Abstract
MicroRNAs (miRNAs) are evolutionarily conserved, small, non-coding RNAs that have emerged as key regulators of myogenesis. Here, we examined the miRNA expression profiles of developing sheep skeletal muscle using a deep sequencing approach. We detected 2,396 miRNAs in the sheep skeletal muscle tissues. Of these, miR-192 was found to be up-regulated in prenatal skeletal muscle, but was down-regulated postnatally. MiR-192 expression also decreased during the myogenic differentiation of sheep satellite cells (SCs). MiR-192 overexpression significantly attenuated SCs myogenic differentiation but promoted SCs proliferation, whereas miR-192 inhibition enhanced SCs differentiation but suppressed SCs proliferation. We found that miR-192 targeted retinoblastoma 1 (RB1), a known regulator of myogenesis. Furthermore, knockdown of RB1 in cultured cells significantly inhibited SCs myogenic differentiation but accelerated SCs proliferation, confirming the role of RB1 in myogenesis. Taken together, our findings enrich the ovine miRNA database and outline the miRNA transcriptome of sheep during skeletal muscle development. Moreover, we show that miR-192 affects SCs proliferation and myogenic differentiation via down-regulation of RB1.
Similar content being viewed by others
Introduction
Sheep are important farm animals worldwide, producing high quality meat with the ability to adapt to different climatic and grazing conditions. An improved understanding of the molecular mechanisms of muscle development (myogenesis) in ovine may increase meat productivity in the sheep and lamb industry. In adults, the cells responsible for producing myoblasts in skeletal muscle are satellite cells (SCs), which are located beneath the basal lamina of the myofibre. After skeletal muscle suffers injury, SCs fuse together to allow new myotube formation, or fuse to injured myofibres to replace lost myonuclei1. As myonuclei are post-mitotic, SCs serve as an excellent model to study the molecular mechanisms underlying muscle development. Previous studies show that in order for skeletal muscle regeneration to take place, SCs must express Pax7, a transcription factor that regulates cell proliferation2,3,4,5. Moreover, it was recently suggested that microRNA-1 (miR-1) and miR-206 regulate the SCs proliferation and myogenic differentiation by repressing Pax76. Therefore, microRNAs (miRNAs) appear to be important for SCs proliferation and differentiation.
MiRNAs are short non-coding RNAs that modulate gene expression through translational repression and mRNA decay7 and have recently been profiled in a variety of tissues from different animals8,9,10. However, few studies have focused on the identification and profiling of miRNAs in sheep skeletal muscle. Such profiling of miRNAs in skeletal muscle could enhance our understanding of the effect of miRNAs on myogenesis. Indeed, recent studies have identified several miRNAs involved in myogenesis based on miRNAs expression profiling of skeletal muscle. For example, in mouse, miR-1 and miR-206 are now considered as muscle-specific miRNAs, which promote muscle differentiation by targeting histone deacetylase 4 (HDAC4) and DNA polymerase α (Pola1), respectively11,12. These miRNAs are also implicated in sheep myogenesis. For example, in Texel sheep, a G to A transition in the three prime untranslated region (3′-UTR) of myostatin (GDF8) creates a target site for miR-1 and miR-206 and in turn, these miRNAs inhibit the expression of GDF8 resulting in muscular hypertrophy13. In mouse, skeletal myogenesis is also regulated by several other ubiquitously expressed miRNAs, including: miR-125b14, miR-26a15, miR-2716, miR-2917, miR-48618, miR-37819 and miR-15520.
In sheep, miR-192 was previously found to be expressed in skeletal muscle21, although its precise function remains unclear. Several studies indicate that miR-192, which was first cloned from mouse and human22, is involved in disease pathogeneses, including atrial fibrillation, aristolochic acid nephropathy and diabetic kidney glomeruli23,24,25. In humans, miR-192 is also known as a tumor-related miRNA: up-regulation of miR-192 represses breast cancer cell proliferation26 and inhibits lung cancer cell proliferation, as well as inducing cell apoptosis27. Moreover, in colon cancer, miR-192 regulates dihydrofolate reductase and cellular proliferation through the p53 tumor suppressor network28. However, little is known about the function of miR-192 in skeletal myogenesis.
In addition to the above miRNAs, retinoblastoma 1 (RB1) is also implicated in skeletal myogenesis29. Retinoblastoma 1 protein (pRb) belongs to a family of three proteins, which also includes retinoblastoma-like 1 (RBL1/p107) and retinoblastoma-like 2 (RBL2/p130)30. pRb critically regulates cell cycle progression by interacting with the E2F transcription factor family of proteins31,32; however, it can also control cell cycle progression via E2F-independent ways33. RB1 is also known as a tumor suppressor gene, as it is functionally inactivated in most human cancers34. However, increasing evidence suggests that pRb is also a key regulator of murine muscle development29. pRb is a tumor suppressor protein that also restricts the cell’s ability to replicate DNA, so inhibition of this protein will decrease myogenesis.
Here, we examined miRNA expression during ovine skeletal muscle development by deep sequencing of skeletal muscle obtained from local Chinese Duolang sheep that is bred for meat and fat. We investigated the significantly differentially expressed miRNAs at each distinct stage of muscle development (from fetal to 3 years postnatal). We then further explored the functional mechanisms of one differentially expressed miRNA (miR-192) that has previously been implicated in myogenesis and found that it targeted RB1. The overall aim of this study was to improve our understanding of miRNA function during muscle development in ovine.
