MicroRNA hsa-miR-613 Targets the Human LXRα Gene and Mediates a Feedback Loop of LXRα Autoregulation

Zhimin Ou; Taira Wada; Roberto Gramignoli; Song Li; Stephen C Strom; Min Huang; Wen Xie

doi:10.1210/me.2010-0360

. 2011 Feb 10;25(4):584–596. doi: 10.1210/me.2010-0360

MicroRNA hsa-miR-613 Targets the Human LXRα Gene and Mediates a Feedback Loop of LXRα Autoregulation

Zhimin Ou ¹, Taira Wada ¹, Roberto Gramignoli ¹, Song Li ¹, Stephen C Strom ¹, Min Huang ^1,^✉, Wen Xie ^1,^✉

PMCID: PMC3063084 PMID: 21310851

MicroRNA hsa-miR-613 targets human LXRα, which represents a mechanism of negative regulation to prevent the over-activation of LXRα.

Abstract

The nuclear receptor liver X receptor (LXR) is a ligand-dependent transcription factor that plays an important role in the metabolism and homeostasis of cholesterol, lipids, bile acids, and steroid hormones. MicroRNAs (miRNAs) are recently recognized important negative regulators of gene expression. In this report, we showed that miRNA hsa-miR-613 played an important role in the autoregulation of the human LXRα gene. hsa-miR-613 targeted the endogenous LXRα through its specific miRNA response element (613MRE) within the LXRα 3′-untranslated region. Interestingly and paradoxically, the expression of hsa-miR-613 itself was induced upon the activation of LXR. However, hsa-miR-613 did not appear to be a direct LXR target gene. Instead, the positive regulation of hsa-miR-613 by LXR was mediated by the sterol regulatory element binding protein (SREBP)-1c, a known LXR target gene. Promoter analysis revealed an SREBP response element in the hsa-miR-613 gene promoter. Treatment with insulin also induced the expression of hsa-miR-613 in an SREBP-1c-dependent manner, further supporting the role of SREBP-1c in the positive regulation of this miRNA species. Finally, the autoinduction of LXRα by a LXR agonist was enhanced when hsa-miR-613 was inhibited or SREBP-1c was down-regulated. hsa-miR-613 appeared to specifically target the human LXRα. We propose that the negative regulation mediated by hsa-miR-613 and SREBP-1c and the previously reported positive regulation mediated by an LXR response element in the LXRα gene promoter constitute a ying-yang mechanism to ensure a tight regulation of this nuclear receptor of many metabolic functions.

Liver X receptor (LXR) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. There are two LXR isoforms. LXRα has a tissue-specific distribution pattern with relatively high expression in the liver, kidney, intestine, fat tissues, macrophages, lung, and spleen. LXRβ is ubiquitously expressed (1). LXR can be activated by many endogenous or synthetic ligands, such as GW3965 (2). After ligand binding, LXR forms a heterodimer with the retinoid X receptor (RXR) that binds to LXR response elements (LXREs) in the promoters of LXR target genes. LXRs exhibit effects on diverse physiological functions, ranging from cholesterol (3) and lipid metabolism (4, 5) to antiinflammation (6, 7), hepatobiliary diseases (8, 9), and steroid hormone biosynthesis and metabolism (10, 11).

Interestingly, the expression of LXRα itself was induced in macrophages in response to LXR agonists (12). It was reported that this induction was mediated by the direct binding of LXR/RXR heterodimers to an LXRE in the LXRα gene promoter (12). This LXRα autoregulation seemed to be species specific because it was observed only in human cell lines but not in murine cells. Although LXRα has many beneficial function, overexpression and/or activation of LXRα can be harmful in certain tissue and cellular context, such as the lipogenic side effect in the liver (4, 5) and sensitization of mice to cholesterol gallstone disease (9). As such, it is interesting to know whether there is a negative regulatory pathway that can function as checks and balances to maintain a proper concentration of LXRα.

MicroRNAs (miRNAs), first described in 1993 by Lee et al. (13), represent a family of short, noncoding RNAs that control gene expression (14 –16). miRNAs are initially transcribed as long primary transcripts (17) and then undergo sequential processing by the ribonuclease III endonucleases Drosha and Dicer to yield the mature 20- to 23-nucleotide species (18). The mature miRNAs function through base pairing to partially complementary segments in the 3′-untranslated region (UTR), termed miRNA response elements (MREs), of their target mRNAs, leading to mRNA cleavage and/or translational inhibition and ultimately the negative regulation of target gene expression (19). miRNAs exhibit unique expression patterns in specific tissues or cell types, at certain developmental stages, or in response to distinct cellular stressors (20). Among the nuclear receptor superfamily, it has been reported that the expression of pregnane X receptor (21), estrogen receptor-α (22), and tailess (23) can be subjected to the regulation of miRNAs. However, whether the expression and activity of LXRα can be regulated by miRNAs has not been reported.

In this study, we showed that hsa-miR-613 targeted and down-regulated the expression of human LXR (hLXR)-α. Interestingly, the expression of hsa-miR-613 can be transcriptionally activated by LXRα, which was mediated by sterol regulatory element binding protein (SREBP)-1c, a known LXRα target gene. A functional SREBP response element (SRE), but not an LXRE, was found in the hsa-miR-613 gene promoter. We propose that the negative regulation mediated by hsa-miR-613 and SREBP-1c and the previously reported positive regulation mediated by an LXRE constitute a ying-yang mechanism to ensure a tight regulation of LXRα.

Results

Bioinformatic analyses predicted that hsa-miR-613 is a human LXRα targeting miRNA

To investigate the potential regulation of LXRα by miRNA, we first used multiple algorithms, including Microcosm Targets, TargetScan, and Probability of Interaction by Target Accessibility, to screen for human miRNAs that may target the 3′-UTR of the human LXRα gene. Both Microcosm Targets and TargetScan analyses scored hsa-miR-613 as the best LXRα 3′-UTR targeting miRNA (Supplementary Table 1, published on The Endocrine Society's Journals Online web site at http://jcem.endojournals.org). PITA analysis also suggested hsa-miR-613 as a top candidate by having a favorable target-site accessibility, which included a relatively low interaction free energy; a low free energy lost by opening the target site; and a low free binding energy (data not shown). Based on these bioinformatic results, we chose hsa-miR-613 for further study.

