MiRNA-6870-3p regulates LPS-induced epicardial adipose tissue inflammatory genes via targeting Tollip-mediated JNK and NF-κB signaling in coronary artery disease

doi:10.21203/rs.3.rs-1522099/v2

Download PDF

Research Article

MiRNA-6870-3p regulates LPS-induced epicardial adipose tissue inflammatory genes via targeting Tollip-mediated JNK and NF-κB signaling in coronary artery disease

https://doi.org/10.21203/rs.3.rs-1522099/v2

This work is licensed under a CC BY 4.0 License

Version 2

posted

You are reading this latest preprint version

Objective

MiR-6870-3p acts as a crucial regulator of gene expression at the posttranscriptional level and participates in immune responses. However, the roles of miR-6870-3p and its target genes and their underlying mechanisms in the inflammatory responses of epicardial adipose tissues (EATs) are unknown.

Methods

MiRNA microarray was used to collect miRNA expression profiles in EATs from five patients with coronary artery disease (CAD) and four individuals without CAD (n-CAD). Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to check the expression of miR-6870-3p in CAD and n-CAD EATs. The mRNA and protein expression levels of Tollip and the key genes of the TLR4 signaling pathway were examined by qRT-PCR and Western blot. The levels of inflammatory factors in the cell supernatant were measured by enzyme-linked immunosorbent assay. Bioinformatics algorithms were used to predict the target gene of miR-6870-3p, and the result was validated by dual-luciferase reporter assay. The protein expression levels of JNK and NF-κB were measured by Western blot.

Resules:

Our results showed that miR-6870-3p was remarkably higher in CAD EATs than in n-CAD EATs. MiR-6870-3p was positively correlated with TLR4, IL-6, JNK, NF-κB (p65), and TNF-α in CAD EAT samples. Lipopolysaccharide (LPS) treatment upregulated miR-6870-3p mRNA expression and downregulated Tollip mRNA and protein expression in macrophages. MiR-6870-3p upregulation also aggravated the production of proinflammatory cytokines, such as TNF-α and IL-6, when the macrophages were stimulated with LPS. The result of luciferase reporter assays confirmed that miR-6870-3p directly targets Tollip. Moreover, miR-6870-3p upregulation in macrophages resulted in the activation of the JNK/NF-κB pathway.

Conclusion

Our study concluded that miR-6870-3p regulates human EAT inflammation by targeting Tollip-mediated JNK and NF-κB signaling events.

miR-6870-3p

Tollip

coronary artery disease (CAD)

epicardial adipose tissue (EAT)

inflammation

Cardiovascular disease is a common disease and the major cause of death worldwide. Some studies confirmed that atherosclerosis is closely related to coronary artery disease (CAD) ^{[1, 2]}. Recent studies have found that visceral adipose tissue (VAT) plays a vital role in CAD pathogenesis through the paracrine or endocrine pathway to secrete some adipokines, including pro-inflammatory and anti-inflammatory cytokines^{[3, 4]}. In addition, the volume or thickness of cardiac fat is associated with cardiovascular disease^[5]. Epicardial adipose tissue (EAT) is a subtype and one of the important components of VAT. EATs are close to the myocardium and warp coronary arteries without fibrous layer separation. EATs can secrete cytokines and chemokines when CAD occurs, which indicates that microcirculation changes take place during the development and progression of CAD^[6]. The microcirculation connects EATs and the coronary adventitia via the vasa vasora^[7]. A growing body of evidence supports that the inflammatory signaling pathway is activated in the EATs of patients with CAD[8]. According to clinical studies, inflammatory cytokines, particularly T-lymphocytes and proinflammatory M1 macrophages, have higher expression in CAD EATs than in non-CAD (n-CAD) EATs^[9]. In addition, some changes, like oxidative stress and gene transcription regulation, have been found in CAD EATs and affect the pathophysiological processes of CAD.Although increasing evidence indicates the correlations of EATs and inflammation with CAD,the underlying molecular mechanisms remain to be further explored.

MiRNAs are a type of small, non-protein-coding RNAs. The function of miRNAs is to interfere with protein translation or reduce transcript levels^[10]. MiRNAs can modulate multiple target genes by binding to the complementary sequences of mRNAs at 3′ untranslated regions (UTRs)^[11]. Many studies found that miRNAs are involved in human diseases, including inflammation^{[12, 13]} Furthermore, some miRNAs play substantial roles in immune responses^{[14, 15]}. MicroRNA-301a and microRNA-101-3p are downregulated to regulate Toll-like receptor (TLR)-triggered inflammatory response in rheumatoid arthritis monocytes or THP-1 cells^[16],[17]. The upregulation of miR-92a and miR-146 can enhance LPS-stimulated inflammatory response in mouse macrophages or PC‑12 cells^[18]. EAT-related CAD physiology and pathology are associated with two types of miRNAs: circulating miRNAs and adipose tissue miRNAs^[19]. CAT EATs contain lower miR-103-3p levels than subcutaneous adipose tissues in metabolically healthy cases as revealed by miRNA microarray analysis^[19]. Conversely, inflammation-related miR-135b-3p is elevated in CAD EATs according to transcriptomic and miRNome analyses. Despite these strong associations between adipose tissue miRNAs and CAD, whether dysfunctional miRNAs take part in TLR-induced inflammatory responses remains unclear.

