Identifying deer antler proliferation and mineralization genes using comparative RNA-seq
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ABSTRACT: Purpose: The goal of this study is to compare (RNA-seq) transcriptomes of in vitro cultured human bone marrow-derived mesenchymal stem cells (hMSCs) and fallow deer antler-derived skeletal progenitors (FD RM Cells) under multiple conditions to identify candidate proliferation and mineralization genes responsible for fast antler regeneration Methods: hMSCs and FD RM Cells were cultured in vitro under 1) serum-free (0% serum) or serum (10% serum) conditions for 2.5 days or 2) Control (0 ng/mL BMP-2 and 0 nM dexamethasone) and osteogenic (100 ng/mL BMP-2 and 100 nM dexamethasone) media for 24 days. mRNA profiles were generated by deep sequencing, in duplicate, using Illumina HiSeq 2000. The sequence reads were analyzed at the transcript isoform level STARS followed by Cufflinks. Validation for genes of interest was performed using immunofluorescence staining. Results: Comparison of human and fallow deer skeletal progenitor datasets yielded proliferation and mineralization gene candidates Conclusions: Our study represents the first detailed analysis of human and fallow deer transcriptomes of skeletal progenitor cells under proliferation and mineralization conditions, with biologic replicates, generated by RNA-seq technology. Our in vitro comparative approach circumvent some of the logistical and technical challenges in identifying candidate proliferation and mineralization genes responsible for rapid deer antler regeneration. We conclude that in vitro comparison of RNA-seq based transcriptomes identified candidate proliferaiton and mineralization genes to advance bone biology and holds promise to rapidly regenerate large bone volumes for regenerative medicine. The comparative approach utilized here can be adapted for almost any tissue to study a specific phenomenon of interest.
Project description:Rib bone growth in red deer stags - Abstract: In 'The Bone and Joint Decade' interest is focused on genetic factors causing bone disorders. Osteoporosis, attacking 10% of the population worldwide, is the most common metabolic bone disease, which is mimiced by several ovarectomised or genetically modified 'cascadeur' animal species, but none of them is able to remedy its pathologically porous bone tissue. Regeneration in skeletal elements is the curiosity of our newly investigated osteoporosis animal model, red deer (Cervus elaphus). The cyclic physiological osteoporosis in red deer stag is a consequence of the annual antler cycle. This phenomenon raises the possibility to explore new genes involved in regulating bone mineral density (BMD) and recovery of bone resorption on the basis of comparative genomics between deer and human. Here we compared the gene expression activities of osteoporotic and regenerating flying rib bone samples versus late autumn dwell control in red deer by heterologous microarray hybridization. Identified genes were tested on human femoral bone tissue from postmenopausal osteoporotic and non-osteoporotic patients. Expression data were evaluated by Principal Components Analysis and Discriminant Analysis. Keywords: Gene Expression experiment Approximately 2-3 g flying rib bone pieces in the entire cross section of bony rib were surgically removed from 3 anaesthetized [SBH-Ketamine (2.5 mg/kg live weight) combined with Xylazine (0.2 mg/kg live weight) i.m. injection] 6, 7 and 8 year old Cervus elaphus stags. (Cast antler pairs weighed 7-8 kg for each animal.) Removed rib pieces were extensively washed in PBS for eliminating blood and marrow contamination, than immediately frozen in liquid nitrogen. The time of tissue collections were (i) within the period of the active mineralization of antler, at the beginning of June when skeletal osteoporosis takes place, (ii) in the fitness improvement period with velvet shedding in late July, that is the 'regenerating time' and (iii) in the period of late autumn dwell at the end of November when in the skeleton the mineral mobilization and deposition are dynamically equilibrated (BMD is in steady state). Each comparison performed on Platforms GPL4052 and GPL5352.