hsa-miR-613 directly targeted hLXRα through a MRE (613MRE) on the hLXRα 3′-UTR

Figure 1A shows the putative 613MRE on the human LXRα 3′-UTR as predicted by Microcosm Targets and TargetScan analyses. Interestingly, although a mouse homolog of hsa-miR-613 was not found in any of the miRNA databases we have searched, an apparent conserved 613MRE was found in the 3′-UTR of the mouse LXRα (mLXRα) gene that differed with its human counterpart by a single nucleotide (Fig. 1A). However, PITA analysis predicted that the free binding energy (ddG), which determines the equilibrium constant of the binding reaction (24), was more than twice higher for the binding of hsa-miR-613 to the mouse 613MRE than to the human 613MRE (Supplementary Table 1). To determine whether hsa-miR-613 can down-regulate LXRα by targeting 613MRE, we constructed the hsa-miR-613 expression vector pCMX-miR-613, and the LXRα 3′-UTR reporter gene pCMX-Luc-LXRα 3′-UTR that contained LXRα 3′-UTR. CMX-miR-613 and CMX-Luc-LXRα 3′-UTR were transiently transfected into HepG2 cells, and luciferase activities were measured 48 h after transfection. Results in Fig. 1B showed that cotransfection of pCMX-miR-613 reduced the pCMX-Luc-LXRα 3′-UTR reporter activity but had little effect on the parent pCMX-Luc reporter without the LXRα 3′-UTR (Fig. 1B). Mutation of 613MRE abolished the inhibitory effect of hsa-miR-613, demonstrating that 613MRE was necessary for the inhibition. As a positive control, we cloned a luciferase reporter gene carrying the complementary sequence of mature hsa-miR-613, pCMX-Luc-613CS, which allowed a perfect binding by hsa-miR-613. As expected, hsa-miR-613 effectively inhibited the activity of this control reporter (Fig. 1B). The overexpression of hsa-miR-613 in pCMX-miR-613-transfected cells was confirmed by real-time PCR analysis (Fig. 1C). Taken together, these results indicated that hsa-miR-613 targeted LXRα 3′-UTR through 613MRE.

Fig. 1. — hsa-miR-613 directly targeted hLXRα through a MRE (613MRE) on the hLXRα 3′-UTR. A, A putative hsa-miR-613 MRE located within the 3′-UTR of hLXRα (*boxed*), as predicted by bioinformatic analyses. The sequence of 613MRE mutant is also shown, with the mutated nucleotides in *italics* and *underlined*. Also shown is the alignment of the mLXRα and hLXRα 3′-UTRs. B, Luciferase reporter gene assays showed that hsa-miR-613 directly targeted the 3′-UTR of hLXRα. HepG2 cells were transiently transfected with pCMX-Luc-LXR 3′-UTR 613MRE wild-type, pCMX-Luc-LXR 3′-UTR 613MRE mutant, pCMX-Luc, or pCMX-Luc-613CS, together with the control vector (pS-Neg) or pCMX-miR-613 and the transfection efficiency control pCMX-β-gal. Cells were harvested and assayed for luciferase and β-gal activities 48 h after transfection. The transfection efficiency was normalized against the β-gal activity. Ctrl, Control. C, The expression of hsa-miR-613 in pCMX-miR-613 transfected cells was confirmed by real-time PCR analysis. *, P < 0.05 (n = 3 for each group).

hsa-miR-613 negatively regulated the expression and activity of endogenous hLXRα but not the mouse LXRα

We then determined the effect of hsa-miR-613 on the endogenous LXRα by transfecting pCMX-miR-613 into HepG2 cells. As shown in Fig. 2A, transfection of pCMX-miR-613 decreased the mRNA expression of endogenous LXRα in a dose-dependent manner. The down-regulation of LXRα by hsa-miR-613 at the protein level was confirmed by Western blot analysis (Fig. 2B). Under the same experimental conditions, the mRNA (Fig. 2A) and protein (Fig. 2B) expression of LXRβ was not affected, suggesting that the hsa-miR-613 effect was specific for LXRα. The bioinformatic analysis also suggested that hsa-miR-613 does not target LXRβ (data not shown). The effect of hsa-miR-613 on endogenous LXRα was also observed in reporter gene assays, in which the activation of tk-LXRE-Luc reporter gene by the LXR ligand GW3965 was abolished in pCMX-miR-613 cotransfected cells (Fig. 2C). tk-LXRE-Luc is a LXR responsive reporter gene that contains three copies of the LXRE derived from the stearoyl-coenzyme A desaturase 1 (SCD-1) gene promoter (25). As a negative control, hsa-miR-328, a non-LXRα targeting miRNA based on the algorithm analysis, had little effect on the tk-LXRE-Luc reporter (Fig. 2D).

Interestingly, the effect of hsa-miR-613 appeared to be specific for hLXRα despite an apparent conserved 613MRE in the 3′-UTR of mLXRα (Fig. 1A). As shown in Fig. 2E, transfection of hsa-miR-613 into the mouse hepatoma Hepa1 cells had little effect on the mRNA expression of endogenous mLXRα. Overexpression of hsa-miR-613 in the mouse liver by a hydrodynamic gene delivery method also had no effect on the expression of mLXRα (Fig. 2F). The overexpression of hsa-miR-613 in transfected Hepa1 cells and mouse livers was confirmed by real-time PCR analysis (data not shown). The lack of hsa-miR-613 effect on mLXRα was consistent with the bioinformatically predicted unfavorable targeting of hsa-miR-613 to mLXRα 3′-UTR (Supplementary Table 1).

LXRα positively regulated the expression of hsa-miR-613

Having demonstrated that hsa-miR-613 targeted LXRα, we were surprised to find that LXRα can positively regulate the expression of hsa-miR-613. Treatment with GW3965 induced the expression of mature hsa-miR-613 in primary human hepatocytes as well as in human hepatoma HepG2 and Huh7 cells (Fig. 3A). Among these three cell types, HepG2 cells appeared to have the highest basal and GW3965-inducible expression of mature hsa-miR-613. Mature miRNAs are processed from pre-miRNAs. The induction of pre-hsa-miR-613 in GW3965-treated primary hepatocytes was confirmed by real-time PCR analysis (Fig. 3B). The expression of SCD-1, a known LXR target gene (5), was induced in the same GW3965-treated HepG2 cells and primary hepatocytes as expected (Fig. 3C). The GW3965-responsive induction of both hsa-miR-613 (Fig. 3D) and SCD-1 (Fig. 3E) in HepG2 cells was abolished when the expression of LXRα and LXRβ was knocked down by small interfering RNA (siRNA), suggesting that the GW3965 effect was LXR dependent. The knockdown effects of LXR siRNAs were confirmed by Western blot analysis (Fig. 3F).