Results
Differential expression profile of microRNAs from developing sheep skeletal muscle
To explore the potential function of miRNAs in sheep skeletal myogenesis, we examined the expression of miRNAs using deep sequencing at four distinct stages of muscle development: fetus 90 days (F90), fetus 110 days (F110), lamb 40 days (L40) and adult 3 years (A3Y). A total of 15,711,484, 14,075,573, 15,452,905 and 15,832,379 reads were obtained for the F90, F110, L40 and A3Y libraries, respectively. The analysis of length distribution showed that the majority of the reads had lengths of 21–23 nt (Figure S1). Among these reads, 14,746,030 at F90, 13,223,764 at F110, 11,043,952 at L40 and 11,290,187 at A3Y were mappable to the Ovis aries genome. In total, 2,396 miRNAs were identified in the four small RNA libraries. Of the total miRNAs, 80.1% (1920/2396 miRNAs) were predicted to be new miRNAs that were not deposited in the miRBase database. Of the predicted new miRNAs, 37.0% (711/1920 miRNAs) were conserved in other species (cow, human, mouse, etc.) and 63.0% (1209/1920 miRNAs) were unannotated miRNAs (Table S1). The most abundant known sheep miRNAs in ovine skeletal muscle are listed in Table 1.
Next, we compared the miRNA expression profiles at the four developmental stages (F90, F110, L40 and A3Y). In total, 15 significantly differently expressed miRNAs (SDEmiRs) were identified between F90 vs. F110 (Table S2), 290 SDEmiRs were identified between F90 vs. L40 (Table S3), 343 SDEmiRs were identified between F90 vs. A3Y (Table S4), 207 SDEmiRs were identified between F110 vs. L40 (Table S5), 209 SDEmiRs were identified between F110 vs. A3Y (Table S6) and 293 SDEmiRs were identified between L40 vs. A3Y (Table S7). Furthermore, to validate the miRNA deep sequencing data (according to Table S1), quantitative real-time PCR (qPCR) was performed to verify 10 differently expressed miRNAs, including: miR-127, miR-495-3p, miR-503, miR-3958-3p, miR-433-3p, miR-382-5p, miR-299-3p, miR-125b, miR-1 and miR-206. The relative expression of these miRNAs was highly correlated with the sequence data (Fig. 1).
MiRNA target prediction, gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of target genes
The TargetScan program was applied to predict the targets of the SDEmiRs (Table S8,S9,S10,S11,S12–S13). Gene ontology term and KEGG pathway enrichment analyzes were conducted for the targets of SDEmiR. The targets of SDEmiRs were grouped into three gene ontology categories: biological process, cellular component and molecular function (Figure S2). Furthermore, KEGG pathway analyzes identified some significantly enriched pathways, such as focal adhesion, glioma, PI3K-AKT signaling pathway and oxidative phosphorylation (Figure S3).
MiR-192 may be involved in sheep skeletal muscle development
According to our miRNA profiling in sheep, we found miR-192 was down-regulated by 5-fold in postnatal skeletal muscle of A3Y compared with prenatal skeletal muscle of F90. The expression of miR-192 in developing sheep skeletal muscle was confirmed by qPCR (Fig. 2A). Tissue expression profiling of miR-192 showed that miR-192 was ubiquitously expressed in different tissues, including skeletal muscle (semitendinosus and longissimus dorsi) (Fig. 2B). Moreover, miR-192 was up-regulated in skeletal muscle from Lamin A/C (LMNA)-mutated patients with abnormal muscle development35, indicating that it was closely related to myogenesis. Ovine mir-192 is an intergenic miRNA gene, which is adjacent to mir-194 and located between EHD1 and ATG2A at chromosome 21 (Fig. 2C). The 20 nucleotides comprising the sheep mature miR-192 are completely conserved across species, although the sheep miR-192 lacks an additional “C” and the cattle miR-192 has an extra “AG” (Fig. 2D). Altogether, these results indicate that miR-192 decreases during muscle growth in the developing sheep and is an attractive candidate that may be involved in myogenesis.
MiR-192 is down-regulated during myogenic differentiation of sheep SCs and murine C2C12 myoblasts
To further research the possible role of miR-192, we separated sheep skeletal muscle SCs. We collected leg muscle samples of ovine fetus and then carefully separated SCs according to a previous study36. The expression of Pax7 in the SCs was detected by immunofluorescence and the SCs were consistently >90% pure (Figure S4). The SCs formed significant myotubes by day 3 after they were induced to differentiate (Fig. 3A,B). Moreover, myogenin (MyoG), an early myogenic marker and myosin heavy chain (MHC), a late myogenic marker, were both normally expressed during SCs differentiation (Fig. 3C). To explore the expression of miR-192 during SCs differentiation, we examined miR-192 levels during SCs differentiation. The results showed that the miR-192 levels in SCs declined on days 1 and 3 of differentiation (Fig. 3D), which was a period of myotube formation (Fig. 3A–C). However, as miR-192 expression increased again on days 5 and 7 during the myogenic differentiation of sheep SCs (Fig. 3D), additional mechanisms may control miR-192 levels. Similarly, miR-192 was also down-regulated in C2C12 myoblasts on day 3 of differentiation (Fig. 3E,F). These results indicate that miR-192 is down-regulated during SCs and C2C12 myoblasts differentiation.
