The world’s population has increased very rapidly in recent years. According to an estimate, the world’s population in 1915 was approximately 1.8 billion. A recent survey by the United Nations (UN) estimated the world’s population at 7.7 billion in 2019 will reach 9.7 billion by 2050 [1]. Over the years, the dietary habit of most people has changed owing to increase in population and changes in economy, especially in developing countries. This accounts for a high increase in demand for animal meat and protein. To meet the ever-increasing food demand requires more research for improving the existing food production system. The development of skeletal muscle is a complicated process comprised of embryonic myogenesis to postnatal muscle growth. The postpartum muscle growth in swine is dependent on the total number of myofibers which are generated in two waves before birth. Pig myogenesis occurs in two phases, the first phase includes the formation of primary myofibers from 35 to 55 days post coitus (dpc), followed by the formation of secondary myofibers around each primary myofiber between 50 and 90 dpc [2]. Postpartum muscle growth is generally considered from birth to 60 days. Postnatal muscle growth mostly includes the conversion from slow-oxidative to fast glycolytic fiber types [3].

Meat quality is an important commercial trait in domestic animals, which is mainly estimated by intermuscular fat (IMF), pH value, tenderness, flavor substance, and water holding capacity. Further, meat quality is significantly affected by myofibers composition [4]. Quality of pork is mainly based on different kinds of myofibers and different contents of IMF present in different swine breeds. The pork industry mainly focuses on enhancing the muscle mass and development rate [5]. This goal achieved in various breeds of swine through genetic selection and cross-breeding [6]. Through these tactics, the Landrace swine breed has been developed, which has high body weight, rapid growth ability, and high lean meat percentage [7]. Despite Landrace, some local breeds have less weight and a slow growth rate, but these local breeds have high IMF content that affects the flavor of meat [8].

MicroRNAs are short (approximately 22 to 25 nucleotide), non-coding, single-stranded RNA molecules that are responsible for controlling gene expression through translational degradation, repression, and deadenylation of target mRNA. Studies present strong evidence that miRNAs play important roles in porcine skeletal muscle development [9–18]. The most important miRNA in myogenesis is miR-133a [19], which plays regulatory roles in muscle tissues [20–22]. Both miR-1 and miR-133 have multiple functions in myogenesis primarily, they are responsible for skeletal muscle development, and secondarily, they also act as myocyte enhancers [20]. Due to the advancement in sequencing technologies the numbers of miRNA discovered increased day by day [23]. Different type of miRNAs found to play regulatory roles in pig skeletal muscle development includes miR-1, miR-21, miR22, miR-27, miR-34, miR-127, miR-133, miR-143, miR-155, miR-199, miR-206, miR-208, miR-378, and miR-432 [12,14,15,17,20,23–26]. Here, we will discuss the regulatory roles of different miRNAs in myogenesis and meat quality regulation in pigs.

Prenatal swine myogenesis depends on the number of factors including nutrient accessibility, hormones, and growth factors that influence the changes in metabolism at the transcriptional and translational level of regulatory genes. These factors, linked with the myogenic regulatory factors, play a significant role in controlling muscle-specific gene expression [27]. Skeletal muscle development in pigs occurs in two phases, the first phase occurs from 35 to 55 dpc in which the primary myofibers are formed and the formation of secondary myofibers around each primary myofiber occurs between 50 and 90 dpc [2,28]. In prenatal development of muscles, the content of fibers depends on the propagation and apoptosis of stem cell differentiation and the conjoining of myoblasts accompanied by the ultimate maturation of myofibers. Myostellite cells, with multipotency, can differentiate to muscle cells, but are not able to form the myofibers. During postpartum growth, these cells act as the source of new myonuclei which play a major role in fiber size and regeneration process. Despite this contribution, myonuclei remain mitotically dormant. The number of fibers is determined during the prenatal development stage in most of the mammalian species and remain fixed throughout life. Albeit, under the influence of the genetic and physiological factors, the size of the fibers increases without an increase in the number of fibers [29]. During post-partum muscular development, if the fiber numbers are more, then the individual fiber grows slowly and vice versa [29]. In an adult, the muscle’s cross-sectional area directly relates to the number and size of fibers. One such example is of massive loin area which is composed of, in most cases, the low fiber size and high fiber number and vice-versa. However, the best loin area can be achieved through an optimal combination of fiber size and fiber number.

