Department of Ophthalmology, Indiana University School of Medicine, 1160 W Michigan St, Indianapolis, IN, 46202, USA.
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN, 46202, USA.
BMC Genomics. 2024 Aug 12;25(1):782. doi: 10.1186/s12864-024-10679-3.
The LinSca1c-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.
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.
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.
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.
骨髓（BM）中的 LinSca1c-Kit（LSK）分数包含多能造血干细胞（HSCs），这对组织内稳态和血管修复至关重要。虽然糖尿病总体上影响 HSC 的内稳态，但在长期 2 型糖尿病（T2D；>6 个月）条件下的 mRNA 和 miRNA 转录组的分子特征仍未得到探索。
在这项研究中，我们评估了 db/db 小鼠（一种用于 T2D 的著名且广泛使用的模型）中 HSCs 的转录组特征。使用细胞分选器富集 db/db 小鼠的 LSK 细胞，然后对其进行配对末端 mRNA 和单端 miRNA seq 文库测序，并在 Illumina NovaSeq 6000 上进行测序。使用 STAR（拼接转录物到参考的比对）将 mRNA 序列读数映射，使用 Qiagen GeneGlobe RNA-seq Analysis Portal 将 miRNA 序列读数映射到指定的参考基因组，使用 miRNA 的默认参数。
我们发现 2076 个 mRNA 和 35 个 miRNA 在 db/db 动物中表达显著；引人注目的是，以前未报道的 miRNA（miR-3968 和 miR-1971）在 db/db 小鼠中下调。此外，我们观察到糖尿病中 HSCs 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。途径映射表明，趋化因子、细胞因子和血管生成介导的炎症是 db/db 小鼠中转录本数量明显增加的主要途径之一。这些分子变化反映在骨髓中 LSK 迁移的明显缺陷中。miRNA 下游靶分析揭示了几种针对白细胞迁移、小胶质细胞激活、吞噬体形成和巨噬细胞激活信号的 mRNAs 作为其主要途径，表明向炎症表型转变。
我们的研究结果表明，慢性糖尿病在转录水平上不利地改变 HSCs 的内稳态，从而可能导致长期糖尿病下 HSCs 的炎症表型。我们还认为，在 miRNA 或 mRNAs 中识别 HSCs 相关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记物和潜在治疗靶点。