The present study aimed to elucidate the underlying target of miR-6870-3p and how Tollip regulates EAT inflammation and verify the potential inflammatory signaling pathway. The expression levels of miR-6870-3p and key genes in inflammatory signaling pathways were first determined. MiR-6870-3p and the key genes in inflammatory signaling pathways had higher expression levels in CAD EATs than in n-CAD EATs, which indicates that miR-6870-3p has a strong association with inflammation. Importantly, the results are consistent with the correlation analysis results. LPS treatment upregulated miR-6870-3p expression and downregulates Tollip mRNA in cells. MiR-6870-3p positively regulated LPS-stimulated inflammatory cytokine production by targeting Tollip-mediated JNK and NF-κB signaling pathways, which can promote inflammation accumulation in EATs.

Human samples

We collected sample of EAT biopsy samples (average weight of 0.5–1.0 g) from 12 newly diagnosed CAD and n-CAD and 12 individuals with n-CAD according to coronary angiography from January 2019 to December 2019. The patients with CAD who underwent on-pump coronary artery bypass grafting (CABG), and the patients with n-CAD who underwent valvular replacement surgery. The recruitment criteria were as follows: all patients didn’t diagnose acute myocardial infarction and severe heart failure, also didn’t receive any radiotherapy or chemotherapy therapy before surgery. Patients were excluded from the study if they had other diseases, for example：active phase of infectious, hematologic diseases, rheumatologic diseases, steroid or immunomodulatory drug use. After the samples were collected from patients, we used cold PBS to wash blood in samples and putted in liquid nitrogen, and then stored at −80 °C until analysis. The human subject studies were approved and performed according to the standards established by the Ethics Committee on Human Subject Research at Central people's Hospital of Zhanjiang, Guangdong Medical University. We have complied with all relevant ethical regulations. Written informed consent was received from all human participants prior to inclusion in the study.

Cell culture

Human monocytes (THP-1) and human embryonic kidney (HEK) 293T cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). THP-1 cells and HEK 293T cells were grown in RPMI 1640 medium with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 mg/mL) at 37 °C in a humidified atmosphere containing 5% CO2. Before the THP-1 cells were differentiated into macrophages, the cells were seeded in 6-well plates according to protocol and used 100 ng/mL phorbol-12-myristate 13-acetate (Sigma) for 24 h to differentiated into macrophages. After that the cells were incubated with 20 ng/mL IFN-γ (Gibco) and 10 ng/mL LPS(Sigma) for 24 h to further induce M1 macrophages before their use in experiments.

Quantitative real-time PCR

Total RNAs from human adipose tissue samples or cells were extracted using Trizol reagent (Invitrogen), then reverse-transcribed into complementary DNA (cDNA) using PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio) according to the manufacturer’s protocol. qRT-PCR was performed using Universal SYBR Green qPCR Supermix (US EVERBRIGHT INC) using Bio-Rad PCR System. The primers used for mRNA quantification were follows miR-6870-3p, forward 5’-ACGCTCATCCCCATCTCC-3’, reverse 5’-CAGTGCGTGTCGTGGAGT-3’; IL-6, forward 5’-CTCTCCGCAAGAGACTTCCAG-3’, reverse 5’-TGTGGGTGGTATCCTCTGTGA-3’; TNF-α, forward 5’-GTGACAAGCCTGTAGCCCAT-3’, reverse 5’-TATCTCTCAGCTCCACGCCA-3’; TLR4, forward 5’-AATGCCAGGATGATGCCTCT-3’, reverse 5’-AGGAAGTACCTCTATGCAGGG-3’; JNK, forward 5’-ACACCACAGAAATCCCTAGAAG-3’, reverse 5’-CACAGCATCTGATAGAGAAGGT-3’; NF-kB (p65), forward 5’-CTGAGTCCTGCTCCTTCCA-3’, reverse 5’-CTTCGGTGTAGCCCATTTGT-3’; Tollip, forward 5’-GGACAACGGTCAGCGACGCA-3’, reverse 5’-CATAGCCCAGACGCAGGCGG-3’; β-actin, forward 5’-AGGCCGGCTTCGCGGGCGAC-3’, reverse 5’-CAGGGGAGCACACGCAGCTC-3’; U6, forward 5’- GCTTCGGCAGCACATATACTAAAAT-3’, reverse 5’- CGCTTCACGAATTTGCGTGTCAT-3’. The relative expression levels of the genes were calculated using the 2^−ΔΔCT method.

