MiR-29a Family as a Key Regulator of Skeletal Muscle Dysplasia in a Porcine Model of Intrauterine Growth Retardation

Abstract

MicroRNAs (miRNAs) play an essential role in many biological processes. In this study, miRNAs in the skeletal muscle of normal and intrauterine growth retardation (IUGR) neonatal piglets were identified by sequencing, and canonical miRNAs were functionally validated in vitro. A total of 403 miRNAs were identified in neonatal piglet skeletal muscle, among them 30 and 46 miRNAs were upregulated and downregulated in IUGR pigs, respectively. Upregulated miRNAs were mainly enriched in propanoate metabolism, endocytosis, beta-Alanine metabolism, gap junction, and tumor necrosis factor signaling pathway. Down-regulated miRNAs were mainly enriched in chemical carcinogenesis-receptor activation, endocytosis, MAPK signaling pathway, insulin resistance, and EGFR tyrosine kinase inhibitor resistance. Co-expression network analysis of umbilical cord blood and skeletal muscle miRNAs showed that the miR-29 family is an essential regulator of IUGR pigs. The dual-luciferase reporter system showed that IGF1 and CCND1 were target genes of the miR-29 family. Transfection of IUGR pig umbilical cord blood exosomes and miR-29a mimic significantly inhibited cell proliferation and promoted the expression of cellular protein degradation marker genes Fbxo32 and Trim63. In summary, these results enrich the regulatory network of miRNAs involved in skeletal muscle development in IUGR animals.

Keywords: CCND1; IGF1; intrauterine growth retardation; miR-29 family; skeletal muscle.

Figure 1

IUGR pigs with skeletal muscle dysplasia. (A) Birth to adult weight of normal and IUGR pigs; (B) Daily weight gain of normal and IUGR pigs at different stages; (C) Loin muscle area of 170–day–old normal and IUGR pigs, measurement using B-mode ultrasound; (D) Photographs of the legs of newborn normal and IUGR pigs; (E,F) HE (Hematoxylin and eosin) staining of longissimus dorsi muscle and muscle fiber diameter analysis. (A–C): Normal group, N = 149; IUGR group, N = 75; (D–F): N = 3. * p–value ≤ 0.05.

Figure 2

Expression characteristics of miRNAs in porcine skeletal muscle. (A) Species characterization of miRNAs in normal and IUGR pig skeletal muscle; (B) Top 10 miRNAs expressed in normal and IUGR pig skeletal muscle; (C) Length distribution of miRNAs in normal and IUGR pig skeletal muscle; (D) Volcano plot of differential miRNAs in normal and IUGR pig skeletal muscle; (E) RT-qPCR validation of partial sequencing results; (F) Correlation analysis between sequencing results and RT-qPCR results.

Figure 3

Functional enrichment analysis of differential miRNA in normal and IUGR pigs. (A) GO results of highly expressed miRNAs in the normal group; (B) GO results of highly expressed miRNAs in the IUGR group; (C) KEGG pathway analysis of highly expressed miRNAs in the normal group; (D) KEGG pathway analysis of highly expressed miRNAs in the IUGR group.

Figure 4

Expression network of differential miRNAs in neonatal pig skeletal muscle and umbilical cord blood. Red is the highly expressed miRNAs in IUGR pig skeletal muscle (IUGR-M group), blue is the highly expressed miRNAs in normal pig skeletal muscle (Normal-M group), pink is the highly expressed miRNAs in IUGR pig umbilical cord blood (IUGR-B group), light Blue is the highly expressed miRNAs in normal pig umbilical cord blood (Normal-B group), gray is the co-highly expressed miRNAs of IUGR-M group and Normal-B group, purple is the co-highly expressed miRNAs of Normal-M group and IUGR-B group.

Figure 5

miR-29 family is central regulators of IUGR pig development. (A) Venn plots showing miRNA profiles in cord blood and skeletal muscle of normal and IUGR pigs; (B) Variation patterns of the miR-29 family during porcine skeletal muscle development; (C–F) Correlation analysis of relative expression levels of skeletal muscle miR-29 family with pig embryo weight; miR-29a (C), miR-29b (D), miR-29c (E), correlations among miRNAs within the miR-29 family (F). Data from GSE169093; (G) GO analysis results of common target genes of the miR-29 family; (H) KEGG analysis results of common target genes of the miR-29 family.

Figure 6

IGF1 and CCND1 as common target genes of miR-29a. (A) Relative expression of the miR-29 family in skeletal muscle of neonatal piglets; (B) Relative expression of IGF1 and CCND1 in skeletal muscle of neonatal piglets; (C) Potential binding sites of the miR-29 family to the 3’UTR of IGF1 and CCND1; (D) TargetScan results showed the conservation of potential binding sites of the miR-29 family to the 3’ UTR of IGF; (E,F) The dual-luciferase reporter system showed the binding relationship of miR-29a to the ① (E) and ② (F) of the 3’UTR of IGF1 and to the 3’UTR of CCND1 (G). N = 3. * p-value < 0.05.

Figure 7

miR-29a is involved in the regulation of umbilical cord blood miRNAs in skeletal muscle cells in vitro. (A) The relative expression of miR-29a in pig primary skeletal muscle cells after transfection with miR-29a mimic or miR-29a inhibitor; (B) The relative expression of IGF1, CCND1 CCNB1, and CDK4 in pig primary skeletal muscle cells after transfection with miR-29a mimic or miR-29a inhibitor; (C) The relative expression of Fbxo32 and Trim63 in pig primary skeletal muscle cells after transfection with miR-29a mimic or miR-29a inhibitor; (D) EdU staining results of porcine primary skeletal muscle cells after transfection with miR-29a mimic or miR-29a inhibitor; (E) The relative expression of miR-29a in porcine primary skeletal muscle cells after co-treatment with normal porcine umbilical cord blood exosomes (exo-Normal) or IUGR porcine umbilical cord blood exosomes (exo-IUGR) and miR-29a inhibitor (exo-I-miR-29I); (F,G) The relative expression of IGF1, CCND1 CCNB1, CDK4, Fbxo32, and Trim63 in porcine primary skeletal muscle cells after co-treatment with exo-Normal or exo-IUGR and exo-I-miR-29I; (H) EdU staining results of porcine primary skeletal muscle cells after co-treatment with exo-Normal or exo-IUGR and exo-I-miR-29I. N = 3. * p-value < 0.05 vs. NC, # p-value < 0.05 vs. Mimic.

Figure 8

The miR-29 family is involved in the regulation of skeletal muscle development in IUGR pigs. This section may be divided into subheadings; it should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.