MicroRNA-23a suppresses the apoptosis of inflammatory macrophages and foam cells in atherogenesis by targeting HSP90

Journal Pre-proofs Research paper MicroRNA-23a suppresses the apoptosis of inflammatory macrophages and foam cells in atherogenesis by targeting HSP90 Yu Qiao, Chuxuan Wang, Jiayuan Kou, lujing Wang, Dong Han, Da Huo, Fuyan Li, Xiaoxi Zhou, Dehao Meng, Jiaran Xu, Ghulam Murtaza, Bobkov Artyom, Ning Ma, Shanshun Luo PII: DOI: Reference:

S0378-1119(19)30978-3 https://doi.org/10.1016/j.gene.2019.144319 GENE 144319

To appear in:

Gene Gene

Received Date: Accepted Date:

17 December 2019 20 December 2019

Please cite this article as: Y. Qiao, C. Wang, J. Kou, l. Wang, D. Han, D. Huo, F. Li, X. Zhou, D. Meng, J. Xu, G. Murtaza, B. Artyom, N. Ma, S. Luo, MicroRNA-23a suppresses the apoptosis of inflammatory macrophages and foam cells in atherogenesis by targeting HSP90, Gene Gene (2019), doi: https://doi.org/10.1016/j.gene.2019.144319

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MicroRNA-23a suppresses the apoptosis of inflammatory macrophages and foam cells in atherogenesis by targeting HSP90 Yu Qiao a,b,e,

a,b,e,*,

Da Huo

Chuxuan Wang

a,b,e,

Fuyan Li

a,b,c,e,*,

a,b,e,

Jiayuan Kou a,b,e , lujing Wang

Xiaoxi Zhou

a,b,e,

Dehao Meng

a,b,e,

a,b,e,

Dong Han

Jiaran Xu

a,b,e,

Ghulam Murtaza a,b,e, Bobkov Artyom a,b,e, Ning Ma a,b,e,#, Shanshun Luo d,#. a Department of Biochemistry and Molecular Biology, Harbin Medical University, Harbin, China b Key Laboratory of Cardiovascular Medicine Research (Harbin Medical University), Ministry of Education, China c Translational Medicine Center of Northern China, Harbin Medical University, Harbin, China d Department of Gerontology, The First Hospital of Harbin Medical University, Harbin, China e Medical Science Institute of Hei Longjiang Province, China * These authors contribute equally to this work # corresponding author MicroRNA-23a suppresses the apoptosis of inflammatory macrophages and foam cells in atherogenesis by targeting HSP90 Yu Qiao a,b,e,

a,b,e,*,

Da Huo

Chuxuan Wang

a,b,e,

Fuyan Li

a,b,c,e,*,

a,b,e,

Jiayuan Kou a,b,e , lujing Wang

Xiaoxi Zhou

a,b,e,

Dehao Meng

a,b,e,

a,b,e,

Dong Han

Jiaran Xu

a,b,e,

Ghulam Murtaza a,b,e, Bobkov Artyom a,b,e, Ning Ma a,b,e,#, Shanshun Luo d,#. a Department of Biochemistry and Molecular Biology, Harbin Medical University, Harbin, China b Key Laboratory of Cardiovascular Medicine Research (Harbin Medical University), Ministry of Education, China c Translational Medicine Center of Northern China, Harbin Medical University, Harbin, China d Department of Gerontology, The First Hospital of Harbin Medical University, Harbin, China