Plasmid construction

For Tollip overexpression, Tollip was directly cloned from human genomic DNA into pcDNA3.1 expression vector at the Hind III and Not I sites. The Tollip-3′-UTR was cloned from genomic DNA into psiCHECK-2 luciferase reporter vector at the Not I and XhoI sites. The Tollip-3′-UTR mutant was generated using four primers according to an overlapping PCR. All constructs were verified through plasmid DNA sequencing.

Transfection

MiRNA negative control (miR-con and anti-miR-con), miR-6870-3p mimics, and anti-miR-6870-3p were obtained from GenePharma (Shanghai, China). The macrophage cells were transfected with miR-6870-3p mimics, anti-miR-6870-3p, negative control and lipofectamine 3000 (Invitrogen, Germany) in accordance with the manufacturer’s protocol.

Luciferase reporter assay

HEK 293T cells were cultured in a 24-well plate at a density of 70% and then the cells were co-transfected with miRNA-6870-3p mimics or controls and luciferase reporter plasmids using lipofectamine 3000 reagent (Invitrogen). Cell extracts were prepared 24 h after transfection, and the relative luciferase activities were measured using a Dual Luciferase Reporter Assay Kit (Promega).

Western blotting

Total proteins were extracts from cells and tissue were prepared using PMSF、 phosphatase inhibitors and RIPA lysis buffer. We used bicinchoninic acid method to detected the protein concentrations. Approximately 30 µg to 50 µg of total protein was separated using SDS‑PAGE on 10% gels, and then transferred onto polyvinylidene fluoride membranes. Membrane was blocked with 5% skim milk for 2 h at room temperature and incubated at 4 °C overnight with primary antibodies were followed by incubations with the appropriate horseradish peroxidase-conjugated secondary antibodies. Antibodies used in the study were β-actin (ZSGBBIO, China), Tollip (Abcam, USA), p-IκBα (Cell Signal Technology, USA), JNK (Cell Signal Technology, USA), p-JNK (Cell Signal Technology, USA), p-p65 (Cell Signal Technology, USA), mouse IgG and rabbit IgG (ZSGBBIO, China).

Enzyme-linked immunosorbent assay (ELISA)

After the cells stimulation with LPS at the indicated time points, the cell culture supernatants were collected and then assessed the level of TNF-α and IL-6 using human IL-6 and TNF-α, human IL-6 ELISA and TNF-α kits (Invitrogen). Assays were performed in accordance with the manufacturer’s protocols.

Statistical Analysis

All the data were expressed as mean ± SEM from at least three independent experiments. The differences between two groups were analyzed by Student’s t-test. Correlations in gene expression was assessed by Spearman’s rank correlation coefficient for non-normally distributed data. GraphPad Prism 8 statistical software package (GraphPad Software, USA) and SPSS version 20 were used for data analysis. P <0.05 was considered as statistically significant.

Differential profiling of EAT by miRNA microarray analysis

A microarray analysis of EATs was performed to identify the miRNA expression patterns from five patients with CAD and four matched n-CAD individuals (controls). The most diferentially expressed miRNAs were selected according to the following criteria: −1.5 ≥ fold change ≥ 1.5 and false discovery rate-adjusted P≤0.05. Based on this analysis, 12 differentially expressed miRNAs were selected (Fig. 1A). Among them, five (has-miR-6870-3p, has-miR-4429, has-miR-4687-3p, has-miR-5703, and has-miR-630) were obviously upregulated and seven (has-miR-3651, has-miR-664b-3p, has-miR-619-5p, has-miR-564, has-miR-8485, has-miR-125-5p, and has-miR-574-3p) were downregulated in CAD EATs compared with n-CAD EATs (P<0.05). Moreover, pathway analysis (Fig. 1B) and Gene Ontology (GO) analysis (Fig. 1C) were also applied to analyze the differentially expressed mRNAs. GO analysis indicated the most substantial biological processes, cellular components, and molecular function. Pathway analysis indicated that the most substantial pathways consisted of the TLR signaling pathway, TGF-beta signaling pathway, and mTOR signaling pathway. According to the pathway analysis results, we selected the classical pathway related to inflammatory response: “TLR signaling pathway.” In summary, according to the microarray results and bioinformatics analysis, we identified signal molecules related to the TLR signaling pathway as the target of interest.

