Comparison of microRNAs in adipose and muscle tissue from seven indigenous Chinese breeds and Yorkshire pigs

Elucidation of the pig microRNAome is essential for interpreting functional elements of the genome and understanding the genetic architecture of complex traits. Here, we extracted small RNAs from skeletal muscle and adipose tissue, and we compared their expression levels between one Western breed (Yorkshire) and seven indigenous Chinese breeds. We detected the expression of 172 known porcine microRNAs (miRNAs) and 181 novel miRNAs. Differential expression analysis found 92 and 12 differentially expressed miRNAs in adipose and muscle tissue respectively. We found that different Chinese breeds shared common directional miRNA expression changes compared to Yorkshire pigs. Some miRNAs differentially expressed across multiple Chinese breeds, including ssc‐miR‐129‐5p, ssc‐miR‐30 and ssc‐miR‐150, are involved in adipose tissue function. Functional enrichment analysis revealed that the target genes of the differentially expressed miRNAs are associated mainly with signaling pathways rather than metabolic and biosynthetic processes. The miRNA–target gene and miRNA–phenotypic traits networks identified many hub miRNAs that regulate a large number of target genes or phenotypic traits. Specifically, we found that intramuscular fat content is regulated by the greatest number of miRNAs in muscle tissue. This study provides valuable new candidate miRNAs that will aid in the improvement of meat quality and production.     

Altered microRNA expression in bovine skeletal muscle with age

Age‐dependent decline in skeletal muscle function leads to several inherited and acquired muscular disorders in elderly individuals. The levels of microRNAs (miRNAs) could be altered during muscle maintenance and repair. We therefore performed a comprehensive investigation for miRNAs from five different periods of bovine skeletal muscle development using next‐generation small RNA sequencing. In total, 511 miRNAs, including one putatively novel miRNA, were identified. Thirty‐six miRNAs were differentially expressed between prenatal and postnatal stages of muscle development including several myomiRs (miR‐1, miR‐206 and let‐7 families). Compared with miRNA expression between different muscle tissues, 14 miRNAs were up‐regulated and 22 miRNAs were down‐regulated in the muscle of postnatal stage. In addition, a novel miRNA was predicted and submitted to the miRBase database as bta‐mir‐10020. A dual luciferase reporter assay was used to demonstrate that bta‐mir‐10020 directly targeted the 3′‐UTR of the bovine ANGPT1 gene. The overexpression of bta‐mir‐10020 significantly decreased the DsRed fluorescence in the wild‐type expression cassette compared to the mutant type. Using three computational approaches – miranda , pita and rnahybrid – these differentially expressed miRNAs were also predicted to target 3609 bovine genes. Disease and biological function analyses and the KEGG pathway analysis revealed that these targets were statistically enriched in functionality for muscle growth and disease. Our miRNA expression analysis findings from different states of muscle development and aging significantly expand the repertoire of bovine miRNAs now shown to be expressed in muscle and could contribute to further studies on growth and developmental disorders in this tissue type.     

Identification and differential expression of microRNAs in the ovaries of pigs (Sus scrofa) with high and low litter sizes

Litter size affects profitability in the swine industry. Mammalian ovaries play important roles during reproduction, including ovulation and hormone secretion, which are tightly regulated by specific microRNAs (miRNAs). In this study, we investigated the effects of specific miRNAs on porcine litter size. We compared the ovarian miRNAs of Yorkshire pigs with high (YH) and low (YL) litter sizes using Solexa sequencing technology. We identified 327 and 320 miRNAs in the ovaries of YH and YL pigs respectively. A total of 297 miRNAs were co‐expressed; 30 and 23 miRNAs respectively were specifically expressed in the two libraries. A total of 83 novel miRNAs were predicted; 37 specific miRNAs were obtained, of which 21 miRNAs were upregulated and 16 miRNAs were downregulated in YH compared with YL. Additionally, 19 628 and 19 250 target genes were predicted in the two libraries respectively. The results revealed that specific miRNAs (i.e., miR‐224, miR‐99a, let‐7c, miR‐181c, miR‐214 and miR‐21) may affect porcine litter size. The results of this study will help in gaining understanding of the role of miRNAs in porcine litter size regulation.     

