K. Attwood, Jayme Salsman, Dudley Chung, Sabateeshan Mathavarajah, C. Van Iderstine, G. Dellaire
Promyelocytic leukemia nuclear bodies (PML NBs) are nuclear subdomains that respond to genotoxic stress by increasing in number via changes in chromatin structure. However, the role of the PML protein and PML NBs in specific mechanisms of DNA repair has not been fully characterized. Here, we have directly examined the role of PML in homologous recombination (HR) using I-SceI extrachromosomal and chromosome-based homology-directed repair (HDR) assays, and in HDR by CRISPR/Cas9-mediated gene editing. We determined that PML loss can inhibit HR in an extrachromosomal HDR assay but had less of an effect on CRISPR/Cas9-mediated chromosomal HDR. Overexpression of PML also inhibited both CRISPR HDR and I-SceI-induced HDR using a chromosomal reporter, and in an isoform specific-manner. However, the impact of PML overexpression on the chromosomal HDR reporter was dependent on the intra-nuclear chromosomal positioning of the reporter. Specifically, HDR at the TAP1 gene locus, which is associated with PML NBs, was reduced compared to a locus not associated with a PML NB; yet, HDR could be reduced at the non-PML NB-associated locus by PML overexpression. Thus, both loss and overexpression of PML isoforms can inhibit HDR, and proximity of a chromosomal break to a PML NB can impact HDR efficiency.
{"title":"PML isoform expression and DNA break location relative to PML nuclear bodies impacts the efficiency of homologous recombination.","authors":"K. Attwood, Jayme Salsman, Dudley Chung, Sabateeshan Mathavarajah, C. Van Iderstine, G. Dellaire","doi":"10.1139/bcb-2019-0115","DOIUrl":"https://doi.org/10.1139/bcb-2019-0115","url":null,"abstract":"Promyelocytic leukemia nuclear bodies (PML NBs) are nuclear subdomains that respond to genotoxic stress by increasing in number via changes in chromatin structure. However, the role of the PML protein and PML NBs in specific mechanisms of DNA repair has not been fully characterized. Here, we have directly examined the role of PML in homologous recombination (HR) using I-SceI extrachromosomal and chromosome-based homology-directed repair (HDR) assays, and in HDR by CRISPR/Cas9-mediated gene editing. We determined that PML loss can inhibit HR in an extrachromosomal HDR assay but had less of an effect on CRISPR/Cas9-mediated chromosomal HDR. Overexpression of PML also inhibited both CRISPR HDR and I-SceI-induced HDR using a chromosomal reporter, and in an isoform specific-manner. However, the impact of PML overexpression on the chromosomal HDR reporter was dependent on the intra-nuclear chromosomal positioning of the reporter. Specifically, HDR at the TAP1 gene locus, which is associated with PML NBs, was reduced compared to a locus not associated with a PML NB; yet, HDR could be reduced at the non-PML NB-associated locus by PML overexpression. Thus, both loss and overexpression of PML isoforms can inhibit HDR, and proximity of a chromosomal break to a PML NB can impact HDR efficiency.","PeriodicalId":9524,"journal":{"name":"Canadian journal of biochemistry and cell biology = Revue canadienne de biochimie et biologie cellulaire","volume":"38 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83654562","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Xiujuan Chen, Xu Yu, Xinxiang Li, Li Li, Fang Li, Ting Guo, C. Guan, Li-ping Miao, Guoping Cao
Diabetic retinopathy (DR) is a common complication of diabetes mellitus (DM), which results in vision loss. The present study aimed to explore the role of miR-126 in high-glucose-induced human retinal endothelial cells (HRECs) and its underlying molecular mechanism. The results showed that expression of miR-126 and interleukin-17A (IL-17A) in high-glucose-induced HRECs was downregulated and upregulated, respectively. Functionally, overexpression of miR-126 promoted proliferation and suppressed apoptosis in high-glucose-induced HRECs, while IL-17A reversed the effects induced by miR-126. However, overexpression of IL-17A inhibited the proliferation and induced apoptosis, while knockdown of IL-17A accelerated the proliferation and repressed apoptosis. In addition, miR-126 repressed the expression of IL-17A, Bax, and caspase-3,while promotingthe expression of survivin and phosphorylation of PI3K and AKT;restoration of IL-17A rescued these effects. Furthermore, IL-17A was identified as a target of miR-126. Altogether, miR-126 enhances proliferation and inhibits apoptosis in high-glucose-induced HRECs by activating the PI3K/AKT pathway, increasing survivinand decreasing Bax and caspase-3 expression by targeting IL-17A, suggesting that miR-126 may be a novel target for preventing DR.