Fig. 3. — LXRα positively regulated the expression of hsa-miR-613. A–C, Primary human hepatocytes (HH), HepG2 cells, and Huh7 cells were treated with vehicle or GW3965 (10 μm) for 24 h before total RNA harvesting and real-time PCR analysis to detect the expression of endogenous mature hsa-miR-613 in all three cell types (A), the expression of pre-has-miR-613 in HH (B), and the expression of SCD-1 mRNA in HH and HepG2 cells (C). D and E, HepG2 cells were transfected with control siRNA (scramble siRNA) or LXRα and LXRβ siRNA (10 nm each). Twenty-four hours after transfection, cells were treated with vehicle or GW3965 (10 μm) for 24 h before subjecting to real-time PCR analysis to detect the expression of mature hsa-miR-613 (D) and SCD-1 mRNA (E). The expression level of the DMSO groups was arbitrarily set at 1 in B–E. Ctrl, Control. F, The efficiencies of LXR siRNA knockdown in HepG2 cells were confirmed by Western blot analysis. The relative protein expression was determined by densitometric quantification. G, HepG2 cells were transfected with pGL-miR-613 reporter gene and pCMX-β-gal, in the presence or absence of expression vector for LXRα. Twenty-four hours after transfection, cells were treated with vehicle or GW3965 (10 μm) for 24 h before harvesting for luciferase and β-gal assays. The transfection efficiency was normalized against the β-gal activity. All transfections were performed in triplicate and repeated at least three times.

To further determine whether hsa-miR-613 was subjected to LXRα regulation, we constructed the hsa-miR-613 luciferase reporter gene pGL-miR-613 that contained the 656-bp promoter region of the hsa-miR-613 gene. As shown in Fig. 3G, treatment with GW3965 modestly but significantly activated this reporter gene.

The regulation of hsa-miR-613 by LXRα was mediated by SREBP-1c

We initially hypothesized that the regulation of hsa-miR-613 by LXRα was mediated by an LXRE in the hsa-miR-613 gene promoter. Indeed, a putative direct repeat spaced by four nucleotides (DR4) was identified in the hsa-miR-613 gene promoter (Fig. 4A). DR4 has been reported to function as an LXRE (26). However, the LXRα-RXR heterodimer failed to bind to this putative DR4 as determined by EMSA (data not shown), suggesting that hsa-miR-613 may not be a direct target gene of LXRα. Further analysis of the hsa-miR-613 gene promoter revealed an SRE (GTGGtGTGA) (Fig. 4A). SREBP-1c is a known LXR target gene. Indeed and as expected, the expression of SREBP-1c mRNA was induced in GW3965-treated HepG2 cells and primary human hepatocytes (Fig. 4B). These results led to our new hypothesis that LXR may regulate the expression of hsa-miR-613 by activating SREBP-1c, and subsequent binding of SREBP-1c to SRE on the hsa-miR-613 gene promoter. Consistent with our new hypothesis, cotransfection of SREBP-1c robustly activated the pGL-miR-613 reporter gene, and the SREBP-1c effect was abolished when the SRE was mutated (Fig. 4C). The same SRE mutation also abolished the induction of pGL-miR-613 by GW3965 (Fig. 4D).

Fig. 4. — The regulation of hsa-miR-613 by LXRα was mediated by SREBP-1c. A, Putative SRE and DR4 were identified within the hsa-miR-613 promoter region (nt −434 to −652). B, HepG2 cells and primary human hepatocytes (HH) were treated with vehicle or GW3965 (10 μm) for 24 h before total RNA harvesting and real-time PCR analysis to detect the expression of SREBP-1c mRNA. C, HepG2 cells were transfected with pGL-miR-613 containing the wild-type SRE (GTGGTGTGA) or mutated SRE (GTAATATAA), empty vector or pCMX-SREBP-1c, and pCMX-β-gal. Forty-eight hours after transfection, cells were harvested for luciferase and β-gal assays. D, HepG2 cells were transfected with pGL-miR-613 SRE mutant and pCMX-β-gal. Twenty-four hours after transfection, cells were treated with vehicle or GW3965 (10 μm) for 24 h before harvesting for luciferase and β-gal assays. E, Binding of SREBP-1c to ³²P-labeled hsa-miR-613/SRE was demonstrated by EMSA. In the competition lanes, unlabeled probes were added. The binding of radiolabeled FAS/SRE by SREBP-1c was included as a positive control, whereas the lack of binding of radiolabeled mutated 613/SRE by SREBP-1c was included as a negative control. F, ChIP assay to demonstrate the recruitment of SREBP-1c onto the hsa-miR-613 gene promoter. Formaldehyde cross-linked DNA was extracted from Huh-7 cells treated with vehicle or GW3965 (10 μm) for 24 h. ChIP assay was performed using an antibody against SREBP-1c or control IgG. The final DNA extracts were amplified by PCR using the primer pairs encompassing 613/SRE (*left panel*). The recruitment of SREBP-1c onto LDLR/SRE was included as a positive control (*right panel*). G, Huh7 cells were transfected with control siRNA or SREBP-1c siRNA. Forty-eight hours after transfection, cells were harvested for real-time PCR analysis to detect the expression of mature hsa-miR-613. Huh7 cells were chosen for siSREBP-1c experiments because for unknown reasons, siSREBP1c knockdown was not as efficient in HepG2 cells. H, The efficiency of SREBP-1c siRNA knockdown in Huh7 cells was confirmed by Western blot analysis. The relative protein expression was determined by densitometric quantification. *, P < 0.05 (n = 3 for each group).

