MALAT1 overexpression attenuates AS by inhibiting ox-LDL-stimulated dendritic cell maturation via miR-155-5p/NFIA axis

ABSTRACT

MALAT1 is associated with dendritic cells (DCs) maturation in Atherosclerosis (AS). This article aims to demystify the role of MALAT1 in AS. We separated immature DCs (iDCs) from healthy volunteers or ApoE-/- mice. And iDCs were treated with oxidized low density lipoprotein (ox-LDL) to induce DCs maturation. We found that ox-LDL promoted the levels of DCs maturation markers including CD83, CD86, IL-12 and IL-6. MALAT1 and NFIA were down-regulated, whereas miR-155-5p was up-regulated in the ox-LDL-treated iDCs. Furthermore, DCs maturation was notably suppressed by MALAT1 overexpression, NFIA overexpression or miR-155-5p knockdown. Moreover, MALAT1 functioned as a competing endogenous RNA to repress miR-155-5p, which controlled its down-stream target, NFIA. In addition, MALAT1 overexpression inhibited ox-LDL-stimulated DCs maturation by regulating miR-155-5p/NFIA axis. In AS mice, MALAT1 overexpression attenuated ox-LDL-stimulated DCs maturation and reduced atherosclerotic plaque area. In summary, our study demonstrates that MALAT1 overexpression attenuates AS by inhibiting ox-LDL-stimulated DCs maturation via miR-155-5p/NFIA axis. Thus, MALAT1/miR-155-5p/NFIA axis can potentially be used in the treatment of AS.

Introduction

Atherosclerosis (AS) is a common clinical disease, and one of the main pathological foundations of ischemic cardio-cerebrovascular disease. But the pathogenesis of AS still remains unclear. There is currently no effective and specific therapeutic drug in clinical practice. Many basic and clinical studies have shown that AS is a chronic inflammatory disease. The inflammatory response appears in all the stages of AS, and it may be the common link or pathway of many pathogenic factors in AS [1]. However, the mechanism of inflammation in AS has not been fully elucidated. Dendritic cells (DCs) are an important cell type that regulate the immune inflammatory response, researches have gradually recognized their roles in the occurrence and development of AS [2]. There are only a few immature DCs (iDCs) in the healthy vessel wall. In the AS lesion sites, there are plenty of iDCs accumulated in the subendothelial layer. The iDCs can be coaxed into mature DCs (mDCs) and promote the development of AS in different ways [3].

In low-density lipoprotein receptor-deficient mice, lipids promote CD11c⁺ DCs accumulation in the aortic vascular and CD11c⁺ DCs depletion reduces the deposition of lipids in the vascular wall. It indicates that CD11c⁺ DCs are the most important cells in early plaque formation [4]. In the pathogenesis of AS, iDCs differentiate into mDCs under the action of low density lipoprotein, especially oxidized low density lipoprotein (ox-LDL) [5]. Elevated ox-LDL is one of the most important risk factors for AS, which promotes the plaque formation and development of AS [6]. In summary, DCs differentiation plays a vital role in plaque formation and development of AS. However, under the action of ox-LDL, the mechanism by which iDC differentiates into pro-inflammatory mDCs is still unclear.

MALAT1 is a kind of long non-coding RNA, which involves in the occurrence of cancer. MALAT1 affects the levels of inflammatory cytokines such as IL-6 and TNF-α and is involved in the inflammatory progression of endothelial cells caused by diabetes. So far, the molecular mechanism of AS mediated by MALAT1 is still unknown. Some studies have shown that the deficiency of MALAT1 in ApoE^−/- mice promotes the progress of AS [7,8]. In the colon cancer tissues, the expression of MALAT1 in CD11c⁺ DCs affects the function of DCs, and it plays a vital role in the occurrence of colon cancer [9]. Thus, MALAT1 may participate in the progress of AS by regulating inflammation of DCs.