{"title":"MiR-126 targets IL-17A to enhance proliferation and inhibit apoptosis in high-glucose-induced human retinal endothelial cells.","authors":"Xiujuan Chen, Xu Yu, Xinxiang Li, Li Li, Fang Li, Ting Guo, C. Guan, Li-ping Miao, Guoping Cao","doi":"10.1139/bcb-2019-0174","DOIUrl":"https://doi.org/10.1139/bcb-2019-0174","url":null,"abstract":"Diabetic retinopathy (DR) is a common complication of diabetes mellitus (DM), which results in vision loss. The present study aimed to explore the role of miR-126 in high-glucose-induced human retinal endothelial cells (HRECs) and its underlying molecular mechanism. The results showed that expression of miR-126 and interleukin-17A (IL-17A) in high-glucose-induced HRECs was downregulated and upregulated, respectively. Functionally, overexpression of miR-126 promoted proliferation and suppressed apoptosis in high-glucose-induced HRECs, while IL-17A reversed the effects induced by miR-126. However, overexpression of IL-17A inhibited the proliferation and induced apoptosis, while knockdown of IL-17A accelerated the proliferation and repressed apoptosis. In addition, miR-126 repressed the expression of IL-17A, Bax, and caspase-3,while promotingthe expression of survivin and phosphorylation of PI3K and AKT;restoration of IL-17A rescued these effects. Furthermore, IL-17A was identified as a target of miR-126. Altogether, miR-126 enhances proliferation and inhibits apoptosis in high-glucose-induced HRECs by activating the PI3K/AKT pathway, increasing survivinand decreasing Bax and caspase-3 expression by targeting IL-17A, suggesting that miR-126 may be a novel target for preventing DR.","PeriodicalId":9524,"journal":{"name":"Canadian journal of biochemistry and cell biology = Revue canadienne de biochimie et biologie cellulaire","volume":"13 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84787874","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Gang Li, Longfeng Sun, Zhong-yi Mu, Shi-bo Liu, Hong-Chen Qu, Qingpeng Xie, Bin Hu
MicroRNA (miR)-1298 is widely down-regulated in various malignant tumors, which facilitates cell proliferation, invasion and migration. However, the specific biological function of miR-1298 in bladder cancer (BC) is still unknown. Connexin 43 (Cx43) is often up-regulated in different tumors. Identifying miRNAs that target Cx43 in the setting of BC will help to develop Cx43-based therapies for BC. In this study, the results demonstrated that the expression levels of miR-1298 and Cx43 were significantly down-regulated and up-regulated in BC clinical tissues, respectively. The overexpression of miR-1298 inhibited cell proliferation, migration, and invasion in two BC cell lines via MTT assay, cell cycle assay, colony formation assay, transwell assay, gelatin zymography, and western blot, respectively. In addition, it was found that miR-1298 decreased Cx43 expression by directly targeting the 3'-UTR. Following, we observed that the promotion effect of Cx43 on BC cell proliferation, migration, and invasion could be partially attenuated by miR-1298 overexpression. Moreover, the protein expression of p-ERK was ameliorated after transfected with overexpressed-miR-1298. The knockdown of Cx43 reversed the promotion effect of cell migration and invasion due to the decreased miR-1298 expression. All data suggest miR-1298 might be a potential therapeutic agent and diagnostic marker of BC by inhibiting Cx43.
