scholarly journals High-Throughput Identification of MiR-145 Targets in Human Articular Chondrocytes

Life ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 58
Author(s):  
Aida Martinez-Sanchez ◽  
Stefano Lazzarano ◽  
Eshita Sharma ◽  
Helen Lockstone ◽  
Christopher L. Murphy
Keyword(s):  
Cartilage Repair ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Articular Chondrocytes ◽  
Articular Chondrocyte ◽  
Coding Region ◽  
Cartilage Development ◽  
Rna Immunoprecipitation ◽  
Important Regulator ◽  
Mmp13 Expression

MicroRNAs (miRNAs) play key roles in cartilage development and homeostasis and are dysregulated in osteoarthritis. MiR-145 modulation induces profound changes in the human articular chondrocyte (HAC) phenotype, partially through direct repression of SOX9. Since miRNAs can simultaneously silence multiple targets, we aimed to identify the whole targetome of miR-145 in HACs, critical if miR-145 is to be considered a target for cartilage repair. We performed RIP-seq (RNA-immunoprecipitation and high-throughput sequencing) of miRISC (miRNA-induced silencing complex) in HACs overexpressing miR-145 to identify miR-145 direct targets and used cWords to assess enrichment of miR-145 seed matches in the identified targets. Further validations were performed by RT-qPCR, Western immunoblot, and luciferase assays. MiR-145 affects the expression of over 350 genes and directly targets more than 50 mRNAs through the 3′UTR or, more commonly, the coding region. MiR-145 targets DUSP6, involved in cartilage organization and development, at the translational level. DUSP6 depletion leads to MMP13 upregulation, suggesting a contribution towards the effect of miR-145 on MMP13 expression. In conclusion, miR-145 directly targets several genes involved in the expression of the extracellular matrix and inflammation in primary chondrocytes. Thus, we propose miR-145 as an important regulator of chondrocyte function and a new target for cartilage repair.

scholarly journals High through-put identification of miR-145 targets in human articular chondrocytes

2020 ◽  
Author(s):  
Aida Martinez-Sanchez ◽  
Stefano Lazzarano ◽  
Eshita Sharma ◽  
Chris L. Murphy
Keyword(s):  
Cartilage Repair ◽  
Motif Discovery ◽  
Articular Chondrocytes ◽  
Biological Processes ◽  
Articular Chondrocyte ◽  
Coding Region ◽  
Rna Immunoprecipitation ◽  
Important Regulator ◽  
Discovery Method ◽  
Mmp13 Expression

ABSTRACTObjectiveMicroRNAs play a key role in biological processes, including cartilage development and homeostasis and are dysregulated in many diseases, including osteoarthritis. MiR-145 modulation induces profound changes in the human articular chondrocyte (HAC) phenotype, partially through direct repression of SOX9. Since miRNAs can simultaneously silence multiple targets, we aimed to identify the whole targetome of miR-145 in HACs. This information is critical if miR-145 is to be considered a target for cartilage repair.MethodsRIP-seq (RNA-immunoprecipitation plus HT-sequencing) of miRISC (miRNA-induced silencing complex) was performed in HACs overexpressing miR-145 to identify miR-145 direct targets. The motif discovery method cWords was used to assess enrichment on miR-145 seed matches in the identified targets. RT-qPCR, Western (immuno-)blot and luciferase assays were used to validate miRNA-target interactions.ResultsMiR-145 overexpression affects the expression of over 350 genes and directly targets more than 50 mRNAs through the 3’UTR or, more commonly, the coding region.We also demonstrate that miR-145 targets DUSP6, involved in cartilage organization and development, at the translational level. DUSP6 depletion using specific siRNAs lead to MMP13 up-regulation, suggesting that miR-145-mediated DUSP6 depletion contributes to the effect of miR-145 on MMP13 expression.ConclusionWe demonstrate that miR-145 directly targets several genes in primary chondrocytes including those involved in the expression of the extracellular matrix and inflammation. Thus, we propose miR-145 as an important regulator of chondrocyte function and a new target for cartilage repair.

