microRNA-933对LX-2细胞凋亡和增殖的影响及其分子机制  

Effect of miRNA-933 on the apoptosis and proliferation of LX-2 cells and its molecular mechanism

作　　者：海龙 马丽娜[1,2] 雒夏 丁向春[1,2] HAI Long;MA Lina;LUO Xia;DING Xiangchun(Department of Infectious Diseases,General Hospital of Ningxia Medical University,Yinchuan 750004,China;School of Clinical Medicine,Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学总医院感染疾病科,银川750004 [2]宁夏医科大学临床医学院,银川750004

出　　处：《临床肝胆病杂志》2024年第7期1382-1389,共8页Journal of Clinical Hepatology

基　　金：宁夏医科大学总医院创新项目(2023AAC03584)。

摘　　要：目的 探讨microRNA-933(miRNA-933)对人肝星状细胞系LX-2细胞凋亡和增殖的调控作用及其机制。方法 首先以人肝组织为研究对象,利用基因芯片技术,检测肝硬化与慢性乙型肝炎肝组织相较于正常肝组织中差异表达的基因,并从中筛选差异表达显著的miRNA,从而确定研究对象为miRNA-933。进而以人肝星状细胞系LX-2为研究对象,利用mi RNA-933分子模拟剂(mi RNA-933 mimic)与抑制剂(mi RNA-933 siRNA),构建LX-2过表达与敲减模型,并以转染mimic-NC(过表达)或si RNA-NC(敲减)的细胞作为阴性对照。使用实时荧光定量PCR(q PCR)和Western Blot技术,检测mi RNA-933与活化标志蛋白的表达水平;随后采用细胞增殖实验、流式细胞术等技术,检测mi RNA-933对细胞凋亡、增殖和活化的影响,并探讨其机制。计量资料两组间比较采用成组t检验,多组间比较采用单因素方差分析,并经过Bonferroni校正。结果 基于基因芯片检测结果,最终筛选出18个显著差异表达的mi RNA,其中mi RNA-933表达显著下调(P<0.05)。mi RNA-933 mimic与siRNA转染LX-2细胞后,结果显示,与阴性对照组相比,miRNA-933 siRNA显著下调miRNA-933的表达(P=0.000 7),而mi RNA-933 mimic显著上调mi RNA-933的表达(P=0.000 3);Western Blot和q PCR检测结果表明,mi RNA-933 siRNA显著上调胶原蛋白Ⅰ(CollagenⅠ)和α-平滑肌肌动蛋白(α-SMA)的表达(P值均<0.001),而mi RNA-933 mimic显著抑制CollagenⅠ和α-SMA的表达(P值均<0.05)。流式细胞术检测结果显示,与阴性对照组相比,miRNA-933 siRNA显著下调LX-2细胞凋亡率(P=0.031 9),miRNA-933 mimic显著上调LX-2细胞凋亡率(P=0.005 5);Western Blot检测结果表明,与阴性对照组相比,miRNA-933 siRNA可以抑制LX-2细胞中胱天蛋白酶3(Caspase-3)和多聚ADP核糖聚合酶1(PARP-1)的表达(P值分别为0.006 7、0.003 0),上调B淋巴细胞瘤-2(Bcl-2)的表达(P=0.002 0),而miRNA-933 mimic可显著上调Caspase-3(P=0.011 8)和PARP-1(P=0.049 5)的表达,下Objective To investigate the regulatory effect of miRNA-933 on the apoptosis and proliferation of human hepatic stellate cell line LX-2 and its mechanism.Methods Firstly,with human liver tissue for research,gene microarray technology was used to detect the differentially expressed genes in liver tissue between liver cirrhosis/chronic hepatitis B tissue and normal liver tissue,among which the significantly differentially expressed miRNAs were identified,and thus miRNA-933 was determined as the research object.Then,with the human hepatic stellate cell line LX-2 for research,miRNA-933 mimic and inhibitor(miRNA-933 siRNA)were used to construct the LX-2 models of overexpression and knockdown,and the cells transfected with mimic-NC(overexpression)or siRNA-NC(knockdown)were established as the negative control group.Quantitative real-time PCR and Western blot were used to measure the expression levels of miRNA-933 and activation biomarkers;techniques such as cell proliferation assay and flow cytometry were used to investigate the effect and mechanism of miRNA-933 on cell apoptosis,proliferation,and activation.The independent-samples t test was used for comparison of continuous data between two groups;a one-way analysis of variance was used for comparison between multiple groups,and Bonferroni correction was also performed.Results A total of 18 significantly differentially expressed miRNAs were obtained based on the results of gene microarray,among which miRNA-933 was significantly downregulated(P<0.05).After LX-2 cells were transfected with miRNA-933 mimic or siRNA,compared with the negative control group,miRNA-933 siRNA significantly downregulated the expression of miRNA-933(P=0.0007),while miRNA-933 mimic significantly upregulated the expression of miRNA-933(P=0.0003).Western blot and quantitative real-time PCR showed that miRNA-933 siRNA significantly upregulated the expression of collagen I andα-SMA(P<0.001),while miRNA-933 mimic significantly inhibited the expression of collagen I andα-SMA(P<0.05).Flow cytometry s

关 键 词：肝纤维化 微RNAS 肝星状细胞 

分 类 号：R575.2[医药卫生—消化系统]