本文旨在构建容积调控性阴离子通道主要成分LRRC8A的细胞模型,并应用该模型研究LRRC8A的生理特性。构建LRRC8A和YFP-H148Q/I152L真核表达载体,应用脂质体转染、抗生素筛选和有限稀释,获取共表达LRRC8A和YFP-H148Q/I152L的Fisher大鼠甲...本文旨在构建容积调控性阴离子通道主要成分LRRC8A的细胞模型,并应用该模型研究LRRC8A的生理特性。构建LRRC8A和YFP-H148Q/I152L真核表达载体,应用脂质体转染、抗生素筛选和有限稀释,获取共表达LRRC8A和YFP-H148Q/I152L的Fisher大鼠甲状腺滤泡上皮(Fischer rat thyroid, FRT)细胞。倒置荧光显微镜观察目的基因表达情况,荧光淬灭动力学实验检测LRRC8A和YFP-H148Q/I152L的功能。获得用于研究LRRC8A容积调控性阴离子通道的细胞模型,并应用该细胞模型研究LRRC8A的生理特性,包括阴离子转运特性、渗透压对LRRC8A的开放、阴离子转运速度的影响以及氯离子通道抑制剂对LRRC8A的作用。结果显示:(1)成功获得共表达LRRC8A和YFP-H148Q/I152L的FRT细胞,该细胞模型可用于LRRC8A容积调控性氯离子通道生理特性的研究。(2)在低渗状态下,LRRC8A容积调控性阴离子通道激活,可转运阴离子,如:碘离子和氯离子等;YFP-H148Q/I152L可用于研究阴离子的转运速度;渗透压是LRRC8A容积调控性阴离子通道开放的调控因素,其开放与渗透压呈负相关;氯离子通道抑制剂对LRRC8A通道的转运功能具有抑制作用,并呈剂量依赖关系。上述结果提示,本研究成功构建LRRC8A细胞模型,且应用该模型研究显示LRRC8A具有经典的容积调控性阴离子通道的特性。展开更多
胃癌作为全球高发恶性肿瘤,其代谢重编程引起的酸代谢异常在肿瘤进展中具有重要作用,然而其具体分子机制及靶向干预策略仍有待深入探索。本研究结合TCGA和GTEx数据库的转录组数据,系统探究了胃癌细胞酸性代谢的分子调控机制及预后相关...胃癌作为全球高发恶性肿瘤,其代谢重编程引起的酸代谢异常在肿瘤进展中具有重要作用,然而其具体分子机制及靶向干预策略仍有待深入探索。本研究结合TCGA和GTEx数据库的转录组数据,系统探究了胃癌细胞酸性代谢的分子调控机制及预后相关标志物。通过差异表达分析、WGCNA共表达网络构建、随机森林模型和生存分析,筛选出与酸性代谢特征及肿瘤微环境酸碱平衡的调控相关的103个差异表达基因(75个上调,28个下调),其中16个基因与胃癌细胞排酸功能显著相关。WGCNA分析揭示了green模块(模块核心基因包括LRRC8C)与胃癌TNM分期正相关。随机森林模型在胃癌诊断中表现出高灵敏度和特异性,其中LRRC8C基因的特征重要性位于其他基因前列。生存分析进一步鉴定了LRRC8C核心基因能够作为独立预后标志物,且该基因高表达与患者不良预后显著相关。本研究为胃癌的分子分型、预后评估及靶向治疗提供了新视角。Gastric cancer, a prevalent malignancy worldwide, is critically influenced by metabolic reprogramming-driven acid metabolism dysregulation during tumor progression. However, its specific molecular mechanisms and targeted intervention strategies remain underexplored. This study systematically investigated the molecular regulatory mechanisms of acidic metabolism and prognosis-related biomarkers in gastric cancer using transcriptomic data from the TCGA and GTEx databases. Through differential expression analysis, WGCNA co-expression network construction, random forest modeling, and survival analysis, 103 differentially expressed genes (75 upregulated and 28 downregulated) associated with acidic metabolic features and acid-base balance regulation in the tumor microenvironment were identified, including 16 genes significantly linked to acid extrusion in gastric cancer cells. WGCNA revealed the green module (core gene: LRRC8C) to be positively correlated with TNM staging. The random forest model demonstrated high sensitivity and specificity in gastric cancer diagnosis, with LRRC8C ranking high in feature importance. Survival analysis further identified LRRC8C as an independent prognostic biomarker, where its high expression was significantly associated with poor patient outcomes. This study provides novel insights into molecular subtyping, prognostic evaluation, and targeted therapy for gastric cancer.展开更多
