Effects of microRNAs on skeletal muscle development.

MicroRNAs (miRNAs) are small (about 22 nucleotides) noncoding RNAs, which were highly conserved among mammals. They have ushered in a new era in molecular biology over twenty years. They can negatively regulate gene expression at the posttranscriptional level through the principle of complementary base pairing with the 3' untranslated region (UTR) of their target mRNAs and induce their degradation. They involve in tissue morphogenesis, cellular processes like apoptosis, and major signaling pathways. Previous studies have promoted our understanding that miRNAs play an important role in myogenesis and have a big impact on muscle mass, muscle fiber type and muscle diseases. Many researchers have provided evidence of the involvement of muscle-specific and enriched miRNAs in the individual stages of skeletal muscle development as well as of their significant influence on muscle metabolism during quiescence, proliferation, differentiation and regeneration. Here, we focus on the microRNAs that related to the development of skeletal muscle. For example, some microRNAs are upregulated in differentiated skeletal muscle and can promote differentiation, like, miR-1, miR-24, miR-26a, miR-181 and miR-206. However, some microRNAs highly expressed in proliferating myoblasts, downregulated in differentiated and could inhibit differentiation, like MiR-221 and miR-222. Some others not only promote skeletal muscle proliferation, but also promote differentiation, like miR-214. Studying the miRNAs' regulatory mechanisms in skeletal development will help us know more about the knowledge of miRNAs in muscle developmental biology and make us learn more about involved signal pathway.

[Research of the effect of bone mineral density and fracture site of the vertebrae on low back pain in elderly patients with osteoporotic vertebral compression fractures].

OBJECTIVE: To investigate the effect of bone mineral density(BMD) and fracture site of the vertebrae on low back pain in elderly patients with osteoporotic vertebral compression fractures. METHODS: From August 2011 to August 2013, a total of 107 senile patients with osteoporotic vertebral compression fractures underwent percutaneous vertebroplasty were followed up for more than 2 ( average 2.5) years in Department of orthopedics, Xuanwu Hospital, Capital Medical University. The incidence of low back pain after vertebroplasty were analyzed on visual analog scale (VAS), and the relationship between BMD, vertebral fracture site were investigated. RESULTS: A total of 18 cases(16.8%)after vertebroplasty have significant low back pain. Along with BMD decreased [(-2.90±0.91) vs (-4.87±0.52)], the VAS of low back pain increasing, which showed statistical significance difference[(-3.12±0.91) vs (4.03±1.08), P<0.05]. The site of vertebral fracture was lower, the VAS was higher. CONCLUSIONS: About 16.8% patients show obvious low back pain after vertebroplasty. BMD and Vertebral fracture site were important factors of low back pain in elderly patients with osteoporotic vertebral compression fractures after vertebroplasty.

[Identification and verification of the candidate proteins that interact and collaborate with ATF3 in inhibiting hepatocarcinogenesis].

OBJECTIVE: To identify and verify proteins that interact and collaborate with ATF3 in inhibiting hepatocarcinogenesis. METHODS: Immunoprecipitation (IP), co-IP and protein spectrum analysis were used to identify the protein which interacted with ATF3 in HepG2. Immunohistochemistry (IHC) and Western blot (WB) were used to detect the expression pattern of ATF3 and its candidate interacting proteins in liver tissue. RESULTS: The protein expression differences were detected by IP in two HepG2 groups. The experimental group was infected by lentiviral vector with ATF3 over-expression and the control group was infected by mock-vehicle. Several protein bands with expression diversity were analyzed by protein spectrum, which revealed several candidate proteins that may be related with ATF3. Peptide sequences were analyzed by Mascot software and NCBI database. Combined with the existing literature and our study results, Gelsolin (GSN) was identified as a protein closely interacting with ATF3 and confirmed by co-IP, IHC and WB. CONCLUSIONS: GSN is identified and verified as an interacting protein with ATF3. ATF3 may function as a suppressor of liver cancer via protein-protein interactions with Gelsolin.

