MicroRNA-29a regulates pro-inflammatory cytokine secretion and scavenger receptor expression by targeting LPL in oxLDL-stimulated dendritic cells

FEBS Letters 585 (2011) 657–663

journal homepage: www.FEBSLetters.org

MicroRNA-29a regulates pro-inflammatory cytokine secretion and scavenger receptor expression by targeting LPL in oxLDL-stimulated dendritic cells Ting Chen a, Zhoubin Li c, Jing Tu c, Weiguo Zhu a, Junhua Ge a, Xiaoye Zheng a, Lin Yang a, Xiaoping Pan b, Hui Yan a,⇑, Jianhua Zhu a,⇑ a b c

Department of Cardiology, First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, PR China Department of Infectious Disease, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, PR China Department of Cardiothoracic Surgery, First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, PR China

a r t i c l e

i n f o

Article history: Received 18 November 2010 Revised 18 January 2011 Accepted 18 January 2011 Available online 26 January 2011 Edited by Tamas Dalmay Keywords: MicroRNA-29a Oxidized low density lipoprotein Dendritic cell Lipoprotein lipase Immuno-inflammation

a b s t r a c t There is increasing evidence that microRNAs (miRNAs) play important roles in cell proliferation, apoptosis and differentiation that accompany inflammatory responses. However, whether microRNAs are associated with DC immuno-inflammatory responses with oxidized low density lipoprotein (oxLDL) stimulation is not yet known. Our study aims to explore the link of miRNAs with lipidoverload and immuno-inflammatory mechanism for atherosclerosis. In DCs transfected with microRNA-29a mimics or inhibitors, we showed that microRNA-29a plays an important role in proinflammatory cytokine secretion and scavenger receptor expression upon oxLDL-treatment. Furthermore, we suggest an additional explanation for the mechanism of microRNA-29a regulation of its functional target, lipoprotein lipase. We conclude that microRNA-29a could regulate proinflammatory cytokine secretion and scavenger receptor expression by targeting lipoprotein lipase in oxLDL-stimulated dendritic cells. Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction Atherosclerosis is currently the leading cause of human morbidity and mortality in developed countries, and has been established as a chronic inflammatory disease [1]. Oxidized low-density lipoproteins (oxLDLs) remain the most important risk factor for atherosclerosis. Recently, the immuno-inflammatory mechanisms of atherosclerosis have gained tremendous interest and the process has been recognized as a complex interplay between modified lipids, different cells of the immune system, endothelial cells, and smooth muscle cells [2]. Aside from the well-known cell types such as monocytes, T cells, and B cells, the unexpected roles of dendritic cells (DCs) in atherosclerosis have recently been discovered [3]. DCs, specialized antigen-presenting cells, are central to the regulation of immunity because they are at the interface of the innate

Abbreviations: DC, dendritc cell; miRNA, microRNA; OxLDL, oxidized low density lipoprotein; TNF-a, tumor necrosis factor-a; IL, interleukin; LPL, lipoprotein lipase; LOX-1, lectin-like oxidized low-density lipoprotein receptor-1; UTR, untranslated regions; SRA, scavenger receptor A ⇑ Corresponding authors. Fax: +86 21 87236741 (H. Yan), +86 21 87236700 (J. Zhu). E-mail addresses: [email protected] (H. Yan), ting010151452@ yahoo.com.cn (J. Zhu).

and adaptive immune systems. DCs also play an important role in this activation and affect the initiation and progression of atherosclerosis, either pro-atherogenically or anti-atherogenically [4]. However, despite ongoing research regarding the immuneinflammatory mechanisms in atherosclerosis, the intracellular molecular events that induce and regulate the activation have yet to be elucidated. MicroRNAs (miRNAs), a novel class of short (22 nucleotides) non-coding RNAs, have been identified as important posttranscriptional inhibitors of gene expression by base pairing with the 30 untranslated regions (UTRs) of mRNAs and promoting mRNA stability [5,6]. miRNAs are implicated in various physiologic and pathologic processes [7,8], including cardiogenesis, hematopoietic lineage differentiation, and oncogenesis. Accumulating evidence suggest that miRNAs also play an important role in cardiovascular disease [9–15]. The MiR-29 family, predicted to function as inhibitors of numerous mRNAs involved in extracellular matrix (ECM) production and fibrosis, are pathologically associated with tumor and cardiac fibrosis through the regulation of collagens [16,17]. The expression of miR-29a is downregulated after myocardial infarction [18]. Holmstrom et al. [19] revealed the differential expression of miR-29a in mature DCs relative to immature DCs. In this study, the increased expression of miR-29a in oxLDL-stimulated DCs is first established, in