Stanković A’s [10] study shows that miR-155-5p is highly expressed in carotid plaque of patients with AS. In ApoE^−/- mice, the deficiency of miR-155-5p inhibits the progress of AS [11]. In the pathogenesis of AS, miR-155-5p has a crucial in inflammatory reaction. MiR-155-5p affects inflammatory reaction of endothelial cells by regulating the inflammation-related transcription factors [12]. In AS mice, miR-155-5p affects the content of inflammatory factors by regulating the gene expression in macrophages [13]. The expression of miR-155 is up-regulated in the ox-LDL treated DCs, indicating that ox-LDL participates in the pathogenesis of AS by regulating the function of DCs and inflammatory reaction [14]. Nuclear factor I/A (NFIA), as a member of nuclear factor family, is a specific DNA binding protein. NFIA is involved in the growth of various cancer cells, embryonic cell differentiation and myeloid cell differentiation. Studies have shown that in the Grl⁺ CDllb⁺ myeloid cells isolated from mice with terminal sepsis, NFIA deletion promotes the differentiation of macrophages and DCs, and the degree of DCs maturation [15,16]. Therefore, NFIA may participate in the regulation of DCs maturation.

Moreover, prediction software have analyzed that miR-155-5p targets binging with 3ʹ untranslated region of NFIA, and miR-155-5p is the target gene of MALAT1. In this article, we aim to detect the effect of ox-LDL on the expression of MALAT1, miR-155-5p and NFIA in DCs, and investigate the regulatory effects among them. Our research is mainly to explore the role of MALAT1 in ox-LDL-stimulated DCs maturation.

Materials and methods

Experimental animals

Male ApoE^−/- mice with 6 weeks old were purchased from the Laboratory Animal Center of Anhui Provincial Hospital. The mice were raised with high-fat diet (21% lard and 0.15% cholesterol) at a room temperature of 20 ~ 25°C for 10 weeks. No mice were died in this experiment. Then, the lentiviral vector carrying MALAT1 (LV-MALAT1) was purchased from RIBOBIO (Guangzhou, China). The lentiviral vector carrying NC (LV-NC) served as the control. AS mice were injected 100 μL LV-MALAT1 or LV-NC vectors (1 × 10 8PFU/ml) by caudal vein. All protocols were authorized by the Ethics Committee of Anhui Provincial Hospital.

The induction and cultivation of DCs in vitro

The peripheral blood was obtained from healthy volunteers or ApoE^−/- mice. The peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood by ficoll-hypaque density gradient centrifugation. PBMCs were cultured in RPMI-1640 supplemented with rhGM-CSF (1000 U/mL) and rhIL-4 (500 U/mL) with 5% CO₂ in air at 37°C. The medium was changed every 3 days. The iDCs were obtained after being cultured for 6 days. The iDCs were treated with ox-LDL (50 μg/mL, Solarbio, Beijing, China) for 24 or 48 h.

Flow cytometry

The ox-LDL-treated DCs were collected, and the concentration of DCs was adjusted to 1 × 10⁶/mL. The DCs suspension was labeled by antibodies CD83 and CD86, which were the markers of mature DCs. The IgG antibodies against CD83 and CD86 were added to another DCs suspension as control. The suspension was placed in dark room at room temperature for 20 min. DCs suspension was resuspended in the wash buffer, and then the degree of DCs maturation was detected by flow cytometry.

Enzyme-linked immunosorbent assay (ELISA)

The culture supernatant of DCs or the peripheral blood of ApoE^−/- mice was collected to detect the content of TNF-α, hs-CRP, IL-12 and IL-6. The assay was performed using ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. The optical density values of samples were detected at 450 nm wavelength using enzyme-labeled instrument (Thermo Fisher Scientific, Waltham, MA, USA).

Quantitative real-time PCR (qRT-PCR)

QRT-PCR was performed to measure the expression of different genes. Total RNA was extracted from cells using RNAprep Pure Tissue Kit (Tiangen, Beijing, China). The RNA was converted to complementary DNA using PrimeScript™ RT Reagent Kit (Takara, Tokyo, Japan). QRT-PCR was carried out using SYBR Green PCR Mix Kit (Takara) according to the instruction. The results were analyzed using the ∆∆CT (cycle threshold) method for quantification.

