Identification of miR-185 as a regulator of de novo cholesterol biosynthesis and low density lipoprotein uptake.

Dysregulation of cholesterol homeostasis is associated with various metabolic diseases, including atherosclerosis and type 2 diabetes. The sterol response element binding protein (SREBP)-2 transcription factor induces the expression of genes involved in de novo cholesterol biosynthesis and low density lipoprotein (LDL) uptake, thus it plays a crucial role in maintaining cholesterol homeostasis. Here, we found that overexpressing microRNA (miR)-185 in HepG2 cells repressed SREBP-2 expression and protein level. miR-185-directed inhibition caused decreased SREBP-2-dependent gene expression, LDL uptake, and HMG-CoA reductase activity. In addition, we found that miR-185 expression was tightly regulated by SREBP-1c, through its binding to a single sterol response element in the miR-185 promoter. Moreover, we found that miR-185 expression levels were elevated in mice fed a high-fat diet, and this increase correlated with an increase in total cholesterol level and a decrease in SREBP-2 expression and protein. Finally, we found that individuals with high cholesterol had a 5-fold increase in serum miR-185 expression compared with control individuals. Thus, miR-185 controls cholesterol homeostasis through regulating SREBP-2 expression and activity. In turn, SREBP-1c regulates miR-185 expression through a complex cholesterol-responsive feedback loop. Thus, a novel axis regulating cholesterol homeostasis exists that exploits miR-185-dependent regulation of SREBP-2 and requires SREBP-1c for function.


RNA isolation and quantitative real-time PCR
Total RNA was extracted with TRIzol reagent (Invitrogen). cDNA was synthesized from total RNA using the RT Easy First Strand kit (Qiagen). Quantitative real-time-PCR was carried out using a Stratagene MX3005P (Stratagene).The relative mRNA levels were normalized to levels of GAPDH. In addition, small RNA was converted to complimentary DNA from 500 ng of total RNA using the miScript II RT kit (Qiagen). The miR level was determined by quantitative real-time-PCR using miR-specifi c (miR-185) primers (Qiagen) and normalizing to RNU6-2 snRNA levels as a control. For miR-185 levels in mouse liver, miR-185 expression was normalized to SNORD66 level. Data is shown as the ratio of miR-185 expression as compared with either RNU6-2 or SNORD66.

Immunocytochemistry staining
Forty-eight hours after HepG2 cells were transfected with pre-miR-185 and control pre-miR, cells were fi xed and labeled with rabbit anti-LDLR antibody (Cayman Chemicals, Ann Arbor, MI), followed by incubation of Alexa Fluor 488-conjugated anti-rabbit secondary antibody (Invitrogen). After antibody incubation, cells were mounted with fl uorescent mounting medium containing DAPI (Invitrogen) for counterstaining. Fluorescent microscopy was performed using a 20× objective on a Leica DMI6000 confocal microscope, and images were processed using LAS AF software. is cleaved by site-1 protease/site-2 protease and converted to a soluble mature SREBP-2 transcription factor that translocates to the nucleus, where it binds SREs in the promoters of sterol-responsive genes ( 12,13 ).
miR-185 and its role in cell biology fi rst came to light when a connection was discovered between miR-185 expression and cancer progression. For instance, miR-185 overexpression suppressed the migration and invasiveness of LNCaP prostate cancer cells ( 37 ), while its repression led to cisplatin resistance in SKOV3/DDP ovarian cell lines ( 38 ). Moreover, specifi c miR-185 SNPs have an inverse relationship with breast cancer risk, suggesting this miR may have biomarker attributes ( 39 ). miR-185 targets include RhoA, Cdc42, DNA methyltransferase 1, the androgen receptor, and the Six1 oncogene ( 37,(40)(41)(42). While evidence is accumulating establishing the miR-185 cancer connection, its role in regulating lipid metabolism is obscure. Very recently, miR-185 was shown to repress selective HDL-cholesterol uptake through the inhibition of scavenger receptor BI (SR-BI) expression in human hepatic cells ( 43 ). This is the only report linking miR-185 to any type of lipid metabolic regulation. Interestingly, another miR, miR125a-5p, was also found to inhibit SR-BI expression in steroidogenic cells ( 44 ). We tested several miRs (miR-1260, -532, -324, and -185) potentially targeting the 3 ′ UTR of SREBP-2 for the ability to modulate SREBP-2 expression. Of those tested, only miR-185 was found to signifi cantly reduce SREBP-2 expression. Here, studies show that miR-185 regulates SREBP-2 expression, which in turn regulates LDLR expression and LDL uptake. Moreover, evidence is presented for the fi rst time showing that SREBP-1c regulates miR-185 expression, thus there is a feedback loop that precisely regulates miR-185 activity in order to maintain cholesterol homeostasis.