Levels of miR-6870-3p and inflammation-related factors in CAD and n-CAD EATs

MiR-6870-3p levels in human EATs from healthy controls (n-CAD, n = 12) and patients with CAD (n = 12) were examined to determine the relationship between EAT and CAD. The mRNA expression level of miR-6870-3p was markedly elevated in CAD relative to controls (Fig. 2A). The mRNA expression levels of TLR4, IL-6, JNK, NF-κB (p65), and TNF-α were also remarkably higher in CAD than in n-CAD (Figs. 2B–F).

Correlation of miR-6870-3p, TLR4, and EATinflammation indices in CAD

The relationship between miR-6870-3p and inflammation indices was investigated. The results of the correlation analysis showed the remarkably positive correlations of miR-6870-3p level with TLR4, IL-6, TNF-α, JNK, and NF-κB (p65) (Figs. 3A–E). These findings suggest that high miR-6870-3p expression plays an important role in EAT inflammation.

MiR-6870-3p regulates the production of proinflammatory cytokines in macrophages after TLR4 stimulation

Macrophages were transfected with miR-6870-3p mimics, inhibitors, or their controls, and the production of proinflammatory cytokines was analyzed to evaluate the role of miR-6870-3p in the regulation of immune responses to inflammatory stimulation. MiR-6870-3p overexpression remarkably increased LPS-stimulated TNF-α and IL-6 production at the mRNA and protein levels (Figs. 4A–D). By contrast, the mRNA and protein expression levels of TNF-α and IL-6 decreased when miR-6870-3p expression was inhibited (Figs. 4E–H), indicating the suppression of LPS-induced inflammation. Together, these data suggest that miR-6870-3p is a regulator of the TLR4-induced production of inflammatory cytokines, such as IL-6 and TNF-α, in macrophages.

MiR-6870-3p targets Tollip

TargetScan 7.2 (www.targetscan.org) was used to determine the putative target genes of miR-6870-3p to predict the effective target sites of miR-6870-3p (Fig. 5A). Previous studies have shown that Tollip, a negative regulator of the TLR signaling cascade, directly binds to the IL-1RI complex and is implicated in the suppression of the TLR4 pathways^[20]. In the present study, the mRNA and protein expression levels of Tollip were lower in the CAD group than in the n-CAD group (Figs. 5B and C). Tollip protein level in macrophages transfected with miR-6870-3p or anti-miR-6870-3p was examined to assess whether miR-6870-3p regulates Tollip expression. MiR-6870-3p overexpression remarkably reduced Tollip expression (Fig. 5D), whereas anti-miR-6870-3p increased the Tollip protein level (Fig. 5E). Tollip was remarkably negatively correlated with miR-6870-3p in the CAD group (Fig. 5F). Next, a luciferase reporter assay was conducted to obtain a direct evidence that Tollip is targeting the 3′ UTR of miR-6870-3p, Luciferase reporter vectors were generated by cloning the wild-type or mutated 3′ UTR of Tollip mRNA. The reporter plasmids and miR-6870-3p mimics were co-transfected into HEK 293T cells. The results showed that miR-6870-3p overexpression resulted in a remarkable decrease in the luciferase activity of the luciferase reporter that contains the wild-type 3′ UTR of Tollip mRNA compared with the set of cells transfected with miR-NC. However, this effect was abolished in the cells transfected with a luciferase reporter that contains the mutant 3′ UTR of Tollip mRNA (Fig. 5G). The findings suggest that miR-6870-3p binds to the 3′ UTR of Tollip to inhibit its expression.

Effect of miR-6870-3 on the LPS-induced activation of the JNK/NF-κB pathway

Tollip overexpression impedes the TLR4-triggered JNK and NF-κB signaling pathways^[21]. We tested whether LPS stimulation could modulate miR-6870-3p and Tollip levels in macrophages. Interestingly, LPS stimulation suppressed Tollip expression at the mRNA and protein levels in a time-dependent manner (Fig. 6A), whereas the expression of miR-6870-3p increased correspondingly (Figs. 6B and C). The effect of miR-6870-3p mimics on the activation of the JNK/NF-κB pathway was investigated next. The cells were transfected with miR-6870-3p mimic and then stimulated with LPS. JNK, p-JNK, p-IκBα, and p-p65 levels were detected by Western blot analysis. As expected, the cells treated with miR-6870-3p mimics had markedly increased phosphorylation of JNK, p-IκBα, and p-p65 (Figs. 6D–F), which suggests that miR-6870-3p modulates the LPS-activated JNK/NF-κB pathway that contributes to the inflammatory phenotype.