e Medical Science Institute of Hei Longjiang Province, China * These authors contribute equally to this work # corresponding author Abstract： In previous study, we have found that microRNA-23a is down regulated in atherosclerotic tissues. Here we demonstrate that miR-23a directly binds to 3’UTR of HSP90 mRNA to suppress the expression of HSP90. To investigate the potential roles of miR-23a in macrophage, THP-1 macrophages were transfected with miR-23a mimics or inhibitors. Our results showed inflammatory factors IL-6 and MCP-1 concentrations in cell culture medium of macrophage and foam cell transfected with miR-23a mimics were decreased. Furthermore，we find that apoptosis of macrophage and foam cells transfected with miR-23a mimics were inhibited. Over expression of miR-23a in foam cells could reduced lipid intake and accumulation in foam cells. Meanwhile, we found that in inflammatory macrophages and foam cells transfected with miR-23a mimcs, HSP90 and NF-κB proteins are significantly decreased. Our results have suggested a promising and potential therapeutic target for atherosclerosis. Key words: miR-23a, HSP90, inflammatory response, apoptosis. Cardiovascular disease (CVD) is the leading cause of mortality in developed countries and is likely to attain this status worldwide. Coronary artery disease (CAD) and cerebrovascular disease are the most common forms of CVD, whose underlying pathological feature is atherosclerosis

[1].

The excessive production of oxidized low

density lipoprotein (ox-LDL) in the blood leads to the adequate removal of fats and cholesterol from macrophages, and consequently promotes the formation of multiple atheromatous plaques within the arteries[2]. During the formation of the atherosclerotic lesions, macrophages are the first inflammatory cells of the invastion and the main component of atherosclerotic plaques[3]. Inflammatory cytokines produced by macrophages stimulate the generation of endothelial adhesion molecules, proteases, and other mediators, which may enter systemic circulation in soluble forms[4]. The presence of foam cells is a hall mark of an atherosclerotic lesion. Apoptotic foam cells are the source of pro-inflammatory cytokines and proteases,

which cannot be coupled with proper efferocytosis because the phagocyte nearby the apoptotic cells are vulnerable to death[5,6]. Apoptosis of macrophages and foam cells has become the significant event leading to the plaque formation and instability[7-9]. miRNAs are highly conserved single-stranded noncoding small RNAs that control cellular functions by either degrading mRNAs or inhibiting their translation. The involvement of miRNAs in different aspects of cardiovascular diseases has emerged as an important research field. The dysregulation of many individual miRNAs has been linked to the development and progression of cardiovascular diseases. The forced expression or suppression of a single miRNA is enough to cause or alleviate pathological changes. The roles of miRNAs in the pathogenesis of heart and vascular diseases suggest the possibility of using miRNAs as a potential diagnostic biomarker and/or therapeutic target for cardiovascular diseases. One of the miRNAs reported to have a critical role in development and prognosis of heart disease is miR-23a. The expression of miR-23a has been evaluated in diseases such as rheumatoid arthritis[10] and anoxia[11], and we have investigated its expression in apolipoprotein E (apoE) knockout mice (apoE−/−), we have also demonstrated that circulating miR-23a can serve as a potential biomarker in predicting and monitoring patient in coronary artery disease[12]. However, there are no reports describing the function of miR-23a in atherosclerosis. Little is known about the abundance and molecular function of miR-23a in macrophage in normal conditions or in ox-LDL conditions induced foam cells. We hypothesized that in the physiological activities of atherosclerosis, miR-23a might function as a modulatory molecule. 2. Materials and Methods 2.1. Cells culture and building models of macrophage and foam cells PMA (phorbol myristate acetate) were bought from Sigma Chemicals (St. Louis, MO, USA). Ox-LDL was obtained from LU WEN Biotechnologies Company (Shang Hai, China). The THP-1 cell line was maintained in our lab. Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 200 U/ml penicillin G and 200

μg/ml streptomycin. Prior to all experiments, the cells were differentiated into macrophages by preincubation with 100 ng/ml PMA for 48h. Thereafter, cells were further incubated with the different stimuli. Cells classified as foam cells were further incubated with ox-LDL under concentration of 80 μg/ml for 48h. 2.2. RNA Isolation and Real-Time RT-PCR Total RNA was extracted using TRIzol reagent (Invitrogen, USA) in accordance with the manufacturer’s protocol. Reverse transcription of this RNA was performed with a characteristic miR-23a reverse transcription primer, on a 7500 fast Real-Time PCR System (ABI, USA). U6 was used as internal control for normalization. 2.3. TUNEL staining Apoptosis in macrophage or foam cells was detected by performing TUNEL (dUTP Nick-End Labeling) assay, using Apoptosis Detection Kit (Roche, MD, USA), as per the manufacturer’s protocol. 2.4. Antibodies and western blotting Total protein samples from macrophage and foam cells were extracted as described previously and were separated by SDS-PAGE followed by blotting on a PVDF film. The PVDF membrane was incubated with primary antibody, followed by incubation with secondary antibody conjugated to peroxidase. The samples were analyzed for the level of GAPDH, HSP90 or NF-kB (antibodies purchased from Cell Signal Technology, USA). Image J software was used for quantifying protein bands by optical density. 2.5. Prediction of target gene Target gene HSP90A was predicted online through http://www.microrna.org/ . 2.6. Dual luciferase reporter assay Confirmation of target gene HSP90A was accomplished using dual luciferase reporter