Western blot (WB)

The total proteinwas extracted from cells using Tissue or Cell Total Protein Extraction Kit (Sangon Biotech, Shanghai, China). Equivalent protein from different samples were separated by protein electrophoresis and transferred on PVDF membranes. The membranes were incubated with anti-NFIA after treated with blocking buffer. After the membranes were washed with TBST for several times, secondary antibodies labeled with horseradish peroxidase were incubated with the membranes. β-actin was used as a reference protein for normalization. The gray level of the protein bands was examined by Image J software. The experimental procedure was performed according to the protocol previously reported [17].

Cell transfection

Plasmid vectors, pcDNA3.1-MALAT1 and pcDNA3.1-NFIA, which could consistently express MALAT1 or NFIA was constructed by RIBOBIO via standard molecular cloning approaches. MiR-155-5p mimic, miR-155-5p inhibitor, small interfering RNAs specific targeting NFIA (si-NFIA) and its negative control (NC) were purchased from RIBOBIO. They were transfected into iDCs using Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Luciferase reporter gene assay

Luciferase reporter vector for NFIA regulated by miR-155-5p was constructed successfully. The miR-155-5p mimic or mimic NC was co-transfected with the wild type or mutant vectors of NFIA. The luciferase activity of the cells was detected after 48 hours of transfection using the luciferase assay system (Ambion, Austin, TX, USA).

RNA pull-down

RNA pull-down assay was performed to detect the interaction between MALAT1 and miR-155-5p. MALAT1 was labeled using Pierce™ RNA 3ʹ End Desthiobiotinylation Kit (Thermo Fisher Scientific). The labeled RNA was captured with streptavidin magnetic beads. Magnetic beads-RNA was incubated with total RNA. Then the RNA-RNA complex was eluted. QRT-PCR was performed to detect the expression of miR-155-5p to analyze the interaction between MALAT1 and miR-155-5p. The experimental procedure was performed according to the protocol previously reported [18].

Oil Red O staining

Oil Red O staining was performed to measure the atherosclerotic plaque lesions in the vessels wall of ApoE^−/-mice. Frozen sections of aorta roots were dyed with Oil Red O. The photomicrographs of the atherosclerotic plaques were taken by microscope. The ImageJ software was used to analyze the ORO-positive areas.

Statistical analysis

All experiments were independently repeated at least 3 times. All values were exhibited as mean ± standard deviation and analyzed by SPSS 22.0 statistical software (IBM, Armonk, NY, USA). For comparison of two groups, a two-tailed Student’s t-test was used. Comparison of multiple groups was made using a one- or two-way ANOVA. P < 0.05 was considered statistically significant.

Results

MALAT1 and NFIA are down-regulated, and miR-155-5p is up-regulated in the mDCs

PBMCs were separated from healthy peripheral blood to induce DCs. The phenotype of iDCs was observed by inverted microscope. There were few spiculate cytosol protuberances extended from the cell surfaces, indicating that PBMCs were successfully induced into iDCs (Figure 1(a)). The iDCs were stimulated by ox-LDL for 24 or 48 h, and then some molecules related to DCs maturation were detected, including co-stimulatory molecules CD83 and CD86, and cytokines IL-12 and IL-6. Flow cytometry data showed that the expression of CD83 and CD86 was up-regulated in ox-LDL-treated iDCs (Figure 1(b)). The levels of IL-12 and IL-6 were detected by ELISA and qRT-PCR. We found that levels of IL-12 and IL-6 were significantly increased in the iDCs after ox-LDL treatment for 24 or 48 h (Figure 1(c–e). Furthermore, we performed qRT-PCR and WB to detect the gene or protein expression of MALAT1, miR-155-5p and NFIA. Ox-LDL-treated iDCs exhibited a decrease in the gene or protein expression of MALAT1 and NFIA (Figure 1(f,g)). The expression of miR-155-5p was increased with the increasing of ox-LDL treatment duration (Figure 1(f)). Therefore, these data indicate that MALAT1 and NFIA are down-regulated, and miR-155-5p is up-regulated in the mDCs. Above all, MALAT1, miR-155-5p and NFIA are related to ox-LDL-stimulated DCs maturation.