0014-5793/$36.00 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2011.01.027

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addition, the interaction between miR-29a and lipoprotein lipase (LPL) and their functional roles in the lipid uptake and proinflammatory cytokine secretion by oxLDL-stimulated DCs were determined. Furthermore, miR-29a is shown to decrease the proinflammatory cytokine secretion and enhance scavenger receptors expression efficiently by targeting LPL, evidence that regulation of LPL is associated with miR-29a in DCs is also provided.

2. Materials and methods The investigation conformed with the principles outlined in the Declaration of Helsinki for the use of human blood and was approved by the Ethics Committee of Experimental Research of Zhejiang University. 2.1. Human primary peripheral blood DC culture Peripheral human blood was obtained from healthy donors. Mononuclear cells were isolated by centrifugation through a Ficoll-isopaque (Sigma) density gradient [20]. To obtain the monocytes, the mononuclear cells were allowed to adhere to 6wells plates with 5% autologous serum for 2 h at 37 °C, in a 5% CO2 incubator. Non-adherent cells were removed and the adherent cells (monocytes) were co-cultured with with 1000 units/ml granulocyte GM-CSF and 1000 units/ml interleukin-4 for 5–7 days to obtain the immature DCs, monocytes were cultured in a complete medium (RPMI 1640 with 10% fetal calf serum). OxLDL (30 ug/ml) was added at day 6 and cells were harvested at day 7. 2.2. Small RNA transfection by electroporation treatment Pre-miR™ precursor molecules for miR-29a, anti-miR™ inhibitor for miR-29a were obtained from Dharmacon. SiLPL and siNS (not significant) duplex were synthesized by Dharmacon RNAi Technologies. DCs were harvested and resuspended in Opti-MEM (Invitrogen Life Technologies) at a concentration of 4  107 cells/ ml. Next, 7.5 ug miRNA inhibitor/mimic and siRNA (Dharmacon) were transferred to a 4-mm cuvette (Peqlab Biotechnologie) and filled to a final volume of 100 ul with Opti-MEM. Cell suspension was added and immediately pulsed in a Gene Pulser II apparatus (Bio-Rad) at 300 V, 150 uF, and 100 ohms. To confirm the efficiency of transfection, the same amount of Cy3-labeled negative control was also transfected and the efficiency of miR-29a overexpression and inhibition were at least 90%.

2.5. Cloning of 30 UTR of LPL mRNA and reporter gene assay The 293T were cotransfected with p-LPL UTR miRNA luciferase reporter vector and miR-29a mimic/inhibitor using lipofectamine 2000 (Invitrogen). Cells were also transformed with the PGL3-control vector, which is useful for monitoring transfection efficiency. MiRNA mimic/inhibitor negative, an miRNA non-homologous to the human genome, was used as a control. All cells were also transfected with pRL-TK (Promega) for normalization control. After 24 h, firefly luciferase activities were determined using the dualluciferase reporter assay system. Relative reporter activity was obtained by normalization to the Renilla control. The ratio of the luciferase activity of each construct was calculated using a luminometer. 2.6. mRNA real time quantitative PCR This step was done after 24-h incubation of DCs with oxLDL. The mRNA levels were analyzed using the SYBR-GREEN reagent kits with gene specific primers on the Applied Biosystems 7000 real time PCR system according to the manufacturer’s instruction. Specific fragments were amplified and human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was also amplified to serve as internal standard, the primer sequences are listed in Supplementary Table 1. 2.7. Western blot analysis Protein extracts were denatured and the solubilized proteins (20 lg) were subjected to electrophoresis on 10% polyacryl amide SDS gels, This was followed by probing with antibodies (Abcom) for rabbit anti-human-LOX1, scavenger receptor A (SRA), CD36 (diluted 1:1000 in TBST), or rabbit anti-actin (diluted 1:5000 in TBST), and then by goat anti-rabbit secondary antibody labeled with far-red-fluorescent Alexa Fluor 680 dye. All signals were detected by Odyssey (Li-cor, USA). Densitometric analysis was performed using Quantity One (Bio-Rad) to scan the signals. 2.8. Statistical analysis Data is presented as mean ± S.D. of at least three independent experiments and were compared using the two-tailed paired Student’s t-test or one-way ANOVA, a difference with a P < 0.05 was considered to be statistically significant.