Project description:The therapeutic functions of stem cells were mainly depending on paracrine effects rather than their own proliferation and differentiation on site. In advantage of easy to collected from conditioned medium, have contents of cytokines, growth factors, signaling lipids, mRNAs, and regulatory miRNAs, exosomes from stem cells were considered to be a promising material for stem cell-based cell-free therapies. In traditional Chinese medicine, antler extracts have been used to alleviates aging and aging related diseases. We have demonstrated that exosomes from antler stem cells (ASCs), the source of the annual full regeneration of deer antler, could alleviate cellular senescence and aging related diseases. With exosomes treatment, the senescent phenotype of primary MSCs and hMSCs at late passage were significantly mitigated. When injected into articular cavity, exosomes from ASCs markedly promote joint function recovery and bone and cartilage regeneration. By LC-MS/MS assay, we found that exosomes from ASCs contain various proteins which in the regulation of biological processes including cell proliferation and migration, immunoregulation, wound healing and angiogenesis. With the advantages of abundant sources, low risk of tumorigenicity and immunogenicity, exosomes from ASCs may consider as a promising bio-material for cell-free therapies in the future.
Project description:we used proteomic technology to disclose the difference of antler regeneration between red deer and sika deer. Through functional analysis, we obtained differentially expressed proteins and the pathway involved in antler regeneration between two groups
Project description:To elucidate the complex physiological process of the growth, development and immunity response of Sika Deer, this study evaluated the changes of miRNA profiles in the four developmental stages (juvenile, adolescence, adult and aged) of ten tissue (adrenal, antler, brain, heart, kidney, lung, liver, skeletal muscle, spleen and testes). The results showed that a total of 306 known miRNAs and 143 novel miRNAs were obtained. Many miRNAs displayed organ-specificity and age-specificity. The largest number of miRNAs were enriched in the brain, some of which were shared only between the brain and adrenal. These miRNAs were involved in maintaining specific functions within the brain and adrenal. Additionally, the adolescence-adult transition of Sika Deer was a crucial stage in its life cycle. In conclusion, our study provided abundant data support for the current research Sika Deer. It also contributes to understand the role of miRNAs play in regulating the growth, development and immunity response of Sika Deer.
Project description:Deer antlers are amazing natural appendages that grow faster than any other known mammalian bone. Antler growth occurs at the tip and is initially cartilage, which is later replaced by bone tissue. However, little is known regarding the precise role of cooperation between cell lineages and functional genes in regulating antler growth, and molecular mechanisms responsible for rapid growth remain elusive. In this study, we use an RNA-Seq approach to identify miRNA expression patterns during antler growth.
Project description:Rib bone growth in red deer stags - Abstract: In 'The Bone and Joint Decade' interest is focused on genetic factors causing bone disorders. Osteoporosis, attacking 10% of the population worldwide, is the most common metabolic bone disease, which is mimiced by several ovarectomised or genetically modified 'cascadeur' animal species, but none of them is able to remedy its pathologically porous bone tissue. Regeneration in skeletal elements is the curiosity of our newly investigated osteoporosis animal model, red deer (Cervus elaphus). The cyclic physiological osteoporosis in red deer stag is a consequence of the annual antler cycle. This phenomenon raises the possibility to explore new genes involved in regulating bone mineral density (BMD) and recovery of bone resorption on the basis of comparative genomics between deer and human. Here we compared the gene expression activities of osteoporotic and regenerating flying rib bone samples versus late autumn dwell control in red deer by heterologous microarray hybridization. Identified genes were tested on human femoral bone tissue from postmenopausal osteoporotic and non-osteoporotic patients. Expression data were evaluated by Principal Components Analysis and Discriminant Analysis. Keywords: Gene Expression experiment
Project description:We investigate cellular responses to traditional bioactive mineral-based nanomaterials, such as hydroxyapatite (nHA), whitlockite (nWH), silicon-dioxide (nSiO2), and the emerging synthetic 2D nanosilicates (nSi), on human mesenchymal stem cells (hMSCs). Specifically, using next-generation transcriptome sequencing (RNA-seq), we uncover key signaling pathways and transcriptome dynamics due to exposure to these inorganic nanomaterials. Whole-transcriptome sequencing identified a stabilized skeletal progenitor state in stem cells treated with nanosilicates that suggests endochondral differentiation of hMSCs, resulting in increased deposition of matrix mineralization compared with control or other treatment groups.