The binding of SREBP-1c to the SRE was first confirmed by EMSA. SREBP-1c bound to the ³²P-labeled wild-type (wt) 613SRE but not the ³²P-labeled mutant 613SRE (Fig. 4E, lines 1 and 13, respectively). An SRE derived from the fatty acid synthase (FAS) gene (27) was included as a positive control (Fig. 4E, line 7). The binding of ³²P-labeled wt 613SRE to SREBP-1c can be efficiently competed away by excess unlabeled (cold) 613SRE (Fig. 4E, lines 2 and 3), or FAS SRE (Fig. 4E, lines 4 and 5) in a dose-dependent manner. Unlabeled 613SRE could also compete away the binding of SREBP-1c to FAS SRE (Fig. 4E, lanes 10 and 11). Chromatin immunoprecipitation (ChIP) assay was then used to demonstrate the binding of SREBP-1c to 613SRE in vivo. In this experiment, the human hepatoma Huh7 cells were treated with vehicle [dimethylsulfoxide (DMSO)] or GW3965 for 24 h before ChIP analysis using an anti-SREBP1 antibody or the control mouse IgG. As shown in Fig. 4F, treatment with GW3965 resulted in the recruitment of SREBP-1c to 613SRE, whereas no obvious recruitment was observed in DMSO-treated cells (Fig. 4F, left panel). The recruitment of SREBP-1c to the SRE of the low-density lipoprotein receptor (LDLR) gene (28) was included as a positive control (Fig. 4F, right panel). Together these results suggested that hsa-miR-613 is a direct transcriptional target of SREBP-1c.

We further investigated the role of SREBP-1c in hsa-miR-613 regulation by SREBP-1c siRNA knockdown. In contrast to its induction of mature hsa-miR-613 in control-siRNA transfected cells, GW3965 failed to induce the expression of mature hsa-miR-613 in SREBP-1c siRNA transfected cells (Fig. 4G). The efficiency of SREBP-1c siRNA knockdown was confirmed by Western blot analysis (Fig. 4H).

hsa-miR-613 is an actively transcribed miRNA that exhibits unique tissue distribution

miRNAs are known to be transcribed by the RNA polymerase II (PolII) (17). To support that hsa-miR-613 is a bona fide miRNA, we used ChIP analysis to determine whether PolII can be recruited to the hsa-miR-613 gene promoter. As shown in Fig. 5A, treatment with GW3965 enhanced the recruitment of PolII to the proximal promoter of hsa-miR-613, suggesting that hsa-miR-613 is an actively transcribed miRNA. No PCR products were amplified from the PolII-ChIP DNA when primers encompassing the SRE were used (data not shown). In humans, the expression of LXRα is high in the liver and modest in the kidney and skeletal muscle (29). When the expression of pre-hsa-miR-613 was profiled, we found the expression of pre-hsa-miR-613 was higher in the liver and lower in the kidney and skeletal muscle (Fig. 5B). In cell lines, the expression of mature hsa-miR-613 in Huh7 cells was higher than the human embryonic kidney 293 cells (Fig. 5C). The tissue distribution of hsa-miR-613 supported the functional relevance of this miRNA species in vivo.

Fig. 5. — hsa-miR-613 is an actively transcribed miRNA that exhibits unique tissue distribution. A, ChIP assay to demonstrate the recruitment of RNA PolII onto the hsa-miR-613 gene promoter. Formaldehyde cross-linked DNA was extracted from Huh-7 cells treated with vehicle or GW3965 (10 μm) for 24 h. ChIP assay was performed using an antibody against PolII or control IgG. The final DNA extracts were amplified by PCR using a primer pairs encompassing nt −495 to −99 that contain the predicted PolII binding site. B, The expression of pre-hsa-miR-613 in the total RNA of human liver, kidney, and skeletal muscle was measured by real-time PCR analysis. The expression level in the liver was arbitrarily set at 1. C, The expression of mature hsa-miR-613 in Huh7 and HEK293 cells was measured by real-time PCR analysis. The expression level in Huh7 cells was arbitrarily set at 1. Real-time PCRs were performed in triplicate. HEK293, Human embryonic kidney 293 cells. *, P < 0.05.

Insulin also up-regulated the expression of hsa-miR-613 in an SREBP-1c-dependent manner

SREBP-1c can be activated by either LXR or insulin (30 –32). Having demonstrated that the LXR-SREBP-1c pathway can activate hsa-miR-613 gene expression, we went on to determine whether insulin could also positively regulate hsa-miR-613 through the activation of SREBP-1c. Indeed, treatment of HepG2 cells with insulin activated the pGL-miR-613 reporter gene, whereas the activation was not seen in cells transfected with the SRE mutant reporter gene (Fig. 6A). Treatment of primary human hepatocytes with insulin induced the expression of mature hsa-miR-613 (Fig. 6B). In the same insulin-treated primary human hepatocytes, the expression of known SREBP-1c target genes, such as SCD1 (33) and SREBP-1c itself (34), was induced as expected (Fig. 6C). Treatment of HepG2 cells with insulin also induced the expression of hsa-miR-613, and this effect was abolished in SREBP-1c siRNA transfected cells (Fig. 6D). These results suggested that the insulin effect on the expression of hsa-miR-613 was SREBP-1c dependent.

Fig. 6. — Insulin also up-regulated the expression of hsa-miR-613 in an SREBP-1c-dependent manner. A, HepG2 cells were transfected with the wild-type or SRE mutant pGL-miR-613 and pCMX-β-gal. Twenty-four hours after transfection, cells were treated with vehicle or insulin (100 nm) for 16 h before harvesting for luciferase and β-gal assays. The transfection efficiency was normalized against the β-gal activity. B and C, Primary human hepatocytes were treated with vehicle or insulin (100 nm) for 16 h before real-time PCR analysis to detect the expression of mature hsa-miR-613 (B) and SCD-1 and SREBP-1c mRNA (C). D, Huh7 cells were transfected with siControl or siSREBP-1c. Transfected cells were treated with vehicle or insulin (100 nm) for 16 h before real-time PCR analysis to detect the expression of mature hsa-miR-613. The expression level of the DMSO groups was arbitrarily set at 1 in C and D. *, P < 0.05 (n = 3 for each group).

Inhibition of hsa-miR-613 or down-regulation of SREBP-1c enhanced LXR agonist-responsive induction of LXR

LXR is known to play a positive role in the autoregulation of LXRα through its binding to an LXRE in the LXRα gene promoter (12). We proposed that the negative regulation mediated by hsa-miR-613 and SREBP-1c helped to constitute a ying-yang mechanism to ensure a tight regulation of LXRα. To test whether a disruption of this ying-yang balance will affect the outcome of LXRα autoregulation, we measured the mRNA expression of LXRα in either anti-613 or SREBP-1c siRNA transfected cells treated with GW3965. Anti-613 is a synthetic and hsa-miR-613 specific miRNA inhibitor. As shown in Fig. 7, compared with control transfected cells, inhibition of hsa-miR-613 by anti-613 (Fig. 7A) or down-regulation of SREBP-1c by siRNA (Fig. 7B) enhanced the GW3965-responsive induction of LXRα mRNA. The GW3965-responsive induction of the tk-LXRE reporter gene was also increased significantly in anti-613 transfected cells (Fig. 7C).