Cell lines and reagents
THLE-2, HepG2, and 293T cells were obtained from ATCC. THLE-2 cells were cultured in BEGM medium with supplements sites shown in underline, mutated bases shown in bold text). Electrophoretic mobility shift assay (EMSA) binding reactions were performed at room temperature for 20 min and consisted of NE in 1× binding buffer (50% glycerol, 100 mM MgCl 2 , 1 g/ l poly(dI-dC), 1% NP-40, 1 M KCl, 200 mM EDTA, and 5 M DNA probe). The mixture was run on 6% nondenaturing polyacrylamide gels in 0.5× Tris borate-EDTA buffer. Protein-DNA complexes were then transferred to Hybond-N+ nylon membrane using the Trans-Blot semi-dry method (Bio-Rad, CA), and cross-linked using the Spectrolinker XL-1000 UV cross-linker (Spectronics Corporation, Westbury, NY). Detection of biotinlabeled DNA was performed using the LightShift chemiluminsecent EMSA kit (20148; Thermo Scientifi c) and visualized by exposure to a charge couple device camera (GE ImageQuant LAS 4000).
For EMSA competition studies, 20-fold molar excess of WT nonbiotin labeled site 1 forward and reverse oligonucleotides were added to the EMSA reaction mix. For the gel super-shift assay, following the incubation of the NEs with site 1 WT miR-185 promoter probes, 4 g of SREBP-1c mouse antibody (Santa Cruz) or 4 g of GAPDH mouse control antibody (GeneTex Inc., Irvine, CA) were added to the reaction mixture, and incubated at room temperature for 30 min. The mixture was fractionated on a 5% nondenaturing polyacrylamide gel. Transfer and detection were performed as described above.

Liver X receptor activation assays
HepG2 cells were treated with 1% hydroxypropyl-␤ -cyclodextrin (HPCD) in serum-free medium for 1 h at 37°C to remove intracellular cholesterol ( 47 ). To activate liver X receptor (LXR) signaling, cells were incubated with 2 g/ml 25-HC in serum-free medium for 6 h at 37°C. quantitative real-time-PCR was then used to compare SREBP-1c , ABCA1 , ABCG1 , SREBP-2 , and miR-185 expression levels. GAPDH and RNU6-2 were used as internal controls for mRNA and miR, respectively. 25-HC was dissolved in ethanol and control cells were added with the same volume of ethanol as control.

Insulin treatment
HepG2 cells were grown in serum-free medium with or without 100 nM insulin overnight at 37°C. SREBP-1c , SREBP-2 , and miR-185 expression levels were determined by quantitative realtime-PCR. GAPDH and RNU6-2 were used as internal controls for mRNA expression and miR-185 expression, respectively. The SREBP-1c protein level was examined by Western blotting. GAPDH was used as internal control. Densitometry was used to normalize both full-length and mature forms of SREBP-1c protein normalized to GAPDH protein level. The values are the average of three independent experiments.

Mouse feeding and lentiviral injection studies
WT male C57BL/6J (B6) mice were purchased from Jackson Laboratories and housed at Temple University, Philadelphia, PA. Temple University Institutional Animal Care and Use Committee (IACUC) approved all experimental procedures. Male B6 mice (6-8 weeks old) were fed either a normal diet (7% fat; BioServ, Frenchtown, NJ) or a high-fat diet (21% fat; BioServ) for 16 weeks. Fasted blood samples were taken every 4 weeks. Blood serum was obtained and used to measure total cholesterol using a total cholesterol kit (Stanbio, Boerne, TX) following the manufacturer's protocol. Mice from each group were also euthanized at weeks 4, 8, 12, and 16, and liver tissue was collected for protein and miR analysis.

LDL uptake assay
Twenty-four hours after HepG2 cells were transfected with pre-miR-185 and control miR, cells were washed with PBS and incubated overnight in lipoprotein-defi cient medium (LPDS) to induce the expression of LDLR. LDL uptake was initiated by incubating cells that were grown in serum-defi cient medium with 5 g/ml BODIPY-LDL (Invitrogen). Uptake of BODIPY-LDL was measured after a 30 min incubation at 37°C. Cells were fi xed and mounted with fl uorescent mounting medium containing DAPI (Invitrogen) for counterstaining. Intercellular BODIPY-LDL was visualized using fl uorescence microscopy.