Figure 1.

MALAT1 and NFIA are down-regulated, and miR-155-5p is up-regulated in the mDCs. (a) The PBMCs were obtained from healthy peripheral blood and incubated with rhGM-CSF and rhIL-4 to induce iDCs. The phenotype of iDCs was observed by inverted microscope. (b) The iDCs were treated with ox-LDL for 24 or 48 h. The expression of CD83 and CD8 in the iDCs was measured by flow cytometry. The levels of IL-12 (c) and IL-6 (d) in the culture supernatant of iDCs were assessed by ELISA. (e) The expression of IL-12 and IL-6 was detected by qRT-PCR. (f) QRT-PCR was performed to detect the expression of MALAT1, miR-155-5p and NFIA in the iDCs. (g) WB was performed to measure the protein expression of NFIA in the iDCs. (*P < 0.05 compared with the 0 h group, **P < 0.01 compared with the 0 h group).

MALAT1 up-regulation represses the ox-LDL-stimulated DCs maturation

To investigate the role of MALAT1 in DCs maturation, iDCs were transfected with pcDNA3.1-MALAT1 to induce MALAT1 overexpression. QRT-PCR data showed that MALAT1 overexpression significantly enhanced MALAT1 expression in the iDCs (Figure 2(a)). Then, the modified iDCs were treated with ox-LDL for 48 h. And we estimated the expression of MALAT1, miR-155-5p and NFIA in the modified iDCs. The expression of MALAT1 and NFIA was decreased, whereas miR-155-5p was up-regulated in the ox-LDL-treated iDCs. However, MALAT1 overexpression enhanced the expression of MALAT1 and NFIA, and lead to a decrease of miR-155-5p expression in the ox-LDL-treated iDCs (Figure 2(b)). Furthermore, the protein expression of NFIA was decreased in the ox-LDL-treated iDCs, which was abolished by MALAT1 up-regulation (Figure 2(c)). In addition, we assessed the levels of CD83, CD86, IL-12 and IL-6 in the iDCs by ELISA and flow cytometry. Compared with the control group, the levels of CD83, CD86, IL-12 and IL-6 were increased in the ox-LDL-treated iDCs. These molecules were notably repressed by MALAT1 up-regulation in the ox-LDL-treated iDCs (Figure 2(d–f)). Therefore, these findings suggest that MALAT1 up-regulation represses the ox-LDL-stimulated DCs maturation.

Figure 2.

MALAT1 overexpression inhibits ox-LDL-stimulated DCs maturation. IDCs were transfected with pcDNA3.1-MALAT1 or pcDNA3.1-NC. (a) The expression of MALAT1 in the modified iDCs was detected by qRT-PCR. The modified iDCs was treated with ox-LDL for 48 h. (b) QRT-PCR was performed to measure the expression of MALAT1, miR-155-5p and NFIA in the modified iDCs. (c) WB was performed to measure the protein expression of NFIA in the modified iDCs. The levels of IL-12 (d) and IL-6 (e) in the modified iDCs were detected by ELISA. (f) The expression of CD83 and CD86 in the modified iDCs was measured by flow cytometry. (**P < 0.01 compared with the NC group, ^$$P < 0.01 compared with the control group, ^##P < 0.01 compared with the ox-LDL+NC group).