Blocking Tollip abolishes LPS-induced inflammatory responses

Our results showed that the mechanisms by which miR-6870-3p regulates LPS-triggered proinflammatory cytokine production include targeting Tollip in macrophage and knocking down Tollip expression through lentivirus transfection. Tollip expression was remarkably reduced at the mRNA and protein levels (Figs. 7A and B). Interestingly, Tollip downregulation considerably increased the LPS-triggered expression of IL-6 and TNF-α at the mRNA and protein levels in macrophages (Figs. 7C–F). The result suggests that Tollip plays a vital role in the regulation of inflammatory response. The cells were transfected with control mimics combined with vector, miR-6870-3p mimics combined with vector, or miR-6870-3p mimics combined with Tollip to establish the relationship between miR-6870-3p and Tollip(Figs7.G-H). The results suggest that the proinflammatory cytokines were remarkably increased by miR-6870-3p mimics and that this effect could be blocked by Tollip upregulation.

This study demonstrated that miR-6870-3p regulates inflammatory response via targeting Tollip-mediated inflammatory signaling pathways in human macrophages. We found that miR-6870-3p was upregulated after LPS stimulation, which selectively downregulated Tollip in macrophages and resulted in the overactivation of JNK/NF-κB p65 and the consequent inflammatory cytokine production. MiR-6870-3p expression was remarkably higher in EAT from the CAD group compared with the n-CAD group. Moreover, miR-6870-3p upregulation aggravated the production of proinflammatory cytokines, such as TNF-α and IL-6, when the macrophages were stimulated with LPS. Conversely, miR-6870-3p inhibition attenuated the production of proinflammatory cytokines in macrophages after LPS stimulation. Furthermore, Tollip expression was lower in CAD EATs than in n-CAD EATs. The inverse correlation between miR-6870-3p and Tollip in CAD EAT indicates that miR-6870-3p may regulate Tollip expression in EATs from patients with CAD. Moreover, miR-6870-3p overexpression suppressed Tollip expression in macrophages, whereas miR-6870-3p knockdown remarkably increased Tollip expression. The result of luciferase reporter assays confirmed that miR-6870-3p directly targets Tollip. Moreover, miR-6870-3p upregulation in macrophages resulted in the activation of the JNK/NF-κB pathway and the subsequent inflammatory cytokine production. Finally, Tollip overexpression in macrophages remarkably decreased the LPS-triggered production of TNF-α and IL-6. Overall, our study revealed that LPS-induced miR-6870-3p expression regulates inflammatory responses by targeting the Tollip/JNK/ NF-κB signaling axis in macrophages.

The differentially expressed miRNAs, including upregulated miRNAs (miR-6870-3p, miR-4429, miR-4687-3p, miR-5703, and miR-630) and downregulated miRNAs (miR-3651, miR-664b-3p, miR-619-5p, miR-564, miR-8485, miR-125-5p, and miR-574-3p), were identified according to the microarray analysis data of EATs from the CAD and n-CAD groups. Interestingly, the pathway analysis revealed that miR-6870-3p is involved in the TLR signaling pathway. Expectedly, TLR4 and the key genes of the inflammatory signaling pathway were higher in CAD EATs. This result is similar to the results of previous studies^{[21],[22, 23]}. MiR-6870-3p was positively correlated with TLR4, IL-6, JNK, NF-κB (p65), and TNF-α in EAT samples from CAD patients, which indicated that miR-6870-3p plays a critical role in TLR-induced inflammatory response signaling, especially in the TLR4 signaling pathway. The TLR4 signaling pathway was activated and then triggered the translocation of NF-𝜅B into the nucleus, which induced the overexpression of proinflammatory factors[24]. Faccini et al. applied circulating miRNAs, including miR-155, miR-145, and let-7c, to distinguish patients with CAD from n-CAD control subjects and identified the potential diagnostic value of circulating miRNA with CAD^[25]. Inflammation-related miRNAs (miR-155, miR-146a, and miR-124) can regulate the inflammatory response in LPS-treated macrophages^[26]. Currently, the roles and functions of miRNAs during disease development and progression remain unknown. In our study, miR-6870-3p overexpression using miR-6870-3p mimics further increased the production of proinflammatory cytokines in macrophages after LPS stimulation. Conversely, miR-6870-3p knockdown eliminated the inflammatory response of macrophages to LPS stimulation. Furthermore, miR-6870-3p expression increased after LPS stimulation in macrophages. Our results suggested that LPS stimulation increased the levels of miR-6870-3p, which resulted in the accumulation of TNF-α and IL-6 in macrophages.