In the late 1980s, the revolutionary discovery of the myoblast differentiation (MyoD) family of basic-helix-loop-helix transcription factors responsible for regulating the molecular mechanism of muscle development has opened a new arena of discussion. The four master members of the MyoD family, myogenic regulatory factors (MRFs), MRF4, myogenin (MyoG), and myogenic factor 5, play key roles in myogenesis. Regarding their origin, different parts of the muscles have different origins. For example, trunk and limb muscles are engendered from somite-derived dermomyotome. While extraocular, neck, and head muscles originate from cranial mesoderm [30]. During myogenesis, microsatellite cells pass through several processes regulated by vital transcriptional factors giving rise to myoblast cells which, ultimately, differentiates to form myofibers [31]. From the surrounding environment, extracellular signaling molecules released are bone morphogenetic protein (BMPs), wingless and Int (Wnts), and sonic hedgehog (Shh) responsible for sclerotome differentiation and determination [32]. Transcription factors produced from these signaling proteins affect stem cells responsible for the determination of the myogenic fate. These signaling molecules are released from different parts, but all have effects on sclerotome development. BMP and the Shh are released from the notochord through transcription factor paired box protein (Pax1), which plays a significant role in sclerotome development. Pax1 is responsible for the changes in sclerotome progenitor cell’s epithelium, which ultimately differentiate into chondrocytes. Wnt1 and Wnt3a determine the myogenic fate of epaxial dermomyotome, both of which are secreted along with Shh. Shh is secreted from the floor plate of the neural tube and the notochord, while Wnt1 and Wnt3a are released from the dorsal neural tube. Wnt7a is responsible for specifying the myogenic fate of hypaxial dermomyotome which is released from the dorsal ectoderm [33].

The quality of meat mainly depends on the type of myofibers. A myofiber is divided into four types due to the presence of the phenomenon of polymorphism in the myosin heavy chain. The type is I with myosin heavy chain (MyHC) I, type IIa with MyHC IIa, type IIx with MyHC IIx, and type IIb with MyHC IIb [34]. The type I and IIa fibers are included in slow and fast oxidative muscle types, while type IIx and IIb fibers are considered intermediate and fast glycolytic myofibers. If we compare the properties of both fiber types, then we can conclude that type I and type II have higher myoglobin content, more mitochondria, low glycolytic capacity, and less myosin ATPase activity. The type IIb fibers have more glycogen, longer fiber size, and they show quick contractions. The pork flavor, tenderness, water holding capacity, and IMF are determined by type I and II type fibers, and these fibers also reduce the risk of soft, exudative, and pale pork efficiently [35,36].

China is the biggest producer and consumer of pork. Pork consumers tend to consume pork with good IMF which is also a key indicator of pork quality [98,99]. The contents of IMF are used as a trait for quantifying the percentage of fat content in myofibers and it acts as an economic factor in the pork industry. It impacts the overall carcass quality not only through marbling appearance but also through affecting the tenderness and juiciness of meat [100]. Thus, the researchers are showing great interest to produce pork with high IMF contents. Thus far, the molecular mechanisms underlying the deposition of IMF are not clear. In recent years, some progress has been made in exploring the role of miRNAs in intramuscular preadipocyte development [101]. IMF deposition is a complex process that could be diverse among different species, breeds, age, and depends upon the nutrition [102–104]. The IMF content may vary from 2% to 10% in different breeds of swine [105]. But the understanding of the genetic and molecular players in IMF deposition could help in the selection and breeding of high IMF content animals. Therefore, in recent years, researchers have scratched the surface of this field for understanding the molecular mechanisms governing IMF deposition, but much more work is needed to establish the paradigm-shifting impact in improving the quality of pork production.

The expression of miRNAs was analyzed by Liu et al [99] in the longissimus dorsi muscle of Yorkshire (YY, lean-type) and Chinese Wannanhua (WH, fatty) pigs. They found 598 miRNAs from which 42 were differentially expressed between YY and WH pigs. Two miRNAs, miR-196a/b (miR-196a, miR-196b-5p), were highly expressed in WH pigs giving the clue about their influence in porcine adipogenesis an adipocytokine signaling pathways [99]. The differential expression of miRNAs between IM and subcutaneous (SC) adipocytes from Jiaxing black pigs, one of the Taihu pig breeds well known for its high IMF and excellent pork flavor [106], showed 155 miRNAs with significant differential expression (SDE). Target genes of SDE miRNAs were enriched in categories and pathways related to fatty acid biosynthesis, transcriptional regulation, and MAPK as well as PI3K-Akt pathways. The expression of miR-206 was 36-fold higher in IM adipocytes as compared to those of SC adipocytes. The role of miR-206 is to regulate adipocyte proliferation through star-related lipid transfer domain containing 7 (STARD7) and it represses kruppel-like factor 4 (KLF4) expression which results in inhibition of adipogenesis. Thus, miR-206 acts as a suppressor of adipogenesis, and its attenuation can help in the production of pork with higher IMF [107]. Recently, Sun et al [108] analyzed the miRNA sequencing of high (2.94% ± 0.04%) and low (1.62%±0.02%) IMF group samples from Yorkshire pigs. From a total of 268 identified miRNAs, 28 were differentially expressed between two groups. From these 28 differentially expressed miRNAs, 13 were upregulated and 15 were downregulated. Top five differentially expressed miRNAs included miR-365-3p, miR-208b, miR-206, miR-126-5p, and miR-10a-5p, from which only miR-365-3p was highly expressed in low IMF group while others were high in high IMF group. Additionally, the authors reported that hsa-miR-208a-3p only expressed in the high IMF group, whereas has-miR-500a-5p was found only in the low IMF group [108].