MiR-155-5p silencing inhibits the ox-LDL-stimulated DCs maturation

To explore the role of miR-155-5p in DCs maturation, we silenced miR-155-5p in iDCs by cell transfection. The expression of miR-155-5p was severely decreased in the iDCs after transfected with miR-155-5p inhibitor (Figure 3(a)). Then, miR-155-5p silenced iDCs were treated with ox-LDL for 48 h. After ox-LDL treatment, the expression of miR-155-5p was up-regulated in the ox-LDL-treated iDCs, whereas NFIA was severely down-regulated in the ox-LDL-treated iDCs. MiR-155-5p silencing repressed miR-155-5p expression and enhanced NFIA expression in the ox-LDL-treated iDCs. (Figure 3(b)). Furthermore, ox-LDL-treated iDCs displayed a decrease of NFIA protein expression, which was rescued by miR-155-5p knockdown (Figure 3(c)). Moreover, ELISA and flow cytometry were performed to explore the levels of CD83, CD86, IL-12 and IL-6 in the iDCs. Compared with the control group, the levels of CD83, CD86, IL-12 and IL-6 were increased in the ox-LDL group. The knockdown of miR-155-5p led to a reduction of these molecules (Figure 3(d–f)). Taken together, the down-regulation of miR-155-5p inhibits the ox-LDL-stimulated DCs maturation.

Figure 3.

The knockdown of miR-155-5p inhibits ox-LDL-stimulated DCs maturation. IDCs were transfected with miR-155-5p inhibitor or inhibitor NC. (a) The expression of miR-155-5p in the modified iDCs was detected by qRT-PCR. The modified iDCs was treated with ox-LDL for 48 h. (b) QRT-PCR was performed to measure the expression of miR-155-5p and NFIA in the modified iDCs. (c) WB was performed to measure the protein expression of NFIA in the modified iDCs. The levels of IL-12 (d) and IL-6 (e) in the modified iDCs were detected by ELISA. (f) The expression of CD83 and CD86 in the modified iDCs was measured by flow cytometry. (**P < 0.01 compared with the NC group, ^##P < 0.01 compared with the control group, ^$P < 0.05 compared with the ox-LDL+inhibitor NC group, ^$$P < 0.01 compared with the ox-LDL+inhibitor NC group).

NFIA up-regulation suppresses the ox-LDL-stimulated DCs maturation

Next, we up-regulated NFIA in iDCs, and NFIA was obviously increased in iDCs after transfected with pcDNA3.1-NFIA (Figure 4(a)). Then, the modified iDCs were treated with ox-LDL for 48 h. The gene and protein expression of NFIA were down-regulated in ox-LDL-treated iDCs. Compared with the ox-LDL+NC group, the gene and protein expression of NFIA were notably increased in the ox-LDL+NFIA group (Figure 4(b,c)). Furthermore, the levels of CD83, CD86, IL-12 and IL-6 in the iDCs were examined by ELISA and flow cytometry. The levels of CD83, CD86, IL-12 and IL-6 were increased in the ox-LDL-treated iDCs. NFIA overexpression significantly repressed the levels of CD83, CD86, IL-12 and IL-6 in the ox-LDL-treated iDCs (Figure 4(d–f)). Thus, these results show that the up-regulation of NFIA inhibits the ox-LDL-stimulated DCs maturation.

Figure 4.

NFIA overexpression inhibits ox-LDL-stimulated DCs maturation. IDCs were transfected with pcDNA3.1-NFIA or pcDNA3.1-NC. (a) The expression of NFIA in the modified iDCs was verified by qRT-PCR. The modified iDCs was treated with ox-LDL for 48 h. QRT-PCR (b) and (c) WB were performed to detect the gene and protein expression of NFIA in the modified iDCs. The levels of IL-12 (d) and IL-6 (e) in the modified iDCs were detected by ELISA. (f) The expression of CD83 and CD86 in the modified iDCs was measured by flow cytometry. (**P < 0.01 compared with the NC group, ^##P < 0.01 compared with the control group, ^$P < 0.05 compared with the ox-LDL+NC group, ^$$P < 0.01 compared with the ox-LDL+NC group).

