the 3 miRNAs in the PMA-stimulated THP-1 cells was about one order of magnitude 295
higher than that in the untreated THP-1 cells (Fig. 6A). The expression levels of the 3 miRNAs in 296
peritoneal macrophages from mice, as well as in mouse macrophage RAW 264.7 cells, were also 297
detected. The results showed that the miRNA levels were much lower than that from 298
PMA-induced THP-1 cells, which might be attributed to the species difference (Supplemental 299
Figure 3). The inhibitory effect of these miRNAs on SR-BI expression was detected in PMA 300
induced THP-1 cells. As shown in Fig. 6B, the transfection of miR-185 and miR-96 mimics 301
markedly decreased SR-BI mRNA levels, while miR-223 exerted little effect. Further analysis 302
showed that miR-185 significantly repressed DiI-HDL uptake, as expected (Fig. 6C), indicating 303
that miR-185 regulated the DiI-HDL uptake by targeting SR-BI in PMA induced THP-1 cells. 304
Surprisingly, miR-96 increased DiI-HDL uptake (Fig. 6C), giving a hint that miR-96 might also 305
regulate other pathways involved in cholesterol delivery in macrophages. 306
307
Discussion 308
SR-BI, the first molecularly defined and physiologically relevant HDL receptor, is abundantly 309
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expressed in the liver, the organ occupying a central position in the reverse cholesterol transport 310
(RCT) pathway. Numerous studies have established that hepatic SR-BI is a major lipoprotein 311
receptor mediating the selective uptake of periphery-derived cholesterol from HDL particles and 312
possesses atheroprotective activity (8). A better understanding of the molecular mechanisms that 313
regulate SR-BI expression in the liver may provide new insight in the global modulation of HDL 314
metabolism. In addition to elaborate regulation at the transcriptional level, SR-BI regulation in the 315
liver is also exerted at the posttranscriptional level, allowing fast and adaptable modifications in 316
the amount of available SR-BI mRNA and/or protein (20). A wide variety of this type of regulation 317
of SR-BI has been reported in liver in previous studies involving the alternative splicing of 318
pre-mRNA, protein stabilization and subcellular translocation (13,21,22). However, the repression 319
of mRNA transcripts by miRNAs, one centrally important mode of posttranscriptional regulation, 320
has been poorly studied in hepatic SR-BI modulation. 321
Bioinformatic predictions generate dozens of potential miRNAs targeting the hSR-BI 322
3’-untranslated region (3’UTR), suggesting that miRNAs are involved in the complex mechanism 323
to ensure the appropriate SR-BI gene expression regulation. In this work, we first examined the 324
potential involvement of miRNAs by knocking down Dicer and Drosha, which are two important 325
enzymes required for the miRNA maturation. The results provided primary evidence that miRNAs 326
are associated with the regulation of the HDL receptor SR-BI in hepatic cells in either a direct or 327
an indirect manner. Next, we presented the first experimental evidence of the involvement of 328
miR-185, miR-96 and miR-223 in modulating cellular HDL cholesterol delivery by repressing the 329
expression of SR-BI in different human liver cell lines. We also demonstrated that the miRNAs 330
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identified above regulated endogenous SR-BI expression and DiI-HDL uptake by treating the cells 331
with antisense inhibitors. Furthermore, the inhibitory effect of miR-185 on the selective uptake of 332
lipids from HDL was diminished when SR-BI was expressed in CHO cells with mutated miR-185 333
target sites in its 3’UTR. Together with the application of SR-BI siRNA, it was indicated that 334
SR-BI is the functional target of miR-185 in this process. However, it was found in some assays 335
that the effect of the anti-miRNAs did not achieve statistical significance, while the miRNA 336
mimics significantly repressed the SR-BI expression and function. This phenomenon may be 337
attributed to the low endogenous miRNA expression level in the cell lines. In addition, the 338