scholarly journals Episo: quantitative estimation of RNA 5-methylcytosine at isoform level by high-throughput sequencing of RNA treated with bisulfite

2019 ◽  
Vol 36 (7) ◽  
pp. 2033-2039 ◽  
Author(s):  
Junfeng Liu ◽  
Ziyang An ◽  
Jianjun Luo ◽  
Jing Li ◽  
Feifei Li ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Quantitative Estimation ◽  
Supplementary Information ◽  
Biological Processes ◽  
Single Nucleotide ◽  
Rna Immunoprecipitation ◽  
Nucleotide Resolution ◽  
Human And Mouse ◽  
Single Nucleotide Resolution

Abstract Motivation RNA 5-methylcytosine (m5C) is a type of post-transcriptional modification that may be involved in numerous biological processes and tumorigenesis. RNA m5C can be profiled at single-nucleotide resolution by high-throughput sequencing of RNA treated with bisulfite (RNA-BisSeq). However, the exploration of transcriptome-wide profile and potential function of m5C in splicing remains to be elucidated due to lack of isoform level m5C quantification tool. Results We developed a computational package to quantify Epitranscriptomal RNA m5C at the transcript isoform level (named Episo). Episo consists of three tools: mapper, quant and Bisulfitefq, for mapping, quantifying and simulating RNA-BisSeq data, respectively. The high accuracy of Episo was validated using an improved m5C-specific methylated RNA immunoprecipitation (meRIP) protocol, as well as a set of in silico experiments. By applying Episo to public human and mouse RNA-BisSeq data, we found that the RNA m5C is not evenly distributed among the transcript isoforms, implying the m5C may subject to be regulated at isoform level. Availability and implementation Episo is released under the GNU GPLv3+ license. The resource code Episo is freely accessible from https://github.com/liujunfengtop/Episo (with Tophat/cufflink) and https://github.com/liujunfengtop/Episo/tree/master/Episo_Kallisto (with Kallisto). Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals High-throughput sequencing screen reveals novel, transforming RAS mutations in myeloid leukemia patients

Blood ◽  
2009 ◽  
Vol 113 (8) ◽  
pp. 1749-1755 ◽  
Author(s):  
Jeffrey W. Tyner ◽  
Heidi Erickson ◽  
Michael W. N. Deininger ◽  
Stephanie G. Willis ◽  
Christopher A. Eide ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Myeloid Leukemia ◽  
High Throughput Sequencing ◽  
Point Mutations ◽  
Causative Role ◽  
Coding Region ◽  
Ras Mutations ◽  
Oncogenic Ras ◽  
Frequent Mutations

Abstract Transforming mutations in NRAS and KRAS are thought to play a causative role in the development of numerous cancers, including myeloid malignancies. Although mutations at amino acids 12, 13, or 61 account for the majority of oncogenic Ras variants, we hypothesized that less frequent mutations at alternate residues may account for disease in some patients with cancer of unexplained genetic etiology. To search for additional, novel RAS mutations, we sequenced all coding exons in NRAS, KRAS, and HRAS in 329 acute myeloid leukemia (AML) patients, 32 chronic myelomonocytic leukemia (CMML) patients, and 96 healthy individuals. We detected 4 “noncanonical” point mutations in 7 patients: N-RasG60E, K-RasV14I, K-RasT74P, and K-RasA146T. All 4 Ras mutants exhibited oncogenic properties in comparison with wild-type Ras in biochemical and functional assays. The presence of transforming RAS mutations outside of positions 12, 13, and 61 reveals that alternate mechanisms of transformation by RAS may be overlooked in screens designed to detect only the most common RAS mutations. Our results suggest that RAS mutations may play a greater role in leukemogenesis than currently believed and indicate that high-throughput screening for mutant RAS alleles in cancer should include analysis of the entire RAS coding region.

scholarly journals Genome re-sequencing and reannotation of the Escherichia coli ER2566 strain and transcriptome sequencing under overexpression conditions