HMGCR activity assay
HMGCR activity assay was performed as described previously ( 45,46 ). Briefl y, 24 h after HepG2 cells were transfected with pre-miR-185 or control pre-miR, cells were incubated with 5% LPDS overnight to stimulate HMGCR activity. After lysing the cells, 100 g of total protein was incubated at 37°C for 30 min in reaction buffer (20 mM glucose-6-phophate, 0.7 units glucose-6phosphate dehydrogenase, 3 mM NADPH, and 5 mM DTT). The reaction was started by addition of 30 M 14 C-HMG-CoA (American Radiolabeled Chemicals). After a 2 h incubation at 37°C, the reaction was stopped by addition of 5 N HCl (EMD) and 3 M 3 H-mevolonolactone (American Radiolabeled Chemicals). 14 Cmevalonolactone was separated from unreacted 14 C -HMG-CoA by column chromatography using AG1-X8 resin (200-400 mesh; Millipore). After the samples were added to the resin bed, seven 1 ml aliquots of water were used to elute 14 C-mevalonolactone. The fi rst 2 ml of aliquots were discarded, and the next 5 ml of elute were quantifi ed using a liquid scintillation counter (Beckman Coulter). HMGCR activity was determined by normalizing isolated 14 C-mevalonolactone from the internal control, 3 Hmevolonolactone.

Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) was performed using the Imprint ChIP kit (Sigma) following the manufacturer's instructions. Immunoprecipitation was performed using anti-polyclonal rabbit SREBP-1c (Santa Cruz) antibody and rabbit IgG (Santa Cruz) as a control. PCR was used to amplify the promoter region of miR-185 using primers detecting the 50-500 bp upstream from the transcriptional start site (TSS) (forward, ATCCCAGAGTA AA-GGCAGATAAGG; reverse, GCGGAGACATGTCATCTCC).

Nonradiolabeled electrophoretic mobility shift and gel super-shift assays
Nuclear extracts (NEs) were prepared from HepG2 cells using the Nuclear Extract kit (Active Motif, Carlsbad, CA) as described by the manufacturer. Wild-type (WT) and mutant probes were synthesized as single stranded oligonucleotides with biotin 3 ′ -end labeling (Integrated DNA Technologies) from the Ϫ 100 to Ϫ 139 and Ϫ 225 to Ϫ 261 regions of the miR-185 putative promoter. Transfection of pre-miR-185 signifi cantly decreased the cellular luciferase activity of cells carrying a WT SREBP-2 3 ′ UTR reporter plasmid (70%), as compared with cells transfected with only a control miR ( Fig. 3A , D vs. E). The addition of point mutations within the four MREs significantly interfered with miR-185 targeting and silencing of SREBP-2 3 ′ UTR luciferase activity ( Fig. 3A , E vs. G). These results suggested that miR-185 interacted with the MREs found within the SREBP-2 mRNA 3 ′ UTR.
Previous studies have shown that by targeting mRNA 3 ′ UTRs, miRs silence target genes through initiating mRNA degradation or inhibition of translation ( 27,28 ). To retro-orbital injection. Two weeks postinjection, mice were euthanized and liver tissue was collected for miR analysis. Primary miR-185 sequence was cloned into pCDH-CMV-MCS-EF1 lentivector (SBI, Mountain View, CA). Lentivirus was generated by transfection of packaging plasmids (SBI) and expressing the vector into 293T cells. The virus was collected 48 h posttransfection.

Human serum samples
Human normal serum samples (n = 46) and hypercholesterolemia serum samples (n = 40) were collected from Bioreclamation. All serum samples were from patients >50 years old. Mixed sex and race samples were used. Total RNA was extracted with TRIzol reagent (Invitrogen). cDNA was synthesized from total RNA using the RT Easy First Strand kit (Qiagen). In addition, small RNA was converted to complimentary DNA from 100 ng (human serum) of total RNA using the miScript II RT kit (Qiagen). The miR-185 level was determined by quantitative real-time-PCR using miR-specifi c (miR-185) primers (Qiagen) and normalizing to RNU6-2 snRNA level as a control. All patients were being treated with statins. Patients diagnosed with hypercholesterolemia had total cholesterol values >240 mg/dl and LDL levels >160 mg/dl.