transfection of miRNA mimics resulted in hundreds of times overexpression compared to 339
endogenous miRNA level, while the introduction of anti-miRNAs resulted in much less reduction 340
(Supplemental Table 3), suggesting that the effect of the cell enrichment of the miRNA mimics is 341
much higher than that of the anti-miRNAs. Interestingly, transfection with anti-miR-96 resulted in 342
a significant increase in miR-185 levels (data not shown), which might have an inverse effect on 343
SR-BI expression against anti-miR-96. All of these may contribute to the relatively unapparent 344
effect of some of the anti-miRNAs. 345
The direct targets within hSR-BI 3’UTR for miR-185, miR-96 and miR-223 were further 346
confirmed by a detailed analysis of these sites in the luciferase reporter assay. Luciferase 347
repression in HepG2 cells was relieved when the specific target site(s) was deleted, and the results 348
of anti-miRNAs are consistent with the above mentioned regulation of endogenous SR-BI 349
expression. We also observed significantly greater repression when two or three miRNAs were 350
combined, compared to that conferred by each miRNA alone. It seems likely that these miRNAs 351
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simultaneously repress the hSR-BI transcript in human hepatic cells by targeting different regions 352
of the 3'UTR. Other studies have reported the coordinated regulation of a single mRNA transcript 353
by multiple miRNAs under a certain circumstances (23,24). 354
There is a predicted target site for miR-96 in the mouse SR-BI 3'UTR and a target site of 355
miR-185 with a G:U wobble in the seed region located within a stretch of the phylogenetically 356
conserved sequence. The G:U wobble is a thermodynamically favorable base pair in RNA 357
secondary structure, and there are validated examples of miRNA-target interactions with wobbles 358
within the seed region (25). Whether miR-185 is involved in the regulation of mouse SR-BI 359
expression needs to be further investigated. Taken together, it will be of interest to know the 360
changes of SR-BI and expression level of the miRNAs in rodents in vivo. The ApoE KO mice fed 361
with HFD showed a repressed level of miR-96 and miR-185 and a more than two-fold increased 362
level of SR-BI in the liver. A similar increase in SR-BI expression was also observed in 363
HFD-treated ApoE KO mice in other reports (26). The expression of SR-BI might be activated by 364
numerous pathways independent of miRNAs, according to the large changes in the mouse 365
metabolism (Fig. 5B). However, there is a possibility that the decreased expression of miR-96 and 366
miR-185 is involved in the mechanisms to increase the SR-B1 expression and regulate the 367
function of SR-BI in the delivery of excess cholesterol to the liver. The actual involvement of 368
miRNAs in mouse SR-BI regulation may be further investigated by experiments using 369
anti-miRNAs in vivo. As the target site of miR-223 is missing in the rodent SR-BI 3’UTR, the 370
presence of miR-223 in the human SR-BI 3’UTR suggests species specificity and perhaps a more 371
elaborated regulation of SR-BI by miRNAs in primates. 372
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Interestingly, among the three miRNAs identified above, we found that miR-185 suppressed 373
SR-BI expression and selective HDL-C uptake in PMA-induced THP-1 cells, which are cultured 374
human monocyte-derived macrophages. Macrophages play a pivotal role in the deposition of 375
excess cholesterol in atherogenesis. Studies have shown that SR-BI liver-specific conditional 376
knockout mice develop less atherosclerosis than that of the SR-BI global knockout mice, 377
suggesting an atheroprotective role for SR-BI in peripheral tissues (27), although the mechanisms 378
by which SR-BI modulates atherosclerosis are unclear. A recent study in cultured mouse bone 379
marrow-derived macrophages found that SR-BI played a significant role in facilitating 380
macrophage bidirectional cholesterol flux in the absence of lipid loading (28). Taken together, 381
miR-185 may serve as a key regulator of lipid metabolism in normal physiological or pathological 382
