鼠妇miRNA-2863调控Bcl-2/Bax/Caspase-3信号通路抑制肝癌细胞增殖、迁移和侵袭  

Armadillidium vulgare miRNA-2863 inhibits liver cancer cell proliferation,migration and invasion via Bcl-2/Bax/Caspase-3 signaling pathway

作　　者：郭倩倩 王若宇[2] 卢林竹 谭年花 易纯 田雪飞[1] GUO Qianqian;WANG Ruoyu;LU Linzhu;TAN Nianhua;YI Chun;TIAN Xuefei(College of Integrative Chinese and Western Medicine,Hunan Provincial Key Laboratory of Translational Medicine in Chinese Medicine,Key Laboratory of Chinese Medicine Prevention and Treatment of Tumor Mechanism in Hunan,Hunan University of Chinese Medicine,Changsha 410208,China;The First Affiliated Hospital of Hunan University of Chinese Medicine,Changsha 410007,China;Medical College,Hunan University of Chinese Medicine,Changsha 410208,China)

机构地区：[1]湖南中医药大学中西医结合学院,湖南中医药大学方证转化湖南省重点实验室,湖南省中医药防治肿瘤机理重点实验室,湖南长沙410208 [2]湖南中医药大学第一附属医院,湖南长沙410007 [3]湖南中医药大学医学院,湖南长沙410208

出　　处：《中草药》2024年第24期8445-8456,共12页Chinese Traditional and Herbal Drugs

基　　金：2022年度“刘良院士工作站”指导项目(22YS002);中药粉体与创新药物省部共建国家重点实验室培育基地开放基金项目(2022 FTKFJJ16);湖南省自然科学基金青年基金项目(2023JJ40487);湖南省教育厅科学研究项目(21B0392);湖南省教育厅优秀青年项目(22B0392);湖南省中医药科研计划一般项目(B2023009);湖南省研究生科研创新项目(CX20230820)。

摘　　要：目的探讨鼠妇Armadillidium vulgare miRNA-2863(avu-miR-2863)对肝癌HepG2细胞和MHCC97H细胞增殖、迁移、侵袭的影响及相关机制。方法基于small RNA测序筛选鼠妇的miRNA,将HepG2细胞和MHCC97H细胞分为对照组和avu-miR-2863组,采用CCK-8法、划痕实验和Transwell侵袭实验检测肝癌细胞增殖、迁移和侵袭能力;3D肿瘤球模拟体内肿瘤微环境研究肿瘤细胞的增殖;Western blotting法检测细胞周期蛋白D1(cyclin D1)、细胞性骨髓细胞瘤原癌基因(cellular myelocytomatosis oncogene,C-Myc)、E-钙黏蛋白(E-cadherin,E-Ca)、N-Ca、波形蛋白(vimentin,Vim)和基质金属蛋白酶14(matrix metalloproteinase 14,MMP14)的表达;利用生信分析预测avu-miR-2863的靶基因,京都基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析和基因本体论(gene ontology,GO)功能富集分析探讨avu-miR-2863的作用机制,Western blotting法检测活化的半胱氨酸天冬氨酸蛋白酶-3(cleaved cysteine-aspartic protease-3,cleaved Caspase-3)、B细胞淋巴瘤-2(B-cell lymphoma-2,Bcl-2)和Bcl-2关联X蛋白单克隆抗体(monoclonal antibody to Bcl-2 associated X protein,Bax)蛋白的表达;流式细胞术检测细胞凋亡情况;Mito-Tracker Red、Hoechst染色和2,7二氯二氢荧光素二乙酸酯(DCFH-DA)分别进行线粒体荧光和细胞内活性氧(reactive oxygen species,ROS)检测;qRT-PCR检测avu-miR-2863在小鼠血液中的表达情况。结果通过高通量测序技术从鼠妇中挖掘并筛选鉴定出avu-miR-2863;与对照组比较,avu-miR-2863可抑制HepG2和MHCC97H细胞的增殖、迁移和侵袭(P<0.01),抑制3D肿瘤球体积的增大(P<0.01);可显著降低Cyclin D1、C-Myc、N-ca、Vim、MMP14蛋白的表达(P<0.05、0.01)并显著升高E-ca蛋白的表达(P<0.01)。生信分析预测avu-miR-2863可能通过调控Bcl-2家族蛋白介导的凋亡通路发挥抗肝癌的作用;与对照组比较,avu-miR-2863可显著升高cleaved Caspase-3、Bax蛋白的表达(P<0.05、0.01)并降低BObjective To explore effects and underlying mechanisms of miRNA-2863 derived from the traditional Chinese medicine Armadillidium vulgare,on the proliferation,migration,and invasion of HepG2 and MHCC97H liver cancer cells.Methods Small RNA sequencing identified miRNAs from A.vulgare,followed by screening candidate miRNAs.HepG2 and MHCC97H cells were divided into control group and avu-miR-2863 group.Cell proliferation,migration,and invasion were assessed using CCK-8,scratch,and Transwell invasion assays.3D tumor sphere model was used to simulate the tumor microenvironment and evaluate tumor cell proliferation.Western blotting was performed to detect the expression of cyclin D1,cellular myelocytomatosis oncogene(C-Myc),E-cadherin(E-ca),N-ca,vimentin(Vim),and matrix metalloproteinase 14(MMP14).Bioinformatics analysis was conducted to predict target genes of avu-miR-2863,and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment and Gene Ontology(GO)function enrichment analyses were performed to explore its mechanism of action.The protein expression of cleaved cysteine-aspartic protease-3(cleaved Caspase-3),B-cell lymphoma-2(Bcl-2)and monoclonal antibody to Bcl-2 associated X protein(Bax)were also detected by Western blotting.Cell apoptosis was assessed using flow cytometry,while mitochondrial fluorescence and intracellular reactive oxygen species(ROS)were detected using Mito-Tracker Red,Hoechst staining,and DCFH-DA assays.The expression of avu-miR-2863 in blood was measured by qRT-PCR.Results High-throughput sequencing identified and screened avu-miR-2863.Compared with the control group,avu-miR-2863 inhibited the proliferation,migration,and invasion of HepG2 and MHCC97H cells(P<0.01)and reduced the size of 3D tumor spheres(P<0.01).avu-miR-2863 downregulated the expression of Cyclin D1,C-Myc,N-ca,Vim,and MMP14(P<0.05,0.01),while upregulating E-ca expression(P<0.01).Bioinformatics analysis suggested that avu-miR-2863 may exert its anti-liver cancer effects by regulating the Bcl-2 family-mediated apoptosis path

关 键 词：鼠妇 MICRORNA 肝癌 增殖 迁移 凋亡 

分 类 号：R285.5[医药卫生—中药学]