miR-628-5p promotes growth and migration of osteosarcoma by targeting IFI44L.

This study investigated the role of miR-628-5p and interferon-induced protein 44-like (IFI44L) in osteosarcoma (OS) and determined whether miR-628-5p modulated OS growth by regulating IFI44L. Based on the data downloaded from Gene Expression Omnibus (GEO) database, we revealed that the expression of IFI44L was downregulated in OS and low expression of IFI44L was correlated with better prognosis of patients with OS. Biological prediction of its upstream regulatory miRNAs on the miRWalk website found that miR-628-5p is a possible upstream regulatory miRNA of IFI44L. Luciferase activity assay demonstrated that miR-628-5p could bind to the 3' untranslated region (UTR) of IFI44L, which proved the above prediction. The expression of miR-628-5p is upregulated in OS and high expression of miR-628-5p is correlated with poor prognosis of patients with OS. The results of RT-qPCR showed that the expression of miR-628-5p in MG-63, U2OS, Saos-2, and SW1353 cells was significantly higher than that in the hFOB1.19 cells. Downregulation of miR-628-5p by miR-628-5p inhibitor significantly inhibited the proliferation, migration, and invasion of MG-63 cells. By rescue assay, we found that knockdown of IFI44L rescued the proliferation and motility of miR-628-5p depleted MG-63 cells. Collectively, our present data illustrated that miR-628-5p promoted the growth and motility of OS at least partly by targeting IFI44L. Moreover, miR-628-5p and IFI44L might be proposed as promising biomarkers in OS diagnosis and treatment.

Haplotype combination of SREBP-1c gene sequence variants is associated with growth traits in cattle.

The aim of this study was to examine the association of the SREBP-1c polymorphism with growth traits in cattle breeds. Five sequence variants (SVs) were identified within the bovine sterol regulatory element-binding protein-1c gene (SREBP-1c), using DNA sequencing, PCR, PCR­RFLP, and forced PCR­RFLP methods. These polymorphisms include three missense mutations (SV1, SV4, and SV5) in exons 7, 9, and 12, a silent mutation (SV3) in exon 9, and a large deletion (SV2) in intron 7. Overall, we report the validation of polymorphisms within the bovine SREBP-1c gene, and the haplotype variability and extent of linkage disequilibrium (LD) in 1061 individuals representing the five main cattle breeds from China. We also investigated haplotype frequencies and LD coefficients for five SVs in all study populations. LD and haplotype structure of SREBP-1c were different between breeds. The result of haplotype analysis of five SVs showed that 27 different haplotypes were identified by all breeds. Two haplotypes (Hap1 and Hap2) shared by all five populations accounted for 42.75%, 35.68%, 36.44%, 25.43%, and 96.26% of all haplotypes observed in the cattle breeds Nanyang, Qinchuan, Jiaxian, Jinnan, and Chinese Holstein, respectively. The statistical analyses indicated that one single SV and 38 combined haplotypes were significantly associated with growth traits in the Nanyang cattle population (P < 0.05 or P < 0.01). The results of this study suggest that the SREBP-1c gene possibly is a strong candidate gene that affects growth traits in the Chinese beef cattle breeding program.

The polymorphisms of bovine VEGF gene and their associations with growth traits in Chinese cattle.

PCR-SSCP and DNA sequencing methods were employed to screen the genetic variation of VEGF gene in 671 individuals belonging to three Chinese indigenous cattle breeds including Nanyang, Jiaxian Red and Qinchuan. Three haplotypes (A, B and C), four observed genotypes (AA, AB, BB and AC) and three new SNPs (6765T>C ss130456744, 6860A>G ss130456745, 6893T>C ss130456746) were detected. The analysis suggested that one SNP (ss130456744) in the bovine VEGF gene had significant effects on birth weight, body weight and heart girth at 6 months old in the Nanyang breed (P < 0.05). The results showed that the SNP (ss130456744) in intron 2 of the VEGF gene is associated with early development and growth of Chinese cattle. These findings raise hope that this polymorphism can be a molecular breeding marker in breeding strategies through marker assisted selection (MAS) in Chinese domestic cattle.