Cloning and characterization of microRNAs from porcine skeletal muscle and adipose tissue

MicroRNAs (miRNAs) are an abundant class of small regulatory RNAs that regulate the stability and translation of cognate mRNAs. Although an increasing number of porcine miRNAs has recently been identified, the full repertoire of miRNAs in pig remains to be elucidated. To identify porcine miRNAs potentially involved in myogenesis and adipogenesis, we constructed small RNA cDNA libraries from skeletal muscle and adipose tissue and identified 89 distinct miRNAs that are conserved in pig, of which 15 were new. Expression analysis of all newly identified and selected known porcine miRNAs revealed that some miRNAs were enriched in a tissue-specific manner, whereas others were expressed ubiquitously in the porcine tissues examined. Our results expand the number of known porcine miRNAs and provide useful information for further investigating the biological functions of miRNAs associated with growth and development of skeletal muscle or adipose tissue in pig.     

Discovery of porcine miRNA‐196a/b may influence porcine adipogenesis in longissimus dorsi muscle by miRNA sequencing

Intramuscular fat (IMF) is one of the fat traits that has economic importance in the pork industry. Longissimus dorsi muscle contains IMF and is suitable for studying adipogenesis. To discover further potential regulatory miRNAs that may influence adipogenesis, we analyzed miRNA in the longissimus dorsi muscle of Yorkshire (YY, lean‐type) and Chinese Wannanhua (WH, fatty) pigs using miRNA sequencing (miRNA‐seq). From this dataset, we identified 598 unique miRNAs comprising 325 pre‐miRNAs and 273 novel pre‐miRNAs through comparison with known miRNAs in miRBase version 21. We found 42 miRNAs including nine up‐ and 33 down‐regulated between the YY and WH pigs. Moreover, we found two miRNAs, miR‐196a/b (miR‐196a, miR‐196b‐5p), that had the highest level of expression in WH pigs, and miR‐196a/b may influence porcine adipogenesis in longissimus dorsi muscle through an adipocytokine signaling pathway.     

Hormone replacement therapy enhances IGF‐1 signaling in skeletal muscle by diminishing miR‐182 and miR‐223 expressions: a study on postmenopausal monozygotic twin pairs

MiRNAs are fine‐tuning modifiers of skeletal muscle regulation, but knowledge of their hormonal control is lacking. We used a co‐twin case–control study design, that is, monozygotic postmenopausal twin pairs discordant for estrogen‐based hormone replacement therapy (HRT) to explore estrogen‐dependent skeletal muscle regulation via miRNAs. MiRNA profiles were determined from vastus lateralis muscle of nine healthy 54–62‐years‐old monozygotic female twin pairs discordant for HRT (median 7 years). MCF‐7 cells, human myoblast cultures and mouse muscle experiments were used to confirm estrogen's causal role on the expression of specific miRNAs, their target mRNAs and proteins and finally the activation of related signaling pathway. Of the 230 miRNAs expressed at detectable levels in muscle samples, qPCR confirmed significantly lower miR‐182, miR‐223 and miR‐142‐3p expressions in HRT using than in their nonusing co‐twins. Insulin/IGF‐1 signaling emerged one common pathway targeted by these miRNAs. IGF‐1R and FOXO3A mRNA and protein were more abundantly expressed in muscle samples of HRT users than nonusers. In vitro assays confirmed effective targeting of miR‐182 and miR‐223 on IGF‐1R and FOXO3A mRNA as well as a dose‐dependent miR‐182 and miR‐223 down‐regulations concomitantly with up‐regulation of FOXO3A and IGF‐1R expression. Novel finding is the postmenopausal HRT‐reduced miRs‐182, miR‐223 and miR‐142‐3p expression in female skeletal muscle. The observed miRNA‐mediated enhancement of the target genes' IGF‐1R and FOXO3A expression as well as the activation of insulin/IGF‐1 pathway signaling via phosphorylation of AKT and mTOR is an important mechanism for positive estrogen impact on skeletal muscle of postmenopausal women.     

