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 EAT inflammation 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. 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. 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. 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.