2.3. HPLC analysis of lipid levels DCs were transfected with miRNA mimic and inhibitor, oxLDL was added 6 h after transfection, and cells were further incubated for 24 h. The sterol analyses were performed using an HPLC system (model 2790, controlled with Empower Pro software; Waters Corp., Milford, MA). Sterols were detected using a photodiode array detector equipped with a 4-lL cell (model 996; Waters Corp.). Analysis of cholesterol and cholesteryl esters was performed after elution with acetonitrile-isopropanol 30:70 (v/v) [21] and detection by absorbance at 210 nm. 2.4. ELISA assays of inflammatory markers and LPL expression DCs that have undergone 24 h incubation with oxLDL were used for ELISA. Culture supernatants were analyzed to determine the presence of tumor necrosis factor-a (TNF-a), IL-6, LPL using Sandwich Enzyme Immunoassay kits (RD) according to the manufacturer’s instructions.

3. Results 3.1. Increased expression of miR-29a in oxLDL-treated DCs Considering that oxLDL is a major pro-inflammatory factor and auto-antigen in the development of atherosclerosis, oxLDL-stimulated human primary peripheral blood DCs were chosen as the cell model for determining the miRNAs associated with atherosclerosis. In this study, miR-29a was found to be aberrantly expressed in oxLDL-stimulated DCs for the first time using TaqMan Real-time PCR. After stimulation with oxLDL, the miR-29a levels in DCs were increased threefold (Supplementary Fig. 1A), suggesting the role of miR-29a in the immuno-inflammatory response to oxLDLs. The miR-29a levels in DCs were then modulated. Transfection of miRNA-29a mimic resulted in an apparent increase in miR-29a levels (Supplementary Fig. 1B) compared with miR-29a inhibitor and miRNA negative transfection.

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3.2. LPL is a direct target of miR-29a To investigate the mechanism of miR-29a function in DCs, miRanda, TargetScan, and PicTar algorithms were employed to search for putative human protein-coding gene targets of miR-29a. The LPL gene, predicted by both TargetScan and PicTar, was further studied as a potential target (Fig. 1). To determine if miR-29a specifically attenuates LPL expression, the level of endogenous LPL mRNA was measured via qPCR after transfection of miR-29a mimic

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and inhibitor into DCs. Inhibition of miR-29a expression with miR-29a inhibitor increased LPL mRNA levels, whereas enhancing miR-29a expression with miR-29a mimic decreased the LPL mRNA levels relative to that in the control cells transfected with nonspecific miRNA. Consistent with the changes in mRNA levels, the level of LPL protein was also altered by miR-29a inhibitor and miR-29a mimic (Figs. 1A and 2B). To determine if regulation of LPL by miR-29a is specifically mediated by the microRNA mechanism, the 30 -UTR of the LPL gene containing miR-29a recognition

Fig. 1. Verification of the potential target genes (LPL) of miR-29a by luciferase reporter assay coincided with the mRNA and protein expression levels. (A) LPL mRNA expression was measured by real-time PCR. (B) ELISA results of LPL expression. (C) The cells were cotransfected with p-LPL-UTR, pGL-3 control, and with the negative control anti-miR, anti-miR-29a, pre-miR-29a, or negative control pre-miR. Data were obtained from at least three independent experiments. ⁄P < 0.01 compared with the pGL3-LPL– 30 UTR plasmids only group. Data were obtained from three independent experiments. ⁄P < 0.05 versus the ‘‘pre-neg’’ group. #P < 0.05 versus the ‘‘anti-neg’’ group.

