miRNAs generated from Meg3-Mirg locus are downregulated during aging

Abstract

Aging determines a multilevel functional decline and increases the risk for cardiovascular pathologies. MicroRNAs are recognized as fine tuners of all cellular functions, being involved in various cardiac diseases. The heart is one of the most affected organs in aged individuals, however little is known about the extent and robustness to which miRNA profiles are modulated in cardiac cells during aging. This paper provides a comprehensive characterization of the aging-associated miRNA profile in the murine cardiac fibroblasts, which are increasingly recognized for their active involvement in the cardiac physiology and pathology. Next-generation sequencing of cardiac fibroblasts isolated from young and old mice revealed that an important fraction of the miRNAs generated by the Meg3-Mirg locus was downregulated during aging. To address the specificity of this repression, four miRNAs selected as representative for this locus were further assessed in other cells and organs isolated from aged mice. The results suggested that the repression of miRNAs generated by the Meg3-Mirg locus was a general feature of aging in multiple organs. Bioinformatic analysis of the predicted target genes identified Integrin Beta-2 as an aged-upregulated gene, which was thereafter confirmed in multiple mouse organs. In conclusion, our study provides new data concerning the mechanisms of natural aging and highlights the robustness of the miRNA modulation during this process.

Keywords: Meg3-Mirg locus; aging; cardiac fibroblasts; heart ventricles; miRNA.

Figure 1

Characterization of cardiac fibroblasts derived from young and old mice (A) Phase-contrast microscopy of cells after 3 days in culture. No morphological differences were identified between young- and old-derived cells. (B) Flow cytometry analysis of young- and old-derived cells after 5 days in culture revealing the presence of PDGFRα and the absence of CD31, CD45. The histograms illustrate representative results of three experiments. (C) Fluorescent microscopy images illustrating the presence of αSMA, collagen I and collagen III. Note the different patterns of SMA in the two groups (inset). The pictures are representative from three experiments. (D) Cell energy phenotypes of young- and old-derived cardiac fibroblasts obtained by using XF Cell Energy Phenotype Report Generator. (E) Distribution of mean expression levels of miRNAs in young and old cardiac fibroblasts. Red crosses mark mean values. Circles mark outliers. (F) Volcano plot showing 530 sequenced miRNAs as steady or differentially expressed (downregulated or upregulated). MiRNAs with FDR < 0.05 were considered differentially expressed. Note that downregulated and upregulated miRNAs had a variation of at least 25% in old compared to young cardiac fibroblasts. (G) The expression levels of miRNAs in young versus old cardiac fibroblasts. Upregulated miRNAs with a mean RPM value in old cardiac fibroblasts over 370 and downregulated miRNAs with a mean RPM value in young cardiac fibroblasts over 450 were depicted as outliers. FDR, false discovery rate (p-value adjusted for multiple testing by the Benjamini-Hochberg procedure); FC, fold change (miRNA in old compared to young cardiac fibroblasts); RPM, reads per million.

Figure 2

Genomic distribution of miRNAs sequenced in old cardiac fibroblasts (A) The frequency of mature miRNAs mapped to each mouse chromosome or to multiple chromosomes. (B) The expression level of mature miRNAs that mapped to each chromosome or to multiple chromosomes. (C) The genomic location of mature miRNAs represented by the position at which the corresponding gene begins. Mature miRNAs with multiple genomic locations were not depicted due to unknown source of expression given multiple loci. (D) The genomic location and expression level of miRNAs clustered on chromosome 12. (E) The expression of downregulated mature miRNAs mapped to Meg3-Mirg locus in old and young cardiac fibroblasts. When both -3p and -5p strands were detected, the one with greater expression was considered as representative for the respective miRNA gene. MiRNAs were ordered based on their location within the Meg3-Mirg cluster. (F) Schematic representation of the Meg3-Mirg locus as part of the Dlk1-Dio3 paternally imprinted locus. Meg3-Mirg locus is expressed from the maternal allele and comprises only non-coding RNAs, i.e. lncRNA (Meg3, Rian, Mirg), snoRNAs (orange bars) and miRNAs (red bars). MiRNA genes expressed in cardiac fibroblasts are illustrated as arrows: grey arrows depict steady miRNA, and purple arrows show downregulated miRNAs. MiRNAs chosen as representative for Meg3-Mirg cluster are emphasized. Additional information is shown in Supplementary Figure 2.

Figure 3

Validation of age-dependent alterations of representative miRNAs from Meg3-Mirg cluster in cardiac fibroblasts and cardiomyocytes (A, B) The expression of representative miRNAs in cardiac fibroblasts (A) and cardiomyocytes (B) in young, old and very old mice; For each miRNA, the expression is presented relative to the young group. One-way ANOVA for equal variances or Welch’s ANOVA for unequal variances, with Tukey post-hoc test for pairs; * p < 0.05, ** p < 0.01, *** p < 0.001; n=8-10/group. (C) Heat map showing the expression of representative miRNAs normalized to U6 snRNA in cardiac fibroblasts and cardiomyocytes harvested from young, old and very old mice. Note that cardiac fibroblasts showed higher miRNA expression than cardiomyocytes.

Figure 4

Age-dependent alteration of representative miRNAs from Meg3-Mirg cluster in several mouse organs (A–F) The expression of miRNAs in cardiac ventricle (A), skeletal muscle (B), liver (C), pancreas (D), kidney (E) and brain (F) in young and very old mice. For each miRNA, the expression is presented relative to the young group; Two-tailed two-samples T-test for equal or unequal variances; Wilcoxon two-sample test for non-normal distribution; * p < 0.05, ** p < 0.01, *** p < 0.001; n=10/group. (G) Heat map showing the expression of representative miRNAs normalized to U6 snRNA in mouse organs harvested from young and very old mice; Note that different organs showed variable expression of representative miRNAs (brain > skeletal muscle > pancreas > ventricles > kidney > liver).

Figure 5

Genes upregulated in aging which are targeted by miRNAs from Meg3-Mirg locus. (A) Venn diagram illustrating the predicted targets of the 27 downregulated miRNAs. Three databases were interrogated and the overlapping targets were further used for pathway enrichment analysis, which identified 347 unique genes. (B) Graphical illustration of the intersection between the unique genes found in KEGG pathway and the upregulated genes identified in Tabula Muris Senis database. The upregulated genes are highlighted in each organ. Itgb2 emerged as the only gene upregulated in 4 out of 6 organs. (C) Illustration of the recognition sites of miRNAs from the locus targeting the 3`UTR region of Itgb2 mRNA. (D) The expression of the Itgb2 in the brain, ventricle, kidney, liver and cultured cardiac fibroblasts. For each organ, the expression is presented relative to the young group. Two-tailed two-samples T-test for equal or unequal variances; Wilcoxon two-sample test for non-normal distribution; * p < 0.05, ** p < 0.01, *** p < 0.001; **** p<0.0001; n=5-10/group.