Fig 2: Sevoflurane regulates miR-18a-mediated RUNX1/Wnt/β-catenin signal pathway to suppress NSC proliferation and rat neurodevelopment.A The mRNA expression of RUNX1 under different concentrations of sevoflurane treatment detected by RT-qPCR. B The mRNA expression of β-catenin under different concentrations of sevoflurane treatment detected by RT-qPCR. C The protein expression of RUNX1 and β-catenin under different concentrations of sevoflurane treatment detected by Western blot analysis. D The expression of Ki67 protein in NSCs after different concentrations of CWP232228 treatment detected by IHC staining (bar = 25 μm); PI: the nucleus in red fluorescence, Ki67: green fluorescence. E Intuitive and statistical plots of sphere-forming ability of NSCs after sevoflurane, 3 f, miR-18a inhibitor, or CWP232228 treatment (bar = 100 μm). F The effect of sevoflurane, 3 f, miR-18a inhibitor, or CWP232228 treatment on the movement time of rats in the water maze assessed by mirris water maze test. G The effect of sevoflurane, 3 f, miR-18a inhibitor, or CWP232228 treatment on the movement distance of rats in the open field assessed by open field test. H The effect of sevoflurane, 3 f, miR-18a inhibitor, or CWP232228 treatment on the freezing time to cue of rats assessed by conditioned fear test. I The effect of sevoflurane, 3 f, miR-18a inhibitor, or CWP232228 treatment on the freezing time to context of rats assessed by conditioned fear test. J The expression of neural cell marker protein (NF-H/NF-M/NF-L) in cells and tissues detected by Western blot analysis. *p < 0.05 vs. the control/sevoflurane group; #p < 0.05 vs. the sevoflurane + miR-18a inhibitor/sevoflurane + antagomiR-18a group. n = 6. The cell experiment was repeated 3 times independently.

Fig 3: Schematic diagram of the molecular mechanism by which sevoflurane inactivated the RUNX1/Wnt/β-catenin signaling pathway through miR-18a upregulation to regulate NSC proliferation in 9-day-old neonatal rats.Sevoflurane inactivates the RUNX1/Wnt/β-catenin signaling pathway by inducing miR-18a expression, whereby inhibiting NSC proliferation and affecting rat neurodevelopment.

Fig 4: Single-cell RNA sequencing (scRNAseq) in combination with CRISPR activation identifies arterial cell type and functional haematopoietic expansion in association with activation of the nine target genes.(A) Gene expression profile of target genes following target genes’ activation, heatmap shows the expression level of the target genes in the iSAM_NT and iSAM_AGM treated with doxycycline (DOX) following normalisation on the -DOX control. (B) Dimension reduction and clustering analysis of the scRNAseq data following activation, filtered on cells where the gRNA expression was detected. (C) Arterial (GJA4, DLL4), venous (NRP2, APLNR), and haemogenic marker (CD44, RUNX1) expression distribution in the clusters indicated by the colour. (D) Expression distribution visualised on the UMAP plot showing the location of arterial cells marked by DLL4, and haemogenic endothelium marked by CD44 and RUNX1. (E) Heatmap of the top 15 marker genes for each of the clusters. (F) Contribution of the different libraries to the clusters showing that arterial cell cluster is overrepresented in the iSAM_AGM treated with DOX, compared to the other libraries. (G) Expansion of the arterial population assessed by the membrane marker expression of DLL4+ following targets’ activation, quantified by flow cytometry at day 8 of differentiation. (Data are normalised on the iSAM_NT+DOX sample, n=5 independent differentiations, *p=0.0417 paired t-test.) (H) Colony-forming potential of the suspension progenitor cells derived from the two lines treated with or without DOX following OP9 coculture activation, data show the colony obtained for 104 CD34+ input equivalent (n=3 from independent differentiations *p<0.05, Tukey’s two-way ANOVA). Figure 3—source data 1.Spreadsheet source file containing the source data used for the plots in Figure 3.Each tab is labelled to uniquely refer to a specific panel.

Fig 5: RUNX1 knockdown alleviates hypoxia-induced PASMC dysfunction. The regulation of RUNX1 on hypoxic PASMCs was investigated: RT-qPCR (A) and western blotting (B) were used to detect RUNX1 mRNA and protein expression; (C) CCK-8 was used to detect cell proliferation; (D) Transwell assay was performed to detect cell invasion; (E) Scratch assay was performed to detect cell migration; (F) Flow cytometry was used to detect cell apoptosis. N ═ 3; *P < 0.05 and **P < 0.01 vs the control group; #P < 0.05 and ##P < 0.01 vs the hypoxia group. PASMC: Pulmonary artery smooth muscle cell; CCK: Cell counting kit; RT-qPCR: Reverse transcription-quantitative polymerase chain reaction.