Polymorphisms in the bovine ghrelin precursor (GHRL) and Syndecan-1 (SDC1) genes that are associated with growth traits in cattle.

Transgenically expressed Syndecan-1 was found in the hypothalamic nuclei that control energy balance, and was associated with maturity-onset obesity, while ghrelin has been shown to play important roles in the control of food intake, gastric acid secretion, energy homeostasis, and glucose and lipid metabolism. However, the roles of genetic variations of Syndecan-1 and ghrelin on growth trait have few been reported in cattle. Herein, five Chinese cattle breeds were analyzed by PCR-SSCP and DNA sequencing methods. The bovine ghrelin gene showed eleven SNPs g.[267G>A, 271G>A, 290C>T, 326A>G, 327T>C, 420C>A, 569A>G, 945C>T, 993C>T, 4491A>G, 4644G>A] and three SNPs g.[420C>A, 569 A>G, 945C>T] were firstly detected in cattle. The bovine Syndecan-1 gene showed two SNPs. One SNP showed a transition C>G at position 21514, resulting in a synonymous mutation p.G(GGC)169G(GGG) and another showed a transversion C>T at position 22591, resulting in a synonymous mutation p.D(GAC)283D(GAT). In ghrelin gene, no significant associations were revealed between any variant sites and body weight, average daily gain, body sizes for different growth periods (6, 12, 18, and 24 months old), as well as for the milk yield at 305 days, milk protein rate and milk fat percentage. However, the polymorphism of Syndecan-1 gene was significantly associated with bovine birth weight and body length. Hence, we first suggested that Syndecan-1 gene could be regarded as molecular marker for superior birth weight and body length.

Analysis of the genetic variation of vascular endothelial growth factor gene in three Chinese indigenous cattle breeds.

PCR-SSCP and DNA sequencing methods were employed to screen the genetic variation of vascular endothelial growth factor (VEGF) gene in 675 individuals belonging to three Chinese indigenous cattle breeds including Qinchuan (QC), Jiaxian Red (JX) and Nanyang (NY) breed. Three new single nucleotide polymorphisms (SNPs) (g.6765T > C ss130456744, g.6860A > G ss130456745, g.6893T > C ss130456746) were found. One SNP (g.6765T > C) was detected in intron II of VEGF gene in all three breeds and the other two SNPs (g.6860A > G, g.6893T > C) were in exon III of VEGF gene only in NY breed. Among them, two synonymous mutations of exon III were identified: CCA (Pro) > CCG (Pro) at position 65th amino acid (aa) and TGT (Cys) > TGC (Cys) at position 76th aa of VEGF(190aa) in NY breed. Our study revealed that NY breed exhibited the most abundant genetic diversity in VEGF gene within the three cattle breeds. Furthermore, JX cattle breed was more similar to QC breed than to NY breed. Our genetic data in the present study supported the hypothesis that the distribution pattern of Chinese indigenous cattle breeds was closely related to the geographical and climatic background again.

A large indel mutation of the bovine ADD1/SREBP1c gene and its effects on growth traits in some native cattle breeds from China.

Adipocyte determination and differentiation-dependent factor 1/sterol regulatory element-binding protein-1c (ADD1/SREBP1c) is a major determinant of tissue differential lipogenic capacity in mammalian and avian species. The objectives of the present study were to focus on insertion-deletion polymorphism (indel) in the bovine ADD1/SREBP1c gene, and analyzing its effect on growth traits in a sample of 1035 cattle belonging to four Chinese cattle breeds. PCR-SSCP, DNA sequencing and agarose electrophoresis methods were used. The 778 bp PCR products of ADD1/SREBP1c gene exhibited three genotypes and two alleles were revealed: W and D. Frequencies of the W allele varied from 0.8651 to 1.000. The associations of the 84 bp indel mutation of ADD1/SREBP1c gene with growth traits of 265 Nanyang cows were analyzed. The animals with genotype WD had significantly higher birth weight, body weight, average daily gain than those with genotype WW at birth, 6-, 12-, 18-, and 24-month old (P < 0.05 or P < 0.01). These results suggest that the indel mutation of bovine ADD1/SREBP1c gene may influence the growth traits in cattle.

