基于单细胞测序数据分析养阴活胃合剂下调AQP3对MC细胞表型的影响及机制

Effect and mechanism of Yangyin Huowei Mixture(YYHWM)down-regulating Aquaporin 3(AQP3)on MC cell phenotype based on single-cell sequencing data

目的 观察养阴活胃合剂对MC细胞(MNNG诱导人胃黏膜上皮细胞GES-1恶性转化)增殖、迁移、侵袭及凋亡的影响,探讨其下调AQP3抑制IL-10/JAK1/STAT3信号通路激活进而阻断或逆转慢性萎缩性胃炎(CAG)病变的作用机制。方法 选取GEO数据库中的CAG单细胞转录组测序数据,绘制数据的表达矩阵。采用R语言的Seurat 4.3.0包进行处理后分群绘制UMAP可视化图谱。将MC细胞分为养阴活胃合剂组(YYHWM组)、AQP3低表达慢病毒转染组(AQP3低表达组)、养阴活胃合剂+AQP3高表达慢病毒转染组(YYHWM+AQP3高表达组)并以MC细胞和正常人胃黏膜上皮细胞GES-1分别作为模型组(MC组)和空白对照组(Control组)。采用平板克隆、划痕实验、Transwell及流式细胞术检测细胞表型变化情况,采用ELISA检测细胞培养上清IL-10表达水平,Western blot检测酪氨酸激酶1(JAK1)、信号转导和转录激活因子3(STAT3)蛋白表达水平。结果 单细胞测序数据集分析显示AQP3在CAG样本及上皮细胞群中表达升高,AQP3可能通过IL-10抗炎性信号通路影响胃癌前病变病理进程。与MC组相比,AQP3低表达组和YYHWM组细胞侵袭、迁移、增殖能力减弱,凋亡率增加,细胞培养上清中的IL-10水平降低,细胞中JAK1、STAT3蛋白表达下调(P<0.05或P<0.01),YYHWM+AQP3高表达组差异均不显著(P>0.05)。结论 养阴活胃合剂可通过下调AQP3表达抑制IL-10/JAK1/STAT3信号通路激活进而减弱MC细胞侵袭、迁移及增殖能力,促进细胞凋亡进而阻断或逆转CAG病理进程。

Objective To observe the effect of Yangyin Huowei Mixture(YYHWM)on MC cell(MNNG induces malignant transformation of GES-1 in human gastric mucosal epithelial cells)proliferation,migration,invasion and apoptosis,and to explore the mechanism of its downregulating AQP3 to inhibit the activation of IL-10/JAK1/STAT3 signaling pathway to block or reverse CAG lesions.Methods Selected CAG single cell transcriptome sequencing data from GEO database and drawn the expression matrix of the data.UMAP visualization was processed using Seurat 4.3.0 package of R language.MC cells were divided into YYHWM group,Lentiviral transfection group with low expression of AQP3(AQP3 low-expression group),YYHWM and Lentiviral transfection group with high expression of AQP3(YYHWM+AQP3 high-expression group).MC cells and normal human gastric mucosa epithelial cells GES-1 were used as model control group(MC group)and blank control group(control group).Cell phenotype changes were detected by plate cloning,scratch assay,transwell and flow cytometry.IL-10 expression levels were deter-mined by ELISA and the expression levels of JAK1 and STAT3 were determined by western blot.Res-ults Analysis of single-cell sequencing datasets revealed the expression level of AQP3 elevated in CAG samples and epithelial cell populations.AQP3 may affect the pathological process of precancerous GC through IL-10 anti-inflammatory signaling pathway.Compared with MC group,the AQP3 low-expression group and YYHWM group showed reduced cell invasion,migration and proliferation,increased apoptosis rate,decreased IL-10 levels in the cell culture supernatant,and decreased JAK1 and STAT3 protein ex-pression levels in the cells(P<0.05 or P<0.01),YYHWM+AQP3 high-expression group showed no significant differences(P>0.05).Conclusion YYHWM can inhibit the activation of IL-10/JAK1/STAT3 signaling pathway by down-regulating the expression of AQP3,thus weakening the invasion,mi-gration and proliferation of MC cells,promoting cell apoptosis and thus blocking or reversing the patholog-ical process of CAG.