MALAT1 overexpression represses ox-LDL-stimulated DCs maturation by regulating miR-155-5p/NFIA axis

The relationship between MALAT1 and miR-155-5p was verified by RNA pull-down assay. There were binding sites between MALAT1 and miR-155-5p (Figure 5(a)). The luciferase assay data revealed that miR-155-5p targeted binging with 3ʹ untranslated region of NFIA (Figure 5(c)). Furthermore, ox-LDL-treated iDCs were co-transfected with pcDNA3.1-MALAT1 or pcDNA3.1-NC and miR-155-5p mimic or mimic NC. MiR-155-5p overexpression notably enhanced the expression of miR-155-5p in the iDCs (Figure 5(b)). The gene and protein expression of NFIA was examined by qRT-PCR and WB. Compared with the NC+mimic NC group, the gene and protein expression NFIA was increased in the MALAT1+ mimic NC group. MiR-155-5p overexpression significantly repressed NFIA expression. The influence conferred by MALAT1 up-regulation was abolished by miR-155-5p overexpression (Figure 5(d,e)). Moreover, ox-LDL-treated iDCs were co-transfected with pcDNA3.1-MALAT1 or pcDNA3.1-NC and si-NFIA or scramble. The gene and protein expression of NFIA was enhanced by MALAT1 overexpression and repressed by NFIA knockdown. The influence conferred by MALAT1 overexpression was abolished by NFIA silencing (Figure 5(f,g)). Therefore, these data suggest that MALAT1 functions as a competing endogenous RNA to repress miR-155-5p, which controls its down-stream target, NFIA.

Figure 5.

MALAT1 modulates NFIA via competitively binding miR-155-5p. (a) The relationship between MALAT1 and miR-155-5p was verified by RNA pull-down assay. IDCs were transfected with miR-155-5p mimic or mimic NC. (b) QRT-PCR was performed to assess the expression of miR-155-5p in the modified iDCs. (c) The luciferase assay was performed to explore the relationship between miR-155-5p and the NFIA. Ox-LDL-treated iDCs were co-transfected with pcDNA3.1-MALAT1 or pcDNA3.1-NC and miR-155-5p mimic or mimic NC. QRT-PCR (d) and WB (e) were performed to detect the gene and protein expression of NFIA in the modified iDCs. Ox-LDL-treated iDCs were co-transfected with pcDNA3.1-MALAT1 or pcDNA3.1-NC and si-NFIA or scramble. QRT-PCR (f) and WB (g) were performed to detect the gene and protein expression of NFIA in the modified iDCs. (**P < 0.01 compared with the Bio-NC group, ^##P < 0.01 compared with the mimic NC group, ^$$P < 0.01 compared with the NC+mimic NC group, ^&P < 0.05 compared with the MALAT1+ mimic NC group, ^&&P < 0.01 compared with the MALAT1+ mimic NC group, ^@@P < 0.01 compared with the MALAT1+ scramble group, ^%%P < 0.01 compared with the MALAT1+ scramble group).

Subsequently, we investigated the effect of MALAT1 on the expression of CD83, CD86, IL-12 and IL-6 in the ox-LDL-treated iDCs by flow cytometry and ELISA. MALAT1 overexpression led to a decrease in the levels of CD83, CD86, IL-12 and IL-6 in the ox-LDL-treated iDCs, which was effectively abolished by miR-155-5p up-regulation. The levels of CD83, CD86, IL-12 and IL-6 were significantly enhanced by miR-155-5p overexpression in the ox-LDL-treated iDCs (Figure 6(a–c)). Furthermore, MALAT1 overexpression repressed the levels of CD83, CD86, IL-12 and IL-6, whereas NFIA silencing promoted the levels of CD83, CD86, IL-12 and IL-6 in the ox-LDL-treated iDCs. The influence conferred by MALAT1 overexpression was abolished by NFIA knockdown (Figure 6(d–f)). Taken together, our data show that MALAT1 overexpression represses ox-LDL-stimulated DCs maturation by regulating miR-155-5p/NFIA axis.

Figure 6.

