PDF(Pubmed)
Abstract：
BACKGROUND: The Lin-Sca1+c-Kit+ (LSK) fraction of the bone marrow (BM) comprises multipotent hematopoietic stem cells (HSCs), which are vital to tissue homeostasis and vascular repair. While diabetes affects HSC homeostasis overall, the molecular signature of mRNA and miRNA transcriptomic under the conditions of long-standing type 2 diabetes (T2D;>6 months) remains unexplored.
METHODS: In this study, we assessed the transcriptomic signature of HSCs in db/db mice, a well-known and widely used model for T2D. LSK cells of db/db mice enriched using a cell sorter were subjected to paired-end mRNA and single-end miRNA seq library and sequenced on Illumina NovaSeq 6000. The mRNA sequence reads were mapped using STAR (Spliced Transcripts Alignment to a Reference), and the miRNA sequence reads were mapped to the designated reference genome using the Qiagen GeneGlobe RNA-seq Analysis Portal with default parameters for miRNA.
RESULTS: We uncovered 2076 out of 13,708 mRNAs and 35 out of 191 miRNAs that were expressed significantly in db/db animals; strikingly, previously unreported miRNAs (miR-3968 and miR-1971) were found to be downregulated in db/db mice. Furthermore, we observed a molecular shift in the transcriptome of HSCs of diabetes with an increase in pro-inflammatory cytokines (Il4, Tlr4, and Tnf11α) and a decrease in anti-inflammatory cytokine IL10. Pathway mapping demonstrated inflammation mediated by chemokine, cytokine, and angiogenesis as one of the top pathways with a significantly higher number of transcripts in db/db mice. These molecular changes were reflected in an overt defect in LSK mobility in the bone marrow. miRNA downstream target analysis unveils several mRNAs targeting leukocyte migration, microglia activation, phagosome formation, and macrophage activation signaling as their primary pathways, suggesting a shift to an inflammatory phenotype.
CONCLUSIONS: Our findings highlight that chronic diabetes adversely alters HSCs\' homeostasis at the transcriptional level, thus potentially contributing to the inflammatory phenotype of HSCs under long-term diabetes. We also believe that identifying HSCs-based biomarkers in miRNAs or mRNAs could serve as diagnostic markers and potential therapeutic targets for diabetes and associated vascular complications.
METHODS: In this study, we assessed the transcriptomic signature of HSCs in db/db mice, a well-known and widely used model for T2D. LSK cells of db/db mice enriched using a cell sorter were subjected to paired-end mRNA and single-end miRNA seq library and sequenced on Illumina NovaSeq 6000. The mRNA sequence reads were mapped using STAR (Spliced Transcripts Alignment to a Reference), and the miRNA sequence reads were mapped to the designated reference genome using the Qiagen GeneGlobe RNA-seq Analysis Portal with default parameters for miRNA.
RESULTS: We uncovered 2076 out of 13,708 mRNAs and 35 out of 191 miRNAs that were expressed significantly in db/db animals; strikingly, previously unreported miRNAs (miR-3968 and miR-1971) were found to be downregulated in db/db mice. Furthermore, we observed a molecular shift in the transcriptome of HSCs of diabetes with an increase in pro-inflammatory cytokines (Il4, Tlr4, and Tnf11α) and a decrease in anti-inflammatory cytokine IL10. Pathway mapping demonstrated inflammation mediated by chemokine, cytokine, and angiogenesis as one of the top pathways with a significantly higher number of transcripts in db/db mice. These molecular changes were reflected in an overt defect in LSK mobility in the bone marrow. miRNA downstream target analysis unveils several mRNAs targeting leukocyte migration, microglia activation, phagosome formation, and macrophage activation signaling as their primary pathways, suggesting a shift to an inflammatory phenotype.