Fig. 2. Effects of miR-29a on DCs phagocytosis and surface scavenger receptors. DCs were incubated with or without miR-29a mimic/inhibitor for 6 h, and then with oxLDL for 24 h. CD36, LOX-1, and SRA expression were regulated after incubation with miR-29a mimic and inhibitors. (A) The relative mRNA level of CD36, LOX-1, and SRA. (B) The respective densitometric measurement results are shown. The band density of the native cells was defined as the control. (C) The levels of LOX-1, CD36 and SRA were detected by western blot. ⁄P < 0.05 versus the ‘‘pre-neg’’ group. ⁄⁄P < 0.01 versus the ‘‘pre-neg’’ group #P < 0.05 versus the ‘‘anti-neg’’ group. ##P < 0.01 versus the ‘‘anti-neg’’ group.

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site was cloned and inserted downstream to a luciferase reporter. The reporter gene assay showed that, compared with the pGL3LPL-3UTR plasmid cotransfected cells, there was a statistically significant increase and decrease in the activity of the cells cotransfected with miR-29a inhibitor and miR-29a mimic (Fig. 1C, P < 0.01). This suggests that miR-29a directly targets LPL. 3.3. miR-29a in oxLDL-induced DCs interferes with pro-inflammatory cytokine production To investigate the functional consequences of miR-29a expression in oxLDL-induced DCs, pro-inflammatory cytokine production was analyzed. For this purpose, we elected to use the chemically synthesized miR-29a inhibitor or miR-29a mimic, in which the expression of mature miR-29a can be readily inhibited or augmented. As shown in Table 1, miR-29a mimic decreased the inflammatory cytokines IL-6 secretion significantly (more than fourfold) and miR-29a inhibitor increased its secretion (about twofold), meanwhile the level of TNF-a was downregulated by miR29a mimic slightly (40% off)and upregulated by miR-29a inhibitor (about two-fold). This indicates that miR-29a transfection could affect the inflammatory response of oxLDL-treated DCs. 3.4. Effects of miR-29a on the expression of surface scavenger receptors and lipid uptake in oxLDL-stimulated DCs Scavenger receptors family are multiligand, multifunction receptor system which can be either internalized by endocytosis or phagocytosis, or remain at the cell surface and mediate adhesion or lipid transfer. Under physiological conditions, scavenger receptors serve to scavenge or clean up cellular debris and other related materials, and they play a role in host defence. In pathological states, they mediate the recruitment, activation and transformation of macrophages and other cells which may be related to the development of atherosclerosis [22]. To study the effects of miR29a on lipid uptake by DCs, associated scavenger receptors LOX1, SRA, and CD36 were analyzed in oxLDL stimulated DCs. Interestingly, miR-29a clearly enhanced the expression of SRA and a slight increase in CD36 expression, both at the mRNA and protein level. Meanwhile, LOX1 expression was upregulated, but no change was observed in the LOX1 mRNA level in the oxLDL-stimulated DCs, whereas the miR-29a inhibitor treated groups yielded the opposite effect (Fig. 2). These results indicate that miR-29a regulates SRA and CD36 expression at the transcriptional and translational levels and affects LOX1 expression at the posttranscriptional level. Since miR-29a could regulate the expression of scavenger receptor, we further observed whether miR-29a could effect the li-

Table 2 Effects of miR-29a mimcs/inhibitor and siLPLon the level of total, free and esterified cholesterol in oxLDL treated DCs. Total cholesterol

Free cholesterol

Esterified cholesterol

Control Pre-neg Pre-miR-29a Anti-neg Anti-miR-29a Anti-miR-29a + siLPL

500 ± 28 534 ± 48 383 ± 43* 535 ± 35 647 ± 72 580 ± 44

195 ± 16 199 ± 27 188 ± 18 201 ± 34 305 ± 36# 243 ± 23

304 ± 17 335 ± 24 224 ± 31* 335 ± 2.5 342 ± 38 335 ± 20

Control si-neg si-LPL

651 ± 58 686 ± 70 493 ± 63c

297 ± 32 324 ± 33 218 ± 29&

354 ± 41 362 ± 36 276 ± 34&

Values are represented as (mean ± S.D.) mg/g, n = 3. * P < 0.05 compared to pre-neg group. # P < 0.05 compared to anti-neg group. & P < 0.05 compared to si-neg group.