MiR-192 negatively regulates myogenic differentiation of sheep SCs and murine C2C12 myoblasts
To examine the function of miR-192 in myogenic differentiation, we introduced miR-192 mimics and negative control (NC) into SCs. The SCs were transfected with miR-192 mimics or NC in growth medium (GM) (Fig. 4A). Then the cells were induced to differentiate in differentiation medium (DM). The introduction of miR-192 decreased the protein levels of MyoG and MHC by days 2 and 3 of differentiation (Fig. 4B). Similarly, a significant decrease in the mRNA expression levels of MyoG and MHC was observed on days 2 and 3 of differentiation (Fig. 4C). Moreover, overexpression of miR-192 dampened myotube formation (Fig. 4D), as confirmed by a decreased differentiation index (Fig. 4E).
We also introduced the 2′-O-methyl antisense oligonucleotides against miR-192 (anti-miR-192) and single-stranded anti-NC into SCs (Fig. 4A). Inhibition of miR-192 enhanced the protein and mRNA expression of myogenic markers (Fig. 4B,C) and this was accompanied by enhanced myotubes formation (Fig. 4D,E). Next, we explored the role of miR-192 in murine C2C12 myoblasts differentiation. Similarly, overexpression of miR-192 inhibited C2C12 myoblasts differentiation, while inhibition of miR-192 promoted differentiation (Fig. 4F,G). Taken together, our results indicate that miR-192 plays a negative role in the myogenic differentiation of SCs and C2C12 myoblasts.
MiR-192 accelerates proliferation of sheep SCs
Next, we examined the role of miR-192 on proliferation of sheep SCs using 5-Ethynyl-2′-deoxyuridine (EdU) cell proliferation assay. We found that overexpression and inhibition of miR-192 increased and decreased the proportion of EdU-positive cells (Fig. 5A), respectively, indicating that miR-192 promoted proliferation of SCs. Moreover, the MTT assay further confirmed that miR-192 enhanced SCs proliferation (Fig. 5B). Furthermore, we analyzed the cell cycle by flow cytometry after transfection and found that the introduction of miR-192 decreased the G0/G1 population of cells but increased the S population of cells, suggesting that miR-192 overexpression inhibited cell cycle arrest (Figs 5C and S5). Taken together, these results suggest that miR-192 accelerates SCs proliferation.
MiR-192 directly targets 3′-UTR of RB1
pRb plays a critical role in the differentiation of myoblasts; pRb-deficient myoblasts lose the ability to form multinucleated myotubes29. Using TargetScan and PicTar, we found that the sheep RB1 3′-UTR has a highly conserved binding site for miR-192 (Fig. 6A,B). To examine whether miR-192 targets the RB1 3′-UTR, we constructed luciferase reporters that included a fragment of either the wild-type or mutant RB1 3′-UTR (Fig. 6A). The miR-192 mimics or NC were co-transfected with the reporters into human HEK293 cells, a model cell line which has stable efficiency of transfection. MiR-192 significantly reduced the activity of wild-type reporter of RB1 3′-UTR; however, no reduction in activity was observed with the mutant reporter of RB1 3′-UTR (Fig. 5C). This confirms that miR-192 directly targets the 3′-UTR of RB1. Furthermore, the introduction of miR-192 repressed the RB1 mRNA and protein expression in SCs (Fig. 6D,E). Moreover, the expression level of pRb was significantly higher in L40 than in F90, as well as higher in A3Y than in F110 (Fig. 6F). These results showed that the expression of pRb has a negative correlation with miR-192 expression in developing animal (Figs 2A and 6F), further confirming that RB1 is a target gene of miR-192. Together, these results suggest that the protein and mRNA expression of RB1 are directly inhibited by miR-192.
Knockdown of RB1 inhibits myogenic differentiation but promotes proliferation of cultured sheep SCs
To examine the role of pRb on myogenic differentiation and proliferation of sheep SCs, we silenced endogenous RB1 of SCs by siRNA. SiRNA against sheep RB1 (si-RB1) significantly diminished its protein expression (Fig. 7A). Moreover, the reduction of pRb significantly repressed SCs differentiation, as indicated by the inhibition of MHC protein expression and formation of myotubes (Fig. 7B–D). The fact that miR-192 overexpression resulted in down-regulation of MyoG protein expression (Fig. 4B), but that si-RB1 increased MyoG protein expression (Fig. 7B), implies that other target genes of miR-192 may be involved in MyoG expression. Indeed, the increased MyoG expression caused by si-RB1 was consistent with a previous study reporting pRb-deficient myoblasts up-regulated MyoG expression29. Moreover, si-RB1 accelerated SCs proliferation (Fig. 7E), which was very similar to the impact of the overexpression of miR-192. Thus, RB1 knockdown significantly represses SCs myogenic differentiation, but accelerates SCs proliferation, confirming the role of pRb in myogenesis.
Discussion
MiRNA profiling analysis based on deep sequencing is a powerful tool for functional studies of miRNA. In this study, 2,396 miRNAs, including 1,920 novel miRNAs, were identified from four developmental stages of sheep skeletal muscles. Among these miRNAs identified, we found some that were differentially expressed, including miR-192. Further study demonstrated that miR-192 was down-regulated during the myogenic differentiation of sheep SCs and murine C2C12 myoblasts. We found that miR-192 repressed myogenic differentiation but accelerated proliferation of sheep SCs. Dual-luciferase reporter assay confirmed that RB1 was a direct target of miR-192. Moreover, knockdown of endogenous RB1 by siRNA promoted proliferation but inhibited myogenic differentiation of sheep SCs, confirming the role of RB1 in myogenesis.
