Am J Physiol Endocrinol Metab 307: E84–E92, 2014. First published May 13, 2014; doi:10.1152/ajpendo.00542.2013.

miR-24 regulates menin in the endocrine pancreas Jyothi Vijayaraghavan, Elaine C. Maggi, and Judy S. Crabtree Department of Genetics, School of Medicine, Louisiana State University Health Sciences Center, New Orleans, Louisiana Submitted 30 September 2013; accepted in final form 9 May 2014

Vijayaraghavan J, Maggi EC, Crabtree JS. miR-24 regulates menin in the endocrine pancreas. Am J Physiol Endocrinol Metab 307: E84 –E92, 2014. First published May 13, 2014; doi:10.1152/ajpendo.00542.2013.—Menin, the product of the MEN1 gene, functions as a tumor suppressor and was first identified in 1997 due to its causative role in the endocrine tumor disorder multiple endocrine neoplasia, type 1 (MEN1). More recently, menin has been identified as a key player in pancreatic islet biology with the observation of an inverse relationship between menin levels and pancreatic islet proliferation. However, the factors regulating menin and the MEN1 gene in the pancreas are poorly understood. Here, we describe the regulation of menin by miR-24 and demonstrate that miR-24 directly decreases menin levels and impacts downstream cell cycle inhibitors in MIN6 insulinoma cells and in ␤lox5 immortalized ␤-cells. This regulation of menin impacts cell viability and proliferation in ␤lox5 cells. Furthermore, our data show a feedback regulation between miR-24 and menin that is present in the pancreas, suggesting that miR-24 regulates menin levels in the pancreatic islet. menin; MEN1; pancreatic islet; miR-24; miRNA

is responsible for maintaining normal glucose homeostasis through the tightly controlled, regulated release of insulin from the pancreatic islet. Pancreatic islets have the unique ability to dynamically and reversibly expand to adapt to changes in insulin demand through careful balance of cell growth and renewal (including ␤-cell replication, neogenesis, transdifferentiation, and hypertrophy), and cell death (apoptosis, atrophy, and autophagy). When this process becomes compromised, it leads to the clinical manifestation of gestational diabetes or type 2 diabetes (T2D). However, detailed knowledge of the mechanisms underlying this process is lacking. MicroRNAs (miRNAs), an evolutionarily conserved class of posttranscriptional regulators, are short ⬃22-nucleotide noncoding RNA sequences that regulate the expression of mRNA targets by binding to the 3=-UTR and inhibiting translation and/or targeting the mRNA for degradation. There is growing evidence that miRNAs regulate key biological processes in the pancreatic islet (13, 14, 17, 27, 33, 49). For example, miR-375, the first miRNA identified in ␤-cell function, regulates insulin secretion and is essential for normal glucose homeostasis and turnover of ␣- and ␤-cells (40, 41). Other recent studies indicate a role for miR-338-3p and miR-181a in ␤-cell differentiation and insulin sensitivity, respectively (7, 23). p38 (MAPK) is downregulated by miR-24 through the insulinresponsive glucose transporter 4 (GLUT4) to affect peripheral insulin resistance (21). miR-24 also regulates insulin production by targeting the insulin transcriptional repressors Sox6 and Bhlhe22 (35). THE ENDOCRINE PANCREAS

Address for reprint requests and other correspondence: J. S. Crabtree, Louisiana State Univ. Health Sciences Center, School of Medicine, Dept. of Genetics, 533 Bolivar St., New Orleans, LA 70112 (e-mail: [email protected]). E84

Menin, the product of the MEN1 gene, is a ubiquitous nuclear protein that has been shown through the identification of binding partner proteins to participate in cell cycle regulation, apoptosis, and transcriptional regulation (1, 6, 18, 24). Of particular interest is the role played by menin in the pancreatic islet, where it is a key player in islet expansion, adaptation, and tumorigenesis (22, 25, 26, 37). Menin knockout mice exhibit pancreatic islet hyperplasia and tumorigenesis (9, 10), whereas mice overexpressing menin in the pancreas develop gestational diabetes because the islets lack proliferative ability to adapt to increased insulin demand (25). miR-24 is the only conserved miRNA predicted to target menin in human, mouse and rat. Recently, miR-24 targeting of menin was reported in the parathyroid (32). Therefore, we sought to determine whether miR-24 also impacts menin expression in the pancreatic islet. Here, we demonstrate that miR-24 directly decreases menin levels in cell lines derived from the endocrine pancreas and demonstrate a feedback loop similar to that identified in the parathyroid, between miR-24 and menin. Furthermore, we demonstrate that this mechanism impacts the production of the cell cycle inhibitors p27kip1 and p18ink4c, suggesting that miR-24 may play a key role in islet proliferation. MATERIALS AND METHODS