2020 ◽  
Author(s):  
Lizhi Zhou ◽  
Kaihang Wang ◽  
Tingting Chen ◽  
Yue Ma ◽  
Yang Huang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Speed ◽  
High Throughput Sequencing ◽  
Recombinant Protein Expression ◽  
External Pressure ◽  
Universal Method ◽  
Coding Region ◽  
Protein Coding ◽  
Human Papillomavirus 16

Abstract Background The Escherichia coli ER2566 strain (NC_CP014268.2) was developed as a BL21 (DE3) derivative strain and has been widely used in recombinant protein expression. However, like many other current RefSeq annotations, the annotation of the ER2566 strain is incomplete, with missing gene names and miscellaneous RNAs, as well as uncorrected annotations of some pseudogenes. Here, we performed a systematic reannotation of the ER2566 genome by combining multiple annotation tools with manual revision to provide a comprehensive understanding of the E. coli ER2566 strain, and used high-throughput sequencing to explore how the strain adapts under external pressure. Results The reannotation included noteworthy corrections to all protein-coding genes, led to the exclusion of 120 hypothetical genes or pseudogenes, and resulted in the addition of 65 coding sequences and 230 miscellaneous noncoding RNAs and 2 tRNAs. In addition, we further manually examined all 194 pseudogenes in the Ref-seq annotation and directly identified 144 (74%) as coding genes. The remaining pseudogenes without explicit function were removed. We then used whole-genome sequencing and high-throughput RNA sequencing to assess mutational adaptations under consecutive subculture or overexpression burden. Whereas no mutations were detected in response to consecutive subculture, overexpression of the human papillomavirus 16 type capsid led to the identification of a mutation (position 1,094,824 within the 3’ non-coding region) positioned 19-bp away from the lac I gene in the transcribed RNA, which was not detected at the genomic level by Sanger sequencing. Conclusion The ER2566 strain is used by both the general scientific community and the biotechnology industry. Reannotation of the E.coli ER2566 strain not only improved the RefSeq data but uncovered a key site that might involve in the transcription and translation of genes encoding the lactose operon repressor. We propose that our pipeline might offer a universal method for the reannotation of other bacterial genomes with high speed and accuracy. This study may facilitate a better understanding of gene function for the ER2566 strain under external burden and provide more clues to engineer bacteria for biotechnological applications.

scholarly journals Genome re-sequencing and reannotation of the Escherichia coli ER2566 strain and transcriptome sequencing under overexpression conditions

2020 ◽  
Author(s):  
Lizhi Zhou ◽  
Kaihang Wang ◽  
Tingting Chen ◽  
Yue Ma ◽  
Yang Huang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Speed ◽  
High Throughput Sequencing ◽  
Recombinant Protein Expression ◽  
External Pressure ◽  
Universal Method ◽  
Coding Region ◽  
Protein Coding ◽  
Human Papillomavirus 16

Abstract Background The Escherichia coli ER2566 strain (NC_CP014268.2) was developed as a BL21 (DE3) derivative strain and has been widely used in recombinant protein expression. However, like many other current RefSeq annotations, the annotation of the ER2566 strain is incomplete, with missing gene names and miscellaneous RNAs, as well as uncorrected annotations of some pseudogenes. Here, we performed a systematic reannotation of the ER2566 genome by combining multiple annotation tools with manual revision to provide a comprehensive understanding of the E. coli ER2566 strain, and used high-throughput sequencing to explore how the strain adapts under external pressure. Results The reannotation included noteworthy corrections to all protein-coding genes, led to the exclusion of 120 hypothetical genes or pseudogenes, and resulted in the addition of 65 coding sequences and 230 miscellaneous noncoding RNAs and 2 tRNAs. In addition, we further manually examined all 194 pseudogenes in the Ref-seq annotation and directly identified 144 (74%) as coding genes. The remaining pseudogenes without explicit function were removed. We then used whole-genome sequencing and high-throughput RNA sequencing to assess mutational adaptations under consecutive subculture or overexpression burden. Whereas no mutations were detected in response to consecutive subculture, overexpression of the human papillomavirus 16 type capsid led to the identification of a mutation (position 1,094,824 within the 3’ non-coding region) positioned 19-bp away from the lac I gene in the transcribed RNA, which was not detected at the genomic level by Sanger sequencing.Conclusion The ER2566 strain is used by both the general scientific community and the biotechnology industry. Reannotation of the E.coli ER2566 strain not only improved the RefSeq data but uncovered a key site that might involve in the transcription and translation of genes encoding the lactose operon repressor. We propose that our pipeline might offer a universal method for the reannotation of other bacterial genomes with high speed and accuracy. This study may facilitate a better understanding of gene function for the ER2566 strain under external burden and provide more clues to engineer bacteria for biotechnological applications.