Analysis of POU1F1 gene DdeI polymorphism in Chinese goats.

As a member of the POU-domain family, the POU1F1 is a positive regulator for growth hormone, prolactin and thyroid-stimulating hormone b, by binding to target DNA promoters as a dimer in mammals. This study described the polymorphisms at the goat POU1F1-DdeI locus and analyzed the distribution of alleles in 15 indigenous Chinese goat breeds. The PCR-RFLP analysis showed a predominance of the D1D1 genotype and the D1 allele, with average frequencies of 0.550 and 0.790, respectively, irrespective of goat utility type. The D1D2 genotype was the second most frequent, with a mean frequency of 0.371. The distributions of genotypic and allelic frequencies at this locus were found to be significantly different among populations based on a Chi square test (P < 0.001), suggesting that the breed factor significantly affected the molecular genetic character of the POU1F1 gene. The genetic diversity analysis revealed that Chinese indigenous populations had a wide spectrum of genetic diversity at the goat POU1F1-DdeI locus. However, an ANOVA analysis revealed no significant differences in gene homozygosity, gene heterozygosity, effective allele numbers, or polymorphism information content among meat, dairy, and cashmere utility types (P > 0.05). This suggests that the goat utility types had no significant effect on the spectrum of genetic diversity.

Global transcriptional profiling of longissimus thoracis muscle tissue in fetal and juvenile domestic goat using RNA sequencing.

Domestic goats are important meat production animals; however, data from transcriptional profiling of skeletal muscle tissue in goat have thus far been scarce. We used comparative transcriptional profiling based on RNA sequencing of longissimus thoracis muscle tissue obtained from fetal goat muscle tissue (27 512 850 clean cDNA reads) and 6-month-old goat muscle tissue (27 582 908 reads) to identify genes that are differentially expressed, novel transcript units and alternative splicing events. Gene annotation revealed that 15 960 and 14 981 genes were expressed in the fetal and juvenile libraries respectively. We detected 6432 differentially expressed genes and, when considering GO terms, found 34, 27 and 55 terms to be significantly enriched in molecular function, cellular component and biological process categories respectively. Pathway analysis revealed that larger numbers of differentially expressed genes were enriched in fetal myogenesis or cell proliferation and differentiation-related pathways (such as Wnt), genes involved in the cell cycle and the Notch signaling pathway, and most of the differentially expressed genes involved in these pathways were downregulated in the juvenile goat library. These genes may be involved in various regulation mechanisms during muscle tissue differentiation between the two development stages examined herein. The identified novel transcript units, including both non-coding and coding RNA, as well as alternative splicing events increase the level of complexity of regulation mechanisms during muscle tissue formation and differentiation. Our study provides a comparative transcriptome analysis on goat muscle tissue, which will provide a valuable genomic resource for future studies investigating the molecular basis of skeletal muscle development.

Analysis of the polymorphisms in the caprine Gli3 gene and their associations with production traits in goats.

Gli3 is a zinc finger transcription factor which plays a critical role in regulating animal development, metabolism and energy partitioning and thus has the potential to influence economical important traits in farm animals. In this study, we screened the complete exons of the caprine Gli3 gene using PCR-SSCP methods in 430 individuals from three goat breeds to identify sequence variants that might be associated with growth traits. Six novel mutations (GU363952:g.739C>G, 749A>T, 1636C>A, 1982delT, 1983T>C, 2856T>C) were identified. Significant associations were observed between the mutations GU363952:g.739C>G and g.749A>T with body height, chest circumference and canon circumference. Individuals with genotype G4-CC/AA and G4-CG/AT were significantly higher than individuals with genotype G4-GG/TT in body height, chest circumference and canon circumference. The results of this study suggested that the Gli3-gene-specific SNP could be a useful marker for growth traits in future marker-assisted selection programs in goat.