Transfections and luciferase assay. Luciferase constructs were made by cloning a 1600-bp fragment of the human MEN1 3=-UTR into the pmirGLO vector (resulting in pmirGLO-MEN1). Pre-miR-24 (assay no. PM10737), anti-miR-24 (assay no. AM10737), and premiR negative control miRNA (denoted as scrambled, or SCR, in figures; assay no. AM17110) were obtained from Ambion (Life Technologies, Grand Island, NY). MIN6 cells (38) were maintained in DMEM with 15% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, and Pen-Strep (100 U/100 ␮g). ␤lox5 cells (12) were maintained in low-glucose DMEM with 10% heat-inactivated FBS and Pen-Strep (50 U/50 ␮g). All media and components were from GIBCO (Life Technologies, Grand Island, NY) except FBS, which was from Hyclone (Thermo Scientific, Logan, UT). Cells were seeded in a 24-well plate at a density of 100,000 cells/well. After 24 h, the MEN1 luciferase construct was cotransfected into cells by using Lipofectamine 2000 (Invitrogen, Grand Island, NY) at 150 ng/well along with pre- or anti-miR-24, or pre-miR negative control miRNA at 5 pmol/0.5 ml medium. After 24 h, luciferase levels were measured using Dual-Glo Stop & Glo per the manufacturer’s instructions (Promega, Madison, WI). For RNA and protein analyses, cells were cotransfected as above, except with a plasmid expressing GFP replacing the luciferase reporter plasmid as a measure of transfection efficiency. For the menin overexpression studies, cells were transfected with empty FLAGCMV4-vector, or FLAG-CMV4-menin plasmids at 10 ng for MIN6 cells and 50 ng for ␤lox5 cells, using Lipofectamine 2000 (Invitrogen), and harvested 24 h posttransfection (1). Animals. All mice were purchased from The Jackson Laboratories (Bar Harbor, ME) and were bred and maintained in the AAALACcertified animal facility at the Louisiana State University Health Sciences Center, New Orleans, Louisiana. Floxed Men1 mice [no.

0193-1849/14 Copyright © 2014 the American Physiological Society

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miR-24 REGULATES MENIN IN THE ENDOCRINE PANCREAS

005109 129S(FVB)-Men1⬍tm1.2Ctre⬎/J] were crossed with Ins2Cre mice [no. 003573B6.Cg-Tg(Ins2-cre)25Mgn/J] resulting in mice lacking Men1 in the ␤-cell of the pancreatic islet. Mice were housed under a 12:12-h light-dark cycle, fed standard rodent chow, and provided water ad libitum. Genotyping was performed as previously published (10, 43). All animal studies had prior approval by the LSUHSC Institutional Animal Care and Use Committee (protocols no. 3053 and no. B3054) and were performed in accordance with federal guidelines and institutional policies. Murine pancreatic islet isolation. Pancreatic islets were isolated from mouse pancreas according to the protocol published by Zmuda et al. (59) with slight modifications. Briefly, the pancreas was infused with collagenase P solution (0.5 mg/ml; Roche, San Francisco, CA) in HBSS (Invitrogen) through the common bile duct, removed from the body, and further digested by collagenase P at 37°C for 16 min. At the end of incubation, the collagenase digestion was stopped by adding RPMI 1640 (Caisson Labs, North Logan, Utah) with 10% serum. After two rounds of washing, the islets were separated using Histopaque 1077 (Sigma-Aldrich, St. Louis, MO) and serum-free RPMI. Islets were collected from the Histopaque-medium interface, washed, and passed through a nylon mesh to increase the purity of islets with respect to exocrine cell contamination. Purified islets were then hand-picked and used directly for RNA isolation or were stored at ⫺80°C until analyses. Quantitative RT-PCR. Total RNA was extracted from cultured cells or pancreatic islets by use of the mirVana miRNA isolation kit (Ambion) according to instructions. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) for mRNA and the Taqman miRNA reverse transcription kit (Applied Biosystems, Foster City, CA) for miRNA. qRT-PCR was performed with a Bio-Rad CFX96, using commercially available MEN1, p27, p18, and miR-24 primer/probe sets (Applied Biosystems) and Universal Master Mix with no UNG (Applied Biosystems). Reactions were normalized to GAPDH and/or 18S rRNA for mRNA and small nucleolar (sno) RNA202 and/or U6 small nuclear (sn) RNA for miRNA qRT-PCR. Western blotting. Protein was extracted from cultured cells in RIPA buffer containing 1⫻ RIPA lysis buffer along with phenylmethylsulfonyl fluoride, sodium orthovanadate, and protease inhibitor cocktail solution (Santa Cruz, Dallas, TX). Separation was on 4 –20% Trisglycine gels (Novex, Life Technologies, Grand Island, NY), and proteins were transferred to a nitrocellulose membrane using the iBlot transfer system (Invitrogen). Blots were blocked for 45 min to an hour with TBST-milk, incubated with rabbit anti-menin primary antibody (1:2,000; Bethyl Laboratories, Montgomery, TX), rabbit anti-p27 antibody (1:200 sc-528; Santa Cruz, Dallas, TX for ␤lox5 cells, and 1:750 no. 2552, Cell Signaling for MIN6 cells) or rabbit anti-p18 antibody (1:750 no. NBP1-67654; Novus Biologicals, Littleton, CO) overnight at 4°C, washed with TBST, and incubated with anti-rabbit IgG goat HRP-labeled secondary antibody (1:5,000; PerkinElmer, Waltham, MA) for 2 h. Protein levels were detected using the ECL detection system (Thermo Scientific). ␤-Actin rabbit antibody (1: 3,000, Cell Signaling) was used as the loading control. Blots were performed on at least three biological replicate experiments, and all three blots were used for densitometric analyses using Image J software. Chromatin immunoprecipitation. Chromatin immunoprecipitation (ChIP) was performed using the Millipore ChIP assay (Billerica, MA). Each assay was performed on 1 ⫻ 106 cells in 1⫻ PBS (GIBCO, Life Technologies) containing Halt protease inhibitor cocktail (Thermo Scientific). Assays used either a menin (Bethyl Laboratories A300-105A) or MLL (Bethyl Laboratories A300-086A) antibody for the immunoprecipitation. Standard PCR was used to determine which targets were isolated. Primers used for ChIP from MIN6 cells (mouse): miR-24-1 promoter Fwd 5=-GGC TAA GTT CTG CAC CTG AA-3= Rev 5=-CCT TGA TGC ACC TGG GTT TA-3=; miR-24-2 promoter Fwd 5=-GGA TGG GAT TTG ATG CCA GT-3= Rev 5=-CGG AAC TTA GCC ACT GTG AA-3=; p18 positive control