{"title":"MicroRNA-1298-5p inhibits cell proliferation and invasion of bladder cancer via downregulating connexin 43.","authors":"Gang Li, Longfeng Sun, Zhong-yi Mu, Shi-bo Liu, Hong-Chen Qu, Qingpeng Xie, Bin Hu","doi":"10.1139/bcb-2019-0137","DOIUrl":"https://doi.org/10.1139/bcb-2019-0137","url":null,"abstract":"MicroRNA (miR)-1298 is widely down-regulated in various malignant tumors, which facilitates cell proliferation, invasion and migration. However, the specific biological function of miR-1298 in bladder cancer (BC) is still unknown. Connexin 43 (Cx43) is often up-regulated in different tumors. Identifying miRNAs that target Cx43 in the setting of BC will help to develop Cx43-based therapies for BC. In this study, the results demonstrated that the expression levels of miR-1298 and Cx43 were significantly down-regulated and up-regulated in BC clinical tissues, respectively. The overexpression of miR-1298 inhibited cell proliferation, migration, and invasion in two BC cell lines via MTT assay, cell cycle assay, colony formation assay, transwell assay, gelatin zymography, and western blot, respectively. In addition, it was found that miR-1298 decreased Cx43 expression by directly targeting the 3'-UTR. Following, we observed that the promotion effect of Cx43 on BC cell proliferation, migration, and invasion could be partially attenuated by miR-1298 overexpression. Moreover, the protein expression of p-ERK was ameliorated after transfected with overexpressed-miR-1298. The knockdown of Cx43 reversed the promotion effect of cell migration and invasion due to the decreased miR-1298 expression. All data suggest miR-1298 might be a potential therapeutic agent and diagnostic marker of BC by inhibiting Cx43.","PeriodicalId":9524,"journal":{"name":"Canadian journal of biochemistry and cell biology = Revue canadienne de biochimie et biologie cellulaire","volume":"2 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82355695","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Z. Belak, Joshua A Pickering, Z. Gillespie, G. Audette, M. Eramian, Jennifer A. Mitchell, J. Bridger, A. Kusalik, C. Eskiw
We previously demonstrated that genome reorganization, through chromosome territory repositioning, occurred concurrently with significant changes in gene expression in normal primary human fibroblasts treated with the drug rapamycin, or stimulated into quiescence. Although these events occurred concomitantly, it is unclear how specific changes in gene expression relate to reorganization of the genome at higher resolution. Using computational analyses, genome organization assays and microscopy, the relationship between chromosome territory positioning and gene expression was investigated. We determined that despite relocation of chromosome territories, there was no substantial bias in the proportion of genes changing expression on any one chromosome, including chromosomes 10 and 18. Computational analyses identified that clusters of serum deprivation and rapamycin-responsive genes along the linear extent of chromosomes. Chromosome conformation capture (3C) analysis demonstrated the strengthening or loss of specific long-range chromatin interactions in response to rapamycin and quiescence induction, including a cluster of genes containing Interleukin-8 and several chemokine genes on chromosome 4. We further observed that the LIF gene, which is highly induced upon rapamycin treatment, strengthened interactions with up- and down-stream intergenic regions. Our findings indicate that the re-positioning of chromosome territories in response to cell stimuli, this does not reflect gene expression changes occurring within physically clustered groups of genes.
