摘要
目的 通过生物信息学和实验来分析和验证宣肺平喘胶囊治疗COPD潜在的自噬相关基因.方法 将HBE细胞分为空白对照组、模型组、实验组,每组重复3次.通过CCK8检测筛选最佳给药时间,将干预样本送深圳华大基因公司进行转录组测序,获取了 mRNA的表达数据,随后进行差异表达、靶基因预测和功能富集分析,最后对支气管上皮细胞样本进行RT-qPCR和Western Blot验证.结果 经过差异表达分析,预测得其125个靶基因主要与丝裂原活化蛋白激酶激酶结合、白细胞介素-33结合、1-吡咯啉-5-羧酸脱氢酶活性以及MAPK信号通路相关.在125个靶基因中经最大邻域分量(MNC)算法获得与自噬相关的核心基因为CSF1、AREG、MAPK9、MAP3K7、AKT3.qRT-PCR与 Western Blot结果显示CSF1、MAPK9、MAP3K7和AKT3在实验组和模型组的表达水平与生物信息学分析结果一致.结论 通过qRT-PCR和WB验证了转录组测序分析的结果,并确定了 4个潜在的自噬相关基因.宣肺平喘胶囊可能通过下调CSF1、MAPK9、MAP3K7和AKT3的表达来调节自噬,从而达到治疗慢性阻塞性肺疾病的目的.
Abstract
Objective We analyze and validate the potential autophagy related genes of Xuanfei Pingchuan Capsule in treating COPD through bioinformatics and experiments.Methods HBE cells were divided into a blank control group,a model group,and an experimen-tal group,with each group repeated 3 times.Screen the optimal administration time through CCK8 detection.The intervention samples were sent to Shenzhen Huada Gene Company for transcriptome sequencing to obtain mRNA expression data,followed by differential ex-pression,target gene prediction,and functional enrichment analysis.Finally,the bronchial epithelial cell samples were validated by RT-qPCR and Western Blot.Results The result of differential expression analysis displayed that 125 target genes of HBE cells were main-ly related to mitogen-activated protein kinase(MKK)binding,interleukin 33 binding,1-Pyrroline-5-carboxylate dehydrogenase activity,and the mitogen-activated protein kinase(MAPK)signal pathway.Among 125 target genes,the core genes related to autoph-agy were obtained through the Maximum Neighborhood Component(MNC)algorithm,including CSF1,AREG,MAPK9,MAP3K7,and AKT3.The qRT PCR and Western Blot results showed that the expression levels of CSF1,MAPK9,MAP3K7,and AKT3 in the experi-mental and model groups were consistent with the results of bioinformatics analysis.Conclusion We validated the results of transcriptome sequencing analysis using qRT PCR and WB,and identified four potential autophagy related genes.Xuanfei Pingchuan Capsule may reg-ulate autophagy by downregulating the expression of CSF1,MAPK9,MAP3K7,and AKT3,thereby achieving the goal of treating chronic obstructive pulmonary disease.
维普
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