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Fwd 5=-TTC AAG CCT GAG TAA TCC AT-3= Rev 5=-CAG CTG AAA GGT AGG TTT TG-3=. Primers used for ChIP from ␤lox5 cells (human): miR-24-1 promoter Fwd 5=-AGGATGCACTGTCTCCTGCT-3= Rev 5=-TATTTGCAGTCCAGGCCTTCGC-3=; miR-24-2 promoter Fwd 5=-ACCCCTGTTCCTGCTGAACT-3= Rev 5=-CTCACAAGCAGCTAAGCCCT-3=; p18 positive control Fwd 5=-CATTTTGACCACTGGGTGCAT-3= Rev 5=-ACTTCGGCAACCAAGAAATG-3=. Negative control sequences (derived from human chromosome 14) used for both MIN6 and ␤lox5: Fwd 5=-GTTGTTGGATTTGGCTTGCT-3= Rev 5=-GGACCAGATGGCATCATAGC-3=. Viability assays. MIN6 cells (7.5 ⫻ 104) and ␤lox5 cells (2 ⫻ 104) were cotransfected with a GFP plasmid (for transfection efficiency) along with pre- or anti-miR-24 or pre-miR negative control miRNA at 5 pmol/0.5 ml medium. Twenty-four hours posttransfection, cell viability was measured for 4 days using the Cell Counting kit-8 (Dojindo Molecular Technologies, Rockville, MD) per the manufacturer’s instructions. Briefly, 10 ␮l of CCK-8 solution was added per 100 ␮l of cell suspension to each well and incubated for ⬃1 h, and absorbance was measured at 450 nm using a microplate reader. Experiments were performed in triplicate. Proliferation assays. Proliferation was measured using the Click-iT EdU Microplate Assay (Life Technologies) per the manufacturer’s instructions. In brief, MIN6 (1 ⫻ 104) and ␤lox5 (4 ⫻ 103) cells were cotransfected with GFP and pre- or anti-miR-24 or pre-miR negative control miRNA at 1 pmol/100 ␮l medium in 96-well plates. EdU at a final concentration of 10 ␮M was added to the transfected cells. Twenty-four hours posttransfection and EdU treatment, proliferation was measured for 4 days by fixing and incubating with Oregon Green 488 azide followed by incubation with anti-Oregon Green antibody conjugated to HRP. The signal was then amplified using Amplex Ultra Red substrate. Fluorescence was measured at 530 and 590 nm using a microplate reader. Experiments were performed in triplicate. Statistical analyses. qRT-PCR data were analyzed by Student’s t-test using GraphPad Online (www.graphpad.com/quickcalcs/ttest1). All other data were analyzed by Student’s t-test using Excel; *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.005. Data shown are means ⫾ SE. RESULTS

