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Transcriptome sequencing in HL-60 induced neutrophils between the stimulation of AGEs and high-glucose

ABSTRACT: We report the high-throughput sequencing results in mammalian cell lines. Neutrophils were induced by HL-60 with 1umol/L ATRA(all-trans retinoid acid ) stimulation for 7days. Neutrophils were then stimulated with 0.05ug/mL AGEs，30mmol/L D-glucose, AGEs+D-glucose and 0.05ug/mL BSA (control) for 24-hours, then mRNA profiles were extracted and tested using Illumina Hiseq 4000. This study provides a prospective work for the influence of AGEs and high-glucose in human neutrophils.

ORGANISM(S): Homo sapiens

PROVIDER: GSE163687 | GEO | 2022/12/09

REPOSITORIES: GEO

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Project description:Tendon pathologies affect a large portion of people with diabetes. This high rate of tendon pain, injury, and disease appears to manifest independent of well-controlled HbA1c and fasting blood glucose. Advanced glycation end products (AGEs) are elevated in the serum of those with diabetes. In vitro, AGEs severely impact tendon fibroblast proliferation and mitochondrial function. However, the extent that AGEs impact the tendon cell transcriptome has not been evaluated. The purpose of this study was to investigate transcriptome-wide changes that occur to tendon-derived fibroblasts following treatment with AGEs. We propose to complete a descriptive approach to pathway profiling to broaden our mechanistic understanding of cell signaling events that may contribute to the development of tendon pathology. Rat Achilles tendon fibroblasts were treated with glycolaldehyde-derived AGEs (200μg/ml) for 48 hours in normal glucose (5.5mM) conditions. In addition, total RNA was isolated, and the PolyA+ library was sequenced. We demonstrate that tendon fibroblasts treated with 200μg/ml of AGEs differentially express 2,159 gene targets compared to fibroblasts treated with an equal amount of BSA-Control. Additionally, we report in a descriptive and ranked fashion 21 implicated cell-signaling pathways. We demonstrate that tendon fibroblasts treated with 200μg/ml of AGEs differentially express 2,159 gene targets compared to fibroblasts treated with an equal amount of BSA-Control. Additionally, we report in a descriptive and ranked fashion 21 implicated cell-signaling pathways. Our findings suggest that AGEs disrupt the tendon fibroblast transcriptome on a large scale and that these pathways may contribute to the development and progression of diabetic tendinopathy. Specifically, pathways related to cell cycle progression and extracellular matrix remodeling were affected in our data set and may play a contributing role in the development of diabetic tendon complications.

Project description:2×10^7 HL60 cells were treated with 10 uM ATRA for 2 and 5 days, and conducted Hi-C to study the chromosomal architecture. Cells were fixed by 1% formaldehyde (v/v) for 15 min at room temperature (RT) with slowly rotation. Cells were lysed by 550 μl lysis buffer (500 μl 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630 and 50 μl protease inhibitors) using a homogenizer, and harvested the chromatin at 5,000 rpm and washed twice. The pellet was added 1% SDS and incubated at 65 oC for 10 min, then quenched by Triton X-100. The chromatin was digested by 600 U MboI (NEB, Ipswich, USA) at 37 oC overnight. The next day, enzymes were inactivated by 10% SDS at 65 oC for 30 min, and the restriction fragment overhangs were filled in using biotin-14-dCTP and Klenow at 37 oC for 45 min, and the ligation was conducted in 7.61 ml ligation mix (745 μl 10% Triton X-100, 745 μl 10x ligation buffer (500 mM Tris-HCl pH 7.5, 100 mM MgCl2, 100 mM DTT), 80 μl 10 mg/ml BSA, 80 μl 100 mM ATP and 5.96 ml water) for 4 h at 16°C. The blunt-end Hi-C ligation was sonicated and pull down by streptavidin beads, then purified by ethanol. Hi-C library was prepared for paired ending sequencing using NEBNext® UltraTM DNA Library Prep Kit for Illuina® (NEB). For regular 3C ligation, after enzymes inactivation, 10 μl 1 U/μl T4 DNA ligase was added to incubate at 16°C overnight. The interaction was detected by qPCR using the specific 3C primers (Table 1) followed by DNA purification. Hi-C was conducted in PE-150. An iterative method for Hi-C mapping with initial length 30bp, if it fails to map uniquely, the next round of mapping for additional 20 bp continued, this procedure lasts until the full read length reaches 150bp. The Hi-C reads were mapped to the human reference genome (assembly GRCh38) using bowtie2 (v2.2.9). The reads mapped to the same restriction fragment, the reads less than 500bp and PCR duplicates were all removed. Hi-C contact maps were normalized using ICE.

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Transcriptome sequencing in HL-60 induced neutrophils between the stimulation of AGEs and high-glucose

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