MiR-192 is an attractive candidate for regulating skeletal muscle development. It was previously thought to be a regulator of p53, a human tumor suppressor and is overexpressed in gastric cancer28,37. Moreover, miR-192 has been identified as a potential therapeutic target or biomarker for drug-induced liver damage38. MiR-192 targets various genes in different cells and organs24,25,26,27,28, indicating that it is extensively involved in biological processes. Indeed, this is consistent with our tissue expression profile showing that miR-192 was ubiquitously expressed in different sheep tissues. However, whether miR-192 regulates myogenesis has not been clarified.
A previous study demonstrated that miR-192 expression was higher in the muscle of Small Tail Han sheep, a Chinese breed with a slower growth rate, than in muscle from Dorset sheep that yield a large and lean carcass21. Studies have revealed that fetus 80–120 days is the vital stage for ovine myofibre formation and proliferation39,40. In our study, we found that miR-192 expression was significantly higher in prenatal skeletal muscle of fetus 90 days, a period which would expect a significant amount of muscle progenitor cell proliferation occurring, than in postnatal muscle of adult 3 years, a period which myofibres were well established. These facts indicated that miR-192 could be involved in skeletal muscle development.
We then confirmed that miR-192 regulated proliferation and differentiation of SCs by targeting RB1. RB1 is not only implicated in childhood retinoblastoma, bladder cancer and lung cancer, but acts as a negative regulator of the cell cycle and is involved in cell senescence, growth arrest, apoptosis and differentiation30. Recently, several miRNAs were found to participate in the regulation of cancer cell proliferation via targeting RB1. In gastric cancer cells, miR-215 was able to target RB1 to modulate gastric cancer cell proliferation41 and miR-132 also promoted gastric cancer cell proliferation via targeting RB142. In meningioma cells, miR-335 enhanced cell proliferation through targeting RB143. Furthermore, a number of studies indicate RB1 is critical for muscle development. For example, myoblasts expressing low levels of RB1 fail to develop normally and accumulate large polyploid nuclei44 and mice deficient for the RB1 gene die at mid-gestation45,46. Moreover, RB1 was previously shown to be required during myogenic differentiation and modulate the progression of differentiation29. Indeed, a lack of RB1 accelerates cell cycle re-entry of quiescent SCs and increases the number of SCs; however, SCs terminal differentiation was greatly diminished47. Similarly, we demonstrated here that miR-192 attenuated sheep SCs differentiation but accelerated proliferation, which was characterized by directly repressing the expression of RB1.
Another study found that miR-192 was markedly up-regulated in skeletal muscle from Lamin A/C (LMNA)-mutated patients with skeletal muscle dystrophy35. In addition, LMNA null muscle shows delayed expression of RB1, suggesting that the dominant LMNA mutations may alter RB1 function48. However, the mechanism of delayed expression of RB1 in LMNA null muscle is elusive. Here, we demonstrated that miR-192 negatively regulates myogenic differentiation through repressing the expression of RB1. Thus, we speculate that in LMNA-related muscular dystrophy, the up-regulation of miR-192 delays the expression of RB1 by directly targeting its 3′-UTR and in turn, this may lead to abnormalities in skeletal muscle development.
In conclusion, the results of our study indicate that miR-192 is important for myogenic differentiation and proliferation and its up-regulation can negatively affect myogenesis. Myogenic differentiation was delayed by the introduction of miR-192 and promoted by miR-192 inhibition. We also found that miR-192 directly targets the RB1 gene at the post-transcriptional level and that RB1 knockdown significantly represses SCs myogenic differentiation but accelerates SCs proliferation. These results reveal a miRNA-mediated regulation mechanism, by which miR-192 regulates myogenic differentiation and proliferation of skeletal muscle SCs through the repression of RB1.
Methods
Animals and tissue samples collection
All animal experimental procedures (i.e., on Duolang sheep from Sinkiang, China) were performed according to the guidelines for the care and use of experimental animals established by the Ministry of Agriculture of People’s Republic of China. All experimental protocols were approved by Science Research Department of the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences (CAAS) (Beijing, China). The fetal longissimus dorsi were collected from three fetuses of Duolang sheep at 90 days post coitus and from another three fetuses at 110 days post coitus. The lamb longissimus dorsi were collected from three male Duolang lambs at 40 days postnatal. The adult longissimus dorsi were collected from three male Duolang sheep at 3 years postnatal. Nine different tissues (including semitendinosus, longissimus dorsi, small intestine, liver, spleen, lung, heart, kidney and stomach) were collected from the three male Duolang sheep at 40 days postnatal. All animals were euthanized. All tissues were immediately frozen in liquid nitrogen after harvesting.
Library construction and miRNA deep sequencing
RNAs were extracted with TRIzol (Invitrogen, USA) from the longissimus dorsi of Duolang sheep fetuses at 90 days (F90) and 110 days (F110), lambs at 40 days (L40) and adults at 3 years (A3Y), according to the manufacturer’s protocols. The quality (RIN ≥ 8 and 28S/18S ≥ 1.0) and quantity of the RNA samples were detected on a Bioanalyzer 2100 system using an RNA 6000 Nano kit (Agilent Technologies, USA). The RNA from the three Duolang sheep in each group were pooled to generate the four libraries (F90, F110, L40 and A3Y), which were prepared according to Illumina’s instructions (Illumina GAIIx, Illumina, USA) and sequenced by Shanghai Biotechnology Corporation.