conditions by targeting SR-BI, not only in the liver but also in the macrophages. Surprisingly, 383
miR-96 increased DiI-HDL uptake in PMA-induced THP-1 cells (Fig. 6C), suggesting that 384
miR-96 might also be involved in other cholesterol delivery pathways in macrophages. 385
Interestingly, there is a predicted target site of miR-96 in the 3'UTR of the human ABCA1 gene, 386
encoding an ATP-binding cassette transporter, which plays a pivotal role in the process of 387
cholesterol efflux from macrophages (29). 388
Since the first two miRNAs, lin-4 and let-7, were discovered in nematode Caenorhabditis 389
elegans (30,31), more than a thousand human miRNAs have been identified. miRNAs have been 390
found to be molecular “rheostats” or “switches” that modulate multiple facets of biological 391
processes, including differentiation, development, proliferation and apoptosis (32-34). In the 392
previous study, some miRNAs have been described to regulate lipid metabolism related genes and 393
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pathways in the liver. miR-122 is most highly expressed in the liver, and silencing of miR-122 in 394
mice altered the gene expression involved in cholesterol synthesis and fatty acid oxidation, 395
resulting in the reduction of total plasma cholesterol, liver triglyceride content and an increase in 396
the rate of fatty acid β oxidation (35). Another well-known miRNA involved in lipid regulation is 397
miR-33. It was independently reported by several groups that miR-33a/b represses multiple genes 398
involved in cellular cholesterol trafficking, especially ABCA1, a key transporter in the generation 399
of HDL and removal of excess cholesterol from peripheral tissues, the first step of RCT (36,37). 400
Recently, miR-758 was discovered as another regulator directly targeting the ABCA1 mRNA 401
3’UTR (38). Interestingly, HDL was reported to mediate miRNA transport (including miR-223) in 402
plasma and delivery to recipient cells depending on SR-BI (39). As hSR-BI harbors a putative 403
binding site for miR-223, the Renilla-SR-BI-3’UTR luciferase reporters in SR-BI-expressing 404
hamster kidney (BHK) cells were used to demonstrate that the SR-BI dependent HDL-miRNA 405
delivery may result in the direct targeting of specific mRNAs. During the preparation of this 406
manuscript, there was a report on miRNA-125a-5p and miRNA-455 repressing SR-BI in 407
steroidogenic cells (40). It was mentioned that miRNA-125a-5p inhibited the expression and 408
function of SR-BI in Hepa 1-6 cells, a mouse hepatoma cell line. We examined whether 409
miRNA-125a-5p regulated the expression level of hSR-BI in HepG2 cells; however, no effect was 410
found on either the hSR-BI 3’UTR reporter construct or on hSR-BI mRNA levels (Supplemental 411
Figure 4). These findings suggest that the effects of miR-125a-5p on SR-BI are species and/or cell 412
specific. 413
In this work we identified that miR-185, miR-96 and miR-223 are involved in HDL-cholesterol 414
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selective uptake as the posttranscriptional regulators of hepatic SR-BI. Gene analysis identified the 415
sequences encoding miR-185 within the first intron of the unknown gene C22orf25 (homo sapiens 416
chromosome 22 open reading frame 25), which is located in the 22q11.21 chromosome region. 417
miR-96 and miR-223 are located intergenically on chromosome 7 and X, respectively. Previous 418
studies have shown that miR-185 could regulate multiple cellular processes relevant to cancer 419
initiation and progression, and some miR-185 targeted genes have been reported (41,42). miR-96 420
was reported to be expressed in neurosensory cells and to be involved in the progression of 421
cochlear hair cells differentiation in the mammalian auditory system (43). miR-223 was shown to 422
be highly expressed in myeloid cells of the bone marrow, acting as ‘‘a fine tuner’’ of granulocytic 423
differentiation and maturation (44). miR-223 has also been reported to be a potent regulator of 424
some inflammatory responses (44,45). Both miR-96 and miR-223, similar to miR-185, have been 425
reported to regulate the genes involved in the proliferation of different tumor cells (46-49). 426