Profiling highly conserved microrna expression in recombinant IgG‐producing and parental Chinese hamster ovary cells

MicroRNAs (miRNAs) play important roles in global gene regulation. Researchers in recombinant protein production have proposed miRNAs as biomarkers and cell engineering targets. However, miRNA expression remains understudied in Chinese Hamster Ovary cells, one of the most commonly used host cell systems for therapeutic protein production. To profile highly conserved miRNA expression, we used the miRCURY? miRNA array for screening miRNAs in CHO cells. The selection criteria for further miRNA profiling included positive hybridization signals and experimentally validated predicted regulatory targets. On the basis of screening, we selected 16 miRNAs for quantitative RT‐PCR profiling. We profiled miR expression in parental CHO DG44 and CHO K1 cell lines as well as four recombinant DG44‐derived CHO lines producing a recombinant human IgG. We observed that miR‐221 and miR‐222 were significantly downregulated in all IgG‐producing cell lines when compared with parental DG44, whereas miR‐125b was significantly downregulated in one IgG‐producing line. In another IgG‐producing line, miR‐19a was significantly upregulated. miRNA expression was also profiled in two of these lines that were amplified by stepwise increase of methotrexate. In both amplified cell lines, let‐7b and miR‐221 were significantly downregulated. In parental CHO K1, let‐7b, miR‐15b, and miR‐17 were significantly downregulated when compared with DG44. The results reported here are the first steps toward profiling highly conserved miRNAs and studying the clonal difference in miRNA expression in CHO cells and may shed light on using miRNAs in cell engineering. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Identification of microRNAs regulated by activin A in human embryonic stem cells

Human embryonic stem (hES) cells have the capacities to propagate for extended periods and to differentiate into cell types from all three germ layers both in vitro and in vivo. These characteristics of self‐renewal and pluripotency enable hES cells having the potential to provide an unlimited supply of different cell types for tissue replacement, drug screening, and functional genomics studies. The hES‐T3 cells with normal female karyotype cultured on either mouse embryonic fibroblasts (MEF) in hES medium (containing 4 ng/ml bFGF) (T3MF) or feeder‐free Matrigel in MEF‐conditioned medium (supplemented with additional 4 ng/ml bFGF) (T3CM) were found to express very similar profiles of mRNAs and microRNAs, indicating that the unlimited self‐renewal and pluripotency of hES cells can be maintained by continuing culture on these two conditions. However, the expression profiles, especially microRNAs, of the hES‐T3 cells cultured on Matrigel in hES medium supplemented with 4 ng/ml bFGF and 5 ng/ml activin A (T3BA) were found to be different from those of T3MF and T3CM cells. In T3BA cells, four hES cell‐specific microRNAs miR‐372, miR‐302d, miR‐367, and miR‐200c, as well as three other microRNAs miR‐199a, miR‐19a, and miR‐217, were found to be up‐regulated, whereas five miRNAs miR‐19b, miR‐221, miR‐222, let‐7b, and let‐7c were down‐regulated by activin A. Thirteen abundantly differentially expressed mRNAs, including NR4A2, ERBB4, CXCR4, PCDH9, TMEFF2, CD24, and COX6A1 genes, targeted by seven over‐expressed miRNAs were identified by inverse expression levels of these seven microRNAs to their target mRNAs in T3BA and T3CM cells. The NR4A2, ERBB4, and CXCR4 target genes were further found to be regulated by EGF and/or TNF. The 50 abundantly differentially expressed genes targeted by five under‐expressed miRNAs were also identified. The abundantly expressed mRNAs in T3BA and T3CM cells were also analyzed for the network and signaling pathways, and roles of activin A in cell proliferation and differentiation were found. These findings will help elucidate the complex signaling network which maintains the self‐renewal and pluripotency of hES cells. J. Cell. Biochem. 109: 93–102, 2010. © 2009 Wiley‐Liss, Inc.     