Each one of certain histidine residues in G-protein-coupled receptor GPR4 is critical for extracellular proton-induced stimulation of multiple G-protein-signaling pathways.

GPR4, previously proposed as the receptor for sphingosylphosphorylcholine, has recently been identified as the proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways, including the G(s)-protein/cAMP, G(12/13)-protein/Rho, and G(q)-protein/phospholipase C pathways. In the present study, we examined whether extracellularly located histidine residues of GPR4 sense extracellular protons and, if so, whether a certain histidine residue is critical for coupling to the single or multiple signaling pathway(s). We found that the mutation of histidine residue at 79, 165, or 269 from the N-terminal of GPR4 to phenylalanine shifted the half-maximal effective concentration (EC(50)) of proton-induced signaling activities to the right, including cAMP accumulation, SRE promoter activity reflecting Rho activity, and NFAT promoter activity reflecting phospholipase C signaling activity, without an appreciable change in the maximal activities. These results suggest that the protonation of each one of histidine residues at 79, 165, and 269 in GPR4 may be critical for conformational change of the receptor for coupling to multiple intracellular signaling pathways through G-proteins.

Two novel coding SNPs of SREBP1c gene are associated with body weight and average daily gain in bovine.

It is known that the SREBP1c gene is an important gene responsible for adipogenesis and regulation of the expression of genes controlling fatty acid biosynthesis. Its expression levels increase in parallel with obesity. Therefore, the present study focused on screening the genetic variation within bovine SREBP1c gene and analyzing its effect on growth traits in 1035 individuals belonging to four Chinese cattle breeds (QC, NY, JX, CH) using PCR-SSCP, DNA sequencing, and forced PCR-RFLP methods. The results revealed two novel mutations: NC_007317: g. 10781 C > A (457aa), 10914 G > A (502aa). Association analysis with growth traits in the Nangyang breed indicated that: The SNPs in the bovine SREBP1c gene had significant effects on body weight and average daily gain at birth, 6 and 12 months old (P < 0.05 or P < 0.01). Therefore, these results suggest that the SREBP1c gene is a strong candidate gene that affects growth traits in cattle.

Associations of polymorphism within the GHSR gene with growth traits in Nanyang cattle.

GH secretagogue receptor (ghrelin receptor, GHSR) is known to be involved in the control of GH release by mediating the strong stimulatory effect of the endogenous ligand, ghrelin, on GH secretion. Associations between the GHSR gene polymorphism and the growth traits were revealed in Nanyang cattle. The mutations at nt456(G > A) and nt667(C > T) were complete linkage and located in exon 1 of the coding region of the GHSR gene. Least squares analysis revealed a significant statistical effect (P < 0.05) of the GHSR gene different genotypes on body weight and average daily gain at 6 months of age in Nanyang cattle. Individuals with GHSR-MM genotype showed higher body weight and average daily gain than individuals with GHSR-MN genotype.

[The polymorphism of bovine POMC gene and its association with the growth traits of Nanyang cattle].

Pro-opiomelanocortin (POMC) plays an important role on animal ingestive behavior and energy homeostasis. Genetic variation of POMC was detected by PCR-SSCP and DNA sequencing techniques in 480 individuals from seven cattle breeds (i.e., Qinchuan, Nanyang, Jiaxian Red cattle, Jinnan, Luxi, Angus, and Chinese Holstein), and association analyses were carried out to evaluate the effects of genotypes of the candidate genes on growth traits in Nanyang cattle. Three SNPs (811845 C>T, 811821 T>C and 811797 A>G, ref. NW_928357) in linkage were detected in P3 locus, which were located at the 3' flanking region of bovine POMC gene. The body weight at 6-month-old and the average daily gain of 0~6-month-old were higher in Nanyang cattle with the genotype BB than with the genotype AA (P<0.05).

Previously postulated "ligand-independent" signaling of GPR4 is mediated through proton-sensing mechanisms.

