Research Article: Discovery of MicroRNAs Associated with Myogenesis by Deep Sequencing of Serial Developmental Skeletal Muscles in Pigs

Date Published: December 21, 2012

Publisher: Public Library of Science

Author(s): Xinhua Hou, Zhonglin Tang, Honglin Liu, Ning Wang, Huiming Ju, Kui Li, Andre Van Wijnen.


MicroRNAs (miRNAs) are short, single-stranded non-coding RNAs that repress their target genes by binding their 3′ UTRs. These RNAs play critical roles in myogenesis. To gain knowledge about miRNAs involved in the regulation of myogenesis, porcine longissimus muscles were collected from 18 developmental stages (33-, 40-, 45-, 50-, 55-, 60-, 65-, 70-, 75-, 80-, 85-, 90-, 95-, 100- and 105-day post-gestation fetuses, 0 and 10-day postnatal piglets and adult pigs) to identify miRNAs using Solexa sequencing technology. We detected 197 known miRNAs and 78 novel miRNAs according to comparison with known miRNAs in the miRBase (release 17.0) database. Moreover, variations in sequence length and single nucleotide polymorphisms were also observed in 110 known miRNAs. Expression analysis of the 11 most abundant miRNAs were conducted using quantitative PCR (qPCR) in eleven tissues (longissimus muscles, leg muscles, heart, liver, spleen, lung, kidney, stomach, small intestine and colon), and the results revealed that ssc-miR-378, ssc-miR-1 and ssc-miR-206 were abundantly expressed in skeletal muscles. During skeletal muscle development, the expression level of ssc-miR-378 was low at 33 days post-coitus (dpc), increased at 65 and 90 dpc, peaked at postnatal day 0, and finally declined and maintained a comparatively stable level. This expression profile suggested that ssc-miR-378 was a new candidate miRNA for myogenesis and participated in skeletal muscle development in pigs. Target prediction and KEGG pathway analysis suggested that bone morphogenetic protein 2 (BMP2) and mitogen-activated protein kinase 1 (MAPK1), both of which were relevant to proliferation and differentiation, might be the potential targets of miR-378. Luciferase activities of report vectors containing the 3′UTR of porcine BMP2 or MAPK1 were downregulated by miR-378, which suggested that miR-378 probably regulated myogenesis though the regulation of these two genes.

Partial Text

Myogenesis is a complex process that includes proliferation, differentiation, and formation of myotubes and myofibers. In the developing vertebrate embryo, mononuclear proliferative myoblasts exit the cell cycle irreversibly and enter the differentiation phase. Several myoblasts subsequently fuse and form multinuclear myotubes. Finally, myofibers are formed following the distribution of nuclei to the edge of membrane. These molecular events are orchestrated by myogenic regulatory factors and miRNAs. The miRNAs that are expressed abundantly in skeletal muscle cells or myocardial cells are called myomiRs. MiR-1, miR-206 and miR-133 are classified as myomiRs, as they play important roles in the regulation of muscle development and differentiation. Previous experiments showed that miR-1 and miR-206 promoted the differentiation of myoblasts, while miR-133 promoted proliferation [1], [2]. Chromatin immunoprecipitation showed that the myogenic regulatory factors MyoD and myogenin upregulated the expression of miR-1-1, miR-1-2, miR-133a-1, miR-133a-2 and miR-206 in myotubes [3]. The myocyte enhancer factor-2 (MEF2) regulated the expression of myomiRs via a muscle-specific enhancer in an intron lying between the coding regions of miR-1-2 and miR-133a-1. There were also MEF2 and bHLH binding sites in a muscle-specific enhancer separating the miR-1-1 and miR-133a-2 coding regions [4]. Thus, the expression of myomiRs is regulated by myogenic factors, such as MyoD, and the myomiRs mediate muscle development or differentiation by regulating downstream target genes. Also, miRNAs have counter-effects on myogenic factors. Naguibneva et al. reported that miR-181 alleviated the repression of MyoD by downregulating Hox-A11, which represses the expression of MyoD, which, in turn, triggered the expression of muscle markers [5]. Pax3, which plays a role in a number of aspects of embryonic myogenesis, can be targeted by miR-27b, leading to its downregulation and early differentiation. Pax3 levels are maintained when miR-27b is inhibited, and inhibition of miR-27b also leads to more proliferation and delays the onset of differentiation [6]. It seems that miR-24 has a functional role in the regulation of differentiation, as it can be upregulated during the early stages of differentiation but is maintained in adult terminally differentiated cardiac and skeletal muscles. This miRNA is also upregulated during cardiac hypertrophy and induces cardiomyocyte hypertrophy [7]. MiRNAs can also control the fiber type of skeletal muscles. Mice that have a double deletion of miR-208b and miR-499 show a substantial loss of slow myofibers, while overexpression of miR-499 under control of the MCK promoter converts all of the fast fibers to the slow phenotype in the soleus muscles, resulting in an enhanced endurance phenotype, with miR-499 transgenic mice running more than 50% longer than their wild-type littermates [8]. Many other miRNAs also take part in the regulation of muscle development. MiR-221 and miR-222 are downregulated during the transition from proliferation to differentiation of both primary and established myogenic cells. The cell cycle inhibitor p27 can be targeted by both miR-221 and miR-222, and overexpression of these miRNAs delays withdrawal from the cell cycle and differentiation, due to a reduction in sarcomeric proteins [9]. MiR-155 can regulate the expression of Olfactomedin-like 3 (OLFML3), which may affect porcine prenatal skeletal muscle development [10]. Although an increasing body of evidence shows that miRNAs play important roles in the regulation of skeletal muscle development, precise regulatory mechanisms of the biological functions of most miRNAs remain unclear, and most research is restricted to myomiRs, such as miR-1, miR-133 and miR-206.

Solexa sequencing was performed to identify miRNAs in porcine skeletal muscles. A total of 197 known and 78 potential novel miRNAs were identified. Next, we analyzed isomiRs of known miRNAs in our results, as isomiRs might have functions in animals [20]. Nearly 27.7% miRNAs had isomiRs, and only 0.14% were in the seed region, showing that low mutant frequency and high conservation existed in this region. There were always 1∼2 nucleotide differences in length at the 5′ or 3′ terminal of isomiRs for the same miRNA, which might be attributable to variable cleavage of pri- or pre-miRNA by Drosha or Dicer. Potential novel miRNAs were identified by genome location and secondary structure predictions. The number of reads for these miRNAs were usually indicative of low abundance, which might suggest that these low-abundance novel miRNAs had little effect on skeletal muscle myogenesis. Nielsen et al. [21] showed that ssc-miR-1 and ssc-miR-206 are expressed at extremely high levels in the longissimus muscles of Danish Landrace/Yorkshire crossbred pigs of age 1.5–2 years; however, the abundance of other miRNAs was relatively low. In our results, the abundance of ssc-miR-1 and ssc-miR-206 was extremely high, which was consistent with their results. However, there also existed many miRNAs for which the expression levels were different between the two studies. An example was ssc-miR-378, which we found at a much higher abundance than their study found. In addition, we found that ssc-miR-148a was expressed highly in the longissimus muscles, while it was expressed in an extremely low level in their results. These differentially expressed miRNAs may be highly expressed during fetal stages and play vital roles in the regulation of fetal myogenesis. These results also demonstrate that including many developmental stages in our analysis was beneficial to discovering the range of differentially expressed miRNAs among serial development periods.