Statistical analyses
The data shown are the average of at least three independent experiments. Data are expressed as mean ± SEM. Statistical analysis was performed using Student's t -test.

miR-185 expression is regulated by changes in cholesterol level
To begin to elucidate the role of miR-185 in cholesterol homeostasis, it was fi rst addressed whether miR-185 expression was regulated in response to cholesterol level. HepG2 cells were treated with and without the cholesteroldepleting agent, MCD, and SREBP-2 and miR-185 expressions were determined. In the presence of MCD, SREBP-2 expression increased ‫ف‬ 2.5-fold when compared with control cells [

miR-185 represses SREBP-2 expression through targeting SREBP-2 mRNA 3 ′ UTR
There are four predicted MREs within the 3 ′ UTR region of human SREBP-2 mRNA, based on bioinformatics searches using TargetScan 6.1 and miRBase ( Fig. 2A ). To examine whether miR-185 targets to these predicted MREs, a luciferase reporter assay was used. HEK293 cells were transfected with a pSwitchLight WT-SREBP-2 -3 ′ UTR luciferase plasmid or a mutant SREBP-2 -3 ′ UTR luciferase reporter that had C to G point mutations in the four predicted miR-185 seeding sites:  and LDL uptake. In order to examine whether miR-185 affects SREBP-2-dependent gene expression, HepG2 cells were transfected with pre-miR-185 or a control pre-miR and the mRNA expressions of HMGCR , FDFT1 , and LDLR were determined by quantitative real-time-PCR. HepG2 cells expressing pre-miR-185 were treated with MCD. HMGCR , FDFT1 , and LDLR expressions were determined in the absence or presence of MCD.
The levels of HMGCR and FDFT1 expressions did not signifi cantly decrease in HepG2 cells overexpressing miR-185 ( Fig. 4A, C ; Con miR vs. miR-185), while LDLR expression decreased by 60% ( Fig. 4B , Con miR vs. miR-185 ). The addition of MCD resulted in HMGCR upregulation only in control cells ( Fig. 4A ; Con miR vs. Con miR + MCD, >2.5-fold), while miR-185 overexpression abolished MCDstimulated HMGCR upregulation ( Fig. 4A ; Con miR + MCD vs. miR-185 + MCD). In the presence of a control pre-miR, LDLR and FDFT1 gene expressions increased in the presence of MCD by >13-fold and >3-fold, respectively ( Fig. 3B, C ; Con miR vs. Con miR + MCD). The expression of miR-185 abolished MCD-stimulated LDLR and FDFT1 expressions ( Fig. 4B, C ; Con miR + MCD vs. miR185 + MCD). These results indicate that miR-185 has a critical role in regulating cholesterol metabolism-related gene expression via SREBP-2 posttranscriptional repression. determine whether miR-185 targets the 3 ′ UTR of endogenous SREBP-2 mRNA, miR-185 was tested for its ability to attenuate endogenous SREBP-2 mRNA and protein expression. HepG2 and THLE-2 cells were transfected with pre-miR-185 or a control miR, and SREBP-2 mRNA expression was determined by quantitative real-time-PCR. Overexpression of miR-185 signifi cantly decreased the SREBP-2 mRNA level by ‫ف‬ 60% in HepG2 and THLE-2 cells, as compared with miR-control cells ( Fig. 3B, C ). Addition of a miR-185 antagomiR abolished miR-185 targeting of the SREBP-2 3 ′ UTR ( Fig. 3B, C ). The interaction of miR-185 to the endogenous SREBP-2 3 ′ UTR also reduced the level of SREBP-2 full-length and mature proteins ( Fig. 3D ). We do note that in both cell lines the addition of antagomiR plus miR-185 did not cause a further increase in SREBP-2 expression compared with control cells. It does suggest that the endogenous miR-185 level is low in our cell lines. Overall, miR-185 negatively regulated SREBP-2 expression as a result of interaction with the SREBP-2 mRNA 3 ′ UTR region, which leads to reduced SREBP-2 mRNA and protein level.

miR-185 affects SREBP-2-dependent gene expression
SREBP-2 plays a critical role in regulating cholesterol metabolism by controlling the expression of several important genes involved in de novo cholesterol biosynthesis , and pre-miR-185 (miR185) or control pre-miR (Con miR). Luciferase activity was measured in 293T cells as described in the Materials and Methods section. Bar graphs represent mean ± SEM from three independent experiments. B: The fold change in SREBP-2 mRNA was measured by quantitative real-time-PCR in pre-miR-185, Antagamer -miR-185 (Antagomer) (100 nM), or control miR (Con miR) transfected HepG2 cells. Bar graphs represent mean ± SEM from three independent experiments. C: SREBP-2 mRNA was measured by quantitative real-time-PCR in pre-miR-185 or control miR transfected THLE-2 cells. Values were normalized to the level of GAPDH. Bar graphs represent mean ± SEM from three independent experiments. D: SREBP-2 protein level was determined using whole cell lysates by Western analysis in miR-185 overexpressing and control miR transfected HepG2 cells. ␤ -Actin was used as a loading control. FL, full-length SREBP-2; M, mature form of SREBP-2.