MALAT1 overexpression inhibits ox-LDL-stimulated DCs maturation via miR-155-5p/NFIA axis. Ox-LDL-treated iDCs were co-transfected with pcDNA3.1-MALAT1 or pcDNA3.1-NC and miR-155-5p mimic or mimic NC. (a) The expression of CD83 and CD86 in the modified iDCs was measured by flow cytometry. The levels of IL-12 (b) and IL-6 (c) in the modified iDCs were detected by ELISA. Ox-LDL-treated iDCs were co-transfected with pcDNA3.1-MALAT1 or pcDNA3.1-NC and si-NFIA or scramble. (d) The expression of CD83 and CD86 in the modified iDCs was measured by flow cytometry. The levels of IL-12 (e) and IL-6 (f) in the modified iDCs were detected by ELISA. (*P < 0.05 compared with the NC+mimic NC group, **P < 0.01 compared with the NC+mimic NC group, ^#P < 0.05 compared with the MALAT1+ mimic NC group, ^##P < 0.01 compared with the MALAT1+ mimic NC group, ^$P < 0.05 compared with the NC+scramble group, ^$$P < 0.05 compared with the NC+scramble group, ^&P < 0.05 compared with the MALAT1+ scramble group, ^&&P < 0.01 compared with the MALAT1+ scramble group).

MALAT1 overexpression attenuates AS by inhibiting ox-LDL-stimulated DCs maturation in vivo

To explore the role of MALAT1 in AS, we constructed AS model by feeding ApoE^−/- mice with high-fat diet. The AS mice were injected with LV-MALAT1 or LV-NC into caudal vein. The DCs were obtained from peripheral blood of the AS mice. The qRT-PCR results indicated that the expression of MALAT1 and NFIA were significantly increased and the expression of miR-155-5p was decreased in the Model+LV-MALAT1 group (Figure 7(a)). MALAT1 overexpression also notably promoted the protein expression of NFIA in the AS mice (Figure 7(b)). Subsequently, the levels of serum inflammatory cytokines (TNF-α, hs-CRP, IL-12 and IL-6) and mature DCs markers (CD83 and CD86) were measured by ELISA or flow cytometry. Compared with the Model+LV-NC group, the levels of TNF-α, hs-CRP, IL-12, IL-6, CD83 and CD86 were severely decreased in the Model+LV-MALAT1 group (Figure 7(c–g)). In addition, the plaque area of aortic root was analyzed by Oil Red O staining, showing that MALAT1 overexpression significantly reduced the plaque area in AS mice (Figure 7(h)). These data demonstrate that MALAT1 overexpression attenuates AS by inhibiting DCs maturation in vivo.

Figure 7.

MALAT1 overexpression attenuates AS by inhibiting ox-LDL-stimulated DCs maturation in vivo. The AS model established in ApoE^−/- mice by feeding high-fat diet. The AS mice were injected 100 μL LV-MALAT1 or LV-NC vectors (1 × 10 8PFU/ml) by caudal vein. The iDCs were obtained from peripheral blood of the AS mice. (a) QRT-PCR was performed to explore the expression of MALAT1, miR-155-5p and NFIA in the DCs. (b) WB was performed to detect the protein expression of NFIA in the DCs. The levels of TNF-α (c), hs-CRP (d), IL-12 (e) and IL-6 (f) in the DCs were measured by ELISA. (g) The expression of CD83 and CD86 in the DCs was measured by flow cytometry. (h) The plaque area of aortic root was analyzed by Oil Red O staining. (*P < 0.05 compared with the model group, **P < 0.01 compared with the model group).