Fig. 7. — Inhibition of hsa-miR-613 or down-regulation of SREBP-1c enhanced LXR agonist-responsive induction of LXRα. A and B, HepG2 cells transfected with control or anti-613 (A) or Huh7 cells transfected with control siRNA or siSREBP-1c (B) were treated with vehicle or GW3965 (10 μm) for 24 h before being detected for LXRα mRNA by real-time PCR analysis. C, HepG2 cells were transfected with tk-LXRE-Luc, in the absence or presence of anti-613. Twenty-four hours after transfection, cells were treated with vehicle or GW3965 (10 μm) for 48 h before harvesting for luciferase and β-gal assays. *, P < 0.05 (n = 3 for each group).

Discussion

In this study, we demonstrated that hsa-miR-613 played a negative role and mediated a feedback loop in the autoregulation of the human LXRα. As summarized in Fig. 8, when the LXRα gene is transactivated by LXRα due to the LXRE-mediated autoregulation, hsa-miR-613 is simultaneously up-regulated as mediated by SREBP-1c. The SREBP-1c induction can be LXR independent, such as that stimulated by the insulin. The induced hsa-miR-613 will then target and lower the expression of LXRα. The negative regulation mediated by hsa-miR-613 and SREBP-1c, and the positive regulation mediated by LXRE constitute a ying-yang mechanism to ensure a tight regulation of LXRα. The expression of hsa-miR-613 in the human liver was higher than in the kidney and skeletal muscle. The tissue distribution of hsa-miR-613 was consistent with that of hLXRα, supporting the functional relevance of this miRNA species in vivo. hsa-miR-613 appeared to specifically target hLXRα because overexpression of hsa-miR-613 had little effect on the expression of mLXRα.

Fig. 8. — Proposed feedback loop of LXRα autoregulation mediated by LXRα, has-miR-613, and SREBP-1c. In this model, LXRα has a dual role in transactivating its own expression via an LXRE in LXRα gene promoter, as well as inhibiting its own expression via the transactivation of SREBP-1c and has-miR-613. Other SREBP-1c activating mechanisms, such as the insulin stimulation, can also contribute to the negative regulation of LXRα.

LXRα is previously known for its positive autoregulation as mediated by an LXRE in the hLXRα gene promoter (12). The hsa-miR-613-mediated negative regulation of hLXRα, although paradoxical, does make physiological sense. LXRα is a nuclear receptor of diverse functions. The beneficial functions of LXRα range from antiatherosclerosis (1), antiinflammation (6, 7), anti-Alzheimer's disease (35, 36), and anticholestasis (8). However, activation of LXRα has also been linked to some unwanted side effects, such as hepatic steatosis (4, 5), lithogenesis (9), and steroid biosynthesis and metabolism (10, 11). As such, it is necessary to limit the expression and/or activity of LXRα to prevent the overactivation of this receptor. It appears that hsa-miR-613 represents such a mechanism of negative regulation.

Interestingly, the expression of hsa-miR-613 is also subjected to the regulation of LXRα, suggesting a more complex role for LXRα in its autoregulation. An even more intriguing observation is that although its expression is responsive to LXRα, hsa-miR-613 does not appear to be a direct LXRα target gene and no functional LXRE was identified in the hsa-miR-613 gene promoter. Instead, the positive regulation of hsa-miR-613 by LXRα was found to be mediated by SREBP-1c, a well-known LXRα target gene (4, 5). Promoter analysis revealed an SRE in the hsa-miR-613 gene promoter. Treatment with insulin also induced the expression of hsa-miR-613 in an SREBP-1c-dependent manner, further supporting the role of SREBP-1c in the positive regulation of this miRNA species. The insulin-SREBP effect on the expression of hsa-miR-613 suggested that signaling pathways other than a direct LXR activation could also participate in the autoregulation of LXRα. These results also suggested that in addition to its previously recognized role in sterol sensing, LXRα may have a broader function in sensing other cellular signals, such as insulin.

Our results also showed that the balances between ying-yang regulations of LXRα could be shifted by targeted manipulation. Specifically, the LXR agonist responsive induction of LXRα was enhanced when hsa-miR-613 was inhibited by anti-613, or the expression of SREBP-1c was down-regulated by siRNA (Fig. 6). It is interesting to know whether the disruption of the feedback-loop regulation of LXRα is implicated in the pathogenesis of certain human diseases and whether the feedback loop can be explored in establishing LXRα as a therapeutic target.

microRNAs belong to a diverse class of highly conserved small RNA molecules that function as critical regulators of gene expression in eukaryotic cells. The biological effects of miRNAs have been widely appreciated, and dysregulations of miRNA have been linked to a variety of human diseases, such as cancers (37) and heart diseases (38). Recent studies suggest that miRNAs may also impact the signaling of nuclear receptors. In addition to the regulation of LXRα by hsa-miR-613 reported here, the expression and function of pregnane X receptor (21), estrogen receptor-α (22), and tailess (23) can also be regulated by miRNAs. Considering the key functions of nuclear receptors in gene regulation and pathophysiology, it is conceivable that nuclear receptors will have a broader role in mediating the biological effects of miRNAs.

Based on the algorithms, hsa-miR-613 was predicted to be the top hLXRα-targeting miRNA. hsa-miR-206, another miRNA that scored well in the Targetscan analysis (Supplementary Table 1), had little effect on either the expression of endogenous hLXRα or the hLXRα 3′-UTR reporter gene (data not shown). However, we cannot exclude the possibility of the existence of additional hLXRα-targeting miRNAs. Among the limitations of current study, the in vivo relevance of the hsa-miR-613-mediated hLXRα inhibition remains to be established. hsa-miR-613 was found and verified as a candidate novel miRNAs in colorectal cancer, although it was not among the most validated miRNA species in that study (39). Our result that treatment with the LXR agonist GW3965 enhanced the recruitment of PolII to the proximal promoter of hsa-miR-613 (Fig. 5A) further supported that hsa-miR-613 is an actively transcribed miRNA.

In summary, we have revealed an intricate autoregulation of LXRα mediated by hsa-miR-613, LXRα, and SREBP-1c. The feedback loop of LXRα autoregulation can be regulated by not only LXRα but also other signal inputs, such as the insulin stimulation. The complex autoregulation of LXRα is consistent with the widely appreciated metabolic functions of this nuclear receptor.