assay.

For

constructing

the

pmirGLO-WT-3’UTR

and

pmirGLO

-MUT-3’UTR recombination plasmid, 532-bp sequence of HSP90B 3’UTR (carrying the miR-23a-binding sequence) was inserted after the bioluminescence reporter gene in the pmirGLO Dual-Luciferase plasmid (Promega, USA). HEK293 cells were first seeded in 96-well plates at 4×104/well density, and

were then transfected with either luciferase reporter recombinant plasmid carrying 3’UTR sequence or 3’UTR mutant sequence plasmid (20ng), and miR-23a mimics/inhibitors or NCs/inhibitor NCs (140ng) for 48h. Firefly luciferase as well as Renilla luciferase activities were detected using Dual-Glo® Luciferase Assay System (Promega, USA). 2.7. Statistical Analysis Data are presented as mean±S.E.M. Statistical comparisons between groups were made using Anova one-way by LSD method. P ＜ 0.05 was considered statistically significant. All experiments were repeated at least three times. Results 1. Expression of miR-23a is down-regulated and HSP90 is up-regulated in macrophage and foam cells during atherosclerosis. To examine the expression of miR-23a in macrophages and foam cells. We firstly induced macrophage from THP-1 cells by using PMA with different concentration to gain the optimum one (Figure.1A). And then we used ox-LDL treating macrophages to gain foam cells (Figure.1B). Meanwhile, we detect IL-6 and MCP-1 concentration in cell culture medium (Figure.1C). The increasing of these two inflammatory factors also showed successful induction of macrophage and foam cells. The expression of miR-23a was examined in macrophages and foam cells. The results showed that miR-23a was down regulated in foam cells (Figure.1E). And the target gene HSP90 protein expression was up regulated, the NF-kB protein level was also up regulated (Figure.1D). 2. Over-expression and Down-regulation of miR-23a regulate apoptosis and inflammation factors generation of macrophages. To expore the potential role of miR-23a in apoptosis and inflammation response of macrophages, miR-23a mimics/inhibitors or miRNA mimic negative controls (NCs)/ inhibitor negative controls (IN NCs) were used to be transfected in macrophages. Realtime PCR result showed high transfection efficacy of miR-23a mimics and inhibitors(Figure.2A). Potential target gene HSP90 showed decreasing expression in macrophages over expressed miR-23a mimics, as well as increasing