P–556 NAT10-mediated N4-acetylcytidine in RNA regulates mouse oocyte maturation in vitro

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Y Xiang ◽  
C Zhou ◽  
Q Guo ◽  
X Liang
Keyword(s):  
Mass Spectrometry ◽  
Oocyte Maturation ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Regulatory Role ◽  
Maturation In Vitro ◽  
Rna Immunoprecipitation ◽  
Transcriptional Modulation

Abstract Study question Does NAT10-mediated N4-acetylcytidine (ac4C) in RNA, a newly identified mRNA epigenetic modification, participate in modulating in vitro maturation(IVM) of oocytes? Summary answer NAT10-mediated ac4C modification is an important regulatory factor during oocyte maturation in vitro, by regulating genes associated with translation, mitochondrial functions and protein destabilization. What is known already Unlike somatic cells, transcription and translation are uncoupled during oocyte maturation and gene expression is mainly regulated by post-transcriptional modulation, including mRNA degradation, translation and posttranslational modification, which are complex and have not been fully investigated. RNA ac4C is a newly identified mRNA modification and a key determinant of post-transcriptional regulation, which has been shown to promote mRNA stability and translation, and NAT10 is the only known RNA acetyltransferase. Therefore, NAT10-mediated ac4C represents a possible epigenetic regulator in oocyte maturation. Study design, size, duration Oocytes at different stages from mice were collected to detect the changing levels of ac4C and NAT10 during maturation. NAT10 in GV-stage oocytes was knocked down before IVM, to confirm the regulatory role of NAT10-mediated ac4C in meiotic process, followed by further exploration of cellular mechanisms. Each experiment was repeated at least three times, and data were analyzed by chi-square test, one-way ANOVA or unpaired-sample t-test. Participants/materials, setting, methods The expression of ac4C and NAT10 was detected by immunohistochemistry. NAT10 was knocked down in GV-stage oocytes by RNA interference through electroporation. The efficacy of knockdown was confirmed by qPCR and immunohistochemistry targeting ac4C and NAT10, and the percentages of oocytes maturated in vitro were compared among groups. High-throughput sequencing and RNA immunoprecipitation were performed to reveal the modulated genes. Proteins specifically binding to ac4C sites were identified by RNA pulldown and mass spectrometry. Main results and the role of chance We first retrieved publicly available data from GEO and found that transcripts with potential ac4C sites were enriched in genes downregulated during IVM (P < 0.001). The biased distribution of ac4C implicated a possible regulatory role. Then immunohistochemistry revealed significantly decreasing trends of ac4C and NAT10 expression from immature to mature oocytes. With NAT10 knockdown, ac4C modification was reduced and meiotic progression was significantly retarded. Specifically, the rate of first body extrusion was significantly decreased with NAT10 knockdown (34.6%) compared to control oocytes without transfection (74.6%) and oocytes transfected with control siRNA (72.6%) (p < 0.001), while rates of germinal vesicle breakdown were not affected (P = 0.6531). High-throughput sequencing and RNA immunoprecipitation revealed that the modulated genes were enriched in biological processes known to be associated with oocyte maturation, including translation, mitochondrial translational elongation and termination, and protein destabilization. Also, we identified a series of proteins specifically binding to ac4C probes by RNA pulldown and mass spectrometry, through which ac4C modification may exert its function in post-transcriptional modulation. Limitations, reasons for caution This study was performed in vitro. The role of NAT10-mediated ac4C in vivo remains to be elucidated. Also, limited by current techniques, ac4C modification in oocytes cannot be detected. Our exploration of regulated genes and ac4C binding proteins were performed in somatic cell lines. Wider implications of the findings: Post-transcriptional modulation is crucial in oocyte maturation. Our study using in-vitro systems for mouse oocyte identified NAT10-mediated ac4C as an important regulator in IVM. It provided a new insight into the epigenetic mechanisms of IVM, which may lead to improvement of clinical IVM systems. Trial registration number Not applicable