基金supported by the National Natural Science Foundation of China(No.81601234)the Scientific Project of Education Department of Jilin Province,China(No.JJKH20170418KJ,JJKH20191056KJ)+2 种基金the Sanitation and Health Technology Innovation Project,Jilin Province,China(No.2018J113)the Start Funding of Jilin Medical University(No.2017KYQD001)the National Students’ Innovation and Entrepreneurship Training Program of China(No.201613706020,201713743004)
文摘本文旨在构建容积调控性阴离子通道主要成分LRRC8A的细胞模型,并应用该模型研究LRRC8A的生理特性。构建LRRC8A和YFP-H148Q/I152L真核表达载体,应用脂质体转染、抗生素筛选和有限稀释,获取共表达LRRC8A和YFP-H148Q/I152L的Fisher大鼠甲状腺滤泡上皮(Fischer rat thyroid, FRT)细胞。倒置荧光显微镜观察目的基因表达情况,荧光淬灭动力学实验检测LRRC8A和YFP-H148Q/I152L的功能。获得用于研究LRRC8A容积调控性阴离子通道的细胞模型,并应用该细胞模型研究LRRC8A的生理特性,包括阴离子转运特性、渗透压对LRRC8A的开放、阴离子转运速度的影响以及氯离子通道抑制剂对LRRC8A的作用。结果显示:(1)成功获得共表达LRRC8A和YFP-H148Q/I152L的FRT细胞,该细胞模型可用于LRRC8A容积调控性氯离子通道生理特性的研究。(2)在低渗状态下,LRRC8A容积调控性阴离子通道激活,可转运阴离子,如:碘离子和氯离子等;YFP-H148Q/I152L可用于研究阴离子的转运速度;渗透压是LRRC8A容积调控性阴离子通道开放的调控因素,其开放与渗透压呈负相关;氯离子通道抑制剂对LRRC8A通道的转运功能具有抑制作用,并呈剂量依赖关系。上述结果提示,本研究成功构建LRRC8A细胞模型,且应用该模型研究显示LRRC8A具有经典的容积调控性阴离子通道的特性。
文摘胃癌作为全球高发恶性肿瘤,其代谢重编程引起的酸代谢异常在肿瘤进展中具有重要作用,然而其具体分子机制及靶向干预策略仍有待深入探索。本研究结合TCGA和GTEx数据库的转录组数据,系统探究了胃癌细胞酸性代谢的分子调控机制及预后相关标志物。通过差异表达分析、WGCNA共表达网络构建、随机森林模型和生存分析,筛选出与酸性代谢特征及肿瘤微环境酸碱平衡的调控相关的103个差异表达基因(75个上调,28个下调),其中16个基因与胃癌细胞排酸功能显著相关。WGCNA分析揭示了green模块(模块核心基因包括LRRC8C)与胃癌TNM分期正相关。随机森林模型在胃癌诊断中表现出高灵敏度和特异性,其中LRRC8C基因的特征重要性位于其他基因前列。生存分析进一步鉴定了LRRC8C核心基因能够作为独立预后标志物,且该基因高表达与患者不良预后显著相关。本研究为胃癌的分子分型、预后评估及靶向治疗提供了新视角。Gastric cancer, a prevalent malignancy worldwide, is critically influenced by metabolic reprogramming-driven acid metabolism dysregulation during tumor progression. However, its specific molecular mechanisms and targeted intervention strategies remain underexplored. This study systematically investigated the molecular regulatory mechanisms of acidic metabolism and prognosis-related biomarkers in gastric cancer using transcriptomic data from the TCGA and GTEx databases. Through differential expression analysis, WGCNA co-expression network construction, random forest modeling, and survival analysis, 103 differentially expressed genes (75 upregulated and 28 downregulated) associated with acidic metabolic features and acid-base balance regulation in the tumor microenvironment were identified, including 16 genes significantly linked to acid extrusion in gastric cancer cells. WGCNA revealed the green module (core gene: LRRC8C) to be positively correlated with TNM staging. The random forest model demonstrated high sensitivity and specificity in gastric cancer diagnosis, with LRRC8C ranking high in feature importance. Survival analysis further identified LRRC8C as an independent prognostic biomarker, where its high expression was significantly associated with poor patient outcomes. This study provides novel insights into molecular subtyping, prognostic evaluation, and targeted therapy for gastric cancer.