MiRNA profiling and novel miRNA prediction
Clean reads were obtained by removing low quality reads, reads with 5′ adaptor pollution or poly (A) stretches, reads <18 nt and reads without 3′ adaptors. The clean reads were mapped to the Ovis aries genome (version 4.0) using the SOAP program (http://soap.genomics.org.cn/). After mapping, the reads were blasted against the Rfam database (http://www.sanger.ac.uk/software/Rfam) and the GenBank non-coding RNA database (http://blast.ncbi.nlm.nih.gov/). After removal of mRNA, snRNA, tRNA, rRNA and snoRNA, the remaining reads were then searched against the mature miRNAs of chimpanzee, human, cow, horse, mouse, rhesus monkey, western lowland gorilla, bonobo, Tasmanian devil, platypus, pig, gray short-tailed opossum and sheep in miRBase (release 20) to identify known conserved miRNA homologs in the sheep. Only the mature and precursor sequences of small RNAs that perfectly matched to known ovine miRNAs in the miRBase were identified as known ovine miRNAs. To predict potential new miRNAs, the 150 bases upstream and downstream flanking the remaining reads were obtained and secondary structures were detected using the miRDeep program. If a perfect stem-loop structure was formed, the miRNA sequence was located at one arm of the stem, the miRNA sequence ranged between 18–26 nt and its free energy of hybridization was lower than −20 kcal/mol, then the miRNA was considered a potential novel miRNA. The potential novel miRNAs with predicted hairpin structures were mapped to conserved miRNAs of other species (chimpanzee, human, cow, horse, mouse, rhesus monkey, western lowland gorilla, bonobo, tasmanian devil, platypus, gray short-tailed opossum and pig) in the miRBase. The remaining unmapped but hairpin-structured reads were considered as potential new miRNAs without known miRNA annotations.
DEGseq software was applied to identify SDEmiRs and a MA-plot-based random sampling model (MARS) was performed to calculate the p-value. If |log2 (ratio miRNA levels)| > 1 and p < 0.05, the miRNAs were identified as significantly differentially expressed between the two developmental stages of skeletal muscle.
MiRNA targets prediction, gene ontology enrichment and KEGG pathway analyzes
The TargetScan program (http://www.targetscan.org) was employed to predict targets of all the SDEmiRs. The target genes of SDEmiRs were analyzed with gene ontology and association with different pathways was conducted with KEGG (http://www.genome.jp/kegg), Biocarta (www.biocarta.com) and Reatome (http://www.reactome.org/) databases.
Cell culture
Human HEK293 and murine C2C12 myoblast cells were purchased from the China Infrastructure of Cell Line Resource. C2C12 myoblasts were cultured in growth medium (GM) consisting of Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) supplemented with 10% FBS (Gibco, USA) at 37 °C with 5% CO2. Myogenic differentiation was induced by a differentiation medium (DM) with DMEM supplemented with 2% horse serum (Gibco, USA). HEK293 cells were maintained in DMEM with 10% FBS at 37 °C with 5% CO2.
The sheep skeletal muscle SCs were enzymatically isolated from leg muscle tissues obtained from five Duolang sheep fetuses at 90 days, using a two-step digestion method, as described previously36. Briefly, leg muscle tissues were excised, cut into small pieces, digested with 0.1% type I collagenase (Sigma-Aldrich, USA) for 1 h and then digested with 0.25% trypsin (Gibco, USA) for 20 min. The samples were vortexed every 10 min and filtered through a 200-mesh sieve and SCs were purified using the differential adhesion method49. Sheep skeletal muscle SCs were proliferated in GM consisting of DMEM/F-12 (Gibco, USA) supplemented with 20% FBS and 10% horse serum in a collagen type 1-coated plate and then differentiated using DMEM/F-12 with 2% horse serum.
RNA isolation, RT-PCR and quantitative real-time PCR
Total RNA was extracted from the longissimus dorsi muscles, C2C12 myoblasts, or sheep skeletal muscle SCs with TRIzol reagent (Invitrogen, USA), according to manufacturer’s instructions. For mRNA quantification, 1 μL of total RNA (1000 ng/μL) was reverse transcribed into cDNA using the PrimeScript RT reagent Kit (Perfect Real Time; Takara, Japan) for quantitative real-time PCR (qPCR). GAPDH and ACTB genes were used as internal normalization controls. qPCR was carried out in an Applied Biosystems 7500 Real-Time Detection system using the SYBR premix Ex Taq qPCR Kit (Takara, Japan).
For miRNA quantification, a miRNA stem-loop primer and a pair of primers were designed for miRNA reverse transcription and qPCR, respectively. The U6 gene was used as the internal normalization control. MiRNA stem-loop was reverse transcribed with the PrimeScript RT reagent Kit and quantified by qPCR using the SYBR premix Ex Taq qPCR Kit, according to manufacturer’s protocols. Relative gene expression was determined by the 2−ΔΔCt method50,51. The primers used for qPCR are listed in Supplementary Tables S14 and S15.