The role of bone morphogenetic proteins in articular cartilage development, homeostasis and repair

2007 ◽  
Vol 20 (03) ◽  
pp. 151-158 ◽  
Author(s):  
A. O. Oshin ◽  
M. C. Stewart
Keyword(s):  
Articular Cartilage ◽  
Cartilage Repair ◽  
Bone Morphogenetic Proteins ◽  
Genetically Engineered ◽  
Articular Chondrocyte ◽  
Diverse Range ◽  
Joint Formation ◽  
Cartilage Development

SummaryBone morphogenetic proteins (BMPs) are members of the TGF-β superfamily of secreted ligands. BMPs regulate a diverse range of developmental processes during embryogenesis and postnatal development, and control the differentiation of several musculoskeletal tissues including bone, cartilage, tendon and ligaments. The ability of BMPs to modulate the phenotype of cells in these tissue lineages suggests that these factors could be valuable for musculoskeletal tissue regeneration. In fact, BMPs-2 and -7 are already in clinical use for bone regeneration. This review addresses the signaling mechanisms by which BMPs regulate cellular processes, the role of BMPs in articular cartilage development and joint formation, and the data that supports the use of BMPs for in vitro phenotypic support of articular chondrocyte cultures, chondrogenic differentiation of mesenchymal stem cells (MSCs) and articular cartilage repair. Given the documented importance of BMP activity for normal joint formation, articular cartilage development and maintenance, the chondrogenic activity of BMPs when applied to MSC cultures and the encouraging outcomes of several in vivo cartilage repair studies, BMP therapies hold considerable promise for effective cartilage repair and/or regeneration. Future advances in the control of BMP elution from biocompatible matrices and prolonged, dose-controlled BMP expression by genetically engineered cells should substantially improve cartilage repair strategies using BMPs and similar chondro-protective proteins.

P-556 Pre-selected for an award: NAT10-mediated N4-acetylcytidine in RNA regulates mouse oocyte maturation in vitro

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Y Xiang ◽  
C Zhou ◽  
Q Guo ◽  
X Liang
Keyword(s):  
Mass Spectrometry ◽  
Oocyte Maturation ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Regulatory Role ◽  
Maturation In Vitro ◽  
Rna Immunoprecipitation ◽  
Transcriptional Modulation

