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EXPRESSION ANALYSIS OF MIR-1 AND MIR-133 IN THREE DEVELOPMENTAL STAGES OF GOAT SKELETAL MUSCLE.

Byline: Yinghui Ling Lijuan Wang JianpingDing Lisheng Wang Yunhai Zhang and Xiaorong Zhang

ABSTRACT

MiR-1 and miR-133 are evolutionary conserved in most animal species from drosophila to human and have been confirmed to take an important role for skeletal muscle development in mice and pigs. In this study the function of miR- 1 and miR-133 was detected through analysis the expression pattern of the two miRNAs and their predicted target genes in three developmental stages of goat skeletal muscle. HDAC4 and SRF which were the target genes of miR-1 and miR- 133 family respectively were predicted using MiRanda and Target Scan 6.2 software. Then the expression profiles of miR-1 miR-133 HDAC4 and SRF in 10 normal goat tissues (heart liver spleen lung kidney small intestine fat longissimusdorsi muscles pectoral muscles and crural muscles) from three stages (newborn three months eighteen months) of Anhui White goat were characterized by real-time RT-PCR. The expression of miR-1 and miR-133 were varied among different tissues

with most abundant in heart and skeletal muscles (longissimusdori muscles pectoral muscles and crural muscles) in Anhui white goats while there were the least expression in other tissues. However the expression levels of HDAC4 and SRF were on the contrary. The results suggested that miR-1 and miR-133 may enhance myoblast proliferation and differentiation by repressing their predicated target genes thus affecting skeletal muscle growth and development. This study will be a useful reference for further studies about miR-1 and miR-133 for their role in the regulation in animal growth and development processes.

Key words: miR-1 and miR-133 goat target gene growth and development expression profiles.

INTRODUCTION

MicroRNAs (miRNAs) are a class of short (20~25 nucleotides) Single-stranded non-coding RNA that regulate mRNA expression at the post-transcriptional level (Huang et al. 2013). miRNAs negatively regulate gene expression through complementary base-pair binding of the miRNA seed sequence" (nucleotides 2-7) to the 3'untranslated region (UTR) of target mRNA degrading or destabilizing the RNA message or inhibiting protein translation depending on the quality of complimentary base-pair matches or the number of miRNA targeting sites with the 3'UTR (Townley-Tilson et al. 2010). Emerging evidence has demonstrated that miRNAs are essential for normal mammalian development cell proliferation differentiation growth development remodeling in response to stress and many other functions (GA1/4ller and Russell 2010; Lu et al. 2012). Studies from recent years have shown that many miRNAs involved in the regulation of skeletal muscle proliferation and differentiation processes

(Chen et al. 2011; Yan et al. 2012). Subsets of these miRNAs miR-1 and miR-133 are muscle specific and have been the focus of intensive investigation (Lu et al. 2012). Both miR-1 and miR-133 have been found in most animal species from drosophila to human suggesting that they are evolutionary conserved (Chen et al. 2006). In addition Studies have confirmed that miRNAs have an important role for skeletal muscle development in mice and pigs (Mcdaneld et al. 2009; Parra et al. 2010).

According to the foxO1 expression profile of human and mouse indicating that miR-133b may be involved in the regulation of foxO1 in B cell development (Liang et al. 2011). MiR-1 promotes myogenesis by targeting histone deacetylase 4 (HDAC4) a transcriptional repressor of muscle gene expression while miR-133 enhances myoblast proliferation by repressing serum response factor (SRF) (Chen et al. 2006; Rao et al. 2006).Although miR-1 and miR-133 have been extensively studied there is no information about their expression and expression of their predicted target genes (HDAC4 and SRF) of goat skeletal muscle.

In this study real-time quantitative reverse transcription PCR (qRT-PCR) method (Mishina et al. 2007) was used to quantify the expression of miR-1 miR-133 HDAC4 and SRF in the tissues (heart liver spleen lung kidney small intestine fat longissimusdorsi muscles pectoral muscles and crural muscles) from neonatal three-month and eighteen-month Anhui white goats. Studying the regulatory mechanisms of miR-1 and miR-133 and their predicated target genes in muscle proliferation and differentiation will extend our knowledge of miRNAs in muscle biology and will improve our understanding of the myogenesis regulation.