{"title":"Genes responsive to rapamycin and serum deprivation are clustered on chromosomes and undergo re-organization within local chromatin environments.","authors":"Z. Belak, Joshua A Pickering, Z. Gillespie, G. Audette, M. Eramian, Jennifer A. Mitchell, J. Bridger, A. Kusalik, C. Eskiw","doi":"10.1139/bcb-2019-0096","DOIUrl":"https://doi.org/10.1139/bcb-2019-0096","url":null,"abstract":"We previously demonstrated that genome reorganization, through chromosome territory repositioning, occurred concurrently with significant changes in gene expression in normal primary human fibroblasts treated with the drug rapamycin, or stimulated into quiescence. Although these events occurred concomitantly, it is unclear how specific changes in gene expression relate to reorganization of the genome at higher resolution. Using computational analyses, genome organization assays and microscopy, the relationship between chromosome territory positioning and gene expression was investigated. We determined that despite relocation of chromosome territories, there was no substantial bias in the proportion of genes changing expression on any one chromosome, including chromosomes 10 and 18. Computational analyses identified that clusters of serum deprivation and rapamycin-responsive genes along the linear extent of chromosomes. Chromosome conformation capture (3C) analysis demonstrated the strengthening or loss of specific long-range chromatin interactions in response to rapamycin and quiescence induction, including a cluster of genes containing Interleukin-8 and several chemokine genes on chromosome 4. We further observed that the LIF gene, which is highly induced upon rapamycin treatment, strengthened interactions with up- and down-stream intergenic regions. Our findings indicate that the re-positioning of chromosome territories in response to cell stimuli, this does not reflect gene expression changes occurring within physically clustered groups of genes.","PeriodicalId":9524,"journal":{"name":"Canadian journal of biochemistry and cell biology = Revue canadienne de biochimie et biologie cellulaire","volume":"35 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75131702","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Recently, several miRNAs have been suggested to serve as potential therapeutic targets for anesthesia-related diseases. This study was carried out to explore the biological roles of miR-24 in isoflurane-treated rat hippocampal neurons. Isoflurane-treated rat model was established to induce neurotoxicity. Gain- and loss- of function of miR-24 was performed and the size and Ca2+ permeability of mitochondria, cell proliferation and apoptosis and levels of oxidative stress-related factors were measured both in vivo and in vitro. Dual luciferase reporter gene assay was used to identify the target relation between miR-24 and p27kip1. In this study, isoflurane treatment decreased miR-24 expression, after which the neuronal apoptosis and the oxidative-stress-related factors were elevated while the neuronal viability was reduced. Over-expression of miR-24 inhibited oxidative damage and neuronal apoptosis in hippocampus and suppressed the size and Ca2+ permeability of mitochondria of hippocampal neurons. miR-24 enhanced the viability of rat hippocampal neurons by targeting p27kip1. To conclude, this study demonstrated that miR-24 could attenuate isoflurane-induced neurotoxicity in rat hippocampus via anti-oxidative stress function and inhibiting p27kip1 expression.
{"title":"MicroRNA-24 alleviates isoflurane-induced neurotoxicity in rat hippocampus via attenuation of oxidative stress.","authors":"Na Li, L. Yue, Jun Wang, Zhenzhen Wan, Wenhao Bu","doi":"10.1139/bcb-2019-0188","DOIUrl":"https://doi.org/10.1139/bcb-2019-0188","url":null,"abstract":"Recently, several miRNAs have been suggested to serve as potential therapeutic targets for anesthesia-related diseases. This study was carried out to explore the biological roles of miR-24 in isoflurane-treated rat hippocampal neurons. Isoflurane-treated rat model was established to induce neurotoxicity. Gain- and loss- of function of miR-24 was performed and the size and Ca2+ permeability of mitochondria, cell proliferation and apoptosis and levels of oxidative stress-related factors were measured both in vivo and in vitro. Dual luciferase reporter gene assay was used to identify the target relation between miR-24 and p27kip1. In this study, isoflurane treatment decreased miR-24 expression, after which the neuronal apoptosis and the oxidative-stress-related factors were elevated while the neuronal viability was reduced. Over-expression of miR-24 inhibited oxidative damage and neuronal apoptosis in hippocampus and suppressed the size and Ca2+ permeability of mitochondria of hippocampal neurons. miR-24 enhanced the viability of rat hippocampal neurons by targeting p27kip1. To conclude, this study demonstrated that miR-24 could attenuate isoflurane-induced neurotoxicity in rat hippocampus via anti-oxidative stress function and inhibiting p27kip1 expression.","PeriodicalId":9524,"journal":{"name":"Canadian journal of biochemistry and cell biology = Revue canadienne de biochimie et biologie cellulaire","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88616102","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Long non-coding RNAs (lncRNAs) are emerging as vital regulators in various physiological and pathological processes. It was recently found that lncRNA HIF1A-AS2 could play oncogenic roles in several cancers. However, the function and regulatory mechanism of lncRNA HIF1A-AS2 in osteosarcoma (OS) remain largely unclear. In the present study, we demonstrated that HIF1A-AS2 was overexpressed in OS tissues and cells. HIF1A-AS2 downregulation could remarkably affect multiple OS cell biological functions, including cell proliferation, cell cycle progression, cell apoptosis, cell migration and cell invasion. Mechanistic investigations demonstrated that HIF1A-AS2 can interact with miR-33b-5p and negatively regulate its expression, thereby upregulate the protein expression of miR-33b-5p's target SIRT6. Additionally, in vivo experiments using a xenograft tumor mouse model revealed that HIF1A-AS2 downregulation suppressed tumor growth in OS. Taken together, a newly identified regulatory mechanism of lncRNA HIF1A-AS2/miR-33b-5p/SIRT6 axis was systematically studied in OS, which may hold promise as a promising target for treatment.
