MiR-29a Increase in Aging May Function as a Compensatory Mechanism Against Cardiac Fibrosis Through SERPINH1 Downregulation

Abstract

Deregulation of microRNA (miRNA) profile has been reportedly linked to the aging process, which is a dominant risk factor for many pathologies. Among the miRNAs with documented roles in aging-related cardiac diseases, miR-18a, -21a, -22, and -29a were mainly associated with hypertrophy and/or fibrosis; however, their relationship to aging was not fully addressed before. The purpose of this paper was to evaluate the variations in the expression levels of these miRNAs in the aging process. To this aim, multiple organs were harvested from young (2-3-months-old), old (16-18-months-old), and very old (24-25-months-old) mice, and the abundance of the miRNAs was evaluated by quantitative real-time (RT)-PCR. Our studies demonstrated that miR-21a, miR-22, and miR-29a were upregulated in the aged heart. Among them, miR-29a was highly expressed in many other organs, i.e., the brain, the skeletal muscle, the pancreas, and the kidney, and its expression was further upregulated during the natural aging process. Western blot, immunofluorescence, and xCELLigence analyses concurrently indicated that overexpression of miR-29a in the muscle cells decreased the collagen levels as well as cell migration and proliferation. Computational prediction analysis and overexpression studies identified SERPINH1, a specific chaperone of procollagens, as a potential miR-29a target. Corroborating to this, significantly downregulated SERPINH1 levels were found in the skeletal muscle, the heart, the brain, the kidney, and the pancreas harvested from very old animals, thereby indicating the role of the miR-29a-SERPINH1 axis in the aging process. In vitro analysis of miR-29a effects on fibroblast and cardiac muscle cells pointed toward a protective role of miR-29a on aging-related fibrosis, by reducing cell migration and proliferation. In conclusion, our study indicates an adaptive increase of miR-29 in the natural aging process and suggests its role as a transcriptional repressor of SERPINH1, with a potential therapeutic value against adverse matrix remodeling and aging-associated tissue fibrosis.

Keywords: SERPINH1; aging; fibrosis; miR-29a; miRNA; prediction analysis.

Figure 1

Age-related changes in the expression levels of miR-18a, miR-21a, miR-22, and miR-29a in heart and skeletal muscle of C57Bl/6J mice. (A) Schematic illustration of the experimental design. The heart ventricles and left quadriceps muscle were harvested from young (n = 10) and old (n = 4) mice and processed for total RNA isolation and real-time quantitative reverse transcription PCR (RT-qPCR) analysis. (B) The gene expression level of the four miRNAs in the heart. (C) The gene expression level of the four miRNAs in the skeletal muscle. *p < 0.05, **p < 0.01, ***p < 0.005, and **** p < 0.001.

Figure 2

Age-related changes in the expression levels of miR-21a, miR-22, and miR-29a in lymph organs of C57Bl/6 mice. The expressions of (A) miR-22, (B) miR-21a, and (C) miR-29a in the mediastinal lymph nodes, the spleen, and the thymus harvested from the young (n = 10) and old (n = 4) animals. The results are presented as fold changes relative to the young group. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001.

Figure 3

(A,B) The expression levels of miR-21a (A) and miR-29a (B) in multiple organs harvested from young (2–3-months-old) and very old (24–25-months-old) mice. The results are presented as fold changes relative to the young group per each organ (the heart, the skeletal muscle, the brain, the kidney, the pancreas, and the liver); n = 10 animals/group. *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001. (C) Heat map showing the expression of miR-21 and miR-29a normalized to snoRNA/U6 in each organ. Note the higher expression of miR-29a in comparison to miR-21 in the analyzed organs.

Figure 4

Identification of SERPINH1 as a miR-29a target. (A) Venn diagram showing the predicted targets of the miR-29a, obtained by interrogating three computational algorithms i.e., TargetScan, miRDB, and Diana. The targets predicted by at least 2 databases (with numbers illustrated in a triangle) were used in gene ontology (GO) analysis. (B) Enrichment analysis of predicted miR-29a target genes. Note that extracellular matrix (ECM) organization emerged as the most significantly enriched process involving miR-29a target genes. (C) String analysis of genes involved in ECM organization. Node size directly correlates with the number of interactions of each protein (min = 1; max = 18); Edge width denotes the confidence score calculated by STRING: the thinnest = 0.4 (medium confidence); the thickest ≥ 0.9 (the highest confidence); Highlighted in red are the interaction of SERPINH1. (D) Illustration of the 3′ UTR region of the mouse SERPINH1 gene with the predicted site for miR-29a (below). The expression of SERPINH1 mRNA level in HL-1 cells at one, two, and three days after transient transfection of cells with miR-29a mimic; ***p < 0.005.

Figure 5

The expression of SERPINH1 and correlation between SERPINH1 and miR-29a expression levels in various organs. (A) The expression of SERPINH1 in the heart, the skeletal muscle, the brain, the kidney, the pancreas, and the liver. For each organ, the expression is presented relative to the young group. N = 5–10 animals/group; *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001. (B) Pearson's correlations between SERPINH1 and miR-29a expression levels in the six organs mentioned above.

Figure 6

Quantification of collagen proteins in HL-1 cells overexpressing miR-29a and in ventricular lysates obtained from young (2–3-months-old), old (16–18-months-old), and very old (24–25-months-old) animals. (A) Immunofluorescence images of HL-1 cells at 24 h after transfection with mir-29a mimic or scramble miRNA. The histogram shows the quantification of the fluorescence signal produced by collagen III staining, expressed as mean fluorescence intensity. ***p < 0.005. (B) Western blot analysis of the collagen III in miR-29a-overexpressing HL-1 cells. The histogram shows the densitometry quantification normalized to total protein. *p < 0.05. (C) The hydroxyproline levels in tissue lysates of cardiac ventricles were obtained from young, old, and very old animals. ***p < 0.005 and ****p < 0.001.

Figure 7

Impact of miR-29a on muscle cell migration and proliferation. (A) Real-time profiling of HL-1 cell migration in CIM plates, after transfection with miR-29a mimic or scrambled miRNA control. The data represent one representative experiment of three experiments performed with similar results. (B) Real-time profiling of HL-1 cell adherence and proliferation in E-plates, after transfection with miR-29a mimic or scrambled miRNA control. The data represent one representative experiment of three experiments performed in quadruplicates with similar results. (C) Cell-cycle distribution and percentage of cells in G0/G1, S, and G2 /M phases. The data represent the mean +/– SD of 3 independent experiments performed. **p < 0.01 and ***p < 0.005.