Fortunately, miR-185, miR-96 and miR-223 did not influence the growth of hepatic cancer cell 427
lines used in this work (Supplemental Figure 5). Until recently, the roles of miR-185, miR-96 and 428
miR-223 in lipid metabolism had not been systemically explored. 429
In summary, our study demonstrated for the first time that the miR-185, miR-96 and 430
miR-223-mediated repression of hepatic hSR-BI may be an important regulatory mechanism that 431
modulates HDL cholesterol transport. Subsequent studies will further characterize the role of these 432
miRNAs in regulating lipid metabolism pathways and explore their in vivo effects on HDL 433
delivery by applying the antagonism of endogenous miRNAs. These findings will provide new 434
avenues to understand the complex genetic networks that regulate lipid homeostasis and may open 435
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new possibilities to develop potentially useful therapeutic strategies for the treatment of 436
atherosclerosis and cardiovascular disease by enhancing SR-BI expression and HDL-C transport. 437
438
Acknowledgements 439
This work was supported by grants from the National Natural Science Foundation of China 440
(81102442 and 90813027) and by the State Mega Programs (2012ZX09301002-003, 441
2012ZX09301002-001 and 2010ZX09401-403). 442
We thank Jing Zhang for her help in operation of the IN Cell Analyzer 1000. We thank Yi Bao 443
for his help with the discussion. 444
445
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Figure Legends 591
FIG 1. miR-185, miR-96 and miR-223 are involved in SR-BI modulation in HepG2 cells. (A) 592
The transfection of siRNA against Drosha and Dicer results in increased expression of SR-BI in 593
HepG2 cells. *P<0.05, **P<0.01 vs scr-si. (B-D) SR-BI mRNA levels were detected by real-time 594
RT-PCR (B), protein levels were analyzed by western blot (C), and cell surface SR-BI levels were 595
detected by flow cytometry (D) in HepG2 cells transfected with either miRNAs mimics (100 nM) 596
or anti-miRNAs (100 nM) as well as ctl-miR (100 nM) for 72 h. *P<0.05, **P<0.01 vs ctl-miR of 597
miRNA mimics; #P<0.05 vs ctl-miR of anti-miRNAs. All of the data are presented as the mean 598
values ± SEM of 3 independent experiments. A representative result from 3 independent 599
experiments was shown in (C). scr-si, scrambled siRNA; Dicer-si, Dicer siRNA; Drosha-si, 600
Drosha siRNA; miR-185, microRNA-185; miR-96, microRNA-96; miR-223, microRNA-223; 601
ctl-miR, control miRNA. 602
FIG 2. miR-185, miR-96 and miR-223 modulate SR-BI at posttranscriptional level through 603
3’UTR. (A) Human SR-BI harbors two putative miR-185 target sites, a miR-96 site and a 604
miR-223 site within its 3’UTR. Relative positions of their binding sites are indicated by the 605
different colors. Position 1 represents the first nucleotide following the termination codon. The 606
sequence alignment of the human hsa-miR-185, hsa-miR-96 and hsa-miR-223 to the binding sites 607
of the human SR-BI 3'UTR is shown below. (B) The sequence alignment of the seed regions of the 608
binding sites for miR-185, miR-96 and miR-223 within indicated species. The conserved 609
sequences are shown in red. (C) The effect of the 3 miRNAs on the activity of the luciferase 610
reporter constructs with different truncated 3'UTR of hSR-BI (U2~U6) compared with the 611
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luciferase reporter construct containing the full length 3'UTR of hSR-BI (U1). The effect of 612
control miRNA on the luciferase activity of different constructs (U1~U6) was defined as 1. 613
*P<0.05, **P<0.01 vs ctl-miR; #P<0.05, ##P<0.01, n=3. (D) The SR-BI 3'UTR with the deletion of 614
the indicated seed region of the miR-185 binding sites (DEL1, DEL2) or the miR-96 binding site 615
(DEL3). HepG2 cells were transfected with different constructs for 5 h, followed by transient 616
transfection with miR-185/anti-miR-185 (100 nM) or miR-96/anti-miR-96 (100 nM) for 48 h. 617
Firefly luciferase activity was detected and normalized with the Renilla luciferase. The effect of 618
the control miRNA on the luciferase activity of the different constructs was defined as 1. The data 619