pid composition in oxLDL-treated DC, after treatment with miR29a mimic/inhibitor, the levels of cholesterol in DCs were assessed using HPLC. The levels of total and esterified cholesterol significantly decreased in the group with treated with miR-29a mimics compared with the mimic negative group. However, no significant difference was observed between the inhibitor group and the negative group (Table 2). LPL is a functional gene target involved in miR-29a-mediated lipid uptake and pro-inflammatory cytokine secretion on oxLDL-induced DCs. Given the evidence presented above regarding LPL regulation by miR-29a at both the RNA and protein levels, and considering the reported inflammatory effect of LPL, LPL could be a functionally important target of miR-29a. To investigate the biological importance of LPL as a target of miR-29a, the LPL protein in DCs was depleted using siRNA and the effects of miR-29a inhibition were assayed. Knockdown of LPL expression by siRNA treatment efficiently repressed LPL mRNA and protein levels (Fig. 3A and B), decreased inflammatory cytokine secretion, downregulated lipid uptake (Tables 1 and 2), and upregulated scavenger receptor expression (Fig. 3C–E). These effects are similar to that of miR29a mimic in the oxLDL-treated DCs. Furthermore, the miR-29a inhibitor mediated effects on the inflammatory response and scavenger receptor expression of DCs were attenuated by the inhibition of LPL by siLPL in the oxLDL-stimulated cells (Fig. 4). The data therefore suggest an essential role for LPL as a mediator of the biological effects of miR-29a in oxLDL-treated DCs. 4. Discussion

Table 1 Comparison of inflammatory biomarkers secreted by transfecting the miR-29a inhibitor and mimics and siLPL. IL-6 (ng/ml)

TNF-a (ng/L)

Pre-neg Pre-miR-29a Anti-neg Anti-miR-29a Anti-miR-29a+siLPL

0.67 ± 0.21 0.15 ± 0.01* 0.66 ± 0.07 1.28 ± 0.15# 0.81 ± 0.1##

64 ± 4.36 39.8 ± 3.62* 64.06 ± 7.88 130.51 ± 13.6# 90.51 ± 9.6##

si-Neg si-LPL

0.93 ± 0.08 0.51 ± 0.06&

60.42 ± 7.12 35.52 ± 4.8&

Concentration of inflammatory cytokines secreted in 24 h culture supernatants by stimulated human primary dendritic cells from pre-miR, anti-miR, pre-neg, antineg and si-neg, siLPL. * P < 0.05 compared to pre-neg group. # P < 0.05 compared to anti-neg group. ## P < 0.05 compared to anti-miR-29a group. & P < 0.05 compared to si-neg group.

MiRNAs are newly identified, non-coding small RNAs, that are involved in multiple cellular processes [23]. Over the past few years, the vast potential of miRNAs as regulators of cardiovascular diseases 9–15 has fully emerged and has become an intriguing target for therapeutic intervention. However, the signature of miRNA expression and the possible roles of miRNA in atherosclerosis have been less studied. The aim of this study is to link miRNA regulation with lipid overload and the immuno-inflammatory mechanism for atherosclerosis. The effects of miR-29a on oxLDL-stimulated DC activation were characterized in terms of lipid uptake, endocytotic capacity, and pro-inflammatory cytokine secretion to explore the novel functional role of miR-29a in atherosclerosis immunoinflammatory mechanism. Our previous studies [24] have shown that some miRNAs are upregulated in oxLDL-stimulated monocytes/macrophages. In this study, the expression of miR-29a is notably upregulated in

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Fig. 3. Effects of LPL-specific knockdown on DCs. Confirmation of siRNA-induced knockdown of LPL mRNA by qPCR (A) and protein level by ELISA. (B) After transfections of the small interference RNA for LPL (siLPL) or an irrelevant control sequence (sineg) in the oxLDL-induced DCs, the expression of the surface scavenger receptors were regulated. (C) The mRNA levels of CD36, LOX1, and SRA. (D,E) CD36, LOX1, and SRA protein levels were determined by western blot. Data are presented as mean ± S.D., and ⁄ P < 0.05 compared with sineg.