Immunoblotting and immunofluorescence
All protein preparations were quantified using the BCA Protein Assay Kit (Thermo Scientific, USA). Immunoblotting was performed using standard procedures and antibodies against pRb (1:1000, Abcam, UK), MHC (1:200, Developmental Studies Hybridoma Bank, USA), MyoG (1:100, Developmental Studies Hybridoma Bank, USA), β-tubulin (1:2000, Sigma-Aldrich, USA) and GAPDH (1:5000, Sigma-Aldrich, USA). Protein abundance was analyzed by ImageJ tool. Quantification of protein was normalized to β-tubulin.
For immunofluorescence, SCs and C2C12 myoblasts were plated in 6-well plates and induced to differentiate. The cells were harvested, fixed with 4% paraformaldehyde for 20 min, treated with 0.25% Triton X-100 for 15 min and then blocked with goat serum (Bioss, China) for 1 h. After blocking, the cells were incubated with primary antibody against murine and ovine MHC (1:100, Developmental Studies Hybridoma Bank, USA) or Pax7 (1:200, Bioss, China) overnight at 4 °C. The AlexaFluor-488 conjugated mouse IgG (1:1000; Invitrogen, USA) was incubated for 1 h at room temperature. The cell nuclei were stained with DAPI (Sigma-Aldrich, USA). The differentiation index (percentage of nuclei in MHC) was calculated as previously described14.
Cell proliferation assay
Three separate assays were applied to quantify the proliferation of sheep skeletal muscle SCs. First, flow cytometry was used to analyze the phase distribution of the cell cycle. The cultured sheep skeletal muscle SCs in GM were collected 48 h after transfection and fixed in 75% ethanol overnight at −20 °C. Then the cells were incubated with 50 μg/ml propidium iodide (Sigma-Aldrich, USA) containing 10 mg/ml RNase A (Takara, Japan) and 0.2% (v/v) Triton X-100 (Sigma-Aldrich, USA) for 30 min at 4 °C. The cell cycle distribution was analyzed using a CytomicsTM FC 500 flow cytometer (Beckman Coulter, USA). Second, the 5-Ethynyl-2′-deoxyuridine (EdU) (Guangzhou RiboBio, China) cell proliferation assay was used to measure SCs proliferation, according to manufacturer’s protocols. Briefly, after transfection in GM, the sheep skeletal muscle SCs were seeded in 96-well plate and cultured in GM for 24 h. Then, the cells were incubated 2 h with medium containing 50 μM EdU before immunostaining. Images were collected using a fluorescence microscope (Nikon, Japan). The ratio of EdU-positive cells was calculated with (EdU-positive cells/Hoechst stained cells) × 100%. Third, a MTT assay was performed. After transfection with miR-192 mimics or negative control, the sheep skeletal muscle SCs were seeded at 1 × 103 in 96-well plate and cultured in GM for 4 days. Every 24 h, 20 μL (5 mg/mL) of MTT solution (Sigma-Aldrich, USA) was added to the cells for 4 h. After discarding the medium, DMSO was added to the cells to dissolve the crystals. The absorbance of each well was measured on a microplate reader.
RNA oligonucleotides and cell transfection
The siRNA against sheep RB1, an siRNA control (a nonspecific duplex), ovine and murine miR-192 mimics (double-stranded RNA oligonucleotides), double-stranded negative control, 2′-O-methyl antisense oligonucleotides against miR-192 and a single-stranded negative control were all purchased from GenePharma (GenePharma, China). Transfection was performed with the Lipofectamine 3000 reagent (Invitrogen, USA) combined with 200 nM of siRNA, 50 nM of miRNA mimics, or 100 nM of 2′-O-methyl antisense oligonucleotides. All the procedures were performed according to the manufacturer’s protocols.
Dual-luciferase reporter assay
A fragment of 3′-UTR of RB1 (554 bp) containing the binding site of miR-192 was amplified from the Duolang sheep genomic DNA, which was extracted from longissimus dorsi muscle and cloned into the psicheck-2 plasmid (Promega, USA) using the XhoI and NotI restriction sites. RB1 3′-UTR was amplified using the forward primer 5′-GGCGCTCGAGTTAACTTCAGCATGGTCTT-3′ and the reverse primer 5′-AATGCGGCCGCGAAGTTCCTTAAATTCTGA-3′. The PCR product was cloned into the vector downstream of the Renilla Luciferase ORF. The mutant RB1 3′-UTR reporter was obtained by changing the miR-192 binding site from TAGGTCA to ATCCAGT. RB1 3′-UTR binding site mutations were introduced using the KOD-plus mutagenesis kit (Toyobo, Japan) according to the manufacturer’s instructions.
In dual-luciferase reporter (psicheck-2 plasmid), the Renilla luciferase is used as a reporter gene and the firefly luciferase is used as a reference gene for control. Luciferase reporter experiments were performed in HEK293 cells that were co-transfected with 100 nM of the miR-192 mimics or NC and 100 ng of the wild-type or mutant 3′-UTR luciferase reporter using Lipofectamine 3000 reagent in 96-well plates. After transfection for 48 h, the relative activities of luciferase were quantified using a Dual-Luciferase Assay System (Promega, USA), according to the manufacturer’s protocols.
Statistical analysis
All data are shown as mean ± standard deviation (SD) and are based on at least three replicates for each treatment. Unpaired or paired two-tailed t-test was used for individual comparisons. One-way analysis of variance (ANOVA) followed by Duncan’s test was used for multiple comparisons.