{"title":"Long noncoding RNA HIF1A-AS2 facilitates cell survival and migration by sponging miR-33b-5p to modulate SIRT6 expression in osteosarcoma.","authors":"H. Lin, Zhenxu Zhao, Yi Hao, Jun He, Jian He","doi":"10.1139/bcb-2019-0171","DOIUrl":"https://doi.org/10.1139/bcb-2019-0171","url":null,"abstract":"Long non-coding RNAs (lncRNAs) are emerging as vital regulators in various physiological and pathological processes. It was recently found that lncRNA HIF1A-AS2 could play oncogenic roles in several cancers. However, the function and regulatory mechanism of lncRNA HIF1A-AS2 in osteosarcoma (OS) remain largely unclear. In the present study, we demonstrated that HIF1A-AS2 was overexpressed in OS tissues and cells. HIF1A-AS2 downregulation could remarkably affect multiple OS cell biological functions, including cell proliferation, cell cycle progression, cell apoptosis, cell migration and cell invasion. Mechanistic investigations demonstrated that HIF1A-AS2 can interact with miR-33b-5p and negatively regulate its expression, thereby upregulate the protein expression of miR-33b-5p's target SIRT6. Additionally, in vivo experiments using a xenograft tumor mouse model revealed that HIF1A-AS2 downregulation suppressed tumor growth in OS. Taken together, a newly identified regulatory mechanism of lncRNA HIF1A-AS2/miR-33b-5p/SIRT6 axis was systematically studied in OS, which may hold promise as a promising target for treatment.","PeriodicalId":9524,"journal":{"name":"Canadian journal of biochemistry and cell biology = Revue canadienne de biochimie et biologie cellulaire","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84658633","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Qiuwang Zhang, A. Cannavicci, Si-Cheng Dai, Chenxi Wang, M. Kutryk
Human myeloid angiogenic cells (MACs), also termed early endothelial progenitor cells, play an important role in neovascularization and vascular repair. MicroRNAs (miRNAs) are a class of naturally occurring, noncoding, short (~22 nucleotides), single-stranded RNAs that regulate gene expression posttranscriptionally. MiRNAs have been shown to regulate MAC function. A miRNA signature of MACs was described approximately a decade ago, and many new miRNAs have been discovered in recent years. In this study, we aimed to provide an up-to-date miRNA signature for human MACs. MACs were obtained by culture of human peripheral blood mononuclear cells in endothelial medium for 7 days. Using qPCR array analysis we identified 72 highly expressed miRNAs (Ct value < 30) in human MACs. RT-qPCR quantification of select miRNAs revealed a strong correlation between the Ct values detected by the array analysis and RT-qPCR, suggesting the miRNA signature generated by the qPCR array assay is accurate and reliable. Experimentally validated target genes of the 10 most highly expressed miRNAs were retrieved. Only a few of the targets and their respective miRNAs have been studied for their role in MAC biology. Our study therefore provides a valuable repository of miRNAs for future exploration of miRNA function in MACs.
