MiR-208b/miR-21 Promotes the Progression of Cardiac Fibrosis Through the Activation of the TGF-β1/Smad-3 Signaling Pathway: An in vitro and in vivo Study

Abstract

BackgroundRegulatory molecule microRNAs (miRNAs) have been implicated in myocardial fibrosis. However, the specific mechanism by which they lead to myocardial fibrosis remains unclear. This study aimed to explore the roles of miR-208b, miR-21 and transforming growth factor-β1 (TGF-β1)/Smad-3 signaling pathway components in cardiac fibrosis development.Materials and MethodsThirty-six consecutive acute myocardial infarction (AMI) patients were included in this study. Plasma was collected on admission and at 24 h, 48 h and 6 d. The levels of plasma miR-208b, miR-21, TGF-β1, and Smad-3 were measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and cardiac calcium protein T (cTnT) and creatine kinase isoenzyme (CK-MB) were detected by electrochemiluminescence analysis. H9C2 cells were exposed to hypoxia and divided into 4 groups (hypoxia treatment for 6 h, 24 h, 48 h, and 72 h). These stimulated cells were then transfected with miRNA inhibitors and mimics for gene overexpression and inhibition. RT-qPCR was used to detect the expression of miR-208b, miR-21, TGF-β1, and Smad-3, and western blot analysis was used to detect TGF-β1 and Smad-3 protein expression.ResultsThe plasma analysis showed cTnT and CK-MB expression peaked at 24 h after symptom onset; miR-208b, miR-21, TGF-β1, and Smad-3 levels showed no peak and increased gradually with time. Cell experiments revealed that miR-208b and TGF-β1 were upregulated along with increased hypoxia exposure; miR-21 expression peaked at 24 h and 72 h, with the highest peak at 72 h, and Smad-3 expression peaked at 6 h and 72 h, with the highest peak at 72 h. miR-208b and miR-21 expressions were positively correlated with TGF-β/Smad-3 expression. TGF-β1/Smad-3 mRNA and protein levels were elevated in the miR-208b and miR-21 overexpression groups and reduced in the miR-208b and miR-21 inhibition groups.ConclusionMiR-208b and miR-21 promote cardiac fibrosis progression through TGF-β1/Smad-3 signaling pathway activation.

The Role of ncRNAs in Cardiac Infarction and Regeneration

“…miR-208a expression levels are increased in conditions of oxidative stress, fibrosis, and inflammation after MI. An in vitro assay using H9c2 cells cultured under hypoxia showed that miR-208 and TGF-β levels increase with time during incubation, which is in line with similar findings in MI patients [ 133 , 181 ]. Additionally, in a healthy heart, miR-208a overexpression promotes the expression of endoglin, a membrane glycoprotein of the TGF-β receptor signaling pathway, whereas in an MI rat model, particularly high levels of this miRNA can be observed in the fibrotic area [ 182 ].…”

“…miR-21 also regulates other signaling pathways, such as pERK/ERK and Spry1/Erk/Map, with the latter contributing to fibrosis ( Figure 2 ) [ 131 , 132 ]. In H9c2 cells, the expression of TGF-β and miR-21 was increased, establishing that miR-21 promotes fibrosis by regulating TGF-β/Smad3 signaling [ 133 ]. Therefore, miR-21 has anti-inflammatory and pro-fibrotic roles because it participates in inflammation by inhibiting the p38 and NF-kB pathways, thus decreasing pro-inflammatory cytokine production, and in fibrosis by downregulating TGF-β pathway inhibitors ( Figure 2 ).…”

Mechanism of action of non-coding RNAs and traditional Chinese medicine in myocardial fibrosis: Focus on the TGF-β/Smad signaling pathway

“…Some miRNAs promote the development of cardiac fibrosis, by activating the TGF-β/Smad signaling pathway. Zhang et al (2022a) verified that there was a positive correlation between miR-208b and miR-21 and the expressions of TGF-β1 and Smad3. The miR-208b/miR-21 promoted the development of cardiac fibrosis by activating the TGF-β1/Smad3 signaling pathway.…”

“…TGF-β1 is a stimulator which can convert cardiac fibroblasts to myofibroblasts. While classical TGF-β pathway signaling is mediated by the binding of TGF-β1 to transforming growth factor beta receptors II (TGFBR2) ( Huang et al, 2022 ), which subsequently phosphorylates and activates the Smad2 and Smad3, thereby promoting myofibroblast proliferation and migration, and leading to cardiac fibrosis ( Zhang et al, 2022a ). Non-coding RNAs (ncRNAs) can be broadly classified into microRNAs (miRNAs), long-stranded non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) according to their functions ( Iu et al, 2022 ).…”

miRNA Dysregulation in Cardiovascular Diseases: Current Opinion and Future Perspectives

“…Based on these data, the authors concluded that these miRNAs could be used as potential biomarkers for AMI. Zhang et al [ 80 ] demonstrated that miR-21 and MiR-208b promote cardiac fibrosis in AMI patients, activating the TGF- β1/Smad-3 signaling pathway. Wang et al [ 44 ] investigated the role of miR-22 and miR-499 as sensitive biomarkers in the diagnosis of MI, confirming that these miRNAs could play an important role in early diagnosis.…”