miR-24 interacts with the 3=-UTR of menin. Many miRNAs have been identified to play a role in pancreatic biology (5, 11, 12, 19 –21, 28, 35, 39 – 41), including miR-24, which is overexpressed in pancreatic BON1 neuroendocrine cells (32) and downregulated in the skeletal muscle of diabetic rats (21). miR-24 is known to target the MEN1 gene in the parathyroid (32), and on the basis of this information we investigated the role of miR-24 in the endocrine pancreas. To do this we used the MIN6 mouse insulinoma and ␤lox5 immortalized human ␤-cell lines, both of which express endogenous miR-24 (Fig. 1A). To determine whether miR-24 interacts with the 3=-UTR of MEN1 in these cells in vitro, we performed a luciferase assay using a reporter plasmid with a pmiR-Glo backbone containing the firefly luciferase gene fused to the 3=-UTR of MEN1 under the control of the PGK promoter. This construct was transfected into MIN6 or ␤lox5 cells alone or with pre-miR-24, anti-miR-24, or scrambled negative control miRNA. We observed a 34% decrease in luciferase signal in MIN6 and a 27% decrease in ␤lox5 cells 24 h posttransfection of luciferase construct plus pre-miR-24 (Fig. 1B). Transfection of anti-miR-24, a molecule that binds and inhibits endogenous miR-24, produced a signal that was similar to that observed when these cells were transfected with plasmid alone or plasmid plus scrambled negative control miRNA in both cell lines. Given that our reporter construct is specific for MEN1, we

AJP-Endocrinol Metab • doi:10.1152/ajpendo.00542.2013 • www.ajpendo.org

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conclude that miR-24 interacts specifically with MEN1 by targeting the 3=-UTR in MIN6 and ␤lox5 cells. miR-24 affects menin expression levels. To further determine the effect of miR-24 on the expression of menin, we examined MEN1 mRNA and menin protein levels in MIN6 and ␤lox5 cells 24 h posttransfection with pre- or anti-miR-24 (Fig. 1, C and D). As expected, transfection with pre-miR-24 clearly increased miR-24 levels, whereas anti-miR-24 decreased miR-24 levels (Fig. 1C) compared with the scrambled negative control miRNA. Twenty-four hours posttransfection, we observed a 35% reduction in MEN1 mRNA levels in response to pre-miR-24 in MIN6 cells and a 61% reduction in ␤lox5 cells (Fig. 1D). We also measured an increase in MEN1 expression in response to anti-miR-24 in MIN6 cells but not in ␤lox5 cells. Furthermore, we performed Western blotting to determine whether these effects were also present at the protein level. Menin protein levels were reduced by 23% when transfected with pre-miR-24 in MIN6 and 38% in ␤lox5 (Fig. 1, E and F). Transfection with anti-miR-24 did not reveal a statistically significant difference in menin protein levels compared to cells with scrambled negative control miRNA. This seemingly slight reduction in target RNA and protein levels is characteristic of regulation by miRNAs, and is consistent with

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Fig. 1. miR-24 targets the 3=-UTR of the MEN1 gene and decreases MEN1 RNA and menin protein. A: relative levels of miR-24 in MIN6 and ␤lox5 cells as measured by qRT-PCR. B: luciferase assay of MIN6 and ␤lox5 cells transfected with a luciferase plasmid only (CTL), plasmid ⫹ premiR-24 (⫹Pre), plasmid ⫹ anti-miR-24 (⫹Anti), or plasmid ⫹ pre-miR scrambled negative control miRNAs (SCR). Error bars represent SE from 3 independent experiments. ***P ⬍ 0.005 vs. ⫹SCR. C and D: MIN6 or ␤lox5 cells were transfected with pre-miR-24 (Pre), anti-miR-24 (Anti), or pre-miR scrambled negative control miRNAs (SCR). Cells were harvested for RNA 24 h after transfection, and qRT-PCR was performed for miR-24 (C) or MEN1 (D). Representative Western blots for MIN6 (E) or ␤lox5 cells (F) transfected as above are shown, and the relative density of menin protein levels normalized to actin is indicated below. Error bars in E and F represent Image J quantitation of 3 independent blots and show SE. *P ⬍ 0.05, ***P ⬍ 0.005 vs. SCR.

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the function of these molecules as fine tuning mechanisms within the cell. These data indicate that miR-24 impacts expression of menin at both the RNA and protein levels in these cells. miR-24 decreases cell cycle inhibitor levels in vitro. Menin is known to impact cell proliferation by transcriptionally regulating the production of the cell cycle inhibitors p27kip1 and p18ink4c (hereafter p27 and p18, respectively) (37). Therefore, to further understand the impact of miR-24 on downstream targets of menin, we measured the mRNA levels of p27 and p18 in MIN6 and ␤lox5 cells 24 h posttransfection with pre- or anti-miR-24 (Fig. 2). MIN6 and ␤lox5 cells showed a significant 42% and 57% reductions, respectively, in p27 RNA levels on transfection with pre-miR-24, whereas transfection with anti-miR revealed no statistically significant change in p27 levels in either cell line (Fig. 2A). This result is mirrored in the protein data from MIN6 cells, where we saw a 42% reduction in p27 in response to pre-miR-24 and a trending, but nonsignificant, increase in p27 upon treatment with antimiR-24 (based on combined densitometric data from 3 independent experiments; representative blot in Fig. 2B). PremiR-24 did not impact p18 levels in MIN6 cells, but p18 was reduced by a significant 48% at the RNA level in ␤lox5 cells