The potential of microRNAs as human prostate cancer biomarkers: A meta‐analysis of related studies

Prostate cancer (PC) is a very important kind of male malignancies. When PC evolves into a stage of hormone resistance or metastasis, the fatality rate is very high. Currently, discoveries and advances in miRNAs as biomarkers have opened the potential for the diagnosis of PC, especially early diagnosis. miRNAs not only can noninvasively or minimally invasively identify PC, but also can provide the data for optimization and personalization of therapy. Moreover, miRNAs have been shown to play an important role to predict prognosis of PC. The purpose of this meta‐analysis is to integrate the currently published expression profile data of miRNAs in PC, and evaluate the value of miRNAs as biomarkers for PC. All of relevant records were selected via electronic databases: Pubmed, Embase, Cochrane, and CNKI based on the assessment of title, abstract, and full text. we extracted mean ± SD or fold change of miRNAs expression levels in PC versus BPH or normal controls. Pooled hazard ratios (HRs) with 95% confidence intervals (CI) for overall survival (OS) and recurrence‐free survival (RFS), were also calculated to detect the relationship between high miRNAs expression and PC prognosis. Selected 104 articles were published in 2007‐2017. According to the inclusion criteria, 104 records were included for this meta‐analysis. The pooled or stratified analyze showed 10 up‐regulated miRNAs (miR‐18a, miR‐34a, miR‐106b, miR‐141, miR‐182, miR‐183, miR‐200a/b, miR‐301a, and miR‐375) and 14 down‐regulated miRNAs (miR‐1, miR‐23b/27b, miR‐30c, miR‐99b, miR‐139‐5p, miR‐152, miR‐187, miR‐204, miR‐205, miR‐224, miR‐452, miR‐505, and let‐7c) had relatively good diagnostic and predictive potential to discriminate PC from BPH/normal controls. Furthermore, high expression of miR‐32 and low expression of let‐7c could be used to differentiate metastatic PC from local/primary PC. Additional interesting findings were that the expression profiles of five miRNAs (miR‐21, miR‐30c, miR‐129, miR‐145, and let‐7c) could predict poor RFS of PC, while the evaluation of miR‐375 was associated with worse OS. miRNAs are important regulators in PC progression. Our results indicate that miRNAs are suitable for predicting the different stages of PC. The detection of miRNAs is an effective way to control patient's prognosis and evaluate therapeutic efficacy. However, large‐scale detections based on common clinical guidelines are still necessary to further validate our conclusions, due to the bias induced by molecular heterogeneity and differences in study design and detection methods.     

A polymorphism in the porcine miR‐208b is associated with microRNA biogenesis and expressions of SOX‐6 and MYH7 with effects on muscle fibre characteristics and meat quality

MicroRNAs (miRNAs) encoded by the myosin heavy chain (MHC) genes are muscle‐specific miRNAs (myomiRs) and regulate the expression of MHC isoforms in skeletal muscle. These miRNAs have been implicated in muscle fibre types and their characteristics by affecting the heterogeneity of myosin. In pigs, miR‐208b and miR‐499 are embedded in introns of MYH7 and MYH7b respectively. Here, we identified a novel single nucleotide polymorphism (SNP) in intron 30 of MYH7 by which porcine miR‐208b is encoded. Based on the association study using a total of 487 pigs including Berkshire (n = 164), Landrace (n = 121) and Yorkshire (n = 202), the miR‐208b SNP (g.17104G>A) had significant effects on the proportions of types I and IIb fibre numbers (P < 0.010) among muscle fibre characteristics and on drip loss (P = 0.012) in meat quality traits. Moreover, the SNP affected the processing of primary miR‐208b into precursor miR‐208b with a marginal trend towards significance (P = 0.053), thereby leading to significant changes in the levels of mature miR‐208b (P = 0.009). These SNP‐dependent changes in mature miR‐208b levels were negatively correlated with the expression levels of its target gene, SOX‐6 (P = 0.038), and positively associated with the expression levels of its host gene, MYH7 (P = 0.046). Taken together, our data suggest that the porcine miR‐208b SNP differentially represses the expression of SOX‐6 by regulating miRNA biogenesis, thereby affecting the expression of MYH7 and the traits of muscle fibre characteristics and meat quality.