{"title":"MicroRNA profiling of human myeloid angiogenic cells derived from peripheral blood mononuclear cells.","authors":"Qiuwang Zhang, A. Cannavicci, Si-Cheng Dai, Chenxi Wang, M. Kutryk","doi":"10.1139/bcb-2019-0163","DOIUrl":"https://doi.org/10.1139/bcb-2019-0163","url":null,"abstract":"Human myeloid angiogenic cells (MACs), also termed early endothelial progenitor cells, play an important role in neovascularization and vascular repair. MicroRNAs (miRNAs) are a class of naturally occurring, noncoding, short (~22 nucleotides), single-stranded RNAs that regulate gene expression posttranscriptionally. MiRNAs have been shown to regulate MAC function. A miRNA signature of MACs was described approximately a decade ago, and many new miRNAs have been discovered in recent years. In this study, we aimed to provide an up-to-date miRNA signature for human MACs. MACs were obtained by culture of human peripheral blood mononuclear cells in endothelial medium for 7 days. Using qPCR array analysis we identified 72 highly expressed miRNAs (Ct value < 30) in human MACs. RT-qPCR quantification of select miRNAs revealed a strong correlation between the Ct values detected by the array analysis and RT-qPCR, suggesting the miRNA signature generated by the qPCR array assay is accurate and reliable. Experimentally validated target genes of the 10 most highly expressed miRNAs were retrieved. Only a few of the targets and their respective miRNAs have been studied for their role in MAC biology. Our study therefore provides a valuable repository of miRNAs for future exploration of miRNA function in MACs.","PeriodicalId":9524,"journal":{"name":"Canadian journal of biochemistry and cell biology = Revue canadienne de biochimie et biologie cellulaire","volume":"85 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76329100","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pedro Rafael Firmino Dias, P. G. Gandra, R. Brenzikofer, D. Macedo
Cell fractionation can be used to determine the localization and trafficking of proteins between cellular compartments such as cytosol, mitochondria and nuclei. Subcellular fractionation is usually performed immediately after tissue dissection since freezing may fragment cell membranes and induce organellar cross-contamination. Mitochondria are especially sensitive to freezing/thawing and mechanical homogenization. We proposed a protocol to improve soluble proteins retention in the mitochondrial fraction obtained from small amounts of frozen skeletal muscle. Fifty-milligram of red portion of gastrocnemius muscle from Wistar rats were immediately processed or frozen in liquid nitrogen and stored at -80°C for further processing. We compared the enrichment of subcellular fractions from frozen/fresh samples obtained with the modified protocol with those obtained by standard fractionation. Western blot analyses of marker proteins for cytosolic (alpha-tubulin), mitochondrial (VDAC1), and nuclear (histone-H3) fractions indicated that all procedures resulted in enriched subcellular fractions with minimal organellar cross-contamination. Notably, the activity of the soluble protein citrate synthase was higher in mitochondrial fractions obtained with the modified protocol from frozen/fresh samples compared to the standard protocol. Therefore, our protocol improved the retention of soluble proteins in the mitochondrial fraction and may be suitable for subcellular fractionation of small amounts of frozen skeletal muscle samples.