Materials and Methods

Reagents and chemicals

The anti-miR miRNA inhibitor for hsa-miR-613 (anti-613) was custom made by Ambion (Austin, TX). GW3965 was synthesized in-house as previously described (2). All other chemicals, if not specified, were purchased from Sigma (St. Louis, MO).

Bioinformatic analysis

The 3′-UTR sequence of the human LXRα gene was retrieved using Entrez (http://www.ncbi.nlm.nih.gov/entrez/). The sequence was then analyzed for potential targeting miRNAs by using on-line software of PITA (40), TargetScan (41), and miRBase Targets (42).

Plasmid constructs

The hsa-miR-613- and the control hsa-miR-328-expressing vectors were cloned by RT-PCR using the following pairs of primers: hsa-miR-613 forward, 5′-CCGCTCGAGTCTACTAGGTGTGGGCTTTA-3′, and reverse, 5′-CCCAAGCTTCTGTGG CCTTCCTTACTCTT-3′; and hsa-miR-328 forward, 5′-CCCAAGCTTCCCAGCTCCCA CCCCACCCACCTTGTCGAA-3′, and reverse, 5′-CGCGGATCCGTTGGAGAACA GCAGCCGCGAGGAC-3′. The PCR products were digested with XhoI and HindIII (for hsa-mir-613), or BamHI and HindIII (for hsa-miR-328) and inserted into the same enzyme-digested pCMX-PL2 expression vector. The control plasmid, pS-Neg, was a generous gift from Dr. Aiming Yu (University of Buffalo, Buffalo, NY). The 3′-UTR reporters, including LXRα 3′-UTR and hsa-613 complementary sequence, were generated in two steps. First, the luciferase coding region was PCR amplified with primers 5′-CCGCTCGA GATGGAAGACGCCAAAAACAT-3′ and 5′-CCCAAGCTTTTACACGGCGATCTTTC CGC-3′ and then cloned into XhoI- and HindIII-digested pCMX-PL2 vector to generate pCMX-Luc. LXRα 3′-UTR was then PCR amplified with primers 5′-CCCA AGCTTCTGTTCTGTCCCCATATT-3′ and 5′-CGGGATCCACCCTTTGACTCTCTTTT AAT-3′ and then inserted into HindIII- and BamHI-digested pCMX-Luc. The hsa-miR-613 complementary sequence reporter was generated by direct ligation of the annealed oligos 5′-AGCTTGGCAAAGAAGGAACAT TCCTG-3′ and 5′-GATCCAGGAAT GTTCC TTCTTTGCCA-3′ to HindIII- and BamHI-digested pCMX-Luc. The LXRα 3′-UTR mutant reporter was generated by site-directed mutagenesis using primers 5′-GTGGAACAG ACTGAGAAGGGCAAAAATTAATGGGAGCTGGGCAAGGAG-3′ and 5′-CTCCTTG CCCAGCTCCCATTAATTTTTGCCCTTCTCAGTCTGTTCCAC-3′. To generate the hsa-miR-613 promoter reporter gene, the hsa-miR-613 promoter region was PCR amplified with primers 5′-CGCGAGCTCTAGGGAGGATG TACATAGGT-3′ and 5′-CCGCTCGAGTCA CCCTCTAGAGGTAATA-3′, using the human placenta genome DNA as the template. The promoter sequence was then cloned into SacI- and XhoI-digested pGL3-basic vector from Promega (Madison, WI). The 613SRE mutant reporter gene was generated by site-directed mutagenesis using primers 5′-CCCAGGCTGGAGTGCAGTAATATAATCTCGGCTCACTGCAACC-3′ and 5′-GGTT GCAGTGAGCCGAGATTATATTACTGCACTCCAGCCTGGG-3′. The identities of all cloned sequences were verified by DNA sequencing.

Human primary hepatocytes and human tissue total RNA

Normal human hepatocytes were obtained through the Liver Tissue Procurement and Distribution System (Pittsburgh, PA). Human total RNA of the liver (catalog no. 636531), kidney (catalog no. 636529), and skeletal muscle (catalog no. 636534) was purchased from CLONTECH (Madison, WI).

Cell culture and transient transfection

HepG2 cells were transfected using the polyethyleneimine polymer transfection agent as previously described, and the transfection efficiency was normalized against the β-galactosidase (β-gal) activity from the cotransfected pCMX-β-gal vector (43). To evaluate the effect of hsa-miR-613 on endogenous LXRα and for each triplicate transfection, 0.6 μg of tk-LXRE-Luc, 0.3 μg of pCMX-miR-613/328, and 0.3 μg of pCMX-β-gal were transfected on a 48-well plate. Twenty-four hours after transfection, cells were treated with vehicle or GW3965 (10 μm) in medium containing 10% charcoal/dextran-stripped fetal bovine serum for 48 h before being harvested for luciferase and β-gal assays. For LXR 3′-UTR reporter assay and for triplicate transfection, 0.3 μg of pCMX-Luc-LXR 3′UTR, pCMX-Luc-LXR 3′-UTR MRE mutant, pCMX-Luc-613CS, or pCMX-Luc, 0.15 μg of pCMX-miR-613, and 0.3 μg of pCMX-β-gal were used for transfection. Cells were harvested for luciferase and β-gal assays 48 h after transfection. For hsa-miR-613 promoter reporter assay and for triplicate transfection, 0.6 μg of reporter, 0.3 μg of pCMX-SREBP-1c or pCMX-hLXRα, and 0.3 μg of pCMX-β-gal were used for transfection. Twenty-four hours after transfection, cells were treated with vehicle or GW3965 (10 μm) or insulin (100 nm) in serum-free medium for 24 h before being harvested for luciferase and β-gal assays. All transfections were performed in triplicate and repeated at least three times.

Hydrodynamic mouse liver transfection

Hydrodynamic gene delivery was performed following a published protocol (44). Briefly, 6-wk-old female CD-1 mice were injected with pS-Neg or CMX-miR-613 (20 μg DNA per mouse) via the tail vein. Sixteen hours after injection, the mouse livers were harvested and subjected to real-time PCR analysis. The use of mice in this study has complied with all relevant federal guidelines and institutional policies.