Moreover, miR-34a targets the 3′UTR of Acyl-CoA synthetase long-chain family member 4 (ACSL4). Pig preadipocytes show the expression of ACSL4 throughout the differentiation process. The transfection of miR-34a mimic tends to reduce the lipid droplet synthesis during adipogenesis while miR-34a inhibition increases the chances of lipid droplet formation. The expression miR34a target genes which included genes related to lipogeneses such as ACSL4, peroxisome proliferator-activated receptor γ (PPARγ), adipocyte protein 2 (aP2), and sterol regulatory element-binding protein-1C (SREBP-1C) was reduced upon overexpression of miR-34a while, downregulation of miR-34a had opposite results [109]. In addition to this mechanism for regulating the adipogenesis, miR-34a also inhibits the adipogenesis activity of platelet-derived growth factor receptor A (PDGFRα), a positive regulator of adipogenesis and IMF deposition in pigs, and controls the expression of lipogenesis genes PPARg and fatty acid binding protein 4 (FABP4) [110]. The miR-125-5p was found to regulate the proliferation and differentiation of porcine intramuscular preadipocytes. The overexpression of miR-125-5p in porcine preadipocytes enhanced the proliferation and reduced the differentiation of porcine IM preadipocytes while the downregulation of miR-125-5p showed opposite results. This effect was due to the targeting of porcine KLF13 and ELOVL fatty acid elongase 6 (ELOVL6), a regulator of fatty acid composition, through miR-125-5p [101]. Further, miR-146a-5p acts as a potential regulator of porcine IMF adipogenesis. The mimics of miR-146a-5p tend to inhibit the proliferation and differentiation of porcine IM preadipocytes while miR146a-5p inhibitors augment the cell proliferation differentiation of preadipocytes. The proposed mechanism for regulation of preadipocyte proliferation by miR146a-5p is through targeting of SMAD family member 4 which diminishes TGF-β signaling. While the differentiation of adipocytes controlled through targeting tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) attenuates the nuclear factor kappa beta (NF-κB) signaling. This miRNA could also be considered as a biomarker for amending the IM adipogenesis to improve pork quality [111]. The overexpression of miR-181a promoted the accumulation of lipid droplets, raised the amount of triglycerides, and decreased the TNF-α protein expression in porcine preadipocytes, while the inhibition of this miRNA showed opposite effects [112]. Another recent study reported that miR-181a promotes the differentiation of porcine preadipocytes by targeting TGFβR1 [113]. In addition, the expression of miR-185 was found to gradually increase during 3T3-L1 cell differentiation. The overexpression of miR-185 led to the suppression of adipogenic markers, PPARγ, FABP4, fatty acid synthase (FAS), and lipoprotein lipase (LPL), and reduced the lipid accumulation in 3T3-L1 cells. while the inhibition of miR-185 promoted the differentiation of 3T3-L1 cells. This effect was due to the targeting of the SREBP-1 gene by miR-185 [114]. Another miRNA, miR-425-5p also regulates the adipogenic differentiation of IM preadipocytes. The overexpression of miR-425-5p inhibits the IM adipogenic differentiation along with the downregulation of adipogenic marker genes PPARγ, FABP4, and fatty acid synthase. While the inhibitors of miR-425-5p augments adipogenesis. The inhibitory effect of miR425-5p on IM preadipocyte proliferation and adipogenesis is through repression of cyclin B and KLF13 respectively [115]. These findings are in addition to the list of miRNAs for regulating the lipogenesis and ultimately the IMF deposition in skeletal muscles of pig. The summary of roles of aforementioned miRNAs in IMF in porcine skeletal muscles is given in Table 2.

Myogenesis in farm animals is directly linked to the production of meat and its overall quality. To date, the molecular mechanisms involved in governing the muscle development and deposition of fat in muscles to increase the quality of meat produced are not much understood. In recent years, miRNAs were found to play important roles in regulating the expression of some important genes and transcription factors involved in muscle development and IMF deposition in pigs. miRNAs could have significant influences on muscle traits, and play key roles in the phenotypic variation of the porcine muscle. However, underlying genetic factors and functional validation of each miRNA-target relationship remain to be determined. Thus, understanding the molecular mechanisms of myogenesis can turn out to be the breakthrough for increased meat production for meeting global food demands.