Abstract
Tissue fibrosis is a significant health issue associated with organ dysfunction and failure. Increased deposition of collagen and other extracellular matrix (ECM) proteins in the interstitial area is a major process in tissue fibrosis. The microRNA-29 (miR-29) family has been demonstrated as anti-fibrotic microRNAs. Our recent work showed that dysregulation of miR-29 contributes to the formation of cardiac fibrosis in animal models of uremic cardiomyopathy, whereas replenishing miR-29 attenuated cardiac fibrosis in these animals. However, excessive overexpression of miR-29 is a concern because microRNAs usually have multiple targets, which could result in unknown and unexpected side effect. In the current study, we constructed a novel Col1a1-miR-29b vector using collagen 1a1 (Col1a1) promoter, which can strategically express miR-29b-3p (miR-29b) in response to increased collagen synthesis and reach a dynamic balance between collagen and miR-29b. Our experimental results showed that in mouse embryonic fibroblasts (MEF cells) transfected with Col1a1-miR-29b vector, the miR-29b expression is about 1000 times less than that in cells transfected with CMV-miR-29b vector, which uses cytomegalovirus (CMV) as a promoter for miR-29b expression. Moreover, TGF-β treatment increased the miR-29b expression by about 20 times in cells transfected with Col1a1-miR-29b, suggesting a dynamic response to fibrotic stimulation. Western blot using cell lysates and culture media demonstrated that transfection of Col1a1-miR-29b vector significantly reduced TGF-β induced collagen synthesis and secretion, and the effect was as effective as the CMV-miR-29b vector. Using RNA-sequencing analysis, we found that 249 genes were significantly altered (180 upregulated and 69 downregulated, at least 2-fold change and adjusted p-value <0.05) after TGF-β treatment in MEF cells transfected with empty vector. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using GAGE R-package showed that the top 5 upregulated pathways after TGF-β treatment were mostly fibrosis-related, including focal adhesion, ECM reaction, and TGF-β signaling pathways. As expected, transfection of Col1a1-miR-29b or CMV-miR-29b vector partially reversed the activation of these pathways. We also analyzed the expression pattern of the top 100 miR-29b targeting genes in these cells using the RNA-sequencing data. We identified that miR-29b targeted a broad spectrum of ECM genes, but the inhibition effect is mostly moderate. In summary, our work demonstrated that the Col1a1-miR-29b vector can be used as a dynamic regulator of collagen and other ECM protein expression in response to fibrotic stimulation, which could potentially reduce unnecessary side effect due to excessive miR-29b levels while remaining an effective potential therapeutic approach for fibrosis.
Highlights
Formation of tissue fibrosis is a driving force for disease progression that leads to worsened organ function and failure [1, 2]
We showed that transfection of miR-29b mimics or restoring endogenous miR29b by blocking Na/K-ATPase signaling using pNaKtide, a Src inhibiting peptide developed in our laboratory, inhibited collagen synthesis in primary cultured cardiac fibroblasts and attenuated cardiac fibrosis in animal models of uremic cardiomyopathy [25, 28]
The exogenous collagen 1a1 (Col1a1) promoter will stimulate the expression of miR-29b and contra-regulate the elevated collagen expression to reach a dynamic balance between miR-29b and collagen mRNA
Summary
Formation of tissue fibrosis is a driving force for disease progression that leads to worsened organ function and failure [1, 2]. Myocardial infarction (MI) is a major cause of cardiac death and massive cardiac tissue fibrosis. There are two phases of tissue fibrosis after MI [5]. The initial formation of a fibrotic scar at the infarcted area is critical to prevent the heart from rupture, which is defined as reparative fibrosis. The second phase of fibrosis is observed in the remote non-infarcted area, often referred to as reactive fibrosis or interstitial fibrosis. The interstitial fibrosis in the non-ischemic area could significantly deteriorate heart function post MI. Severe cardiac fibrosis can lead to sudden cardiac death even in those without cardiac symptoms or ischemic injury [6,7,8], while reducing cardiac fibrosis in clinical and animal models has been shown to improve cardiac function [9,10,11,12]
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