Western blot analysis

Whole-cell protein extracts were prepared and measured for their protein concentrations with the bicinchoninic assay protein assay kit from Pierce (Rockford, IL). One hundred micrograms of proteins were separated on 10% SDS-PAGE gels and electrotransferred onto polyvinyl difluoride transfer membranes from Invitrogen (Carlsbad, CA). Membranes were then incubated in Tris-buffered saline containing 0.2% (vol/vol) Tween 20 and 5% (wt/vol) fat-free dry milk at room temperature for 1 h before incubation with the primary antibody at 4 C overnight. The primary antibodies used include monoclonal anti-hLXRα antibody (catalog no. PP-PPZ0412-00), monoclonal anti-hLXRβ antibody (catalog no. PP-K8917-10) from R&D Systems (Minneapolis, MN), and polyclonal anti-SREBP-1 antibody (catalog no. sc-366) from Santa Cruz Biotechnology (Santa Cruz, CA). The membranes were then incubated with second antibodies for 1 h before signal detection by using the enhanced chemiluminescence detection system from GE Healthcare (Piscataway, NJ). The second antibodies used include horseradish peroxidase-linked anti-mouse IgG for LXRα and LXRβ, and horseradish peroxidase-linked antirabbit IgG for SREBP-1. Membranes were stripped and reprobed for β-actin for the purpose of loading control.

Real-time RT-PCR analysis

Total RNA was isolated from cell cultures using the miRNeasy mini kit from QIAGEN (Valencia, CA). Stem-loop reverse transcription of mature hsa-miR-613 was conducted as described previously (45), using the Superscript RT III enzyme from Invitrogen and the primer 5′-GTCGTATCCAGTGCAG GGTCCGAGGTATTCGCACTGGATACGACGGCAAA-3′. To detect pre-hsa-miR-613, total RNA was first mixed with reverse transcription primer 5′-GGCAAAGAAGGAACATTCCTG-3′. The reactions were heated to 80 C for 5 min to denature the RNA, followed by a 5-min incubation at 60 C to anneal the primers. The reactions were then cooled to room temperature before adding the remaining reagents (46). Each real-time PCR reaction contained an input of 20 ng of total RNA, in which only samples with cycle threshold (Ct) values higher than 35 were considered undetectable (45, 47). U6 small nuclear RNA was used as the internal control for miRNA. The raw Ct values of miRNA are partially labeled in the figures, and the complete raw and normalized Ct values of miRNA are summarized in Supplementary Table 2. SYBR Green-based real-time PCR was performed with the ABI 7300 real-time PCR system (Applied Biosystems, Foster City, CA). Data were normalized against U6 small nuclear RNA (for miRNAs) or cyclophilin (for non-miRNA genes). The sequences for PCR primers are listed in Supplementary Table 3.

Electrophoretic mobility shift assay

Receptor proteins were prepared using the T7 Quick Coupled Transcription/Translation System in vitro transcription and translation system (Promega). The 20-μl binding reaction contained 2 μl of radioactive probe, 4 μl of SREBP-1c TNT protein, 1 μl of polydeoxyinosine-deoxycytosine, 4 μl of 5× binding buffer, and 5 μl of cold competitor when applicable. Sterile water was used to bring the final volume to 20 μl. The reactions were kept at room temperature for 20 min before electrophoresis through 5% polyacrylamide gel in 0.5× Tris borate-EDTA at 4 C for 1–3 h. For oligonucleotide competition experiments, unlabeled (cold) oligonucleotides were added to the reactions. The EMSA oligonucleotide sequences are: wt 613SRE forward, 5′-CGAGCAGTGGTGTGA TCT-3′, and reverse, 5′-CGAAGATCACACC ACTGC-3′; 613SRE mutant forward, 5′-CGAGCAGTAATATAATCT-3′, and reverse, 5′-CGAAGATTATATTACTGC-3′; FAS SRE forward, 5′-GGAGGCATCACCCCACC GAC-3′, and reverse, 5′-GGAGTCGGTGGG GTGATGCC-3′.

ChIP assay

Huh7 cells treated with vehicle or GW3965 (10 μm) for 24 h were subjected to ChIP assay as described previously (48). Cell lysates were incubated overnight with 1 μg of anti-SREBP1 antibody (Santa Cruz Biotechnology) or anti-RNA PolII antibody (Upstate, Upstate, CA) at 4 C. Parallel samples were incubated with normal IgG as a negative control. The following PCR primers were used: 613/SRE forward, 5′-GACTGACTATATATAGCACA-3′, and reverse, 5′-CATGGTGGCAGGCACCTGC-3′; LDLR/SRE forward, 5′-CACTTTCGAAGGA CTGGAGTGG-3′, and reverse, 5′-CCACGTC ATTTACAGCATTT C-3′; and PolII forward, 5′-CGATCCTGGCCTCAGGTGATCC-3′, and reverse, 5′-TCACCCTCTAGAGGTAATA-3′.

LXR and SREBP-1c RNA interference (siRNA)

The hLXR siRNAs, including the control scrambled siRNA, were purchased from QIAGEN. The sequences of siLXRα and siLXRβ are 5′-AGCAGGGCUGCAAGU GGAA-3′ and 5′-CAG AUCCGGAAGAAG AAGA-3′, respectively. The human SREBP-1 siRNA was purchased from Applied Biosystems (Austin, TX). The sequence of siSREBP-1 is 5′-CCACUCCAUUGAAGAU GCATT-3′. The control scrambled siRNA (catalog no. 1027280) was purchased from QIAGEN. The siRNA transfection was carried out using Lipofectamine 2000 (Invitrogen). The siRNAs were added to the final concentration of 20 nm during transfection. Cells were transfected for 4 h before being replaced with medium containing 10% fetal bovine serum and were maintained in this medium for 24 h before drug treatment.

Statistical analysis

All values were expressed as mean ± sd. Comparisons between groups were performed using a Student t test or one-way ANOVA where appropriate. P < 0.05 was considered statistically significant.

Acknowledgments

This work was supported by National Institutes of Health Grants ES014626, DK076962, and DK083952 (to W.X.). Z.O. is supported by a scholarship from the Government of China's China Scholarship Council (Award 2008638059). Normal human hepatocytes were obtained through the Liver Tissue Procurement and Distribution System (Pittsburgh, PA), which was funded by National Institutes of Health Contract N01-DK-7-0004/HHSN267200700004C.

Disclosure Summary: The authors have nothing to disclose.