Additional Information
How to cite this article: Zhao, Q. et al. Expression profiling and functional characterization of miR-192 throughout sheep skeletal muscle development. Sci. Rep. 6, 30281; doi: 10.1038/srep30281 (2016).
References
Relaix, F. & Zammit, P. S. Satellite cells are essential for skeletal muscle regeneration: the cell on the edge returns centre stage. Development. 139, 2845–2856 (2012).
Moresi, V., Marroncelli, N. & Adamo, S. New insights into the epigenetic control of satellite cells. World J Stem Cells. 7, 945–955 (2015).
Lepper, C., Partridge, T. A. & Fan, C. M. An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration. Development. 138, 3639–3646 (2011).
Murphy, M. M., Lawson, J. A., Mathew, S. J., Hutcheson, D. A. & Kardon, G. Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration. Development. 138, 3625–3637 (2011).
Paldi, A. et al. Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration. Development. 138, 3647–3656 (2011).
Chen, J. F. et al. MicroRNA-1 and microRNA-206 regulate skeletal muscle satellite cell proliferation and differentiation by repressing Pax7. J Cell Biol. 190, 867–879 (2010).
Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell. 136, 215–233 (2009).
Yuan, C. et al. Discovery of cashmere goat (Capra hircus) microRNAs in skin and hair follicles by Solexa sequencing. BMC Genomics. 14, 511 (2013).
Wienholds, E. et al. MicroRNA expression in zebrafish embryonic development. Science. 309, 310–311 (2005).
Li, G. et al. MicroRNA identity and abundance in developing swine adipose tissue as determined by Solexa sequencing. J Cell Biochem. 112, 1318–1328 (2011).
Kim, H. K., Lee, Y. S., Sivaprasad, U., Malhotra, A. & Dutta, A. Muscle-specific microRNA miR-206 promotes muscle differentiation. J Cell Biol. 174, 677–687 (2006).
Chen, J. F. et al. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet. 38, 228–233 (2006).
Clop, A. et al. A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep. Nat Genet. 38, 813–818 (2006).
Ge, Y., Sun, Y. & Chen, J. IGF-II is regulated by microRNA-125b in skeletal myogenesis. J Cell Biol. 192, 69–81 (2011).
Dey, B. K., Gagan, J., Yan, Z. & Dutta, A. MiR-26a is required for skeletal muscle differentiation and regeneration in mice. Genes Dev. 26, 2180–2191 (2012).
Crist, C. G. et al. Muscle stem cell behavior is modified by microRNA-27 regulation of Pax3 expression. Proc Natl Acad Sci USA. 106, 13383–13387 (2009).
Wei, W. et al. MiR-29 targets Akt3 to reduce proliferation and facilitate differentiation of myoblasts in skeletal muscle development. Cell Death Dis. 4, e668 (2013).
Dey, B. K., Gagan, J. & Dutta, A. miR-206 and -486 induce myoblast differentiation by downregulating Pax7. Mol Cell Biol. 31, 203–214 (2011).
Gagan, J., Dey, B. K., Layer, R., Yan, Z. & Dutta, A. MicroRNA-378 targets the myogenic repressor MyoR during myoblast differentiation. J Biol Chem. 286, 19431–19438 (2011).
Seok, H. Y. et al. MiR-155 inhibits expression of the MEF2A protein to repress skeletal muscle differentiation. J Biol Chem. 286, 35339–35346 (2011).
Miao, X., Luo, Q. & Qin, X. Genome-wide analysis reveals the differential regulations of mRNAs and miRNAs in Dorset and Small Tail Han sheep muscles. Gene. 562, 188–196 (2015).
Lagos-Quintana, M., Rauhut, R., Meyer, J., Borkhardt, A. & Tuschl, T. New microRNAs from mouse and human. RNA. 9, 175–179 (2003).
Zhao, Y. et al. Post-transcriptional regulation of cardiac sodium channel gene SCN5A expression and function by miR-192-5p. Biochim Biophys Acta. 1852, 2024–2034 (2015).
Jenkins, R. H. et al. MiR-192 induces G2/M growth arrest in aristolochic acid nephropathy. Am J Pathol. 184, 996–1009 (2014).
Kato, M. et al. MicroRNA-192 in diabetic kidney glomeruli and its function in TGF-β-induced collagen expression via inhibition of E-box repressors. Proc Natl Acad Sci USA. 104, 3432–3437 (2007).
Hu, F. et al. BMP-6 inhibits cell proliferation by targeting microRNA-192 in breast cancer. Biochim Biophys Acta. 1832, 2379–2390 (2013).
Feng, S. et al. MicroRNA-192 targeting retinoblastoma 1 inhibits cell proliferation and induces cell apoptosis in lung cancer cells. Nucleic Acids Res. 39, 6669–6678 (2011).
Song, B. et al. MiR-192 regulates dihydrofolate reductase and cellular proliferation through the p53-microRNA Circuit. Clin Cancer Res. 14, 8080–8086 (2008).
Huh, M. S., Parker, M. H., Scimè, A., Parks, R. & Rudnicki, M. A. Rb is required for progression through myogenic differentiation but not maintenance of terminal differentiation. J Cell Biol. 166, 865–876 (2004).
Giacinti, C. & Giordano, A. RB and cell cycle progression. Oncogene. 25, 5220–5227 (2006).
Frolov, M. V. & Dyson, N. J. Molecular mechanisms of E2F-dependent activation and pRB-mediated repression. J Cell Sci. 117, 2173–2181 (2004).