are shown as the mean values ± SEM and were obtained from three independent experiments 620
performed in triplicate. *P<0.05, **P<0.01 vs ctl-miR; #P<0.05, ##P<0.01 vs WT construct. 621
miR-185, microRNA-185; miR-96, microRNA-96; miR-223, microRNA-223; ctl-miR, control 622
miRNA; WT, wild-type construct; Hsa, Homo sapiens; Ptr, Pan troglodytes; Mml, Macaca 623
mulatta; Rno, Rattus norvegicus; Cpo, Cavia porcellus; Mmu, Mus musculus. 624
FIG 3. miR-185, miR-96 and miR-223 regulate selective HDL-C uptake into hepatic cells 625
depending on SR-BI. (A) Flow cytometry analysis to determine the effect of miR-185, miR-96 626
and miR-223 on lipid uptake in HepG2 cells. HepG2 cells were incubated with 2 μg/mL DiI-HDL 627
for 4 h after treatment with the miRNA mimics (100 nM) or the anti-miRNAs (100 nM) for 72 h. 628
The data are shown as the mean values ± SEM of 3 independent experiments in triplicate. *P<0.05, 629
***P<0.001 vs ctl-miR of miRNA mimics; #P<0.05 vs ctl-miR of anti-miRNA. (B) HepG2 cells 630
were incubated with DiI-HDL after treatment with the ctl-miR or the 3 miRNAs for 72 h and were 631
visualized and photographed using the IN Cell Analyzer 1000 with a 20× objective. ‘Blank’ 632
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indicates cells that were not incubated with DiI-HDL. Scale bar, 50 μm. The experiment was 633
repeated 3 times and the representative result is presented. (C) The effect of miR-185 on cell 634
surface SR-BI protein levels and DiI-HDL selective uptake by HepG2 cells with SR-BI siRNA as 635
analyzed using flow cytometry. (D) The effect of the miR-185 on cellular SR-BI protein levels and 636
DiI-HDL selective uptake in pc-WT or pc-185m-transfected CHO cells. CHO cells were 637
transfected with pc-WT or pc-185m for 6 h, and subjected to miR-185 (100 nM) for 48 h. The 638
cellular SR-BI protein levels were analyzed using western blot. The DiI-HDL uptake was analyzed 639
by incubating the cells with 2 μg/mL DiI-HDL for another 4 h and subjected to flow cytometry. 640
The experiment was repeated 3 times. *P<0.05, **P<0.01, ***P<0.001, NS, not significant. 641
miR-185, microRNA-185; miR-96, microRNA-96; miR-223, microRNA-223; ctl-miR, control 642
miRNA; scr-si, scrambled siRNA; SR-BI-si, SR-BI siRNA. 643
FIG 4. miR-185, miR-96 and miR-223 repressed the luciferase activity and selective HDL-C 644
uptake in a coordinated manner. (A) Activity of the luciferase reporter construct containing the 645
3'UTR of hSR-BI (U1) transfected in HepG2 cells. Different concentrations (50, 100, 200, 250 nM) 646
of the 3 miRNAs were transfected for 48 h. The effect of control miRNA on the luciferase activity 647
was defined as 1 for each concentration. The data are shown as the mean values ± SEM and were 648
obtained from 3-5 independent experiments performed in triplicate. *P<0.05, **P<0.01, ***P<0.001. 649
(B) The luciferase reporter analysis for a coordinated effect of miR-185, miR-96 and miR-223. 650
miRNA mimics (200 nM) were transfected individually or together for 48 h before detection. (C) 651
The selective uptake of DiI-HDL by HepG2 cells transfected with the 3 miRNAs individually or 652
together for 72 h was detected using flow cytometry. All of the data shown are significant 653
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compared to ctl-miR (a), #P<0.05, $P<0.01 compared to miR-185+miR-96+miR-223 (b), n=3. 654
**P<0.01. miR-185, microRNA-185; miR-96, microRNA-96; miR-223, microRNA-223; ctl-miR, 655
control miRNA. 656
FIG 5. The distribution and dietary regulation of miR-96 and miR-185 in mice. (A) 657
Expression profile of miR-96 and miR-185 in selected mouse tissues by quantitative RT-PCR. (B) 658
The measurement of body weight, total cholesterol plasma levels and LDL cholesterol levels of 659
ApoE KO mice fed a chow diet (chow) or high-fat diet (HFD) for 8 weeks. (C) Real-time RT-PCR 660
analysis of the miRNA levels and SR-BI expression in the liver of ApoE KO mice fed a chow diet 661
or HFD for 8 weeks. miRNA levels were normalized to U6. *P<0.05, **P<0.01, n=6 (chow) and 662
n=5 (HFD). miR-96, microRNA-96; miR-185, microRNA-185; U6, U6 small nuclear RNA. 663