{"title":"Subcellular fractionation of frozen skeletal muscle samples.","authors":"Pedro Rafael Firmino Dias, P. G. Gandra, R. Brenzikofer, D. Macedo","doi":"10.1139/bcb-2019-0219","DOIUrl":"https://doi.org/10.1139/bcb-2019-0219","url":null,"abstract":"Cell fractionation can be used to determine the localization and trafficking of proteins between cellular compartments such as cytosol, mitochondria and nuclei. Subcellular fractionation is usually performed immediately after tissue dissection since freezing may fragment cell membranes and induce organellar cross-contamination. Mitochondria are especially sensitive to freezing/thawing and mechanical homogenization. We proposed a protocol to improve soluble proteins retention in the mitochondrial fraction obtained from small amounts of frozen skeletal muscle. Fifty-milligram of red portion of gastrocnemius muscle from Wistar rats were immediately processed or frozen in liquid nitrogen and stored at -80°C for further processing. We compared the enrichment of subcellular fractions from frozen/fresh samples obtained with the modified protocol with those obtained by standard fractionation. Western blot analyses of marker proteins for cytosolic (alpha-tubulin), mitochondrial (VDAC1), and nuclear (histone-H3) fractions indicated that all procedures resulted in enriched subcellular fractions with minimal organellar cross-contamination. Notably, the activity of the soluble protein citrate synthase was higher in mitochondrial fractions obtained with the modified protocol from frozen/fresh samples compared to the standard protocol. Therefore, our protocol improved the retention of soluble proteins in the mitochondrial fraction and may be suitable for subcellular fractionation of small amounts of frozen skeletal muscle samples.","PeriodicalId":9524,"journal":{"name":"Canadian journal of biochemistry and cell biology = Revue canadienne de biochimie et biologie cellulaire","volume":"2 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75345865","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Chunyu Kong, Changlei Wang, Yuquan Shi, Lei Yan, Junhua Xu, W. Qi
Osteoarthritis (OA) is a common joint degenerative disease. Vitamin D (VD) is essential for bone function in human body. We hypothesized that active VD may play key functions in OA treatment. Low level of serum 25-hydroxyvitamin D (25(OH)D) was found in OA patients, and the serum VD level might be supportive for OA diagnosis. OA mouse models were established. HE and SafraninO/Fast Green staining suggested that active VD reduced OA symptoms in mice. VD treatment elevated p-AMPK/AMPK and decreased p-mTOR/mTOR, and it increased LC3II/LC3I, increased the protein level of Beclin-1, but decreased p62 according to Western blot analysis. Besides, VD reduced the contents of tumor necrosis factor-α and interleukin-6 both in cartilage tissues and in chondrocytes. Meanwhile, AMPK inhibitor Compound C and autophagy inhibitor 3-methyladenine (3-MA) reversed these changes following VD treatment. In addition, mRFP-GFP-LC3 transfection identified that active VD led to autophagosome aggregation in OA chondrocytes. 3-MA inhibited cell autophagy and promoted OA inflammation. This study provided evidence that active VD might activate chondrocyte autophagy to reduce OA inflammation via activating the AMPK/mTOR signaling pathway. Active OA treatment might serve as a novel therapeutic option for OA treatment.
{"title":"Active Vitamin D activates chondrocyte autophagy to reduce osteoarthritis via mediating the AMPK/mTOR signaling pathway.","authors":"Chunyu Kong, Changlei Wang, Yuquan Shi, Lei Yan, Junhua Xu, W. Qi","doi":"10.1139/bcb-2019-0333","DOIUrl":"https://doi.org/10.1139/bcb-2019-0333","url":null,"abstract":"Osteoarthritis (OA) is a common joint degenerative disease. Vitamin D (VD) is essential for bone function in human body. We hypothesized that active VD may play key functions in OA treatment. Low level of serum 25-hydroxyvitamin D (25(OH)D) was found in OA patients, and the serum VD level might be supportive for OA diagnosis. OA mouse models were established. HE and SafraninO/Fast Green staining suggested that active VD reduced OA symptoms in mice. VD treatment elevated p-AMPK/AMPK and decreased p-mTOR/mTOR, and it increased LC3II/LC3I, increased the protein level of Beclin-1, but decreased p62 according to Western blot analysis. Besides, VD reduced the contents of tumor necrosis factor-α and interleukin-6 both in cartilage tissues and in chondrocytes. Meanwhile, AMPK inhibitor Compound C and autophagy inhibitor 3-methyladenine (3-MA) reversed these changes following VD treatment. In addition, mRFP-GFP-LC3 transfection identified that active VD led to autophagosome aggregation in OA chondrocytes. 