Sage, J., Miller, A. L., Pérez-Mancera, P. A., Wysocki, J. M. & Jacks, T. Acute mutation of retinoblastoma gene function is sufficient for cell cycle re-entry. Nature. 424, 223–228 (2003).
Indovina, P., Pentimalli, F., Casini, N., Vocca, I. & Giordano, A. RB1 dual role in proliferation and apoptosis: Cell fate control and implications for cancer therapy. Oncotarget. 6, 17873–17890 (2015).
Classon, M. & Harlow, E. The retinoblastoma tumour suppressor in development and cancer. Nat Rev Cancer. 2, 910–917 (2002).
Sylvius, N. et al. MicroRNA expression profiling in patients with lamin A/C-associated muscular dystrophy. FASEB J. 25, 3966–3978 (2011).
Wu, H. et al. In vitro culture and induced differentiation of sheep skeletal muscle satellite cells. Cell Biol Int. 36, 579–587 (2012).
Jin, Z. et al. MicroRNA-192 and -215 are upregulated in human gastric cancer in vivo and suppress ALCAM expression in vitro. Oncogene. 30, 1577–1585 (2011).
Zeng, X. et al. Recombinant adenovirus carrying the hepatocyte nuclear factor-1alpha gene inhibits hepatocellular carcinoma xenograft growth in mice. Hepatology. 54, 2036–2047 (2011).
Ashmore, C. R., Robinson, D. W., Rattray, P. & Doerr, L. Biphasic development of muscle fibers in the fetal lamb. Exp Neurol. 37, 241–255 (1972).
Maier, A. et al. Myosin heavy chain composition of single fibres and their origins and distribution in developing fascicles of sheep tibialis cranialis muscles. J Muscle Res Cell Motil. 13, 551–572 (1992).
Deng, Y. et al. MiR-215 modulates gastric cancer cell proliferation by targeting RB1. Cancer Lett. 342, 27–35 (2014).
Gao, F. Y., Liu, Q. Y., Yuan, L. & Xuan, S. Y. Upregulation of microRNA-132 in gastric cancer promotes cell proliferation via retinoblastoma 1 targeting. Mol Med Rep. 12, 7005–7010 (2015).
Shi, L. et al. MiR-335 promotes cell proliferation by directly targeting Rb1 in meningiomas. J Neurooncol. 110, 155–162 (2012).
Zacksenhaus, E. et al. pRb controls proliferation, differentiation and death of skeletal muscle cells and other lineages during embryogenesis. Genes Dev. 10, 3051–3064 (1996).
Jacks, T. et al. Effects of an Rb mutation in the mouse. Nature. 359, 295–300 (1992).
Wu, L. et al. Extra-embryonic function of Rb is essential for embryonic development and viability. Nature. 421, 942–947 (2003).
Hosoyama, T., Nishijo, K., Prajapati, S. I., Li, G. & Keller, C. Rb1 gene inactivation expands satellite cell and postnatal myoblast pools. J Biol Chem. 286, 19556–19564 (2011).
Melcon, G. et al. Loss of emerin at the nuclear envelope disrupts the Rb1/E2F and MyoD pathways during muscle regeneration. Hum Mol Genet. 15, 637–651 (2006).
Gharaibeh, B. et al. Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique. Nat Protoc. 3, 1501–1509 (2008).
Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25, 402–408 (2001).
Schmittgen, T. D. et al. Real-time PCR quantification of precursor and mature microRNA. Methods. 44, 31–38 (2008).
Acknowledgements
This work was supported by National Natural Science Foundation of China (31201765), The earmarked fund for Modern Agro-industry Technology Research System (CARS-40-01) and The Agricultural Science and Technology Innovation Program of China (ASTIP-IAS01).
Author information
Authors and Affiliations
Contributions
Y.-H.M. and Q.-J.Z. designed the research and participated in its coordination. Q.Z. contributed to the slaughter experiment, data analysis, performed the experiment and wrote the manuscript. Y.K. participated in the data analysis, slaughter experiment and performed the qPCR experiment. H.-Y.W. contributed to the immunoblotting experiment and data analysis. W.-J.G. and X.-C.L. contributed to the cell culture experiment. L.J., X.-H.H. and Y.-B.P. participated in sheep resource population management. J.-L.H. contributed to the drafting of the manuscript.
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Electronic supplementary material
Rights and permissions
This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
About this article
Cite this article
Zhao, Q., Kang, Y., Wang, HY. et al. Expression profiling and functional characterization of miR-192 throughout sheep skeletal muscle development. Sci Rep 6, 30281 (2016). https://doi.org/10.1038/srep30281
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/srep30281
- Springer Nature Limited
This article is cited by
-
Effect of miR-493-5p on proliferation and differentiation of myoblast by targeting ANKRD17
Cell and Tissue Research (2023)
-
Differential expression of miRNAs in skeletal muscles of Indian sheep with diverse carcass and muscle traits
Scientific Reports (2020)
-
Bovine hepatic miRNAome profiling and differential miRNA expression analyses between beef steers with divergent feed efficiency phenotypes
Scientific Reports (2020)
-
Intramuscular preadipocytes impede differentiation and promote lipid deposition of muscle satellite cells in chickens
BMC Genomics (2018)
-
miR-27b regulates myogenic proliferation and differentiation by targeting Pax3 in goat
Scientific Reports (2018)