Transcriptional regulation of miR-185 by SREBP-1c
Transcriptional regulation of miRs is a mechanism by which endogenous miR expression and function are regulated. The TSS of pre-miR-185 was determined using miRStart ( 52 ), and 500 bp upstream of the TSS was chosen to study miR-185 promoter activity . Two putative SREBP-1c binding sites are located within this promoter region ( Fig. 7A ; SRE1 and SRE2); the consensus sequence is TCACNCCAC. Based on the presence of these binding sites, SREBP-1c was tested for its ability to mediate miR-185 promoter activity. Interestingly, siRNA knockdown of SREBP-1c in HepG2 cells ( Fig. 7B ) increased the SREBP-2 mRNA ( Fig. 7C ) and protein ( Fig. 7D ) levels. On the other hand, the mature miR-185 expression level decreased in SREBP-1c knockdown HepG2 (>2-fold, Fig. 7E ) and THLE-2 cells (>2-fold, Fig. 7F ), as compared with control cells. These fi ndings strongly suggested that SREBP-1c negatively regulated SREBP-2 expression through upregulating miR-185, possibly via binding to the promoter of miR-185. To test this hypothesis, ChIP was used to examine the binding of SREBP-1c to the miR-185 promoter. SREBP-1c was found to bind to the 500 bp fragment upstream of the miR-185 TSS ( Fig. 7G ). SREBP-2 binding to these SREs was not detected (not shown).

miR-185 reduces LDLR protein, decreases LDL uptake, and attenuates HMGCR activity
The LDLR plays a critical role in LDL uptake by internalizing LDL-cholesterol via endocytosis ( 3,4 ). In order to explore the physiological signifi cance of miR-185-dependent regulation of SREBP-2 expression, the endogenous level of LDLR protein was examined by Western analysis and indirect immunofl uorescence, and LDL uptake was determined using the fl uorescently-labeled LDL, LDL-BODIPY.
LDLR protein level was decreased in HepG2 cells overexpressing pre-miR-185 as compared with pre-miR control cells ( Fig. 5A ). This result correlates with the expression data obtained in Fig. 3B . Moreover, the endogenous level of LDLR was drastically reduced in these cells ( Fig. 5B ; Con miR/LDLR vs. miR-185/LDLR). Finally, the level of internalized LDL-BODIPY was severely reduced in miR-185 expressing cells ( Fig. 5C ; Con miR/LDL-BODIPY vs. miR-185/LDL-BODIPY). Thus, miR-185 reduces cellular cholesterol level by reducing the uptake of LDL via decreased LDLR expression.
HMGCR is the rate-limiting enzyme of de novo cholesterol biosynthesis and converts HMG-CoA to mevalonate ( 50,51 ). As demonstrated above, miR-185 inhibited MCDinduced HMGCR transcription through repressing SREBP-2 expression ( Fig. 4A ). To determine the physiological signifi cance of this repression, HMGCR activity was determined in HepG2 cells overexpressing miR-185. In the presence of MCD, there was a 1.75-fold increase in HMGCR activity over baseline ( Fig. 6 ; Con vs. Con + MCD). This Fig. 5. The loss of LDLR impairs LDL uptake in miR-185 overexpressing HepG2 cells. A: LDLR protein level was determined by Western analysis in HepG2 cells transfected with control pre-miR (Con miR) or pre-miR-185 (miR-185) using whole cell lysates. ␤ -Actin was used as loading control. B: Densitometry analysis of LDLR protein normalized to actin level. Bar graphs represent mean ± SEM from three independent experiments. C: Cell surface LDLR was determined by immunocytochemistry in HepG2 cells transfected with control pre-miR or pre-miR-185. DAPI was used as counterstain. Uptake of LDL by HepG2 cells was observed by incubating control pre-miR or pre-miR-185 transfected HepG2 cells with LDL-BODIPY. DAPI was used to counterstain for nuclei. A Leica DMI6000 fl uorescent microscope was used to visualize staining.
An EMSA was used to identify the binding site(s) for SREBP-1c. Only a mobility shift of the SRE1 probe was observed using NEs from HepG2 cells, when compared with SRE2 ( Fig. 8A , lane 2 vs. lane 4). Binding was abolished when SRE1 was mutated ( Fig. 8A , lane 2 vs. lane 3). A 20fold cold competitor SRE1 probe also abolished binding ( Fig. 8B , lane 2 vs. lane 3). In order to confi rm that the protein-DNA complex formation was due to a SREBP-1c-SRE1 interaction, a gel super-shift assay was performed using anti-SREBP-1c polyclonal antibodies. Addition of this antibody to the NE resulted in a super shift in the protein-DNA complex ( Fig. 8B , lane 2 vs. lane 3). Thus, SREBP-1c binds to SRE1 in the miR-185 promoter in order to regulate its expression in response to cholesterol level.