CONCLUSIONS: Our findings highlight that chronic diabetes adversely alters HSCs\' homeostasis at the transcriptional level, thus potentially contributing to the inflammatory phenotype of HSCs under long-term diabetes. We also believe that identifying HSCs-based biomarkers in miRNAs or mRNAs could serve as diagnostic markers and potential therapeutic targets for diabetes and associated vascular complications.
摘要：
背景：骨髓（BM）的Lin-Sca1c-Kit（LSK）部分包含多能造血干细胞（HSC），对组织稳态和血管修复至关重要。虽然糖尿病整体影响HSC稳态，在长期2型糖尿病(T2D;>6个月)的条件下,mRNA和miRNA转录组的分子特征仍未被研究.
方法：在本研究中，我们评估了db/db小鼠中HSC的转录组特征，一个众所周知且广泛使用的T2D模型。对使用细胞分选仪富集的db/db小鼠的LSK细胞进行双端mRNA和单端miRNAseq文库,并在IlluminaNovaSeq6000上测序。使用STAR（剪接转录本与参考比对）定位mRNA序列读段，并且使用QiagenGeneGlobeRNA-seq分析门户将miRNA序列读段映射到指定的参考基因组,具有miRNA的默认参数。
结果：我们发现了在db/db动物中显著表达的13,708个mRNAs中的2076个和191个miRNAs中的35个；先前未报道的miRNA（miR-3968和miR-1971）在db/db小鼠中被下调。此外，我们观察到糖尿病HSC转录组的分子变化,促炎细胞因子(Il4,Tlr4和Tnf11α)增加,抗炎细胞因子IL10减少.通路图显示了趋化因子介导的炎症，细胞因子,和血管生成是db/db小鼠中转录物数量明显较高的主要途径之一。这些分子变化反映在骨髓中LSK迁移率的明显缺陷中。miRNA下游靶标分析揭示了几种靶向白细胞迁移的mRNA，小胶质细胞激活，吞噬体形成,和巨噬细胞激活信号作为它们的主要途径，提示向炎症表型的转变。
结论：我们的研究结果强调，慢性糖尿病在转录水平上不利地改变了HSCs的稳态，因此可能导致长期糖尿病下HSC的炎症表型。我们还相信,在miRNA或mRNA中鉴定基于HSC的生物标志物可以作为糖尿病和相关血管并发症的诊断标志物和潜在治疗靶标。
方法：在本研究中，我们评估了db/db小鼠中HSC的转录组特征，一个众所周知且广泛使用的T2D模型。对使用细胞分选仪富集的db/db小鼠的LSK细胞进行双端mRNA和单端miRNAseq文库,并在IlluminaNovaSeq6000上测序。使用STAR（剪接转录本与参考比对）定位mRNA序列读段，并且使用QiagenGeneGlobeRNA-seq分析门户将miRNA序列读段映射到指定的参考基因组,具有miRNA的默认参数。
结果：我们发现了在db/db动物中显著表达的13,708个mRNAs中的2076个和191个miRNAs中的35个；先前未报道的miRNA（miR-3968和miR-1971）在db/db小鼠中被下调。此外，我们观察到糖尿病HSC转录组的分子变化,促炎细胞因子(Il4,Tlr4和Tnf11α)增加,抗炎细胞因子IL10减少.通路图显示了趋化因子介导的炎症，细胞因子,和血管生成是db/db小鼠中转录物数量明显较高的主要途径之一。这些分子变化反映在骨髓中LSK迁移率的明显缺陷中。miRNA下游靶标分析揭示了几种靶向白细胞迁移的mRNA，小胶质细胞激活，吞噬体形成,和巨噬细胞激活信号作为它们的主要途径，提示向炎症表型的转变。
结论：我们的研究结果强调，慢性糖尿病在转录水平上不利地改变了HSCs的稳态，因此可能导致长期糖尿病下HSC的炎症表型。我们还相信,在miRNA或mRNA中鉴定基于HSC的生物标志物可以作为糖尿病和相关血管并发症的诊断标志物和潜在治疗靶标。