Sex Differences in Presynaptic Density and Neurogenesis in Middle-Aged ApoE4 and ApoE Knockout Mice.

Atherosclerosis and apolipoprotein E ε4 (APOE4) genotype are risk factors for Alzheimer's disease (AD) and cardiovascular disease (CVD). Sex differences exist in prevalence and manifestation of both diseases. We investigated sex differences respective to aging, focusing on cognitive parameters in apoE4 and apoE knockout (ko) mouse models of AD and CVD. Presynaptic density and neurogenesis were investigated immunohistochemically in male and female apoE4, apoE ko, and wild-type mice. Middle-aged female apoE4 mice showed decreased presynaptic density in the inner molecular layer of the dentate gyrus of the hippocampus. Middle-aged female apoE ko mice showed a trend towards increased neurogenesis in the hippocampus compared with wild-type mice. No differences in these parameters could be observed in middle-aged male mice. Specific harmful interactions between apoE4 and estrogen could be responsible for decreased presynaptic density in female apoE4 mice. The trend of increased neurogenesis found in female apoE ko mice supports previous studies suggesting that temporarily increased amount of synaptic contacts and/or neurogenesis is a compensatory mechanism for synaptic failure. To our knowledge, no other studies investigating presynaptic density in aging female apoE4 or apoE ko mice are available. Sex-specific differences between APOE genotypes could account for some sex differences in AD and CVD.

The association of bovine T1R family of receptors polymorphisms with cattle growth traits.

The three members of the T1R class of taste-specific G protein-coupled receptors have been proven to function in combination with heterodimeric sweet and umami taste receptors in many mammals that affect food intake. This may in turn affect growth traits of livestock. We performed a comprehensive evaluation of single-nucleotide polymorphisms (SNPs) in the bovine TAS1R gene family, which encodes receptors for umami and sweet tastes. Complete DNA sequences of TAS1R1-, TAS1R2-, and TAS1R3-coding regions, obtained from 436 unrelated female cattle, representing three breeds (Qinchuan, Jiaxian Red, Luxi), revealed substantial coding and noncoding diversity. A total of nine SNPs in the TAS1R1 gene were identified, among which seven SNPs were in the coding region, and two SNPs were in the introns. All five SNPs in the TAS1R2 gene and all three SNPs in the TAS1R3 gene were identified in the coding region. Four SNPs (TAS1R1 g.5081C>T, TAS1R1 g.5110C>A, TAS1R2 g.288A>G, TAS1R2 g.2552T>C) were significantly associated with body height of Qinchuan cattle (P<0.05). The heterozygous genotypes of the four SNPs showed a molecular heterosis on cattle heights at hip cross and sacra. The individuals with different genotypic combinations of the four SNPs had significant association with heights at hip cross and sacra (P<0.05).

Molecular characterization and polymorphisms of the caprine Somatostatin (SST) and SST Receptor 1 (SSTR1) genes that are linked with growth traits.

Somatostatin (SST) and its receptors (SSTR1-5) appear to be important in central regulation of many metabolic systems that affect growth, adiposity and nutrient absorption. In this study, we investigated polymorphisms within the caprine SST and SSTR1 genes and determined their relationship with growth traits. As there were no sequence information of the caprine SST and SSTR1 genes, we explored their DNA sequence and genomic organizations. The caprine SST gene is organized in two exons and is transcribed into an mRNA containing 351 bp of sequence coding for a protein of 116 amino acids. Its protein sequences showed substantial similarity (97-99%) to its respective orthologs from cattle, human and mouse. We also cloned and sequenced a 1.2 kb DNA fragment which contained the major part of the coding region and 3' UTR of the caprine SSTR1 gene. We then detected the polymorphisms in these determined sequences by PCR-SSCP and DNA sequencing methods in 459 goats from four breeds. Four SNPs (GU014693:g.647T>C, GU014693:g.844A>C, GU014693:g.970T>C, GU014693:g.1039T>A), segregating as two haplotypes (T-A-T-T and C-C-C-A), were identified in intron 1 of the caprine SST gene and showed the associations to body length and body height (P < 0.05). Two SNPs (GU014695:g.801 C>T, GU014695:g.948 C>T) were identified in the caprine SSTR1 gene. Significant associations between the three genotypes of GU014695:801 C>T and body length, body height, and chest circumference was observed (P < 0.05). These results suggest that the caprine SST and SSTR1 genes are strong candidate genes that influence growth traits in goat.