miR-185 expression is induced by LXR activation and insulin treatment
LXR activates SREBP-1c expression in the presence of oxidized cholesterol. To determine whether miR-185 expression was regulated by activation of LXR and induction of the SREBP-1c level, HepG2 cells were treated with HPCD to remove cholesterol. They were then incubated with 25-HC and the expressions of several LXR-regulated genes were determined. ABCA1 , ABCG1 , and SREBP-1c expressions were all upregulated upon LXR activation when compared with control cells ( Fig. 9 ). More importantly, miR-185 expression was induced ‫ف‬ 9-fold, while SREBP-2 expression was reduced by nearly 75% ( Fig. 9 ). These results suggest that SREBP-2 expression is regulated by increases in the miR-185 level, brought about by LXR activation and subsequent induction of SREBP-1c transcription factor activity.
Insulin is a major activator of SREBP-1c in the liver ( 53,54 ). To further validate that miR-185 expression was  and control siRNA (Csi). ␤ -Actin was used as a loading control. C, D: SREBP-2 mRNA expression (C) and mature SREBP-2 protein level (D) were determined in HepG2 cells transfected with SREBP-1c siRNA or control siRNA. GAPDH was used as internal control in quantitative real-time-PCR, and ␤ -actin was used as a loading control for Western blot analysis. Expression levels were quantitated by quantitative real-time-PCR. RNU6-2 was used as internal control. E, F: Relative expression of mature miR-185 in SREBP-1c siRNA transfected HepG2 (E) and THLE2 cells (F) compared with control siRNA transfected cells. Expression levels were quantitated by quantitative real-time-PCR. RNU6-2 was used as internal control. G: ChIP assay was used to determine the extent of binding of SREBP-1C to the miR-185 promoter region. Ten percent of the cell extract was used as an input. Rabbit IgG was used as a negative control. Primers detecting 50-500 bp upstream of the miR-185 TSS were used for PCR. FL, full-length SREBP-1c; M, mature form of SREBP-1c.

Human miR-185 expression is elevated in individuals with high cholesterol
Finally, miR-185 expression was determined in individuals with high cholesterol. Control patients had an average miR-185 expression ratio of ‫ف‬ 10:1 (miR-185/RNU-6), while individuals with high cholesterol had an ‫ف‬ 50:1 ratio ( Fig. 13 ). DISCUSSION SREBP-2 acts as a master switch regulating the transcription of an array of genes that are critical for maintaining intracellular cholesterol homeostasis ( 8,13 ). A better knowledge of the molecular mechanisms mediating SREBP-2 expression and function will help further the understanding of the complex systems in place to modulate cholesterol metabolism. Our fi ndings have uncovered a novel mechanism by which SREBP-2 expression is posttranscriptionally repressed by miR-185. By decreasing the SREBP-2 expression level, miR-185 negatively regulated SREBP-2-dependent gene expression, resulting in decreased expression of several genes required for de novo cholesterol biosynthesis, and reduced LDLR protein and LDL uptake. Strikingly, miR-185 expression was regulated by SREBP-1c, thus setting up a possible cholesterol-responsive feedback loop. In vivo high-fat feeding studies showed that high-fat-fed mice had 1 ) an elevated expression of miR-185, and 2 ) reduced SREBP-2 protein. Moreover, mice overexpressing