MATERIALS AND METHODS

Animals tissue sampling and RNA extraction: Anhui white goat was bred in the experimental farm of Hefei Bo Da Livestock Technology Development CO LTD. All goats were allowed free access to water and treated in accordance with the Regulation on Animal Experimentation at Anhui Agricultural University'. Goats from the herd of the farm will be referred to as Anhui white goat in this paper. Generally the goats grow very fast from 0 to three months and grow slowly after eighteen months. Hence three healthy neonatal three- month-old and eighteen-month-old goats were selected from the herd for this study. Heart liver spleen lung kidney small intestine fat and skeletal muscles (longissimusdorsi muscles pectoral muscles and crural muscles) tissues were surgically collected from three different goats and immediately frozen in liquid nitrogen.

Total RNA was extracted from heart liver spleen lung kidney small intestine fat and skeletal muscles (longissimusdorsi muscles pectoral muscles and crural muscles) tissues using TRIzol (TaKaRa) according to the manufacturer's instructions. The quantity and quality of the RNA were measured using SMA 1000 spectrophotometer (Merinton). The integrity of the RNA was assessed by 0.8% electrophoretic analysis of the 28S 18S and 5S rRNA subunits. Total RNAs were stored at - 80 for analyses.

Primer design and synthesize: The mature sequences of miR-1 and miR-133 were from the preliminary Solexa sequencing for Anhui white goats (Ling et al. 2013). The stem-loop primers and specific PCR primers for miR-1 miR-133 and U6 RNA were obtained from RiboBio (RiboBio Guangzhou China). As the goat HDAC4 and SRF gene sequences are not available according to the high homology between bovine and sheep HDAC4 and SRF gene (GenBank accession number XM-002686597 and XM-004003449; NM-001206016 and XM- 004019222) two pairs of primers were designed to amplify goat HDAC4 and SRF gene (Table 1). The primers were synthesized by the BGI (China).

cDNA synthesis: For miR-1 miR-133 and U6 first strand cDNA were synthesized using specific stem-loop RT primers and a cDNA First Strand Synthesis Kit (TaKaRa Code:DRR037S). Briefly the 20L reverse- transcriptase reaction volume contained 1g total RNA 5A-PrimeScript Buffer Prime Script RT Enzyme Mix I 5M specific stem-loop RT primers and RNase free water. The mixture was incubated for 15 min at 42 and 5 sec at 85 . Moreover the 20L reverse-transcriptase reaction volume of HDAC4 and SRF contained 1g total RNA 5A-PrimeScript Buffer Prime Script RT Enzyme Mix I 50M Oligod TPrimer 100M Random 6 mers and RNase free water. The mixture was incubated for 15 min at 37 and 5 sec at 85 . The first-strand cDNAs were then diluted 1:10 with RNase free water and stored at -20 until further use. Table 1. Primer sequence of predicated target genes (HDAC4 and SRF)

Cloning and sequencing of HDAC4 and SRF gene: Of the 2 pairs of primers for sequencing goat HDAC4 and SRF cDNAs were confirmed by subsequent sequencing. The 25L volume contained: 2LcDNA 10M each primer 2.5mMdNTP Mixture 10A-EX Taq Buffer 5UTaKaRaEX Taq HS and ddH2O. The cycling protocol was 30s at 94 35 cycles of 94 for 30s 58 for 30s 72 for 1min with a final extention at 72 for 5min.

The expected sizes of the application can be found in

Table 1. They were confirmed by 1% agarose gel electrophoresis stained with Nucleic Acid Dye (TIAN GEN). The applications were gel-purified (AXYGEN USA) and ligated into the pMD-18T vector (Takara) for sequencing.

Real-Time Quantitative Reverse Transcription PCR (qRT-PCR): Relative quantification of skeletal muscle growth and development-related gene were performed using real-time qRT-PCR assays based on the gene sequences of miR-1 miR-133 HDAC4 and SRF. These qRT-PCR assays were used to assess gene expression in ten normal tissues (heart liver spleen lung kidney small intestine fat longissimusdorsi muscles pectoral muscles and crural muscles) and U6 small RNA was used as an internal reference gene. The specific forward and reverse primers employed for real-time qRT-PCR were designed with Primer Premier 5.0 software and are shown in Table 1. For real-time qPCR a total of 15L mixture composed of 2L of diluted template 10M of each primer and Fast Start Universal SYBR Green Master Mix (Rox) (Roche) was run in a PCR reaction plate in triplicate using Step-One real-time PCR system (Applied Biosystems)

And the thermal cycling conditions were 95 for10 min followed by 40 cycles of a 95 denaturing step for 15 s and a 60 annealing/extending step for1min. The conditions were similar for all of the primer pairs and the dissociation curves were run to ensure single product detection. The reaction conditions of dissociation curves were95 for 15 s;60 for 1 min;95 for 15 s and 60 for 1 min. All of the samples were tested for cDNAs contamination by running no-reverse transcriptase controls.

