蜂斗菜素调控circSETDB1/miR-345-5p对肝癌细胞生物学行为的影响  

Effects of petasin on the biological behaviors of hepatocellular carcinoma cells by regulating circSETDB1/miR-345-5p

作　　者：王锻 周莉 WANG Duan;ZHOU Li(Department of Pharmacy,Northwest University First Hospital,Xi'an,Shanxi 710043,China;Pharmaceutical Science Research Laboratory,Hebei University of Chinese Medicine,Shijiazhuang,Hebei 050200,China)

机构地区：[1]西北大学第一医院药学部,陕西西安710043 [2]河北中医药大学药剂学教研室,河北石家庄050200

出　　处：《湖南中医药大学学报》2024年第11期2014-2023,共10页Journal of Hunan University of Chinese Medicine

基　　金：河北省自然科学基金项目(H2020423006)。

摘　　要：目的本研究旨在评估蜂斗菜素对肝细胞癌细胞系2(hepatocellular carcinoma cell line 2,HepG2)的抗肿瘤活性,并探讨蜂斗菜素通过调控环状SET结构域蛋白1(circular RNA SET domain bifurcated histone lysine methyltransferase 1,circSETDB1)和微小RNA-345-5p(microRNA-345-5p,miR-345-5p)的mRNA表达对细胞增殖、凋亡和迁移的影响。方法将HepG2细胞分为多个处理组。对照组不做任何处理;蜂斗菜素处理包括蜂斗菜素L组(25 mol/L)、蜂斗菜素M组(50 mol/L)、蜂斗菜素H组(100 mol/L)、蜂斗菜素组(100 mol/L);siRNA干扰包含环状SET结构域蛋白1小干扰RNA(small interfering RNA targeting circular SET domain bifurcated histone lysine methyltransferase 1,si-circSETDB1)组、小干扰RNA阴性对照(small interfering RNA negative control,si-NC)组;过表达环状SET结构域蛋白1过表达质粒(overexpression vector for circular SET domain bifurcated histone lysine methyltransferase 1,pcDNA-circSETDB1)组和蜂斗菜素+pcDNA-circSETDB1组。采用qRT-PCR检测肝癌组织及癌旁组织的circSETDB1和miR-345-5p的mRNA表达水平;CCK-8检测细胞增殖活性;流式细胞术检测细胞凋亡情况;Western blot检测半胱氨酸天冬氨酸特异性蛋白酶-3(cleaved cysteinyl aspartate-specific proteinase-3,Cleaved-Caspase-3)蛋白表达水平;克隆形成实验检测细胞集落形成;划痕实验和Transwell实验分别检测细胞愈合率和迁移;双荧光素酶报告基因实验验证circSETDB1与miR-345-5p的靶向关系。结果与癌旁组织相比,肝癌组织中circSETDB1 mRNA表达升高(P<0.05),miR-345-5p mRNA表达降低(P<0.05)。与对照组相比,蜂斗菜素各剂量组HepG2细胞的细胞活性、集落形成数降低(P<0.05),细胞凋亡率和Cleaved-Caspase-3蛋白表达升高(P<0.05),且呈剂量依赖性(P<0.05)。与si-NC组相比,si-circSETDB1组HepG2细胞的circSETDB1表达、细胞活性、集落形成数、迁移细胞数和划痕愈合率降低(P<0.05),miR-345-5p表达、细胞凋亡率和Cleaved-Caspase-3�Objective To evaluate the anti-tumor activity of petasin on hepatocellular carcinoma cell line 2(HepG2)and to investigate its effects on cell proliferation,apoptosis,and migration by regulating the mRNA expressions of circular RNA SET domain bifurcated histone lysine methyltransferase 1(circSETDB1)and microRNA-345-5p(miR-345-5p).Methods Except for control group which received no treatment,HepG2 cells were additionally divided into multiple treatment groups,including petasin L(25μmol/L),petasin M(50μmol/L),and petasin H(100μmol/L)groups,as well as small interfering RNA targeting circular SET domain bifurcated histone lysine methyltransferase 1(si-circSETDB1)group,small interfering RNA negative control(si-NC)group,overexp-ression vector for circular SET domain bifurcated histone lysine methyltransferase 1(pcDNA-circSETDB1)group,petasin+pcDNA-circSETDB1 group,and petasin group(100μmol/L).The mRNA expression levels of circSETDB1 and miR-345-5p in hepatic cancerous tissues and paracancerous tissues were measured by qRT-PCR.Cell proliferation activity was measured using CCK-8,and apoptosis was assessed by flow cytometry.The protein expression level of cleaved cysteinyl aspartate-specific proteinase-3(Cleaved-Caspase-3)was determined by Western blot.Cell colony formation was assessed using the clone formation assay.Cell wound healing rate and migration were checked by wound healing assay and Transwell assay,respectively.A dual-luciferase reporter assay was performed to validate the targeting relationship between circSETDB1 and miR-345-5p.Results Compared with paracancerous tissues,the circSETDB1 mRNA expression was higher(P<0.05)while miR-345-5p mRNA expression was lower(P<0.05)in hepatic cancerous tissues.Compared with control group,the cell viability and number of colonies of HepG2 cells in the petasin L,M,and H groups were lower,while the apoptosis rate and Cleaved-Caspase-3 protein expression were higher(P<0.05),in a dose-dependent manner(P<0.05).Compared with si-NC group,the circSETDB1 expression,cell viability

关 键 词：蜂斗菜素 肝癌 circSETDB1 miR-345-5p 细胞增殖 凋亡 迁移 

分 类 号：R285.5[医药卫生—中药学]