Another interesting finding in this study was the identification of Tollip as a direct target of miR-6870-3p. Tollip is a conserved gene that participates in the TLR signaling pathway and is considered an important negative regulator in host innate immunity^{[27, 28]}. Tollip deficiency could resist apoptosis when primary coelomocytes are stimulated with LPS^[29]. Importantly, the role of Tollip was also reported in inflammation, endoplasmic reticulum stress, and innate immune responses^[30]. In Salmonella infection, SE or LPS induces an increase in miR-1306-5p levels, which activates the production of inflammatory cytokines by inhibiting Tollip^[31]. Moreover, Tollip protects against NLRP3 inflammasome activation by inhibiting TLR2/4-NF-κB signaling, which provides a potential treatment for acute kidney injury after paraquat intoxication^[32]. We found that miR-6870-3p negatively regulates Tollip expression, that is, miR-6870-3p expression increased with the inhibition of Tollip in macrophages. Interestingly, we also found a negative correlation between miR-6870-3p and Tollip in human EATs from patients with CAD. Furthermore, the level of Tollip remarkably decreased in macrophages after LPS stimulation. Indeed, miR-6870-3p overexpression suppressed Tollip expression, whereas miR-6870-3p inhibition increased Tollip expression in macrophages. These results indicated that Tollip levels are regulated by miR-6870-3p.In our present work, we demonstrated that Tollip overexpression negatively regulates the production of TNF-α and IL-6 after LPS treatment in macrophages. The results were consistent with a previous report that Tollip overexpression attenuates PQ-induced lung injury likely via the reduction of inflammation and the suppression of NF-κB signaling pathway activation^[33]. However, the role of Tollip in the production of EAT inflammation has remained unclear. In addition, we found that the expression of proinflammatory cytokine was remarkably increased by miR-6870-3p overexpression and that this effect could be abolished by increasing Tollip. Our results clearly showed that LPS stimulates the proinflammatory mechanisms by stimulating miR-6870-3p expression and inhibiting Tollip-mediated cell signaling, which promote inflammatory production in EATs. Mitogen-activated protein kinases (MAPKs) are key inflammatory pathway signaling molecules associated with cytokines, stress, and inflammation^[34]. The MAPK signaling pathway includes extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 MAPK, which regulate inflammatory response in macrophages in LPS stimulation^[35]. Earlier investigations proved that NF-κB and JNK are upregulated in CAD EATs compared with n-CAD EATs^{[36, 37]}. In support of these findings, our data clearly indicate that LPS stimulates proinflammatory mechanisms via JNK/NF-κB activation in macrophages. Thus, miR-6870-3p likely promotes the LPS-induced production of inflammatory cytokines by targeting Tollip via the JNK/NF-κB pathway. In summary, our study revealed that miR-6870-3p is upregulated in macrophages after LPS stimulation, which resulted in the overactivation of Tollip/JNK/NF-κB signaling and the increase in the production of proinflammatory cytokines. Thus, our data suggest that miR-6870-3p regulates inflammatory genes by targeting Tollip-mediated JNK and NF-κB signaling events in human EATs.

Ethics approval and consent to participate

The human subject studies were approved and performed according to the standards established by the Ethics Committee on Human Subject Research at Central people's Hospital of Zhanjiang, Guangdong Medical University. We have complied with all relevant ethical regulations. Written informed consent was received from all human participants prior to inclusion in the study.All methods were carried out in accordance with relevant guidelines and regulations

Consent for publication

Not Applicable

Availability of data and materials

The datasets used or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

All authors have no competition interests in this article

Funding : This work was supported by Science and Technology Project of Zhanjiang City,Guangdong province (No.2020B101)

Author Contributions:

Study concepts: Jian-Guo Wu,Wenkai Yang

Study design: Jian-Guo Wu,Wenkai Yang

Data acquisition: All authors

Quality control of data and algorithms: Jian-Guo Wu,Hao-Zong Huang

Data analysis and interpretation: Jian-Guo Wu,Hao-Zong Huang

Statistical analysis: Jian-Guo Wu,Kai Tang,Guang-Di Zheng

Manuscript preparation: Jian-Guo Wu,Kai Tang

Manuscript editing: Jian-Guo Wu,Wenkai Yang

Manuscript review: All authors

Acknowledgements: We are grateful to the patients and his family for their participation in this study.