NURSA Molecule Pages:

Coregulators: LXR-α
Ligands: GW3965

Footnotes

Abbreviations:

ChIP: Chromatin immunoprecipitation
Ct: cycle threshold
DMSO: dimethylsulfoxide
DR4: direct repeat spaced by four nucleotides
FAS: fatty acid synthase
β-gal: β-galactosidase
hLXR: human LXR
LDLR: low-density lipoprotein receptor
LXR: liver X receptor
LXRE: LXR response element
mLXR: mouse LXR
MRE: miRNA response element
miRNA: microRNA
PolII: polymerase II
RXR: retinoid X receptor
SCD-1: stearoyl-coenzyme A desaturase 1
siRNA: small interfering RNA
SRE: SREBP response element
SREBP: sterol regulatory element binding protein
UTR: untranslated region
wt: wild type.

References

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[B1] 1. Tontonoz P, Mangelsdorf DJ. 2003. Liver X receptor signaling pathways in cardiovascular disease. Mol Endocrinol 17:985–993 [DOI] [PubMed] [Google Scholar]

[B2] 2. Collins JL, Fivush AM, Watson MA, Galardi CM, Lewis MC, Moore LB, Parks DJ, Wilson JG, Tippin TK, Binz JG, Plunket KD, Morgan DG, Beaudet EJ, Whitney KD, Kliewer SA, Willson TM. 2002. Identification of a nonsteroidal liver X receptor agonist through parallel array synthesis of tertiary amines. J Med Chem 45:1963–1966 [DOI] [PubMed] [Google Scholar]

[B3] 3. Repa JJ, Mangelsdorf DJ. 2002. The liver X receptor gene team: potential new players in atherosclerosis. Nat Med 8:1243–1248 [DOI] [PubMed] [Google Scholar]

[B4] 4. Repa JJ, Liang G, Ou J, Bashmakov Y, Lobaccaro JM, Shimomura I, Shan B, Brown MS, Goldstein JL, Mangelsdorf DJ. 2000. Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRα and LXRβ. Genes Dev 14:2819–2830 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5. Schultz JR, Tu H, Luk A, Repa JJ, Medina JC, Li L, Schwendner S, Wang S, Thoolen M, Mangelsdorf DJ, Lustig KD, Shan B. 2000. Role of LXRs in control of lipogenesis. Genes Dev 14:2831–2838 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6. Zelcer N, Tontonoz P. 2006. Liver X receptors as integrators of metabolic and inflammatory signaling. J Clin Invest 116:607–614 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7. Joseph SB, Bradley MN, Castrillo A, Bruhn KW, Mak PA, Pei L, Hogenesch J, O'Connell RM, Cheng G, Saez E, Miller JF, Tontonoz P. 2004. LXR-dependent gene expression is important for macrophage survival and the innate immune response. Cell 119:299–309 [DOI] [PubMed] [Google Scholar]

[B8] 8. Uppal H, Saini SP, Moschetta A, Mu Y, Zhou J, Gong H, Zhai Y, Ren S, Michalopoulos GK, Mangelsdorf DJ, Xie W. 2007. Activation of LXRs prevents bile acid toxicity and cholestasis in female mice. Hepatology 45:422–432 [DOI] [PubMed] [Google Scholar]

[B9] 9. Uppal H, Zhai Y, Gangopadhyay A, Khadem S, Ren S, Moser JA, Xie W. 2008. Activation of liver X receptor sensitizes mice to gallbladder cholesterol crystallization. Hepatology 47:1331–1342 [DOI] [PubMed] [Google Scholar]

[B10] 10. Cummins CL, Volle DH, Zhang Y, McDonald JG, Sion B, Lefrançois-Martinez AM, Caira F, Veyssière G, Mangelsdorf DJ, Lobaccaro JM. 2006. Liver X receptors regulate adrenal cholesterol balance. J Clin Invest 116:1902–1912 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11. Gong H, Guo P, Zhai Y, Zhou J, Uppal H, Jarzynka MJ, Song WC, Cheng SY, Xie W. 2007. Estrogen deprivation and inhibition of breast cancer growth in vivo through activation of the orphan nuclear receptor liver X receptor. Mol Endocrinol 21:1781–1790 [DOI] [PubMed] [Google Scholar]

[B12] 12. Laffitte BA, Joseph SB, Walczak R, Pei L, Wilpitz DC, Collins JL, Tontonoz P. 2001. Autoregulation of the human liver X receptor alpha promoter. Mol Cell Biol 21:7558–7568 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13. Lee RC, Feinbaum RL, Ambros V. 1993. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75:843–854 [DOI] [PubMed] [Google Scholar]

[B14] 14. Ambros V. 2004. The functions of animal microRNAs. Nature 431:350–355 [DOI] [PubMed] [Google Scholar]

[B15] 15. Bartel DP. 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297 [DOI] [PubMed] [Google Scholar]

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PERMALINK

MicroRNA hsa-miR-613 Targets the Human LXRα Gene and Mediates a Feedback Loop of LXRα Autoregulation

Zhimin Ou

Taira Wada

Roberto Gramignoli

Song Li

Stephen C Strom

Min Huang

Wen Xie

Abstract

Results

Bioinformatic analyses predicted that hsa-miR-613 is a human LXRα targeting miRNA

hsa-miR-613 directly targeted hLXRα through a MRE (613MRE) on the hLXRα 3′-UTR

Fig. 1.

hsa-miR-613 negatively regulated the expression and activity of endogenous hLXRα but not the mouse LXRα

Fig. 2.

LXRα positively regulated the expression of hsa-miR-613

Fig. 3.

The regulation of hsa-miR-613 by LXRα was mediated by SREBP-1c

Fig. 4.

hsa-miR-613 is an actively transcribed miRNA that exhibits unique tissue distribution

Fig. 5.

Insulin also up-regulated the expression of hsa-miR-613 in an SREBP-1c-dependent manner

Fig. 6.

Inhibition of hsa-miR-613 or down-regulation of SREBP-1c enhanced LXR agonist-responsive induction of LXR

Fig. 7.

Discussion

Fig. 8.

Materials and Methods

Reagents and chemicals

Bioinformatic analysis

Plasmid constructs

Human primary hepatocytes and human tissue total RNA

Cell culture and transient transfection

Hydrodynamic mouse liver transfection

Western blot analysis

Real-time RT-PCR analysis

Electrophoretic mobility shift assay

ChIP assay

LXR and SREBP-1c RNA interference (siRNA)

Statistical analysis

Acknowledgments

NURSA Molecule Pages:

Footnotes

References

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