文献类型：期刊文献

中文题名：基于miRNA组学探讨慈菇消脂方调控TGF-β1诱导LX2细胞活化的作用机制

英文题名：Mechanism of Cigu Xiaozhi Formula(慈菇消脂方) in Regulating TGF-β1-Induced LX2 Cell Activation Based on miRNA Omics

作者：任真[1];马燕花[1];杨少军[2];赵秀萍[1];王莉[1];王爱娣[1]

第一作者：任真

机构：[1]甘肃中医药大学,兰州730000;[2]北海市中医医院,北海536000

第一机构：甘肃中医药大学

年份：2023

卷号：39

期号：3

起止页码：32

中文期刊名：中药药理与临床

外文期刊名：Pharmacology and Clinics of Chinese Materia Medica

收录：北大核心:【北大核心2020】；CSCD:【CSCD2023_2024】；

基金：国家自然科学基金地区基金项目(编号:81860821)。

语种：中文

中文关键词：慈菇消脂方;生物信息学;微小RNA-199a-5p;刺猬信号通路;肝星状细胞

外文关键词：Cigu Xiaozhi Formula(慈菇消脂方);Bioinformatics;miRNA-199a-5p;Hedgehog signaling pathway;Hepatic stellate cells

摘要：目的:运用生物信息学方法筛选慈菇消脂方干预人肝星状LX2细胞中差异表达微小RNA(micro RNA,miRNA),并进一步探讨差异miRNA对活化LX2细胞刺猬(Hedgehog, Hh)信号通路的影响及慈菇消脂方含药血清的干预机制。方法:CCK-8法检测不同浓度含药血清,不同时间处理对细胞活性影响,筛选最佳作用时间和作用浓度。6%慈菇消脂方含药血清干预LX2细胞72 h,以空白血清组作为正常对照,进行总RNA提取、RNA质检、文库构建,使用测序平台为illumina HiSeqTM2500进行高通量测序,以P<0.05的标准筛选差异miRNA,利用miRanda和RNAhybrid两个软件进行miRNA靶基因预测,得到miRNA和靶基因间的对应关系。对差异miRNA靶基因分别进行Gene Ontology和KEGG富集分析,筛选Hh信号通路差异表达miRNA。转化生长因子-β1(TGF-β1)诱导建立LX2细胞活化模型。试验随机分为正常对照组、模型对照组、6%慈菇消脂方含药血清组、环靶明脂组、微小RNA-199a-5p(miR-199a-5p)过表达组及其阴性对照组。CCK-8法检测细胞增殖情况;Q-PCR和Western-blot技术检测细胞中miR-199a-5p、α-平滑肌肌动蛋白(α-SMA)、Ⅰ型-胶原(Col-I)及Hh信号通路相关蛋白表达,以此探究miR-199a-5p对活化LX2细胞Hh信号通路的影响及慈菇消脂方含药血清的干预机制。结果:与正常对照组比较,模型对照组细胞活力显著升高(P<0.01),miR-199a-5p表达上调(P<0.01),Sonic刺猬信号通路(Sonic Hedgehog, Shh)、脑胶质瘤相关癌基因1(Glioma related oncogene homology-1,Gli-1)、Gli-2、Col-I、α-SMA蛋白及mRNA表达显著上调(P<0.01);与模型对照组比较,6%慈菇消脂方含药血清组细胞活力显著降低(P<0.01),miR-199a-5p表达显著下调(P<0.01),Gli2、α-SMA蛋白表达显著下调(P<0.05)。结论:慈菇消脂方含药血清可以抑制TGF-β1诱导的LX2细胞活化,其机制可能通过下调miR-199a-5p进而抑制Hh信号通路蛋白表达发挥作用。
Objective:To screen the differentially expressed miRNA in human hepatic stellate cell line(LX2)intervened by Cigu Xiaozhi Formula(慈菇消脂方)using bioinformatics,to further explore the effect of differential miRNA on Hedgehog(Hh)signaling pathway of activated LX2 cells and the intervention mechanism of Cigu Xiaozhi Formula.Methods:Cell counting kit-8(CCK-8)assay was performed to detect the effects of different concentrations of drug-contained serum on cell activity at different treatment times,and to screen the optimal time and concentration.LX2 cells were treated with 6%Cigu Xiaozhi Formula-contained serum for 72 h,and with the blank serum group as normal control,the total RNA extraction,RNA quality inspection and library construction were carried out.High-throughput sequencing was performed using illumina HiSeqTM2500,and P<0.05 was used as the standard for screening differential miRNA.MiRNA candidate genes were predicted by miRanda and RNAhybrid to obtain the relationship between miRNA and candidate genes.GO and KEGG enrichment analysis were performed to screen the miRNA with differential expression in Hh signaling pathway.Activated LX2 cell model was induced by transforming growth factor-β1(TGF-β1),and the experimental cells were randomly divided into normal control group,model control group,6%Cigu Xiaozhi Formula group,cyclopamine group,miR-199a-5p overexpression group,and negative control group.CCK-8 assay was performed to detect cell proliferation in each group.The expressions of miR-199a-5p,α-smooth muscle actin(α-SMA),type I collagen(COL-I)and Hh signaling pathway related proteins in each group were detected by quantitative real time polymerase chain reaction(Q-PCR)and Western blot(WB),to explore the effect of miR-199a-5p on Hh signaling pathway of activated LX2 cells and the intervention mechanism of Cigu Xiazhi Formula.Results:Compared with the normal control group,the model control group had an increase in cell activity(P<0.01)and an up-regulation in the expression of miR-199a-5p(P<0.01)as well as the protein and mRNA expressions of Sonic Hedgehog(Shh),glioma-associated oncogene homology 1(Gli1),Gli2,Col-I andα-SMA(P<0.01).Compared with the model group,the 6%Cigu Xiaozhi Formula group had decreased cell viability(P<0.01)and down-regulated expression of miR-199a-5p(P<0.01)as well as protein expressions of Gli2 andα-SMA(P<0.05).Conclusion:Bioinformatics and cell experiments preliminarily reveal that Cigu Xiaozhi Formula can inhibit the activation of LX2 cells induced by TGF-β1,and the mechanism may be related to down regulating miR-199a-5p and inhibiting Hh signaling pathway proteins.