FIG 6. miR-185 and miR-96 regulate macrophage SR-BI expression and HDL-C uptake. (A) 664
The abundance of miR-185, miR-96 and miR-223 in PMA-induced or non-induced THP-1 cells by 665
quantitative RT-PCR. miRNA levels were normalized by U6. (B) SR-BI mRNA levels in 666
PMA-induced THP-1 cells. THP-1 cells were seeded and stimulated by 100 ng/ml PMA for 24 h 667
before transfection with either the miRNA mimics (100 nM) or the ctl-miR (100 nM) for another 668
48 h. Next, the SR-BI mRNA was analyzed by real-time RT-PCR. (C) Flow cytometry analysis 669
was performed to determine the effect of miR-185, miR-96 and miR-223 on lipid uptake by 670
macrophages. PMA-induced THP-1 cells were incubated with 2 μg/mL DiI-HDL for 4 h prior to 671
detection. The data are presented as the mean values ± SEM of 3 independent experiments. 672
**P<0.01 vs ctl-miR. miR-185, microRNA-185; miR-96, microRNA-96; miR-223, microRNA-223; 673
ctl-miR, control miRNA. 674
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scr-s
i
Dicer
-si
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sha-
si0.0
0.5
1.0
1.5*
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Re
lative
SR
-BI
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er
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ve
l
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β-actin
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β-actin
ctl-m
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-185
anti-
miR
-185
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-96
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-96
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-223
anti-
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-223
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0.5
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*
#
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##
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miR-223
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D
SR-BI 3’UTR (WT)
SR-BI 3’UTR (WT)
SR-BI 3’UTR (DEL1)
SR-BI 3’UTR (DEL2)
...GGACUCUCCCAG...
...GGACUCUCCCAG...
...GGACUCUCCCAG...
...GGACUCUCUCAG...
...GGACUCUCUCAG...
...CUUCUCUCCACG...
...CUUCUCUCCACG...
...CUUCUCUCCACG...
...CUUCUCUCCAAG...
...CCCGCTTCTCCCGGACTCTCCCA...
...CTGTTCTGGAACCTTCTCTCCAC...
...CTGTTCTGGAACCT--------C...
...CCCGCTTCTCCCGGA------CA...
miR-185 Site 2 (274-281 bp)
Seed match
miR-185 Site 1 (84-89 bp)
A
B
CmiR-185 miR-185
(Site 1)SR-BI 3’UTR
(Site 2)
120
miR-96 miR-223
U3
U4
U2
1 498312 664 870
U5
U6
U1
(959 bp)
...CUAAACUGACAU...
...CUAAACUGACAU...
...CUAAACUGACAU...
...CCUGUGCCAAAU...
...CCUGUGCCAAAU...
...CCUGUGCCAAAU...
...CUUGUGCCAAGG...
miR-96 (461-468 bp) miR-223 (694-701 bp)
SR-BI 3’UTR (WT)
SR-BI 3’UTR (DEL3)
...GAGUGCCGCCUUCCUGUGCCAAAU...
...GAGUGCCGCCUUCCU--------U...
hsa-miR-185 AGUCCUUGACGGAAAGAGAGGU...CCCGCUUCUCCCGGACUCUCCCA...SR-BI 3’UTR (84-89 bp)
hsa-miR-185 AGUCCUUGACGGAAAGAGAGGU...CUGUUCUGGAACCUUCUCUCCAC...SR-BI 3’UTR (274-281 bp)
hsa-miR-96 UCGUUUUUACACGAUCACGGUUU...GAGUGCCGCCUUCCUGUGCCAAA...SR-BI 3’UTR (461-468 bp)
hsa-miR-223 ACCCCAUAAACUGUUUGACUGU...UUUCCUCCAGCCUAAACUGACA...SR-BI 3’UTR (694-701 bp)
miR-185 anti-miR-185
0.0
0.5
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Re
lative
lucife
rase
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miR
-96
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-96
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##
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##
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A
B
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0
10
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-18
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ctl-m
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miR
-185
miR
-96
miR
-223
0.0
0.5
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lative
SR
-BI
mR
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le
ve
l
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-96
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-223
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-185/U
6
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