Effect of miRNA26b knockout on vascular calcification [scRNA-seq]

Vascular calcification is the abnormal deposition of calcium phosphates within the blood vessels. Although it significantly contributes to the development of cardiovascular disease, much remains unknown about the mechanisms driving this process. Recent advances have shown that microRNAs (miRNAs) could be critical in regulating the biological processes driving vascular calcification. Here, we investigated whether miRNA-26b (miR-26b) could regulate vascular calcification. By performing scRNA-seq of the aorta of miR-26b knockout (miR-26bKO) mice, we identified cell-specific targets and demonstrated that components of the BMP signalling pathway are dysregulated in the aortic cell types. To investigate the contribution of miRNA26b to the development of vascualr calcification in mice we generated miRNA26b knockout model (miRNA26bKO) . We performed scRNA-seq using the whole aorta from deliniate regulated pathways in individual cell types. The whole mouse aorta was harvested from 6-month-old wild-type and miR-26bKO mice (wildtype n = 3 and miR26bKO n = 3) to determine the transcriptomes. The aorta was dissected from surrounding tissue, and the perivascular fat was removed. Upon digestion of tissue using protocol as reported below, cells were isolated and sorted using a FACS Aria II instrument and BD FACSDiva software (BD Biosciences).血管钙化（Vascular calcification）是磷酸钙在血管内的异常沉积。尽管其显著促进心血管疾病的发生发展，但目前对驱动该过程的分子机制仍知之甚少。近年来的研究进展表明，微小RNA（microRNAs，miRNAs）在调控血管钙化的生物学过程中可能发挥关键作用。本研究旨在探讨miRNA-26b（miR-26b）是否能够调控血管钙化过程。通过对miR-26b基因敲除（miR-26bKO）小鼠的主动脉进行单细胞RNA测序（scRNA-seq），我们鉴定出了细胞特异性靶点，并证实骨形态发生蛋白（Bone Morphogenetic Protein，BMP）信号通路的相关组分在主动脉各类细胞中存在表达失调。为探究miRNA-26b对小鼠血管钙化发生发展的贡献，我们构建了miRNA-26b基因敲除模型（miRNA26bKO）。我们通过对全主动脉进行scRNA-seq，以解析单个细胞类型中的调控通路。我们从6月龄野生型及miR-26bKO小鼠中获取全主动脉样本（野生型n=3，miR-26bKO组n=3）以进行转录组分析。将主动脉从周围组织中分离，并剥离血管周脂肪。采用下述组织消化方案处理样本后，通过FACS Aria II仪器及BD FACSDiva软件（BD Biosciences）分离并分选细胞。