3-MA inhibited cell autophagy and promoted OA inflammation. This study provided evidence that active VD might activate chondrocyte autophagy to reduce OA inflammation via activating the AMPK/mTOR signaling pathway. Active OA treatment might serve as a novel therapeutic option for OA treatment.","PeriodicalId":9524,"journal":{"name":"Canadian journal of biochemistry and cell biology = Revue canadienne de biochimie et biologie cellulaire","volume":"37 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-12-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80403241","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Lili Miao, Fei Su, Yongsheng Yang, Yue Liu, Lei Wang, Y. Zhan, Ronghua Yin, Miao Yu, Changyan Li, Xiaoming Yang, Changhui Ge
Glycerol kinase (GYK) plays a critical role in hepatic metabolism by converting glycerol to glycerol 3-phosphate in an ATP-dependent reaction. GYK isoform b is the only glycerol kinase present in whole cells and has a non-enzymatic moonlighting function in the nucleus. GYK isoform b acts as a co-regulator of nuclear receptor subfamily 4 group A1 (NR4A1) and participates in the regulation of hepatic glucose metabolism by protein-protein interaction with NR4A1. Herein, GYK expression was found to upregulate the expression of NR4A1-mediated lipid metabolism-related genes (SREBP1C, FASN, ACACA, and GPAM) in HEK293T and L02 cells, and in mouse in vivo studies. GYK expression increased blood cholesterol, triglyceride, and high-density lipoprotein cholesterol levels but not low-density lipoprotein cholesterol levels. It enhanced the transcriptional activity of Nr4a1 target genes by negatively cooperating with NR4A1 and its enzymatic activity or by other undefined moonlighting functions. This enhancement was observed in both normal and diabetic mice. We also found a feed-forward regulation loop between GYK and NR4A1, serving as part of a GYK-NR4A1 regulatory mechanism in hepatic metabolism. Thus, GYK regulates the effect of NR4A1 on hepatic lipid metabolism in normal and diabetic mice, partially through the cooperation of GYK and NR4A1.
{"title":"Glycerol kinase enhances hepatic lipid metabolism by repressing nuclear receptor subfamily 4 group A1 in the nucleus.","authors":"Lili Miao, Fei Su, Yongsheng Yang, Yue Liu, Lei Wang, Y. Zhan, Ronghua Yin, Miao Yu, Changyan Li, Xiaoming Yang, Changhui Ge","doi":"10.1139/bcb-2019-0317","DOIUrl":"https://doi.org/10.1139/bcb-2019-0317","url":null,"abstract":"Glycerol kinase (GYK) plays a critical role in hepatic metabolism by converting glycerol to glycerol 3-phosphate in an ATP-dependent reaction. GYK isoform b is the only glycerol kinase present in whole cells and has a non-enzymatic moonlighting function in the nucleus. GYK isoform b acts as a co-regulator of nuclear receptor subfamily 4 group A1 (NR4A1) and participates in the regulation of hepatic glucose metabolism by protein-protein interaction with NR4A1. Herein, GYK expression was found to upregulate the expression of NR4A1-mediated lipid metabolism-related genes (SREBP1C, FASN, ACACA, and GPAM) in HEK293T and L02 cells, and in mouse in vivo studies. GYK expression increased blood cholesterol, triglyceride, and high-density lipoprotein cholesterol levels but not low-density lipoprotein cholesterol levels. It enhanced the transcriptional activity of Nr4a1 target genes by negatively cooperating with NR4A1 and its enzymatic activity or by other undefined moonlighting functions. This enhancement was observed in both normal and diabetic mice. We also found a feed-forward regulation loop between GYK and NR4A1, serving as part of a GYK-NR4A1 regulatory mechanism in hepatic metabolism. Thus, GYK regulates the effect of NR4A1 on hepatic lipid metabolism in normal and diabetic mice, partially through the cooperation of GYK and NR4A1.","PeriodicalId":9524,"journal":{"name":"Canadian journal of biochemistry and cell biology = Revue canadienne de biochimie et biologie cellulaire","volume":"55 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-12-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85082896","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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