HSP90 mRNA in macrophages transfected with miR-23a inhibitors(Figure.2B). Inflammatory response analysis showed that compared with the control group, the experimental group transfected with miR-23a mimics displayed a significantly increasing IL-6 and MCP-1 concentration in cell culture medium, and decreasing concentration in macrophages transfected with miR-23a inhibitors (Figure.2C, D). TUNEL experiment results showed that decreasing number of apoptotic cells in macrophage over-expressed miR-23a compared with control group, macrophage transfected with miR-23a inhibitors showed increasing apoptotic cells compared with inhibitor control group (Figure.2E, F). 3. Overexpression and Down-regulation of miR-23a regulate apoptosis and inflammation factors generation of foam cells. To expore the potential role of miR-23a in apoptosis and inflammation response of macrophages under ox-LDL stimulation condition. miR-23a mimics/inhibitors or miRNA mimic negative controls (NCs)/ inhibitor negative controls (IN NCs) were used to be transfected in macrophages, and then we treated macrophage with ox-LDL to further explore the function of foam cells. Realtime PCR result showed high transfection efficacy of miR-23a mimics and inhibitors in foam cells(Figure.3A). Potential target gene HSP90 showed decreasing expression in foam cells over expressed miR-23a mimics, as well as increasing HSP90 mRNA in foam cells transfected with miR-23a inhibitors (Figure.3B). Inflammatory response analysis showed that compared with the control group, the experimental group transfected with miR-23a mimics displayed a significantly increasing IL-6 and MCP-1 concentration in cell culture medium, and decreasing concentration in macrophages transfected with miR-23a inhibitors (Figure.3C, D). Oil red O staining experiment result showed that compared with control group, foam cells over-expressed miR-23a contain less lipid, and foam cells transfected with miR-23a inhibitors contain more lipid than inhibitor control group(Figure.3E, F). TUNEL experiment results showed that decreasing number of apoptotic cells in macrophage over-expressed miR-23a compared with control group, and macrophage transfected with miR-23a inhibitors showed increasing apoptotic cells compared with inhibitor control group (Figure.3G, H).

4. Identification of HSP90 as a potential target gene of miR-23a and NF-kB pathway. Next, we attempted to elucidate the underlying pathways through which miR-23a inhibits macrophage apoptosis. By using the online software miRanda, we predicted HSP90 as one of the target genes of miR-23a(Figure.4G). We further validated whether miR-23a downregulates the expression of HSP90, by luciferase reporter assay. miR-23a represses HSP90 expression by binding to the 3’UTR of HSP90 mRNA and we observed that miR-23a overexpression inhibits the luciferase activity by 34.2%, as compared to scramble mimic transfected cells (Figure.4H). We further corroborated this result by evaluating the HSP90 mRNA and protein levels. On overexpressing miR-23a we observed reduced protein expression of HSP90A and NF-κB in macrophages. On down regulating miR-23a using miR-23a inhibitors, we detected increasing protein expression of HSP90A and NF-κB in macrophages (Figure. 4A, B, C). Furthermore, we detection the protein expression of HSP90A and NF-κB in foam cells. Overexpression of miR-23a could increase expression of HSP90A and NF-κB protein level in foam cells, inhibition of miR-23a decreaed HSP90A and NF-κB protein level in foam cells(Figure.4D, E, F). These results suggest that one of the pathways through which miR-23a inhibits apoptosis in macrophage and foam cells is by targeting the protective transcription factor HSP90. Discussion: Atherosclerotic vascular disease is the underlying cause of a number of diseases, including claudication, resulting from insufficient blood supply, myocardial infarction, ischemic heart disease and stroke [13]. Notably, the two latter diseases are the top two causes of mortality worldwide [14]. Thus, the pathogenesis of atherosclerosis is always a critical topic, but it is not yet fully understand. Substantial evidence has suggested that the inflammatory response is triggered in atherosclerosis, which was reflected by the leukocyte recruitment from blood into arterial intima [15,16], and many efforts have been done to investigate the involved molecules [17,18]. In the present study, We investigated a novel function of miR-23a as a protective regulator in atherosis. MiR-23a was downregulated in foam cells. MiR-23a inhibits apoptosis in

macrophage as well in foam cells by downregulating the expression of the inflammatory protein HSP90 and influence the NF-kB pathway. miR-23a,

located

on

chromosome

19p13.12,

it

is

involved

in

the

miR-23a~27a~24-2 cluster. The miR-23a~27a~24-2 cluster is found to have altered expression in many diseased states

[19].