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Fig. 2. miR-24 impacts viability, proliferation, and menin downstream targets p27 and p18. MIN6 and ␤lox5 cells were transfected with pre-miR-24 (Pre), anti-miR-24 (Anti) or pre-miR scrambled negative control miRNAs (SCR). Cells were harvested for total RNA 24 h after transfection, and qRT-PCR was performed for p27 (A) or p18 (C). Western blots for MIN6 cells transfected as above are shown, and the relative density of p27 (B) or p18 (D) protein levels normalized to actin is indicated in the bar graphs at the right. Error bars in B and D represent Image J quantitation of 3 independent blots and show SE. Cell viability assays were performed in ␤lox5 (E) or MIN6 cells (F) transfected as above. Cell proliferation assays were performed in ␤lox5 (G) or MIN6 (H) cells transfected as above. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.005 vs. SCR.

(Fig. 2C). Consistent with this result, no change was detected in p18 at the protein level in MIN6 cells treated with pre-miR24, although we did see a slight increase in p18 upon treatment with anti-miR-24, but again, this increase was not statistically significant (based on combined densitometric data from 3 independent experiments; representative blot in Fig. 2D). The reduction in p27 in both cell lines and the decrease in p18 in ␤lox5 cells suggests that miR-24 stimulates ␤-cell proliferation in the pancreatic islet by reducing menin availability. miR-24 impacts cell viability and proliferation in vitro. A reduction in p27 and p18 as a result of increased miR-24 suggests that miR-24 may play a role in regulating cell proliferation via menin. To test this hypothesis, we performed cell viability and proliferation assays in the MIN6 and ␤lox5 cell lines. Transfection of ␤lox5 cells with pre-miR-24 resulted in a significant increase in cell viability (Fig. 2E) and cellular proliferation (Fig. 2G). Anti-miR-24 significantly reduced cell

viability at day 4 but had no effect on proliferation. Transfection of MIN6 cells with pre-miR-24, anti-miR-24, and scrambled negative control miRNA showed no changes in cell viability (Fig. 2F) or proliferation (Fig. 2H), perhaps because the proliferative program in these insulinoma-derived cells was already at such a high level that it obscured any miRNA induced changes. Feedback regulation between mir-24 and menin. In the pancreas, menin is known to exist in a complex with MLL (mixed-lineage leukemia) to modulate the production of cell cycle inhibitors (26, 37). This complex binds upstream of promoters at AT-rich, SET1 binding sites and confers histone lysine methyltransferase activity to regulate target genes (42, 47, 51). This knowledge led to the hypothesis that miR-24 may be a menin/MLL target gene and that a feedback loop may exist between menin and miR-24 (Fig. 3A). Similar feedback loops have been described for a number of miRNAs in a host

AJP-Endocrinol Metab • doi:10.1152/ajpendo.00542.2013 • www.ajpendo.org

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miR-24 REGULATES MENIN IN THE ENDOCRINE PANCREAS

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Fig. 3. miR-24 and menin exist in a feedback loop in the pancreas. A: model of putative feedback regulation of miR-24 by menin. The menin:MLL complex binds upstream of the miR-24 gene region to stimulate production of miR-24 precursor molecules. Mature miR-24 then inhibits menin translation, blocking further menin:MLL complex formation and decreasing miR-24 levels. B: mature miR-24 is produced from 2 chromosomal locations within the mouse and human genomes. Arrowheads indicate location of primers used for chromatin immunoprecipitation (ChIP) in C. ChIP was performed using 1 ⫻ 106 MIN6 cells and immunoprecipitated with antibodies for menin and MLL. Resulting DNA was run in conventional PCR to detect p18 as a positive control, miR-24-1, miR-24-2, or an unrelated region of the genome as a negative control. IN, input; ME, immunoprecipitated with menin antibody; ML, immunoprecipitated with MLL antibody; W, no template control for PCR.