Discussion

AS is one of the common diseases that severely endangers the public health. Clinical studies have shown that AS is a chronic inflammatory disease and an abnormal response of the vascular wall to various injuries [19]. More and more studies have proved that adaptive immune response is one of the important factors that promote the occurrence and development of AS [20]. DCs are the most potent professional antigen-presenting cells. The main function of DCs is to initiate and regulate lymphocyte-mediated immune response. MDCs can migrate into lymph node and activate young and memory T cells [21]. Some researchers have confirmed that there are lots of mDCs aggregate in the atherosclerosis plaque [22]. Thus, the DCs maturation is closely associated with the pathogenesis of AS. Ox-LDL is one of the most important factors that stimulates DCs maturation in the vascular vessels. But the mechanism of iDCs differentiates into pro-inflammatory mDCs under the effect of ox-LDL is still unknown. Our study found that the levels of CD83, CD8, IL-12 and IL-6 were notably increased in the iDCs after treated with ox-LDL. Co-stimulating molecules CD83 and CD86 are markers of DCs maturation, and inflammatory cytokines IL-12 and IL-6 are associated with DCs maturation. It indicates that ox-LDL induces DCs maturation. In addition, MALAT1 and NFIA were down-regulated, and miR-155-5p was up-regulated in the ox-LDL-treated iDCs, showing that MALAT1 and NFIA were associated with DCs maturation.

Previous research has shown that MALAT1 is highly relevant to various cancers [23]. Recent study has proved that the knockdown of MALAT1 in ApoE^−/- mice promotes the progress of AS [8]. In the ox-LDL-treated vascular endothelial cells, the deficiency of MALAT1 promotes DCs maturation in AS [24]. MALAT1 overexpression promotes tolerogenic DCs and immune tolerance in heart transplantation and autoimmune diseases [25]. Our study showed that MALAT1 overexpression significantly promoted miR-155-5p expression and repressed NFIA expression in the ox-LDL-treated iDCs. And MALAT1 up-regulation notably suppressed the levels of CD83, CD8, IL-12 and IL-6 in the ox-LDL-treated iDCs, indicating that MALAT1 overexpression inhibited the ox-LDL-stimulated DCs maturation. Lind’s study indicates that miR-155 affects the function of DCs by negative regulating SHIP1 expression [26]. MiR-155 enhances the maturity of DCs by negative regulating the expression of c-Fos [27]. In our research, the deficiency of miR-155-5p promoted the expression of NFIA and inhibited the DCs maturation. Fatiha et al. find that the expression of NFIA is highly expressed in ApoE^−/- mice than that in the control mice [28]. The up-regulation of NFIA inhibits pro-inflammatory cytokines and alleviates the AS progression [29]. The overexpression of NFIA inhibits the formation of atherosclerotic plaques by promoting the reverse cholesterol transport and inhibiting the level of pro-inflammatory factors in the plasma [30]. Our results demonstrated that NFIA overexpression inhibited the ox-LDL-stimulated DCs maturation. Thus, MALAT1, miR-155-5p and NFIA were closely associated with DCs maturation in AS progression.

Previous studies have demonstrated that MALAT1, miR-155-5p and NFIA are related to DCs maturation. We verified the relationship among MALAT1, miR-155-5p and NFIA. MiR-155-5p was the target gene of MALAT1, and miR-155-5p targeted binging with 3ʹ untranslated region of NFIA. MALAT1 functioned as competing endogenous RNA to repress miR-155-5p, which controlled its down-stream target, NFIA. MALAT1/miR-155-5p/NFIA axis was involved in the ox-LDL-stimulated DCs maturation. MALAT1 overexpression inhibited ox-LDL-stimulated DCs maturation by regulating miR-155-5p/NFIA axis. In AS mice, MALAT1 overexpression inhibited DCs maturation and reduced the plaque area of aortic root. Therefore, these data taken together demonstrate that MALAT1 overexpression attenuates AS by inhibiting ox-LDL-stimulated DCs maturation via miR-155-5p/NFIA axis.

In conclusion, our study shows that ox-LDL-treatment promotes DCs maturation and aggravates AS progression. And MALAT1 overexpression attenuates AS by inhibiting ox-LDL-stimulated DCs maturation via miR-155-5p/NFIA axis. Thus, MALAT1/miR-155-5p/NFIA axis can potentially be used in the treatment of AS or other diseases by inhibiting DCs maturation.

Funding Statement

This project was supported by Natural Science Foundation of Anhui Province (1808085MH281); New Medicine of University of Science and Technology of China (WK9110000046); Anhui Provincial Cardiovascular Institute.

Disclosure statement

All the authors declare no conflict of interest.