GPR4 was initially identified as a receptor for sphingosylphosphorylcholine and lysophosphatidylcholine; however, lipid actions have not always been confirmed. Instead, ligand-independent actions have sometimes been observed in GPR4- and other OGR1 family receptor-expressing cells. Here, we examined the possible involvement of extracellular protons, which have recently been proposed as another ligand for GPR4. At pH 7.4, the epidermal growth factor-induced extracellular signal-regulated kinase activity was lower in GPR4-transfected RH7777 cells, in association with increased cAMP accumulation, than in vector-transfected cells. The serum response element (SRE)-driven transcriptional activity was also clearly higher in GPR4-expressing HEK293 cells than in vector-transfected cells at pH 7.4. These apparent ligand-independent actions were very small at alkalinic 7.8. The SRE activity was further increased by extracellular acidification in a manner dependent on the G13 protein/Rho signaling pathway in HEK293 cells expressing GPR4 or other OGR1 receptor family members. GPR4-expressing cells also showed a calcineurin-dependent nuclear factor of activated T cell (NFAT) promoter activation at pH 7.4, and this activity was further increased by pH below 7.2 in association with inositol phosphate production. In contrast to the cAMP and SRE responses, however, alkalinization to pH 7.8 hardly affected the high basal activity. Finally, the expression of GPR4 hardly modulated the sphingosylphosphorylcholine- or lysophosphatidylcholine-induced action. These results suggest that an extracellular proton play a role as a ligand in some of previously postulated ligand-independent actions through GPR4 receptors. Moreover, GPR4 may be a multi-functional receptor coupling to Gs, G13, and Gq/11 proteins in response to extracellular acidification.

[Association of polymorphisms of DGAT2 gene with growth traits in Jiaxian red cattle].

The polymorphisms of DGAT2 gene in 92 Jiaxian red cattle were analyzed using PCR-RFLP method. Results showed that the PCR products digested with TaqI demonstrated polymorphisms, and showed two kinds of genotype: AA and AB. This polymorphic locus of DGAT2 gene was at Hardy-Weinberg equilibrium (P>0.05). The results of general linear model analysis showed that polymorphisms of DGAT2 gene had significant association (Plt;0.05) with body weight and body length, while had no significant association with others growth traits (P>0.05). Therefore, genotype AB may have positive effect on body weight.

[Polymorphisms of ZAG gene with growth traits in Jiaxian red cattle].

The main function of ZAG gene is to enable the decomposition of fat, and reduced fat content. In this study, polymorphisms of four loci (Z1, Z2, Z3, Z4) at the coding region of the bovine ZAG gene were detected in 145 Jiaxian red cattle, and polymorphisms were found on Z1, Z3, Z4 loci. The fragments showing different SSCP patterns were sequenced, nd a total of six SNPs (C115T, A3257G, A4013G, T4027C, C4032T, and T4120C) were found. The Z3 locus of ZAG gene was at Hardy-Weinberg equilibrium, while Z1 and Z4 loci of ZAG gene were not at Hardy-Weinberg disequilibrium. The association analysis of different genotypes with some of the growth and development traits showed that the individuals with genotype AC had a larger body length, heart girth, circumference of cannon bone, body weight than the individuals with genotypes AA and AB (P<0.05 or P<0.01, respectively) in Z4 locus. It is suggest that Z4 locus may be one of the marker-assisted selection used as growth traits in Jiaxian red cattle.

[Molecular genetic characteristic of bovine HTR1B gene].

The aim of the present study was to identify and characterize polymorphisms within the coding region and the 3' flanking region of the bovine serotonin receptor 1B gene among different cattle breeds. Four DNA fragments were amplified by polymerase chain reaction and then used for polymorphism identification by SSCP. The fragments showing different SSCP patterns were sequenced. And a total of six SNPs (G205T, C507T, C546G, C744T, G816A and G942A) were detected. The SNPs were at Hardy-Weinberg equilibrium except C507T and C546G in all genetic population. The frequencies of allele 205T of Holstein were much higher than that of the other six beef cattle populations. Almost the PIC of all SNPs were not more than 0.10 except that of G205T in Holstein cows, which indicated the bovine HTR1B gene was conserved.

[Polymorphisms of DGAT2 gene and its associations with several growth traits in Nanyang cattle].