Statistical analysis: The relative expression levels of the miR-1 miR-133 HDAC4 and SRF were normalized with internal control (U6 snRNA) expression level which was calculated using the 2_ Ct method (Livak and Schmittgen et al. 2001) among Ct= Ct Target-CtU6; Ct= Ct Target-CtCalibrator. Data were presented as Means SE from at least three separate experiments.

Statistical significance was determined using one-way ANOVA analyses in which a P-value of less than 0.05 was considered to be significant. All statistical analyses were conducted by using SPSS 17.0 software.

RESULTS

Target sequences: MiR-1 and miR-133 were amplified with specific stem-loop RT products as a template. The size of miR-1 and miR-133 products was about 80bp. In addition HDAC4 and SRF were amplified with universal cDNA as a template. The applications were gel-purified (AXYGEN USA) and ligated into the pMD-18T vector (Takara) for sequencing. The fragment length of HDAC4 and SRF products were about 100bp with 2% agarose gel electrophoresis.

Expression profiling of miR-1 and miR-133: To further understand the biological function of miRNAs in goat we first detected the expression profiles of miR-1 and miR-133 identified in ten goat tissues (heart liver spleen lung kidney small intestine fat longissimusdorsi muscles pectoral muscles and crural muscles) by the real-time qRT-PCR. In the gene expression data the expression of miR-1 and miR-133 in skeletal muscles heart tissues and other tissues was found to be significantly different (P less than 0.05) based on comparison with miRNA expression patterns from 0 3 and 18- month-old Anhui white goats. The results showed that both of the two miRNAs appeared to be tissue specific (Figure1). miR-1 and miR-133 were specifically expressed in heart and skeletal muscle (longissimusdorsi muscles pectoral muscles and crural muscles)

Which was reported to be strongly expressed in eighteen-month- old cardiac and three-month-old skeletal muscle tissues and play the important role in regulating muscle development. The results also showed that miR-1 and miR-133 had a similar relative expression pattern in some tissues respectively. In addition miR-1 and miR-133 had anupregulated trend from newborn to three months old and their expression levels were reduced with the individual maturing.

Expression profiling of predicated target genes (HDAC4 and SRF): To validate the differential expression of HDAC4 and SRF in ten normal tissues (heart liver spleen lung kidney small intestine fat longissimusdorsi muscles pectoral muscles and crural muscles) U6 small RNA were selected and their expression were quantified using real-time qRT-PCR (Figure.2). The mean normalized HDAC4 and SRF expression values from each tissue were calculated and expressed as a relative fold change. Both of HDAC4 and SRF could be detected in total tissues. In ten tissues the expression level of HDAC4 and SRF in lung and small intestine tissues were significantly higher than heart and skeletal muscles tissues (Pless than 0.05). In comparison to the high abundance of miR-1 and miR-133 inskeletal muscle tissues goat HDAC4 and SRF had the least expression.

The results also showed that HDAC4 and SRF had anupregulated trend from newborn to three months old and their expression levels were reduced with the individual maturing which was similar with miR-1 and miR-133.

DISCUSSION

Meat production is a vital characteristic of goat thus the exploitation of miRNAs involved in regulation the process of muscle development in goat is very important (Sheng et al. 2010). Numerous cellular and developmental processes in various organisms have been reported to be regarded by miRNAs such as embryonic development cell proliferation and differentiation cell apoptosis and fat metabolism (Kulkarnis et al. 2011; Baley and Li et al. 2012; Nohata et al. 2012; Luo et al. 2013). It is more prevalent for miRNAs to negatively regulate gene expression by promoting degradation of target mRNAs or inhibiting their translation (Podolska et al. 2011). It is a key step to understand miRNAs function by analyzing the expression of tissue-specific miRNAs in different tissues and developmental stages while the quantitative detection of miRNAs is a premise to study miRNAs function. To date there are two ways to detect miRNAs expression such as 3' end plus connectors and stem-loop primers.