The polymorphisms of bovine melanocortin-3 receptor pseudogene.

Mammalian melanocortin-3 receptor (MC3R) plays an important role in the central control of energy homeostasis, and several functional polymorphisms of mc3r have been detected. Interestingly, the bovine mc3r was a pseudogene, and its polymorphisms and function remain to be investigated. Single-strand conformation polymorphism (SSCP) showed 5, 2 and 3 genotypes in fragment F1, F2 and F3 of mc3r in seven cattle breeds, respectively. All genotypes revealed novel sequences. Three SNPs 657G>T, 756C>T, 822T>C were detected in fragment F1, five SNPs 1091T>C, 1133T>C, 1144C>T, 1259T>C and 1319G>A were detected in fragment F2, and two SNPs 1687G>A, 1860C>T were detected in F3. The SNPs in fragment F1 and F2 were located at exon 2. The five SNPs in fragment F2 demonstrated a tight linkage disequilibrium status. Variation detected here might have an impact on the function of bovine mc3r pseudogene.

Novel 12-bp deletion in the coding region of the bovine NPM1 gene affects growth traits.

The nucleophosmin 1 gene (NPM1) encodes a multifunctional nucleolar phosphoprotein that plays a crucial role in the control of various aspects of cell growth and homeostasis. In this study, the coding region of the NPM1 gene was screened in 1035 individuals of 4 Chinese cattle breeds by DNA sequencing and polyacrylamide gel electrophoresis. A novel 12-bp deletion mutation was identified in the coding region of the NPM1 gene. The PCR products of primer NPM1-P2 exhibited 3 genotypes and 2 alleles: 178 bp (denoted as W) and 166 bp (denoted as D). Genotype DD and allele D were predominant in the studied populations. Association analysis with growth traits in the Nanyang breed (N = 265) showed that the animals with genotype DD had significantly greater birth weight, body weight, body length, and heart girth than those with genotype WD (P < 0.01 or P < 0.05) at birth and after 6 months and 12 months, but not at 18 and 24 months of age. Results of this study suggest that the NPM1 gene is a candidate gene for growth traits in cattle.

Analysis of caprine pituitary specific transcription factor-1 gene polymorphism in indigenous Chinese goats.

Since mutations on POU1F1 gene possibly resulted in deficiency of GH, PRL, TSH and POU1F1, this study revealed the polymorphism of goat POU1F1-AluI locus and analyzed the distribution of alleles on 13 indigenous Chinese goat breeds. The PCR-RFLP analysis showed the predominance of TT genotype and the frequencies of allele T varied from 0.757 to 0.976 in the analyzed populations (SBWC, Bo, XH and HM). Further study, distributions of genotypic and allelic frequencies at this locus were found to be significantly different among populations based on a chi(2)-test (P < 0.001), suggesting that the breed factor significantly affected the molecular genetic character of POU1F1 gene. The genetic diversity analysis revealed that Chinese indigenous populations had a wide spectrum of genetic diversity in goat POU1F1-AluI locus. However, the ANOVA analysis revealed no significant differences for gene homozygosty, gene heterozygosty, effective allele numbers and PIC (polymorphism information content) among meat, dairy and cashmere utility types (P > 0.05), suggesting that goat utility types had no significant effect on the spectrum of genetic diversity.