An increase in miR-185 expression level correlates with high blood cholesterol and reduced SREBP-2 protein in mice fed a high-fat diet
To explore the in vivo relevance of miR-185-dependent regulation of SREBP-2 expression, miR-185 expression, cholesterol level, and SREBP-2 level were determined in mice fed a high-fat atherogenic diet (21% fat) or a normal fat diet (7% fat ). Mice were fed each diet for 16 weeks, and blood and organs were harvested at weeks 4, 8, 12, and 16. In mice fed a high-fat diet, there was a time-dependent increase in blood cholesterol level, while control animals fed a normal diet did not show this increase ( Fig. 11A ). The level of SREBP-1c and miR-185 also showed a timedependent increase ( Fig. 11B, C ), while SREBP-2 expression and protein level decreased in mouse livers ( Fig. 11D,  E ). Densitometry analysis of the Western blot verifi ed that SREBP-2 levels decreased in a time-dependent manner ( Fig. 11F ).
To further substantiate these results, mice were injected with miR-185 lentivirus, and miR-185, SREBP-2 , LDLR , PCSK9 , and HMGCR expression levels were determined in the liver at 12 weeks. miR-185 expression was upregulated  effl ux pump, inhibiting its expression, which results in increased intracellular cholesterol concentration under conditions of need ( 58,59 ). Interestingly, miR-33a/b negatively regulate adenosine monophosphate kinase ( 35 ), a kinase that has been shown to phosphorylate and inhibit SREBP-2 activity ( 60 ). Thus, miR-33a/b expression would presumably upregulate SREBP-2 and cholesterol biosynthesis. In our case, miR-185 was upregulated through regulation by SREBP-1c. It targeted SREBP-2 , shutting it down, likely ensuring that SREBP-2 did not increase cholesterol to a toxic level. Interestingly, the miR-185 level was also upregulated in mice fed a high-fat diet, and in human sera from patients with high cholesterol. Thus, it seems that in vivo, miR-185 regulates SREBP-2 activity when cholesterol level is high in order to precisely fi ne-tune the level of this lipid. There are two other miRs, miR-4644 and miR-4306, that are proposed to target the same 3 ′ UTR sequence as miR-185. A scan of their promoter sequences did not show any SREBP-1c binding sites. Thus, if they do regulate SREBP-2 expression, it is likely independent of SREBP-1c regulation.
The removal of cholesterol by MCD resulted in an acute activation of SREBP-2 and sterol gene expression, which was quickly blunted by overexpression of miR-185, suggesting that miR-185 regulation of SREBP-2 transcription may be fast and adaptable. Thus, tight transcriptional regulation of miR-185 expression is likely necessary under conditions where cholesterol level changes are drastic, such as times of fasting or after a high-fat meal. SREBP-1c expression is not regulated by cholesterol level to any measurable extent, thus its constitutive presence would ensure a direct rapid regulation of miR-185 expression. As SREBP-1c function decreases, so should miR-185 expression, resulting in a fi ne-tuning in cholesterol level by the turning on and off of SREBP-2 function. The loss of SREBP-1c function in this scenario presumes that there is some type of modifi cation, either changes in expression or some type of posttranslational modifi cation. Thus, the three components in this loop need to be regulated by the same sensing mechanism. This same scenario is seen with miR-33a/b, as the SREBP-2 promoter regulates miR-33a/b expression ( 59 ). By inhibiting ABCA1 function and cholesterol effl ux, miR-33a/b expression should be turned off, as should SREBP-2 expression, as the cholesterol level rises. It has been shown that nuclear receptors can directly bind to miR promoters and control their expression.
Why would miR-185 be upregulated by a transcription factor (SREBP-1c) involved in fatty acid synthesis, as miR-185 inhibits SREBP-2 expression, thus reducing de novo cholesterol biosynthesis and LDL uptake? SREBP-1c expression is activated during a number of metabolic states (61)(62)(63)(64)(65)(66), insulin treatment (63)(64)(65), and through LXR ␣ activation by oxysterols ( 67,68 ). This latter activation is believed to be required for the production of the fatty acids needed for cholesterol ester (CE) synthesis ( 69 ). Our results indicated that miR-185 expression was induced through LXR activation and the resulting SREBP-1c expression. Based on this data, it is possible that a SREBP-1c/ miR-185 feedback loop exists in order to ensure that the miR-185 had a reduced SREBP-2 expression level and a reduction in the expressions of several SREBP-2-dependent genes. These results strongly suggest that there is a novel miR-185 regulatory axis for the posttranscriptional regulation of SREBP-2 expression, whose activity responds to, and is regulated by, cholesterol level.
It is well established that miRs play an important role in the initiation and/or progression of cardiometabolic disease ( 27,35,36,55,56 ). miR-122 was the fi rst reported miR associated with the regulation of cholesterol homeostasis in the liver ( 27,35,36,57 ). The exact target genes regulated by miR-122, and the mechanism by which miR-122 regulates cholesterol level, remains to be fully elucidated. Another extensively studied group of miRs is the miR-33a/b family, which is found within the introns of SREBP-1 and SREBP-2 . miR-33a/b have several targets that include the ATP binding-cassette (ABC)A1 cholesterol transport by taking up circulating HDL. Although SR-BI knockout mice have increased plasma HDL, reduced hepatic cholesterol uptake, and decreased biliary cholesterol secretion ( 79 ), there are reports demonstrating that SR-BI knockout alone is not suffi cient to induce an atherosclerotic phenotype in mice ( 80 ). It is likely that there is a compensatory ratio of free cholesterol/CEs is maintained. Inhibition of SREBP-2 function by miR-185 may result in a decrease in cholesterol level, allowing for the conversion of the remaining intracellular pool to nontoxic CEs. As the level of free fatty acids increases, SREBP-1c expression would decrease, miR-185 expression would be blunted, and SREBP-2 expression would increase. This would allow for the synthesis of the cholesterol needed to reduce the free fatty acid pool through esterifi cation. It is well-known that the expression of SREBP-1c is negatively regulated by an increase in polyunsaturated fatty acids (PUFAs) (70)(71)(72)(73)(74). Interestingly, ACAT activity is elevated by PUFAs, thus setting up the use of fatty acids as substrates for CE biosynthesis at a time when SREBP-1c activity is being reduced ( 75,76 ). We point out that the hypothesis described is speculative and requires further studies to validate our model.
Our fi ndings showed that SREBP-1c transcriptionally activated miR-185 expression through binding to a specifi c SRE within the promoter region of miR-185, resulting in the suppression of SREBP-2-dependent events. Moreover, the knockdown of SREBP-1c actually resulted in increased SREBP-2 expression and protein. Thus, SREBP-1c inversely regulated SREBP-2 expression. Our fi nding may help to explain why SREBP-1 Ϫ / Ϫ mice show an elevated SREBP-2 expression level in the liver ( 77,78 ). Quite possibly, a decrease in miR-185 expression due to reduced SREBP-1c expression results in elevated SREBP-2 expression, acting as a compensatory mechanism. However, this mechanism may be toxic, as SREBP-1 Ϫ / Ϫ mice have a 3-fold increase in cholesterol biosynthesis in the liver, and a 50% increase in hepatic cholesterol level ( 77 ). Interestingly, the regulation of miR-185 expression was specifi c to SREBP-1c, as siRNA against SREBP-2 had no effect (M. Yang, W. Liu, and J. T. Nickels, unpublished observations). SREBP-1a was not studied, as its expression level is extremely low in liver cells compared with SREBP-1c.
Although SR-BI is targeted by miR-185, how it decreases the SR-BI level and affects cholesterol homeostasis still needs to be elucidated through further in vivo studies. As SR-BI was found to remove CEs from circulating HDL, it was thought to regulate HDL-mediated reverse cholesterol Fig. 11. Mice fed a high-fat diet express a higher level of miR-185. A-D: Mice fed normal (7% fat) or atherogenic (21% fat) diets were euthanized at weeks 4, 8, 12, and 16. Blood was drawn and organs were harvested. Cholesterol level in blood (A), relative SREBP-1c expression in liver (B), relative miR-185 expression in liver (C), SREBP-2 expression (D), SREBP-2 mature protein level in liver (E), and SREBP-2 densitometry measurement (F) were determined at the indicated weeks. Levels were compared with control cells grown in the absence of 25-HC, and the value obtained was set at 1 (n = 3-5 mice).