Due to the stem-loop RT-PCR method is simple fast high accuracy and good specificity that is widely used in miRNAs expression studies. MiR-1 and miR-133 were initially cloned and identified from human and mouse muscle tissues by Lagos-Quintana et al (2001) and were the muscle- specific miRNAs (Sweetman et al. 2008). MiR-1 and miR-133 derived from the same miRNA polycistron and transcribed together can carry out distinct biological functions (Chen et al. 2006). MiR-1 and miR-133 are expressed in muscle tissues and induced during muscle cell differentiation a process that directs myoblasts to differentiate into mature myotubes which are organized into myofibers(Koutsoulidoub et al. 2011). Although miR-1 and miR-133 genes have similar expression patterns they have different biological functions. For example miR-1 was found to promote muscle cell differentiation while miR-133 could promote muscle cell proliferation.

MiR-1 and miR-133 are highly expressed in skeletal muscles implying a critical role in the regulation of muscle developmental process.

At present the functions of most miRNAs remain unclear. Because each miRNA may have many target genes and multiple miRNAs regulate a gene expression at the same time it is urgent to rapidly and accurately predict and identify the target genes of miRNAs which is very important to study miRNAs functions. Studies demonstrated that miRNAs regulated cell proliferation and differentiation by regulating some transcription factors and signal transduction factors (Ge et al. 2011; Huang et al. 2012). It has been proved that miR-1 and miR-133 were involved in cell proliferation and differentiation by regulating the expression of target genes (Chen et al. 2006). Hence to identify target genes that might mediate the observed effects of miR-1 and miR-133 on skeletal muscle proliferation and differentiation we next examined potential targets of these two miRNAs.

Many computational and bioinformatics-based approaches have been used to predict potential target genes of miR-1 and miR-133 such as MiRanda and TargetScan6.2 software. Strikingly many transcription factors and signal transduction factors have been indicated to be target genes of miRNAs raising the possibility that miRNAs might be involved in transcriptional regulation of gene expression. Among the predicated target genes of miR-1 miR-1 was shown to bind to the 3'UTR of HDAC4 an inhibitor of muscle differentiation and suppresses its expression during growth and differentiation conditions (Koutsoulidou et al. 2011). Similarly miR-133-binding sites are found in the 3'UTR of the mammalian gene encoding SRF which has been shown to be important in muscle proliferation and differentiation in vitro and vivo (Chen et al. 2006). qRT-PCR was used to confirm the expression of miR-1 miR-133 and their predicated target genes (HDAC4 and SRF).

Using this method we were able to validate the existence of miR-1 miR-133 HDAC4 and SRF in ten normal tissues (heart liver spleen lung kidney small intestine fat longissimusdorsi muscles pectoral muscles and crural muscles) of Anhui white goats. In this study miR-1 and miR-133 are induced during goat muscle cell differentiation and their expression levels are increased from newborn to three months old and decreased with the individual maturing. That indicated miR-1 and miR-133 was necessary during the muscle rapid growth which could promote bulk copy of fibroblasts. The expression levels of miR-1 and miR- 133 were relatively reduced when the process of myoblasts proliferation and differentiation was weakened. While the expression changes of miR-1 and miR-133 suggested that miR-1 and miR-133 may be related to the development of goat skeletal muscles.

However their predicated target genes were found to be highly expressed in fat and small intestine tissues while there was the least expression in skeletal muscles.

In our study miR-1 and miR-133 and their target genes (HDAC4 and SRF) represented negative correlation of expression patterns by qRT-PCR. MiR-1 and miR-133 was the most abundance in goat skeletal muscles. And the target genes of miR-1 and miR-133 HDAC4 and SRF predicated to be muscle transcription factors which involved in muscle proliferation and differentiation were the least abundant transcript in skeletal muscles. Therefore our study suggested that the increase of miR-1 and miR-133 expression may lead to decrease of HDAC4 and SRF expression. And more studies will need to be done to determine the regulation patterns of miR-1 and miR-133 and their predicated target genes.

Although miR-1 miR-133 HDAC4 and SRF have been extensively studied there is no information about their expression of goat skeletal muscle. Results presented in this study show that miR-1 and miR-133 are induced during goat muscle cell differentiation and their expression levels are increased proportionally to the stage of muscle neonate development. And miR-1 and miR-133 and their predicated target genes (HDAC4 and SRF) represented negative correlation. Since miRNAs regulate important processes during skeletal muscle development such information would be very important for understanding the mechanism of muscle formation during neonate development.

Acknowledgments: Research was supported by the National Natural Science Foundation of China (31301934 31372310)the Natural Science Foundation of Anhui Province (1308085QC54) and China Agriculture Research System and the project Technology resource platform of Ministry of Science and Technology funds. Lijuan WANG and Yinghui LING equally contributed to this paper and both of them be considered as first author.

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Publication:Journal of Animal and Plant Sciences
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