关键词: 血管平滑肌细胞, 成骨分化, 细胞钙化, mRNA测序, 铁死亡

Abstract:

Objective To screen the differentially expressed genes （DEGs） under high phosphate-induced calcification in the vascular smooth muscle cells （VSMCs） by mRNA high-throughput sequencing technology， and to analyze the key genes and signaling pathways involved in the VSMCs calcification. Methods The human VSMCs were divided into control group and model group. The cells in model group was exposed to the high-phosphate medium， while the cells in control group were cultured in DMEM supplemented with 10% fetal bovine serum under the same conditions. The VSMCs in two groups， stably transfected， were cultured for 12 d. The morphology of the cells in two groups were observed and photographed under inverted microscope. The DEGs were selected by Hisat2 software， and Gene Ontology （GO） functional and Kyoto Encyclopedia of Genes and Genomes （KEGG） signaling pathway enrichment analysis were performed by Stringtie software from three aspects， such as biological processes （BP）， molecular functions （MF）， and cellular components （CC）. The calcification of the cells in two groups was observed by Von Kossa staining method. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to analyze the expression levels of alkaline phosphatase （ALP）， bone morphogenetic protein 2 （BMP2）， alpha-smooth muscle actin （α-SMA）， tumor protein 53 （Tp53）， glutathione peroxidase 4 （GPX4）， ferritin light chain 1 （Ftl1）， and glycosylphosphatidylinositol-specific phospholipase D1 （GPLD1） mRNA in the cells in two groups. Results Compared with control group， there were 2 524 DEGs in the cells in model group， and there were 1 368 upregulated DEGs and 1 156 downregulated DEGs. Clustering of DEGs between the cells in two groups was distinct. The GO functional and KEGG pathway enrichment analysis results showed that the upregulated DEGs were primarily involved in regulating the microtubule cytoskeleton， cell polarity， protein localization， and cell cycle regulation among BPs； in constructing cell membrane， microtubule organization， chromosomes， and kinetochore among CCs； and functioning in phosphatidylinositol phosphate， Rho GTPase protein binding， transmembrane transport， and protein kinase regulatory activity among MFs. Downregulated DEGs were mainly involved in cytoplasmic translation， protein membrane localization， mRNA metabolism， and protein endoplasmic reticulum localization among BPs； in forming ribosome subunits， cell membrane， and autophagy among CCs； and functioning in single-stranded DNA， ribonucleoprotein complex， growth factor binding， regulating protein kinase activity， and catalytic activity among MFs. Seven signaling pathways were significantly enriched in upregulated genes， most notably in the biosynthesis of glycosylphosphatidylinositol （GPI） anchors； whereas 18 signaling pathways were significantly enriched in the downregulated genes， most notably in ferroptosis.The RT-qPCR results showed that compared with control group， the expression levels of GPX4， Ftl1， and Tp53 mRNA in the cells in model group were significantly decreased （P<0.01）， while the expression level of GPLD1 mRNA was significantly increased （P<0.01）； compared with control group， the expression level of α-SMA mRNA in the cells in model group was significantly decreased （P<0.01）， and the expression levels of ALP and BMP2 mRNA were significantly increased （P<0.01）. Conclusion The VSMCs underwent calcification and normal cells exhibit the DEGs.The key signaling pathways in the calcification induced by high phosphate in the VSMCs include ferroptosis and GPI anchor biosynthesis， mediated primarily through GPX4， Ftl1， Tp53， and GPLD1.

Key words: Vascular smooth muscle cell, Osteogenic differentiation, Cell calcification, mRNA sequencing, Ferroptosis