Project description:As the only regenerative organ of mammals, antler could grow rapidly without carcinogenesis. To understand the molecular mechanisms of the growth of sika deer antler, we used de novo RNA-seq analyses to determine the differential expression of unigenes and miRNAs from antler at 15, 60, 90, and 110-day. A total of 55004 unigenes, 208 known miRNAs and 38 novel miRNAs were identified. 10182 unigenes and 35 miRNAs were differentially expressed between 60-day and 15-day antler, 13258 unigenes and 53 miRNAs were differentially expressed between 90-day and 60-day antler, and 10740 unigenes and 27 miRNAs were differentially expressed between 110-day and 90-day antler. GO and KEGG analyses showed that DE unigenes and miRNA were mainly related to chondrogenesis, osteogenesis and inhibition of oncogenesis, that were closely associate with antler growth. We also constructed mRNA-mRNA and miRNA-mRNA interaction networks related to chondrogenesis, osteogenesis and inhibition of oncogenesis of antler. The results showed that mRNA (COL2A1, SOX9, WWP2, FGFR1, SPARC, LOX etc.) and miRNAs (miR-145, miR-199a-3p, miR-140, miR-199a-5p etc.) may play important roles in chondrogenesis and osteogenesis of antler, and mRNA (TP53, Tpm3 and ATP1A1 etc.) and miRNAs (miR-106a, miR-145, miR-1260b and miR-2898 etc.) may have key roles in inhibiting the carcinogenesis of antlers. In this study, we identified miRNAs and unigenes related to chondrogenesis, osteogenesis and inhibition of oncogenesis of antler. This will provide a reference for in-depth analysis of the molecular mechanism of antler growth without carcinogenesis, and also provide valuable information for cartilage- and bone-related disease treatment, cancer treatment.
Project description:As the only regenerative organ of mammals, antler could grow rapidly without carcinogenesis. To understand the molecular mechanisms of the growth of sika deer antler, we used de novo RNA-seq analyses to determine the differential expression of unigenes and miRNAs from antler at 15, 60, 90, and 110-day. A total of 55004 unigenes, 208 known miRNAs and 38 novel miRNAs were identified. 10182 unigenes and 35 miRNAs were differentially expressed between 60-day and 15-day antler, 13258 unigenes and 53 miRNAs were differentially expressed between 90-day and 60-day antler, and 10740 unigenes and 27 miRNAs were differentially expressed between 110-day and 90-day antler. GO and KEGG analyses showed that DE unigenes and miRNA were mainly related to chondrogenesis, osteogenesis and inhibition of oncogenesis, that were closely associate with antler growth. We also constructed mRNA-mRNA and miRNA-mRNA interaction networks related to chondrogenesis, osteogenesis and inhibition of oncogenesis of antler. The results showed that mRNA (COL2A1, SOX9, WWP2, FGFR1, SPARC, LOX etc.) and miRNAs (miR-145, miR-199a-3p, miR-140, miR-199a-5p etc.) may play important roles in chondrogenesis and osteogenesis of antler, and mRNA (TP53, Tpm3 and ATP1A1 etc.) and miRNAs (miR-106a, miR-145, miR-1260b and miR-2898 etc.) may have key roles in inhibiting the carcinogenesis of antlers. In this study, we identified miRNAs and unigenes related to chondrogenesis, osteogenesis and inhibition of oncogenesis of antler. This will provide a reference for in-depth analysis of the molecular mechanism of antler growth without carcinogenesis, and also provide valuable information for cartilage- and bone-related disease treatment, cancer treatment.