Abstract
This study examined the effects of Stmn2 on phenotype transformation of vascular smooth muscle in vascular injury via RNA sequencing and experimental validation. Total RNA was extracted for RNA sequencing after 1, 3 and 5 days of injury to screen the differentially expressed genes (DEGs). Western blot was used to detect the protein expression of Stmn2 and its associated targets. The morphological changes of carotid arteries in rats were examined by hematoxylin and eosin (H&E) staining. The expression of vascular smooth muscle cell (VSMC) phenotype markers smooth muscle alpha-actin (α-SMA), vimentin and OPN were detected by immunohistochemistry. DEGs were related to the extracellular matrix and other cell components outside the plasma membrane. They were associated with protein binding, cytoskeleton protein binding, signal receptor binding and other molecular functions, actin cytoskeleton regulation and other Kyoto Encyclopedia of Genes and Genomes pathways. Stmn2 was identified as the hub gene of actin cytoskeleton pathway and vascular disease, and its expression followed the trend of decreasing initially and increasing afterwards during the progress of vascular injury. Western blot assay showed that the expression of Stmn2 and Tubulin decreased immediately after vascular injury; Stmn2 overexpression significantly up-regulated the expression of osteopontin and α-SMA and vimentin in VSMCs. The results of morphology analysis and immunostaining also showed that Stmn2 overexpression promoted the intima thickening and enhanced the proliferating cell nuclear antigen expression in the injured vascular tissues. In conclusion, our results implied that Stmn2 may play a potential role in vascular injury, which may be associated with VSMC phenotype transformation. Further studies are warranted to determine detailed molecular mechanisms of Stmn2 in vascular injury.
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All the data are available upon the request from the corresponding author.
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Funding
This work was supported by the Science and Technology project of Shenzhen of China (JCYJ20180508152222104, JCYJ20180302173909492, KQJSCX20180329104902378), the National Natural Science Foundation of China (81600208, 81970210, 81570256), the Medical Scientific Research Foundation of Guangdong Province of China (A2018019). The study was supported by grants from the Natural Science Foundation of Guangdong Province of China (2018A030313771, 2019A1515010329).
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XK, ZW and MW conceived and supervised the study. ZW and MW made the final approval of manuscript. XK and WG contributed to data interpretation, manuscript writing and performed experiments. YP, ZF, YH and MD contributed to the data collection and analysis. All authors read and approved the final manuscript.
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All the animal experimental procedures were approved by the Animal Ethics Committee of Shenzhen Sun Yat-sen Cardiovascular Hospital.
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Supplementary Information
Supplemental Figure S1.
Schematic diagram of modeling procedures and representative images in the surgical procedures. CCA: common carotid artery; ICA:internal carotid artery; ECA: external carotid artery. (PNG 4469 kb)
Supplemental Figure S2.
The animal grouping and sample size in each group. (PNG 504 kb)
Supplemental Figure S3.
Expression and principal component analysis (PCA) of transcriptome sequencing data at different time points after vascular injury. (A) The results of sequencing showed that fpkm transformed violin map; (B) PCA analysis results of samples; (C) Heatmap of gene expression profile (log2 (fpkm)) in each group. (PNG 284 kb)
Supplemental Figure S4.
Analysis of differentially expressed genes in different groups. (A) The number of differentially expressed genes compared between different treatment groups. (B-F) The differentially expressed genes between different treatment groups were illustrated by volcano plots; (B) D1 group vs con group; (C) D3 group vs con group; (D) D5 group vs con group; (E) D3 group vs D1 group; (F) D5 group vs D1 group; (G) D5 group vs D3 group. (PNG 1159 kb)
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Ke, X., Guo, W., Peng, Y. et al. Investigation into the role of Stmn2 in vascular smooth muscle phenotype transformation during vascular injury via RNA sequencing and experimental validation. Environ Sci Pollut Res 29, 3498–3509 (2022). https://doi.org/10.1007/s11356-021-15846-7
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DOI: https://doi.org/10.1007/s11356-021-15846-7