The associations between SNPs in intron 6 and intron 7 of the bovine diacylglycerolacyltransferase (DGAT2) gene and growth traits in purebred Nanyang cattle (n=131) were reported in this study. Alleles detected in intron 6 were allele A and B with frequencies of 0.875 and 0.125, respectively; alleles detected in intron 7 were allele N and M with frequencies of 0.968 and 0.032, respectively. The animals with genotype AA in intron 6 showed 3.8% increases in body height (P < 0.01), 3.9% increases in body weight (P < 0.05), 3.8% (P < 0.05), 3.6% (P < 0.05), 3.8% (P < 0.01) and 3.1%( P < 0.01) increase in body length at age six-month to two-year compared with genotype AB, respectively. They also showed 3.8 % (P < 0.05), 3.4 % ( P < 0.01), 3.7% (P < 0.01) and 4.3% (P < 0.01) increases in heart girth at age six-month to two-year respectively. Animals with the NM genotype show significantly higher average heart girth at age two years (P < 0.05), and Hucklebone width (P < 0.01) at age eighteen-month (P < 0.01) and two-year (P < 0.01) compared with animals with the genotype NN, respectively.

Low-intensity pulsed ultrasound prompts tissue-engineered bone formation after implantation surgery.

BACKGROUND: A practical problem impeding clinical translation is the limited bone formation seen in artificial bone grafts. Low-pressure/vacuum seeding and dynamic culturing in bioreactors have led to a greater penetration into the scaffolds, enhanced production of bone marrow cells, and improved tissue-engineered bone formation. The goal of this study was to promote more extensive bone formation in the composites of porous ceramics and bone marrow stromal cells (BMSCs). METHODS: BMSCs/ß-tricalcium phosphate (ß-TCP) composites were subcultured for 2 weeks and then subcutaneously implanted into syngeneic rats that were split into a low-intensity pulsed ultrasound (LIPUS) treatment group and a control group. These implants were harvested at 5, 10, 25, and 50 days after implantation. The samples were then biomechanically tested and analyzed for alkaline phosphate (ALP) activity and osteocalcin (OCN) content and were also observed by light microscopy. RESULTS: The levels of ALP activity and OCN content in the composites were significantly higher in the LIPUS group than in the control group. Histomorphometric analysis revealed a greater degree of soft tissue repair, increased blood flow, better angiogenesis, and more extensive bone formation in the LIPUS groups than in the controls. No significant difference in the compressive strength was found between the two groups. CONCLUSION: LIPUS treatment appears to enhance bone formation and angiogenesis in the BMSCs/ß-TCP composites.

SNP identification in FBXO32 gene and their associations with growth traits in cattle.

The F-box protein 32 (FBXO32), also known as Atrogin-1, is one of the four subunits of the ubiquitin protein ligase complex. FBXO32 has been previously shown to be involved in regulation of initiation and development of muscle mass. In the present study, we investigated the polymorphism of FBXO32 gene in 1313 cattle from seven bovine breeds using DNA sequencing, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and PCR-based amplification-created restriction site (PCR-ACRS) methods. Four novel single nucleotide polymorphisms (SNPs) were identified within bovine FBXO32, and were deposited in the GenBank database. The association studies between these four SNPs and growth traits were performed in NanYang cattle. Notably, the SNPs ss411628932 and ss411628936 were shown to be significantly associated with body length of 24-month-old NanYang cattle. Based on the above four SNPs, 16 haplotypes were identified. The main haplotype was AATA, which occurred at a frequency of more than 40%. Additionally, phylogenetic analysis showed that geographical distance was essential to gene flow among seven cattle breeds. Indigenous bovine breeds displayed genetic difference in comparison to hybrid bovine breeds that have foreign origins. We herein describe for the first time a comprehensive study on the variability of bovine FBXO32 gene that is predictive of genetic potential for body length phenotype.

Ovarian cancer G-protein-coupled receptor 1 induces the expression of the pain mediator prostaglandin E2 in response to an acidic extracellular environment in human osteoblast-like cells.