by using antibodies against menin or MLL. In these cells, both proteins are present in the region upstream of miR-24 on both chromosomes (Fig. 3C), suggesting that the formation of a complex containing menin and MLL may be necessary for the production of miR-24 in the pancreatic islet. Overexpression of menin increases mir-24 levels. As further validation of the results obtained by ChIP, we overexpressed menin in MIN6 and ␤lox5 cells and measured changes in miR-24 expression levels. Cells were transfected with pFLAG or pFLAG-menin constructs at 10 ng in MIN6 and 50 ng in ␤lox5 cells (Fig. 4A), and miR-24 expression levels were determined 24 h posttransfection by qRT-PCR. This resulted in a 2.5-fold increase in miR-24 expression in MIN6 (Fig. 4B) and a 0.6-fold increase in ␤lox5 cells upon menin overexpression. These results are consistent with and substantiate our hypothesis that miR-24 controls the availability of menin and that menin in turn impacts miR-24 production via reciprocal regulation in the pancreatic islet. miR-24 is reduced in the hyperplastic islets of Men1 knockout mice. Previously, our laboratory generated the first conditional mouse knockout model where menin was deleted in the pancreatic ␤-cells through the use of Ins2-Cre (10). By 8 wk of age, the absence of menin in these cells resulted in islet hyperplasia, which led to the identification of menin as a key regulator of the proliferative program within the pancreatic islet (36, 37). Therefore, to determine the impact of menin knockdown on miR-24 expression levels in a biological system (as opposed to siRNA in culture systems), we measured the expression levels of miR-24 in the pancreatic islets from mice lacking menin. We isolated total RNA from pancreatic islets of wild-type (⫹/⫹), heterozygous floxed (f/⫹), and homozygous (f/f) floxed Men1 conditional knockout mice with Ins2-Cre at 8 wk of age. We found that the heterozygous and homozygous knockout mice showed the expected decrease in Men1 levels (Fig. 4C), as well as the expected decreases in p27 and p18 (as a result of menin loss; Fig. 4, E and F) by qRT-PCR. Furthermore, associated with this decrease in menin, p27, and p18 levels were also ⬃60% and ⬃90% reductions in miR-24 levels, respectively, compared with wild-type mice (Fig. 4D) that paralleled menin loss. Therefore, the impact of menin on proliferation present in the pancreatic ␤-cell of the mouse was also observed in vitro in ␤lox5 cells due to the reduction of menin by miR-24 (Fig. 2E). This further supports that miR-24 is functioning via menin and is consistent with all other reports wherein decreases in menin also decrease p27 and p18 (22, 26). DISCUSSION

of different biological systems (4, 16, 29, 30, 32, 34, 45, 46, 48, 52, 57) and recently was described for miR-24 and menin in the parathyroid (32). miR-24 is produced from two separate chromosomal locations in both the human and mouse genomes (Fig. 3B), and in both species it exists as part of a miRNA cluster with miR-23a/b and miR-27a/b (8). miR-24-1 is produced from human chromosome 9 (chromosome 13 in mouse), whereas miR-24-2 is made from chromosome 19 (chromosome 8 in mouse), and these two chromosomal locations both produce mature miR-24 (identical in sequence regardless of chromosome of origin). Therefore, to determine if a feedback loop was present in the pancreas, as has been reported for the parathyroid, we performed ChIP from MIN6 and ␤lox5 cells

The endocrine pancreas has the unique and reversible ability to proliferate in response to increased insulin demand. This reversible proliferation of the pancreatic ␤-cells allows for precise, controlled increases in insulin secretion to maintain normal glucose homeostasis. Failure of the ␤-cells to adapt may result in elevated blood glucose and the development of diabetes. Detailed knowledge of the mechanisms involved in ␤-cell expansion will allow a better understanding of the events leading to gestational diabetes and T2D and may provide therapeutic approaches for treating these conditions. Menin, in its role as a tumor suppressor, is well established as a key player in the transcriptional regulation of the cell cycle. In general, the presence of menin increases production

AJP-Endocrinol Metab • doi:10.1152/ajpendo.00542.2013 • www.ajpendo.org

miR-24 REGULATES MENIN IN THE ENDOCRINE PANCREAS

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Fig. 4. Menin availability impacts miR-24 levels. A and B: MIN6 cells were transfected with 10 ng pFLAG or pFLAG-menin plasmids, and ␤lox5 cells were transfected with 50 ng pFLAG or pFLAGmenin. Transfected cells were harvested for RNA 24 h posttransfection, and qRT-PCR was performed for menin (A) or miR-24 (B). Error bars represent SE from 3 independent experiments. *P ⬍ 0.05, ***P ⬍ 0.005 vs. Flag. C and D: mice containing a conditionally floxed Men1 gene were crossed with transgenic Ins2-Cre mice to knock out Men1 in the pancreas. qRT-PCR for Men1 using total RNA from pancreatic islets demonstrates the expected reduction of menin that correlates with genotype (C) as well as demonstrating a dosage-dependent decrease in miR-24 (D), p27 (E), and p18 (F). Error bars represent SE from 3 replicate experiments. *P ⬍ 0.05, ***P ⬍ 0.005 vs. ⫹/⫹. ⫹/⫹ wild-type unfloxed MEN1 mice; f/⫹, heterozygous floxed MEN1 mice; f/f, homozygous floxed MEN1 mice. All mice were hemizygous for the Ins2-cre transgene.