Herrington W, Lacey B, Sherliker P, Armitage J, Lewington S (2016) Epidemiology of Atherosclerosis and the Potential to Reduce the Global Burden of Atherothrombotic Disease. Circ Res 118(4):535–546
Wang QC, Wang ZY, Xu Q, Chen XL, Shi RZ lncRNA expression profiles and associated ceRNA network analyses in epicardial adipose tissue of patients with coronary artery disease. Sci Rep.11(1):1567.
Alexopoulos N, Katritsis D, Raggi P (2014) Visceral adipose tissue as a source of inflammation and promoter of atherosclerosis. Atherosclerosis 233(1):104–112
McLaughlin T, Schnittger I, Nagy A, Zanley E, Xu Y, Song Y, Nieman K, Tremmel JA, Dey D, Boyd J (2021) Relationship Between Coronary Atheroma, Epicardial Adipose Tissue Inflammation, and Adipocyte Differentiation Across the Human Myocardial Bridge. Journal of the American Heart Association 10(22):e021003
Evangelos, Oikonomou, Charalambos, Antoniades (2018) The role of adipose tissue in cardiovascular health and disease. Nature reviews Cardiology.16(2):83–99.
Packer, Milton (2018) Epicardial Adipose Tissue May Mediate Deleterious Effects of Obesity and Inflammation on the Myocardium. Journal of the American College of Cardiology 71(20):2360–2372
Iacobellis G (2015) Local and systemic effects of the multifaceted epicardial adipose tissue depot. Nature Reviews Endocrinology 11(6):363–371
Guauque-Olarte S, Gaudreault N, Piché M-È, Fournier D, Mauriège P, Mathieu P, Bossé Y (2011) The transcriptome of human epicardial, mediastinal and subcutaneous adipose tissues in men with coronary artery disease. PloS one 6(5):e19908
Hirata Y, Tabata M, Kurobe H, Motoki T, Akaike M, Nishio C, Higashida M, Mikasa H, Nakaya Y, Takanashi S (2011) Coronary atherosclerosis is associated with macrophage polarization in epicardial adipose tissue. Journal of the American College of Cardiology 58(3):248–255
Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J, Bartel DP, Linsley PS, Johnson JM (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433(7027):769–773
Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. cell 136(2):215–233
Lu Q, Ma Z, Ding Y, Bedarida T, Chen L, Xie Z, Song P, Zou M-H (2019) Circulating miR-103a-3p contributes to angiotensin II-induced renal inflammation and fibrosis via a SNRK/NF-κB/p65 regulatory axis. Nature communications 10(1):2145
Rasheed Z, Rasheed N, Abdulmonem WA, Khan MI (2019) MicroRNA-125b-5p regulates IL-1β induced inflammatory genes via targeting TRAF6-mediated MAPKs and NF-κB signaling in human osteoarthritic chondrocytes. Sci Rep 9(1):6882
Benameur T, Soleti R, Porro C (2021) The potential neuroprotective role of free and encapsulated quercetin mediated by miRNA against neurological diseases. Nutrients 13(4):1318
Nguyen HT, Dai Phung C, Tran TH, Pham TT, Nguyen TT, Jeong J-H, Choi H-G, Ku SK, Yong CS, Kim JO (2021) Manipulating immune system using nanoparticles for an effective cancer treatment: Combination of targeted therapy and checkpoint blockage miRNA. Journal of Controlled Release 329:524–537
Hsu L-W, Huang K-T, Nakano T, Chiu K-W, Chen K-D, Goto S, Chen C-L (2020) MicroRNA-301a inhibition enhances the immunomodulatory functions of adipose-derived mesenchymal stem cells by induction of macrophage M2 polarization. International journal of immunopathology and pharmacology 34:2058738420966092
Huang T, Yang J, Zhang J, Ke W, Zou F, Wan C, Wang L, Zhang X, Liang F, Mei S (2020) MicroRNA-101-3p Downregulates TLR2 Expression, Leading to Reduction in Cytokine Production by Treponema pallidum–Stimulated Macrophages. Journal of Investigative Dermatology 9(1):6882
Tan Y, Yu L, Zhang C, Chen K, Lu J, Tan L (2018) miRNA–146a attenuates inflammation in an in vitro spinal cord injury model via inhibition of TLR4 signaling. Experimental and therapeutic medicine 16(4):3703–3709
Vacca M, Di Eusanio M, Cariello M, Graziano G, D'Amore S, Petridis FD, D'orazio A, Salvatore L, Tamburro A, Folesani G (2016) Integrative miRNA and whole-genome analyses of epicardial adipose tissue in patients with coronary atherosclerosis. Cardiovascular research 109(2):228–239
Li T, Hu J, Li L (2004) Characterization of Tollip protein upon Lipopolysaccharide challenge. Molecular immunology 41(1):85–92