miR-23a is reported to be universally

expressed in mammalian organ systems, including the heart, brain, colon and small intestine, whereas aberrant expression is observed in cancer, cardiac hypertrophy and muscular atrophy[19]. It is also reported that miR-23a is highly expressed in macrophages [20]. However, few studies have been conducted regarding the expression of miR-23a in macrophages mimicking atherosclerosis condition. In the present study, it was also observed that miR-23a expression was sensitive to ox-LDL in macrophages. miR-23a, as a novel gene regulator, promotes cell proliferation and survival, and inhibits cell apoptosis[21-23]. miR-23a has been observed to regulate cardiomyocyte apoptosis by targeting manganese superoxide dismutase[23]. Knockdown of miR-23a enhanced the antitumor effect of erlotinib by increasing the expression of PTEN. In addition, transfection with miR-23a inhibitors promoted the erlotinib-dependent inhibition of PI3K/AKT pathway, thus, suppressing the proliferation and inducing apoptosis in cancer stem cells[24]. The decrease of miR-23a-5p and miR-28-5p expression promotes protection against fibrosis by decreasing the levels of pro-fibrogenic markers α-SMA and COL1A1 and increasing apoptosis of HSC[25]. miR-23a, miR-24 and miR-27a could protect differentiating embryonic stem cells from BMP4-induced apoptosis[26]. Additionally, stable overexpression of an shRNA targeting miR-23a in U937 lymphoma cells produced stable knockdown of miR-23a and resulted in increased NOXA mRNA and an increased sensitivity to heat-induced apoptosis[27]. In an etoposide-induced in vitro model of apoptosis in primary cortical neurons, miR-23a and miR-27a were markedly downregulated as early as 1 h after exposure, before the upregulation of proapoptotic Bcl-2 family molecules. Administration of miR-23a and miR-27a mimics attenuated etoposide-induced changes in Noxa, Puma, and Bax, reduced downstream markers of caspase-dependent

(cytochrome

c

release

and

caspase

activation)

and

caspase-independent

(apoptosis-inducing factor release) pathways, and limited neuronal cell death[28]. In order to clarify the function of miR-23a in THP-1 cell induced macrophage apoptosis under atherosclerosis mimicking condition in vitro, macrophages were transfected with an miR-23a mimics/inhibitors to over expression/inhibit the expression of miR-23a in the present study. It was shown that over expression of miR-23a in macrophages decreased cell apoptosis and decreased inflammation factor IL-6 and MCP-1 expression, which suggests a role in inhibiting macrophage apoptosis. Apoptosis and inflammation factor concentration was further increased in miR-23a inhibited macrophages. Furthermore, we used ox-LDL to treat with macrophage, in order to investigate the function of miR-23a in foam cells. We find that over expression of miR-23a in foam cells could block the apoptosis of foam cells, and inhibiting of miR-23a expression could promote the apoptosis of foam cells. These results suggest that miR-23a has an antiapoptotic effect on macrophage apoptosis, miR-23a may be a key regulator of macrophage apoptosis. Inflammation and lipid intake dysfunction are also important mechnisms during the process of atherosclerosis. miR-23a is also reported to be deregulated and take part in inflammation

[12,29,30]

or lipid metabolism

[31,32].

However ， the function of

miR-23a through inflammation and lipid metabolism in macrophage during the process of atherosclerosis is unknown. Our finding suggested that over expression of miR-23a in macrophage could decrease inflammation factor IL-6 and MCP-1 concentration, which are important marker factors of inflammation. Meanwhile, after the detection of lipid accumulation, we find that over expression of miR-23a could decrease the lipid accumulation and inhibition of miR-23a could increase the lipid accumulation in foam cells. These results suggest that miR-23a plays an important role in inflammation and lipid metabolism in macrophage under ox-LDL induced condition mimicking the process of atherosclerosis. In the present study, the production of HSP90 protein was higher in foam cells derived from ox-LDL-stimulated macrophages, further validating the positive involvement of HSP90 in the development of atherosclerosis. During the formation of