Fig. 4. LPL is a functional target gene involved in miR-29a-mediated inflammatory and lipid uptake in oxLDL-induced DCs. The miR-29a mediated pro-inflammatory cytokine secretion was attenuated in oxLDL-induced DCs via a small interference RNA for LPL (A and B), and the lipid composition was decreased. (C) The mRNA levels of CD36, LOX1, and SRA. (D and E) CD36, LOX1 and SRA protein levels were observed by western blot. ⁄P < 0.05 compared with pure anti-miR-29a group.

oxLDL-stimulated DCs. Furthermore, the functional role of miR-29a in the lipid uptake of DCs was investigated and the molecular tar-

gets were revealed. On the other hand, little is known regarding the miR-29a–LPL interaction. To our knowledge, LPL may be the

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first identified target gene involved in the regulation of oxLDLstimulated DCs by miR-29a. Understanding the connections between the miRNAs and atherosclerosis has been hampered by the limited knowledge of miRNA target recognition. MiRNAs modulate their biological functions via the mRNA of their multiple target genes [25]. Although their potential gene targets can be predicted by computational analysis, there is a need for experimental approaches to target identification to gain knowledge of the mechanisms and modalities of miRNA target recognition. LPL has emerged as a significant contributor to the pathogenesis of atherosclerosis. LPL belongs to the mammalian triacylglycerol lipase gene family, it and has been shown as a central enzyme in overall lipid metabolism and transport. LPL influences the development of lesions in mouse atherosclerosis models [26–28]. In addition, the mRNA expression levels of LPL in peripheral blood mononuclear cells are strongly correlated with the mutational status of the immunoglobulin V(H) gene. LPL is also a prognostic marker in B-cell chronic lymphocytic leukemia29. All these indicate the pivotal role of LPL in the atherosclerosis immuno-inflammatory mechanism. However, little is known about how LPL transcripts are regulated at the post-transcriptional level, and the regulation of LPL expression by miRNAs has not yet been described. In the current study, computational analysis suggests that LPL may be an miR-29a target. Moreover, miR-29a was upregulated by oxLDL, and exposure of mature macrophages to oxLDL led to a marked reduction of LPL activity [30]. Other research found that miR-29a is downregulated in aggressive chronic lymphocytic leukemia and B-cell chronic lymphocytic leukemia with high LPL expression [29]. To determine whether LPL is an miR-29a target gene in DCs, that the expression of LPL mRNA and protein in DCs was first confirmed to be regulated by miR-29a, as determined by both gain-of-function and loss-of-function approaches. Furthermore, luciferase reporter assay analysis verified that the LPL gene is one of the direct targets of miR-29a. Finally, the miR-29a inhibitor–mediated effects on oxLDL-stimulated DCs were found to be blocked by the siRNA treatment of LPL. These results indicate that LPL is indeed a functional target gene of miR-29a. In our study, oxLDL was used as a stimulus to determine the potential role of miR-29a in DCs under cholesterol-laden conditions, mimicking those often encountered during atherogenesis. Interestingly, upregulation of miR-29a expression inhibits oxLDL-mediated pro-inflammatory cytokine secretion in DCs. In contrast, oxLDL-mediated DCs pro-inflammatory cytokines secretion is exacerbated after downregulation of miR-29a expression. Previously, Qiu G et al. [31] showed that LPL suppression causes decreased expression of pro-inflammatory cytokines. Accordingly, downregulation of LPL through miR-29a overexpression and LPL inhibition with siRNA have similar effects. Furthermore, depletion of LPL by siRNA transfection partly restores pro-inflammatory cytokine secretion induced by miR-29a inhibition in DCs. The results suggest that miR-29a has a negative effect on oxLDL-mediated pro-inflammatory cytokine secretion in DCs, and LPL is an important functional target of miR-29a in this model. Research has indicated that the pronounced oxLDL uptake capacity of DCs is reflected by the expression of scavenger receptors, such as lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and CD36 [32]. At the same time, after suppression of LPL, significantly increased expressions of lipoprotein receptors [31] were observed. Therefore, the expression of scavenger receptors was measured to demonstrate the effect of miR-29a on oxLDLmediated DCs lipid uptake. Transfection with the miR-29a mimic decreased scavenger receptor expression, whereas transfection with the miR-29a anti-sense inhibitors reduced scavenger receptor expression. Furthermore, we explore the effect of miR-29a on the lipid composition of DC, interestingly, increased miR-29a expres-