Burns K, Clatworthy J, Martin L, Martinon F, Plumpton C, Maschera B, Lewis A, Ray K, Tschopp J, Volpe F (2000) Tollip, a new component of the IL-1RI pathway, links IRAK to the IL-1 receptor. Nature cell biology 2(6):346–351
Baker AR, Harte AL, Howell N, Pritlove DC, Ranasinghe AM, Da S, Youssef EM, Khunti K, Davies MJ, Bonser RS Epicardial Adipose Tissue as a Source of Nuclear Factor-B and c-Jun N-Terminal Kinase Mediated Inflammation in Patients with Coronary Artery Disease. Journal of Clinical Endocrinology & Metabolism(1):261–267
Huang W, Wu X, Xue Y, Zhou Y, Wei Y (2020) MicroRNA-3614 regulates inflammatory response via targeting TRAF6-mediated MAPKs and NF-κB signaling in the epicardial adipose tissue with coronary artery disease. International Journal of Cardiology 324: 152–164
Zhu HT, Bian C, Yuan JC, Chu WH, Xiang X, Chen F, Wang CS, Feng H, Lin JK (2014) Curcumin attenuates acute inflammatory injury by inhibiting the TLR4/MyD88/NF-κB signaling pathway in experimental traumatic brain injury. Journal of Neuroinflammation,11:59
Faccini J, Ruidavets JB, Cordelier P, Martins F, Maoret JJ, Bongard V, Ferrières J, Roncalli J, Elbaz M, Vindis C (2017) Circulating miR-155, miR-145 and let-7c as diagnostic biomarkers of the coronary artery disease. Sci Rep 7:42916
Carolina C, Cátia G, Rita VA, Dora B (2016) Exploring New Inflammatory Biomarkers and Pathways during LPS-Induced M1 Polarization. Mediators of Inflammation 2016:6986175
Lu Y, Li C, Wang D, Su X, Jin C, Li Y, Li T (2013) Characterization of two negative regulators of the Toll-like receptor pathway in Apostichopus japonicus: Inhibitor of NF-κB and Toll-interacting protein. Fish & Shellfish Immunology 35(5):1663–1669
Takeda K, Akira S (2004) TLR signaling pathways. In: Seminars in immunology. 16(1):3–9.
Liu J, Zhao X, Duan X, Zhang W, Li C (2021) CircRNA75 and CircRNA72 Function as the Sponge of MicroRNA-200 to Suppress Coelomocyte Apoptosis Via Targeting Tollip in Apostichopus japonicus. Frontiers in immunology 12:770055–770055
Li X, Goobie GC, Gregory AD, Kass DJ, Zhang Y (2021) Toll-Interacting Protein in Pulmonary Diseases. Abiding by the Goldilocks Principle. American journal of respiratory cell and molecular biology 64(5):536–546
Sun W, Liu R, Li P, Li Q, Cui H, Zheng M, Wen J, Zhao G (2019) Chicken gga-miR-1306-5p targets Tollip and plays an important role in host response against Salmonella enteritidis infection. Journal of animal science and biotechnology 10:59
Zheng Q, Zhao H, Jia D, Han X, Liu Z, Zhao M (2021) Overexpression of TOLLIP Protects against Acute Kidney Injury after Paraquat Intoxication through Inhibiting NLRP3 Inflammasome Activation Modulated by Toll-Like Receptor 2/4 Signaling. Mediators of Inflammation 2021:5571272
Zheng Q, Liu Z, Shen H, Hu X, Zhao M (2021) Protective effect of toll-interacting protein overexpression against paraquat-induced lung injury in mice and A549 cells through inhibiting oxidative stress, inflammation, and NF-κB signaling pathway. Respiratory Physiology & Neurobiology 286:103600
Swaroop S, Sengupta N, Suryawanshi AR, Adlakha YK, Basu A (2016) HSP60 plays a regulatory role in IL-1β-induced microglial inflammation via TLR4-p38 MAPK axis. Journal of neuroinflammation 13:27
Abekura F, Park J, Lim H, Kim HD, Choi H, Lee MJ, Kim CH (2022) Mycobacterium tuberculosis glycolipoprotein LprG inhibits inflammation through NF-κB signaling of ERK1/2 and JNK in LPS‐induced murine macrophage cells. Journal of Cellular Biochemistry doi: 10.1002/jcb.30220.
Ansaldo AM, Montecucco F, Sahebkar A, Dallegri F, Carbone F (2019) Epicardial adipose tissue and cardiovascular diseases. International Journal of Cardiology 278:254–260
Huang W, Xue Y, Zhou Y, Wu X, Wu X, Zhang X, Dong X, Wei Y (2020) KLF7 promotes macrophage activation by activating the NF-κB signaling pathway in epicardial adipose tissue in patients with coronary artery disease. Eur Rev Med Pharmacol Sci 24(12):7002–7014

No competing interests reported.

Download PDF

Version 2

posted

You are reading this latest preprint version

MiRNA-6870-3p regulates LPS-induced epicardial adipose tissue inflammatory genes via targeting Tollip-mediated JNK and NF-κB signaling in coronary artery disease

Status:

Version 2

Abstract

Objective

Methods

Resules:

Conclusion

Figures

Introduction

Methods

Results

Discussion

Declarations

References

Additional Declarations

Status:

Version 2