Abstract Study question Does NAT10-mediated N4-acetylcytidine (ac4C) in RNA, a newly identified mRNA epigenetic modification, participate in modulating in vitro maturation(IVM) of oocytes? Summary answer NAT10-mediated ac4C modification is an important regulatory factor during oocyte maturation in vitro, by regulating genes associated with translation, mitochondrial functions and protein destabilization. What is known already Unlike somatic cells, transcription and translation are uncoupled during oocyte maturation and gene expression is mainly regulated by post-transcriptional modulation, including mRNA degradation, translation and posttranslational modification, which are complex and have not been fully investigated. RNA ac4C is a newly identified mRNA modification and a key determinant of post-transcriptional regulation, which has been shown to promote mRNA stability and translation, and NAT10 is the only known RNA acetyltransferase. Therefore, NAT10-mediated ac4C represents a possible epigenetic regulator in oocyte maturation. Study design, size, duration Oocytes at different stages from mice were collected to detect the changing levels of ac4C and NAT10 during maturation. NAT10 in GV-stage oocytes was knocked down before IVM, to confirm the regulatory role of NAT10-mediated ac4C in meiotic process, followed by further exploration of cellular mechanisms. Each experiment was repeated at least three times, and data were analyzed by chi-square test, one-way ANOVA or unpaired-sample t-test. Participants/materials, setting, methods The expression of ac4C and NAT10 was detected by immunohistochemistry. NAT10 was knocked down in GV-stage oocytes by RNA interference through electroporation. The efficacy of knockdown was confirmed by qPCR and immunohistochemistry targeting ac4C and NAT10, and the percentages of oocytes maturated in vitro were compared among groups. High-throughput sequencing and RNA immunoprecipitation were performed to reveal the modulated genes. Proteins specifically binding to ac4C sites were identified by RNA pulldown and mass spectrometry. Main results and the role of chance We first retrieved publicly available data from GEO and found that transcripts with potential ac4C sites were enriched in genes downregulated during IVM (P < 0.001). The biased distribution of ac4C implicated a possible regulatory role. Then immunohistochemistry revealed significantly decreasing trends of ac4C and NAT10 expression from immature to mature oocytes. With NAT10 knockdown, ac4C modification was reduced and meiotic progression was significantly retarded. Specifically, the rate of first body extrusion was significantly decreased with NAT10 knockdown (34.6%) compared to control oocytes without transfection (74.6%) and oocytes transfected with control siRNA (72.6%) (p < 0.001), while rates of germinal vesicle breakdown were not affected (P = 0.6531). High-throughput sequencing and RNA immunoprecipitation revealed that the modulated genes were enriched in biological processes known to be associated with oocyte maturation, including translation, mitochondrial translational elongation and termination, and protein destabilization. Also, we identified a series of proteins specifically binding to ac4C probes by RNA pulldown and mass spectrometry, through which ac4C modification may exert its function in post-transcriptional modulation. Limitations, reasons for caution This study was performed in vitro. The role of NAT10-mediated ac4C in vivo remains to be elucidated. Also, limited by current techniques, ac4C modification in oocytes cannot be detected. Our exploration of regulated genes and ac4C binding proteins were performed in somatic cell lines. Wider implications of the findings Post-transcriptional modulation is crucial in oocyte maturation. Our study using in-vitro systems for mouse oocyte identified NAT10-mediated ac4C as an important regulator in IVM. It provided a new insight into the epigenetic mechanisms of IVM, which may lead to improvement of clinical IVM systems. Trial registration number not applicable

scholarly journals The Study of the Frequency Effect of Dynamic Compressive Loading on Primary Articular Chondrocyte Functions Using a Microcell Culture System

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Wan-Ying Lin ◽  
Yu-Han Chang ◽  
Hsin-Yao Wang ◽  
Tzu-Chi Yang ◽  
Tzu-Keng Chiu ◽  
...  
Keyword(s):  
Cell Culture ◽  
High Throughput ◽  
Culture System ◽  
Articular Chondrocytes ◽  
Compressive Strain ◽  
Compressive Loading ◽  
Cartilage Tissue ◽  
Frequency Effect ◽  
Articular Chondrocyte ◽  
Effective Manner

Compressive stimulation can modulate articular chondrocyte functions. Nevertheless, the relevant studies are not comprehensive. This is primarily due to the lack of cell culture apparatuses capable of conducting the experiments in a high throughput, precise, and cost-effective manner. To address the issue, we demonstrated the use of a perfusion microcell culture system to investigate the stimulating frequency (0.5, 1.0, and 2.0 Hz) effect of compressive loading (20% and 40% strain) on the functions of articular chondrocytes. The system mainly integrates the functions of continuous culture medium perfusion and the generation of pneumatically-driven compressive stimulation in a high-throughput micro cell culture system. Results showed that the compressive stimulations explored did not have a significant impact on chondrocyte viability and proliferation. However, the metabolic activity of chondrocytes was significantly affected by the stimulating frequency at the higher compressive strain of 40% (2 Hz, 40% strain). Under the two compressive strains studied, the glycosaminoglycans (GAGs) synthesis was upregulated when the stimulating frequency was set at 1 Hz and 2 Hz. However, the stimulating frequencies explored had no influence on the collagen production. The results of this study provide useful fundamental insights that will be helpful for cartilage tissue engineering and cartilage rehabilitation.

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