sion was found to be also associated with an altered lipid composition and reduced percentages of cholesterol, but decreased miR29a expression did not obtain the opposite effect. Two reason maybe explain the phenomenon: 1: the functional effect of miRNA was indirectly obtained by base pairing with the 30 UTRs of mRNAs, there are many potential target gene of miR-29a, LPL was just one of them, moreover, LPL could decrease production of cellular lipids in macrophage, but no confirmed evidence in DCs. 2: there are many factors would influence the intracellular lipids composition such as lipid influx and efflux, we just observed the effect of miR-29a on several scavenger receptors0 expression, the complicated mechanisms for these effects of miR-29a on the lipid composition of DC need further study. For the first time, our results strongly indicate that miR-29a acts as an important regulator of the inflammatory response and scavenger receptors expression by targeting LPL. Once effective methods of regulating miR-29a expression are achieved in a clinical setting, it could provide a novel, specific approach to controlling the formation of atherosclerosis. These novel findings may have extensive implications for the diagnosis and therapy of atherosclerosis. Acknowledgements This material is based upon work funded by Zhejiang Provincial Natural Science Foundation of China under Grant No. Y2100372 and National Natural Science Foundation of China (No. 30370570/C03030201, 30670866/C03030201). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2011.01.027. References [1] Libby, P (2002) Inflammation in atherosclerosis. Nature 420, 868–874. [2] Millonig, G., Schwentner, C., Mueller, P., Mayerl, C. and Wick, G. (2001) The vascularassociated lymphoid tissue: a new site of local immunity. Curr. Opin. Lipidol. 12, 547–553. [3] Hansson, G.K. (2001) Immune mechanism in atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 21, 1876. [4] Niessner, Alexander and Weyand, Cornelia M. (2010) Dendritic cells in atherosclerotic disease. Clin. Immunol. 134 (1), 25–32. [5] Cai, X., Hagedorn, C.H. and Cullen, B.R. (2004) Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA 10, 1957–1966. [6] Lee, Y., Kim, M., Han, J., Yeom, K.H., Lee, S., Baek, S.H., et al. (2004) MicroRNA genes are transcribed by RNA polymerase II. EMBO J. 23, 4051–4060. [7] Han, J., Lee, Y., Yeom, K.H., Nam, J.W., Heo, I., Rhee, J.K., et al. (2006) Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell 125, 887–901. [8] Valencia-Sanchez, M.A., Liu, J., Hannon, G.J. and Parker, R. (2006) Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev. 20, 515–524. [9] Cheng, Y. et al. (2007) MicroRNAs are aberrantly expressed in hypertrophic heart: do they play a role in cardiac hypertrophy? Am. J. Pathol. 170, 1831– 1840. [10] Sayed, D., Hong, C., Chen, I.Y., Lypowy, J. and Abdellatif, M. (2007) MicroRNAs play an essential role in the development of cardiac hypertrophy. Circ Res. 100, 416–424. [11] Tatsuguchi, M. et al. (2007) Expression of microRNAs is dynamically regulated during cardiomyocyte hypertrophy. J. Mol. Cell Cardiol. 42, 1137– 1141. [12] Thum, T. et al. (2007) MicroRNAs in the human heart. A clue to fetal gene reprogramming in heart failure. Circulation 116, 258–267. [13] van Rooij, E. and Olson, E.N. (2007) MicroRNAs: powerful new regulators of heart disease and provocative therapeutic targets. J. Clin. Invest. 117, 2369– 2376. [14] van Rooij, E. and Olson, E.N. (2007) MicroRNAs put their signatures on the heart. Physiol. Genomics 31, 365–366. [15] van Rooij, E., Sutherland, L.B., Liu, N., Williams, A.H., McAnally, J., Gerard, R.D., et al. (2006) A signature pattern of stress-responsive microRNAs that can evoke cardiac hypertrophy and heart failure. Proc. Natl. Acad. Sci. USA 103 (48), 18255–18260.

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