***

+/+

of cell cycle inhibitors to stall the G0/G1 to S phase transition and keep cell proliferation in check (44). In the pancreatic islet, this occurs through menin interaction with MLL to cooperatively regulate expression of the cell cycle inhibitors p27 and p18 (37), often through the addition of H3K4me3 marks in the promoters of these genes (26). The impact of menin:MLL complex binding on the methylation status of the miR-24 genomic loci in the pancreas remains to be determined. It is also known that acute loss of menin results in accelerated cell cycle activation through decreases in p27 and p18 and activation of cyclin-dependent kinase-2 activity (44). Our data are consistent with this report. Finally, menin can mediate the G2/M transition by regulating the availability of cyclin B2 in mouse embryonic fibroblasts (50). Experiments performed in mice highlight the importance of menin in regulating proliferation in endocrine tissues including the pancreatic islet. Heterozygous deletion of the Men1 gene in mice leads to a tumor spectrum similar to that of patients with MEN1, including parathyroid, pituitary, and pancreatic adenoma, but few if any tumors of nonneuroendocrine origin (9). Conditional homozygous knockout of Men1 wherein the gene is specifically deleted in parathyroid (31), pancreatic ␤-cell (10), or liver (43) confirms this endocrine-specific impact on proliferation. Overexpression of menin in the ␤-cell of mice reduces the capacity of the islet to respond to increased insulin demand during pregnancy (25), once again highlighting the

f/+

f/f

critical role played by menin in endocrine cell proliferation. Moreover, the expansion of pancreatic islets in pregnancy and obesity is dependent on menin. Both the Ay mouse model of hyperphagic obesity and pregnant wild-type C57Bl/6 mice exhibit decreased menin levels that correlate with expanded pancreatic islets (25). Taken together, these studies imply that menin involvement in ␤-cell proliferation is highly dependent on the presence and regulation of the MEN1 gene itself, which is a relatively understudied area of menin biology. Some studies examining the promoter region of the MEN1 gene suggest that menin functions in a feedback loop to regulate its own transcription via a strong activating promoter element (15, 53). Given that menin is ubiquitously expressed, one of the ongoing challenges is to understand how the levels of this protein specifically impact proliferation in endocrine tissues. One notion is that there are endocrine-specific mechanisms that occur primarily in these tissues to fine-tune menin availability, and the data presented here showing miRNA regulation of menin support this notion. However, the difference, if any, between the regulation of menin in endocrine vs. nonendocrine tissue types remains unknown. Another unknown piece of the puzzle is the impact that loss of miR-24 may have on endocrine or nonendocrine tissue types. In the data presented here, anti-miR-24 does not consistently demonstrate predicted responses compared with the

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scrambled negative control and pre-miR-24. There are three reasons that may explain these results. First, the anti-miR-24 reagent may not be specific, although, as we demonstrate in Fig. 1C, the reagent does indeed reduce the levels of miR-24 in both cell lines studied, but the lack of a response with antimiR-24 is consistent with other literature using this reagent (2, 54). Second, the lack of predicted responses with anti-miR-24 could be due to overall reduced miRNA levels in cell lines vs. primary cells. However, this is unlikely, as we demonstrated in Fig. 1A. miR-24 is present in both of our cell lines, and a higher expression level of miR-24 is observed in ␤lox5 cells compared with MIN6, yet no predicted anti-miR-dependent changes are evident in this cell line even though the miR-24 level was significantly reduced upon transfection with antimiR-24 (Fig. 1C). The third, most plausible explanation is the presence of other miRNAs (in addition to miR-24) that also target MEN1. The human MEN1 3=-UTR contains predicted binding sites for at least seven other miRNAs (miR-28-5p, -125a-5p, -125b, -149, -599, -708, and -876-5p), whereas the mouse Men1 3=-UTR contains sites for at least 11 other miRNAs (miR-17, -20a&b, -33, -93, -106a&b, -138, -340-5p, -365, -486, and -491). None of these miRNAs have been validated in the published literature to impact MEN1, yet these may be playing a role in keeping menin at physiologically relevant levels in the pancreas. The potential, and as of yet unstudied, impact of these other miRNAs cannot be discounted. Conversely, miR-24 has many predicted targets in addition to MEN1. Of particular relevance is a report by Giglio et al., wherein it is reported that miR-24 promotes cellular proliferation in keratinocytes by directly targeting p27 (15a). In contrast to our data, the impact of miR-24 in keratinocytes is restricted to effects at the protein level (not mRNA), suggesting a post transcriptional effect. We measure impacts of miR-24 on p27 at both the protein and mRNA levels, suggesting a different mechanism than that identified in keratinocytes. Based on our data, the decrease in cell cycle inhibitors and increase in proliferation is a direct result of miR-24 regulation of menin availability. However, we cannot fully rule out contribution from direct targeting of p27 by miR-24. This may explain the dramatic decrease in p27 protein levels we observed in MIN6 cells (Fig. 2B) and again highlights the possibility that tissue-specific mechanisms may be relevant. miR-24 is not predicted to directly target p18, and there is no literature evidence disproving this in silico prediction. Our data clearly implicate p18 in ␤lox5 viability and proliferation based on the data in Fig. 2, C and E–H. This suggests a clear role for miR-24 in regulating pancreatic islet proliferation via menin and p18. The role of miR-24 in pancreatic islet proliferation is complemented by studies that implicate this miRNA in other functions of the islet and pathogenesis of T2D, including insulin production (8, 35), peripheral insulin resistance (13), and stimulation by glucose. Indeed, in a study of glucose stimulation performed in MIN6 insulinoma cells, miR-24 was identified as one of many glucose-responsive miRNAs that were significantly upregulated upon treatment with 25 mM glucose (46). In vivo, overexpression of miR-24 was observed in hyperplastic pancreatic islets from mice fed a high-fat diet and in diabetic db/db mice (58). In parallel, glucose-mediated repression of menin has been reported in INS1 cells and primary rat islets and is associated with increased proliferation