Although bone pain in osteoporosis and skeletal metastasis is an expected consequence of fracture, there are other underlying causes responsible. Our study demonstrated that ovarian cancer G-protein-coupled receptor 1 detected extracellular protons in MG63 cells, and regulated osteoblast functions, such as prostaglandin E2 production, in response to acidic circumstances. In this work, we measured inositol phosphate production, intracellular Ca(2+) concentration, prostaglandin E2 production, and cyclic adenosine monophosphate accumulation in MG63 cells exposed to extracellular acidification. Extracellular acidity induced a transient increase in Ca(2+) concentration and inositol phosphate production. Acidification also induced prostaglandin E2 production, resulting in cyclic adenosine monophosphate accumulation. A small interfering RNA specific for the ovarian cancer G-protein-coupled receptor 1 markedly inhibited these proton-induced actions in MG63 cells. These results indicated that the involvement of ovarian cancer G-protein-coupled receptor 1 in acidic extracellular environment may be an underlying mechanism responsible for bone pain in osteoporosis or bone metastasis without clinically proved fractures.

Cyclooxygenase-2 expression and prostaglandin E2 production in response to acidic pH through OGR1 in a human osteoblastic cell line.

Acidosis has been shown to induce depletion of bone calcium from the body. This calcium release process is thought to be partially cell mediated. In an organ culture of bone, acidic pH has been shown to induce cyclooxygenase-2 (COX-2) induction and prostaglandin E(2) (PGE(2)) production, resulting in stimulation of bone calcium release. However, the molecular mechanisms whereby osteoblasts sense acidic circumstances and thereby induce COX-2 induction and PGE(2) production remain unknown. In this study, we used a human osteoblastic cell line (NHOst) to characterize cellular activities, including inositol phosphate production, intracellular Ca(2+) concentration ([Ca(2+)](i)), PGE(2) production, and COX-2 mRNA and protein expression, in response to extracellular acidification. Small interfering RNA (siRNA) specific to the OGR1 receptor and specific inhibitors for intracellular signaling pathways were used to characterize acidification-induced cellular activities. We found that extracellular acidic pH induced a transient increase in [Ca(2+)](i) and inositol phosphate production in the cells. Acidification also induced COX-2 induction, resulting in PGE(2) production. These proton-induced actions were markedly inhibited by siRNA targeted for the OGR1 receptor and the inhibitors for G(q/11) protein, phospholipase C, and protein kinase C. We conclude that the OGR1/G(q/11)/phospholipase C/protein kinase C pathway regulates osteoblastic COX-2 induction and subsequent PGE(2) production in response to acidic circumstances.

Role of lipoprotein-associated lysophospholipids in migratory activity of coronary artery smooth muscle cells.

The migration of vascular smooth muscle cells (SMCs) is a hallmark of the pathogenesis of atherosclerosis and restenosis after angioplasty. Plasma low-density lipoprotein (LDL), but not high-density lipoprotein (HDL), induced the migration of human coronary artery SMCs (CASMCs). Among bioactive lipids postulated to be present in LDL, lysophosphatidic acid (LPA) appreciably mimicked the LDL action. In fact, the LDL-induced migration was markedly inhibited by pertussis toxin, an LPA receptor antagonist Ki-16425, and a small interfering RNA (siRNA) targeted for LPA(1) receptors. Moreover, LDL contains a higher amount of LPA than HDL does. HDL markedly inhibited LPA- and platelet-derived growth factor (PDGF)-induced migration, and sphingosine 1-phosphate (S1P), the content of which is about fourfold higher in HDL than in LDL, mimicked the HDL action. The inhibitory actions of HDL and S1P were suppressed by S1P(2) receptor-specific siRNA. On the other hand, the degradation of the LPA component of LDL by monoglyceride lipase or the antagonism of LPA receptors by Ki-16425 allowed LDL to inhibit the PDGF-induced migration. The inhibitory effect of LDL was again suppressed by S1P(2) receptor-specific siRNA. In conclusion, LPA/LPA(1) receptors and S1P/S1P(2) receptors mediate the stimulatory and inhibitory migration response to LDL and HDL, respectively. The balance of not only the content of LPA and S1P in lipoproteins but also the signaling activity between LPA(1) and S1P(2) receptors in the cells may be critical in determining whether the lipoprotein is a positive or negative regulator of CASMC migration.