atherosclerotic plaques, heat-shock proteins (HSPs) have been considered to play an important role[33,34]. HSP90 is one of the most extensively studied HSP, it is abundantly expressed in the cytoplasm of eukaryotic cells. The pro-inflammatory protein, HSP90, was recently reported to promote atherosclerosis in vitro and in vivo[35,36]. Shimp et al.[37] reported that suppressing HSP90 decreases the expression levels of pro-inflammatory mediator by inhibiting the activation of NF-κB pathways. The role of HSP90 extends beyond heat shock response owing to its chaperoning function to fold and stabilize many client proteins involved in cell proliferation, differentiation, apoptosis, and inflammation, and is therefore proposed as an important target in immunity and inflammation [35]. Macrophage apoptosis has been observed to promote vulnerable atherosclerotic plaque progression.Since it has been identified that miR‑23a is implicated in reducing macrophage apoptosis and foam cells apoptosis, and that miR-23a may inhibit lipid accumulation of foam cells, miR-23a may have a protective role in the development of vulnerable atherosclerotic plaques. Acknowledgments: The present study was supported by research grants from Hei Longjiang Province college youth innovation talent training program (UNPYSCT-2016045), Hei Longjiang Province education department fund (2017JCZX33), Heilongjiang natural science foundation union guiding project(LH2019H031), Heilongjiang natural science foundation general project (H20180006), Application technology research and development plan major project of Hei Longjiang (GA16C105), National Natural Science Foundation of China (81270366; 81401146; 81570534; 81773165). References: [1] Gui T, Shimokado A, Sun Y, et al. Diverse Roles of Macrophages in Atherosclerosis: From Inflammatory Biology to Biomarker Discovery[J]. Mediators of Inflammation, 2012, 2012(1):693083. [2] Kataoka H, Kume N, Miyamoto S, Minami M, Morimoto M, Hayashida K, Hashimoto N and Kita T: Oxidized LDL modulates Bax/Bcl-2 through the lectinlike Ox-LDL receptor-1 in vascular smooth muscle cells[J]. Arterioscler

Thromb Vasc Biol, 2001, 21: 955-960. [3] Gerrity R G, Naito H K, Richardson M, et al. Dietary induced atherogenesis in swine. Morphology of the intima in prelesion stages.[J]. American Journal of Pathology, 1979, 95(3):775. [4] Galkina E, Ley K. Immune and inflammatory mechanisms of atherosclerosis (*).[J]. Annual Review of Immunology, 2009, 27(1):165-197. [5] Wang H, Yang Y, Chen H, et al. The predominant pathway of apoptosis in THP-1

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[33]Pockley A G, Frostegård J. Heat shock proteins in cardiovascular disease and the prognostic value of heat shock protein related measurements.[J]. Heart, 2005, 91(9):1124-6. [34]Pockley A G. Heat Shock Proteins, Inflammation, and Cardiovascular Disease[J]. Circulation, 2002, 105(8):1012. [35]Mu H, Wang L, Zhao L. HSP90 inhibition suppresses inflammatory response and reduces carotid atherosclerotic plaque formation in ApoE mice[J]. Cardiovascular Therapeutics, 2017, 35(2). [36]Taipale M, Jarosz D F, Lindquist S. HSP90 at the hub of protein homeostasis: emerging mechanistic insights[J]. Nature Reviews Molecular Cell Biology, 2010, 11(7):515. [37]Iii S K S, Parson C D, Regna N L, et al. HSP90 inhibition by 17-DMAG reduces inflammation in J774 macrophages through suppression of Akt and nuclear factor-κB pathways[J]. Inflammation Research, 2012, 61(5):521-533. Figures Legends: Figure 1. Expression of miR-23a and HSP90 in macrophage and foam cell during atherosclerosis. (A) THP-1 cells were induced into macrophages by using PMA with different concentration. (n=4). (B) Macrophages were treated with ox-LDL to gain foam cells, which are authenticated by oil red staining and observed under different magnifications. (n=4).

(C) Concentration of IL-6 (ng/ml) and MCP-1 (ng/ml) in cell

culture medium of different period of macrophage and foam cells. (n=4). (D) HSP90 and NF-κB protein expression in macrophage and foam cell. (E) Fold change of miR-23a expression in macrophage and foam cell compared with monocyte. (n= 4); *P < 0.05 & **P < 0.01 versus control; mean ± S.E.M. Note: Scale bar = 50 μm Figure 2. Over-expression and Down-regulation of miR-23a regulate apoptosis and inflammation factors generation of macrophages. (A) Relative expression levels of miR-23a in macrophages transfected with miR-23a mimics or inhibitors compared to control group. They are mimics group (mi), negative group (nc), inhibitors group (in) and inhibitor negative group (in nc). (n=4). (B) HSP90 mRNA was evaluated by real-time PCR in macrophages transfected with miR-23a mimics or inhibitors