that is significantly attenuated by overexpression of menin (3, 55). Therefore, we propose the notion that the regulation of menin in response to elevated glucose may be mediated by miR-24, resulting in increased proliferation and islet adaptation. This hypothesis is consistent with recent literature linking miR-24 to pancreatic ␤-cell function and development of T2D (58), but further experiments are required to elucidate the biological impact of glucose-dependent repression of menin by miR-24. ACKNOWLEDGMENTS We thank Dr. Donna Neumann for technical assistance with the ChIP studies, Dr. Eric Lazartigues for assistance with the islet isolation, Dr. Nicholas Lanson for the GFP plasmid, and the LSUHSC School of Medicine and Department of Genetics for continued financial support. GRANTS This work was supported by the LSUHSC School of Medicine and the Department of Genetics. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: J.V., E.C.M., and J.S.C. conception and design of research; J.V. and E.C.M. performed experiments; J.V., E.C.M., and J.S.C. analyzed data; J.V., E.C.M., and J.S.C. interpreted results of experiments; J.V. and J.S.C. prepared figures; J.V. and J.S.C. drafted manuscript; J.V., E.C.M., and J.S.C. edited and revised manuscript; J.V., E.C.M., and J.S.C. approved final version of manuscript. REFERENCES 1. Agarwal SK, Guru SC, Heppner C, Erdos MR, Collins RM, Park SY, Saggar S, Chandrasekharappa SC, Collins FS, Spiegel AM, Marx SJ, Burns AL. Menin interacts with the AP1 transcription factor JunD and represses JunD-activated transcription. Cell 96: 143–152, 1999. 2. Amelio I, Lena AM, Bonanno E, Melino G, Candi E. miR-24 affects hair follicle morphogenesis targeting Tcf-3. Cell Death Dis 4: e922, 2013. 3. Arumugam R, Fleenor D, Lu D, Freemark M. Differential and complementary effects of glucose and prolactin on islet DNA synthesis and gene expression. Endocrinology 152: 856 –868, 2011. 4. Bonev B, Stanley P, Papalopulu N. MicroRNA-9 Modulates Hes1 ultradian oscillations by forming a double-negative feedback loop. Cell Rep 2: 10 –18, 2012. 5. Cardozo AK, Kruhoffer M, Leeman R, Orntoft T, Eizirik DL. Identification of novel cytokine-induced genes in pancreatic beta-cells by high-density oligonucleotide arrays. Diabetes 50: 909 –920, 2001. 6. Chandrasekharappa SC, Guru SC, Manickam P, Olufemi SE, Collins FS, Emmert-Buck MR, Debelenko LV, Zhuang Z, Lubensky IA, Liotta LA, Crabtree JS, Wang Y, Roe BA, Weisemann J, Boguski MS, Agarwal SK, Kester MB, Kim YS, Heppner C, Dong Q, Spiegel AM, Burns AL, Marx SJ. Positional cloning of the gene for multiple endocrine neoplasia-type 1. Science 276: 404 –407, 1997. 7. Chen H, Gu X, Su IH, Bottino R, Contreras JL, Tarakhovsky A, Kim SK. Polycomb protein Ezh2 regulates pancreatic beta-cell Ink4a/Arf expression and regeneration in diabetes mellitus. Genes Dev 23: 975–985, 2009. 8. Chhabra R, Dubey R, Saini N. Cooperative and individualistic functions of the microRNAs in the miR-23a⬃27a⬃24-2 cluster and its implication in human diseases. Mol Cancer 9: 232, 2010. 9. Crabtree JS, Scacheri PC, Ward JM, Garrett-Beal L, Emmert-Buck MR, Edgemon KA, Lorang D, Libutti SK, Chandrasekharappa SC, Marx SJ, Spiegel AM, Collins FS. A mouse model of multiple endocrine neoplasia, type 1, develops multiple endocrine tumors. Proc Natl Acad Sci USA 98: 1118 –1123, 2001. 10. Crabtree JS, Scacheri PC, Ward JM, McNally SR, Swain GP, Montagna C, Hager JH, Hanahan D, Edlund H, Magnuson MA, GarrettBeal L, Burns AL, Ried T, Chandrasekharappa SC, Marx SJ, Spiegel

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