Fig. 12.
Mice overexpressing miR-185 harbor defects in SREBP-2 signaling. WT male B6 mice (6-8 weeks old) were injected with 5 × 10 9 pfu lentivirus with or without primary miR-185 sequences via retro-orbital injection. Two weeks postinjection, mice were euthanized and liver tissue was collected for miR analysis. The expression levels of miR-185 (A), SREBP-2 (B), and PCSK9 (C). LDLR and HMGCR were determined using quantitative real-time-PCR (n = 3-5 mice). RNU-6 was used as an internal control. SR-BI-independent mechanism for HDL uptake ( 79 ). In vivo studies looking at how miR-185 functions to regulate SR-B1 and how this regulation affects cholesterol homeostasis are necessary to provide more insight concerning miR-185 function and reverse cholesterol transport.
Whether overexpressing miR-185 to inhibit SREBP-2 activity represents a feasible therapeutic for treating cholesterol-related diseases awaits further long-term in vivo mouse studies testing its effi cacy in modulating SREBP-2 expression and lipid levels. It must be kept in mind that negatively regulating SREBP-2 may reduce LDLR expression, possibly leading to increased LDL and free fatty acid levels. However, it would also reduce PCSK9 expression. PCSK9 is involved in LDLR degradation. The loss of PCSK9 function may stabilize LDLR, minimizing the potential deleterious effects caused by loss of LDLR expression by SREBP-2. The idea of targeting SREBP-2 as a means to help treat hypercholesterolemia is beginning to gain traction ( 81 ). Recently, Moon et al. ( 82 ) showed that siSCAP-treated mice showed reduced SREBP-2 expression, but maintained a steady-state LDLR level, most likely due to a reduction in PCSK9 expression. Moreover, SREBP small molecule screening has identifi ed betulin, which enhances the SCAP-INSIG interaction ( 83 ). When administered to mice, betulin reduced hyperlipidemia and insulin resistance, while also decreasing atherosclerotic plaque formation ( 83 ). Thus, targeting SREBP for small molecule therapy warrants exploration. Fig. 13. miR-185 expression is elevated in individuals with high cholesterol. Blood from control individuals (n = 40) or individuals with high cholesterol (n = 46) were obtained and miR-185 expression was determined using quantitative real-time-PCR.