compared to control group. (n=4). (C) Concentration of inflammation factor IL-6 (ng/ml) and (D) MCP-1 (ng/ml) in culture medium of macrophages transfected with miR-23a mimics or inhibitor compared to control group. (n=4). (E) and (F) Fold change of apoptotic cell in macrophages transfected with miR-23a mimics or inhibitors in comparison to control group by TUNEL staining (n=4). *P < 0.05 & **P < 0.01 versus control; mean ± S.E.M. Note: Scale bar = 200 μm Figure 3. Over-expression and Down-regulation of miR-23a regulate apoptosis and inflammation factors generation of foam cells. (A) Relative expression levels of miR-23a in macrophages transfected with miR-23a mimics or inhibitors compared to control group under ox-LDL(80ng/ml) treated condition. They are mimics group (mi), negative group (nc), inhibitors group (in) and inhibitor negative group (in nc). (n=4). (B) HSP90 mRNA was evaluated by real-time PCR in macrophages transfected with miR-23a mimics or inhibitors compared to control group under ox-LDL treated condition. (C) Concentration of inflammation factor IL-6 (ng/ml) and (D) MCP-1 (ng/ml) in culture medium of foam cells transfected with miR-23a mimics or inhibitor compared to control group. (n=4).

(E) and (F) Oil red O staining of foam cells

transfected with miR-23a mimics or inhibitors compared to control group for evaluating lipid intake and accumulation. (G) and (H) Fold change of apoptotic cell in foam cells transfected with miR-23a mimics or inhibitors in comparison to control group by TUNEL (n=4).

*P < 0.05 & **P < 0.01 versus control; mean ± S.E.M.

Note: Scale bar = 200 μm Figure 4. miR-23a targets HSP90 and influence NF-κB pathway in macrophages and foam cells. (A) (B) (C) over-expression of miR-23a reduced protein levels of HSP0 and NF-κB, and inhibition of miR-23a reversed this effect in macrophages. (D) (E) (F) over-expression of miR-23a reduced protein levels of HSP0 and NF-κB, and inhibition of miR-23a reversed this effect in foam cells. (G) and (H) miR-23a targets the HSP90 3’-UTR. Luciferase activity, normalized with Renilla activity, was measured in homogenates of HEK cells transfected with the wild-type or mutated luciferase constructs in combination with miR-23a mimics or inhibitors compared with scrambled control. *P < 0.05 & **P < 0.01 versus control; mean ± S.E.M; n = 4

independent experiments.

Abstract： In previous study, we have found that microRNA-23a is down regulated in atherosclerotic tissues. Here we demonstrate that miR-23a directly binds to 3’UTR of HSP90 mRNA to suppress the expression of HSP90. To investigate the potential roles of miR-23a in macrophage, THP-1 macrophages were transfected with miR-23a mimics or inhibitors. Our results showed inflammatory factors IL-6 and MCP-1 concentrations in cell culture medium of macrophage and foam cell transfected with miR-23a mimics were decreased. Furthermore，we find that apoptosis of macrophage and foam cells transfected with miR-23a mimics were inhibited. Over expression of miR-23a in foam cells could reduced lipid intake and accumulation in foam cells. Meanwhile, we found that in inflammatory macrophages and foam cells transfected with miR-23a mimcs, HSP90 and NF-κB proteins are significantly decreased. Our results have suggested a promising and potential therapeutic target for atherosclerosis.

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for publication elsewhere. The contents of this manuscript will not be copyrighted, submitted, or published elsewhere while acceptance by the manuscript is under consideration. There are no directly related manuscripts or abstracts, published or unpublished, by any author(s) of this paper.

CVD: Cardiovascular disease; CAD: Coronary artery disease;

ox-LDL: oxidized low density lipoprotein; apoE: apolipoprotein E ; PMA : phorbol myristate acetate; HSP90: heat shock protein 90.