芒柄花黄素通过调控miR-140-5p/TIGIT轴影响喉鳞状细胞癌细胞增殖和凋亡  

Effect of formononetin on proliferation and apoptosis of laryngeal squamous cell carcinoma cells and its possible mechanism

作　　者：武川军[1] 彭丽娜[1] 韩海平[1] 冯志星[1] Wu Chuanjun;Peng Lina;Han Haiping;Feng Zhixing(Department of Otolaryngology,Handan Central Hospital,Handan 056002,China)

机构地区：[1]河北省邯郸市中心医院耳鼻咽喉科,邯郸056002

出　　处：《中华细胞与干细胞杂志（电子版）》2023年第1期1-9,共9页Chinese Journal of Cell and Stem Cell（Electronic Edition）

摘　　要：目的:探讨芒柄花黄素对喉鳞状细胞癌细胞增殖、凋亡的影响及其可能的机制。方法:体外培养人喉鳞状细胞癌细胞系Hep-2,以MTT法检测0、5、10、20、40、60μmol/L芒柄花黄素处理24 h后Hep-2细胞活力,根据IC50值筛选出芒柄花黄素合适作用浓度。Hep-2细胞分别采用20、40μmol/L芒柄花黄素,40μmol/L芒柄花黄素+阴性对照(转染mirRNA-140-5p抑制剂阴性对照),40μmol/L芒柄花黄素+miR-140-5p inhibitor干预细胞敲减干预Hep-2细胞。分组转染及药物处理后,采用实时荧光定量PCR(qRT-PCR)实验和免疫印迹检测Hep-2细胞miR-140-5p与T细胞免疫球蛋白和ITIM结构域蛋白(TIGIT)表达;采用MTT法和平板集落形成实验检测Hep-2细胞增殖;采用流式细胞实验检测Hep-2细胞凋亡;采用免疫印迹检测Hep-2细胞增殖(PCNA、Cyclin D1)与凋亡相关蛋白(Bax、cleaved caspase-3)表达。于裸鼠背部皮下注射Hep-2细胞构建裸鼠移植瘤模型,随机分为对照组,25、50 mg/kg芒柄花黄素组,50 mg/kg芒柄花黄素+阴性对照组,50 mg/kg芒柄花黄素+miR-140-5p敲减组,分组处理后,检测各组裸鼠移植瘤质量与体积;采用双荧光素酶报告实验分析Hep-2细胞miR-140-5p对TIGIT的靶向调控。两组间比较采用t检验,多组间比较采用单因素方差分析,进一步两两比较采用SNK-q检验。结果:与对照比较,20、40μmol/L芒柄花黄素干预细胞miR-140-5p表达(1.79±0.16、2.58±0.22比1.04±0.11)、凋亡率[(36.17±8.14)%、(68.65±14.20)%比(2.10±0.65)%]、Bax与cleaved caspase-3蛋白表达升高(P<0.05),TIGIT蛋白(0.56±0.12、0.17±0.01比0.98±0.10)与mRNA表达(0.68±0.10、0.34±0.03比1.05±0.13)、集落生成率[(60.82±12.24)%、(35.36±8.20)%比(100.0±0.00)%]、细胞活力(63.12±11.03、39.75±7.14比100.0±0.00)、PCNA与Cyclin D1蛋白表达降低(P<0.05);与40μmol/L芒柄花黄素比较,芒柄花黄素+miR-140-5p敲减细胞miR-140-5p表达(1.11±0.13比2.58±0.22)、凋亡率[(5.04±1.51)%比(68.65±14.20Objective To investigate the effect of formononetin on the proliferation and apoptosis of laryngeal squamous cell carcinoma cells and its possible mechanism.Methods The human laryngeal squamous cell carcinoma cell line Hep-2 was cultured in vitro,and the cell viability of Hep-2 cells was detected by the MTT method after treatment with 0,5,10,20,40,60μmol/L formononetin for 24 h.The appropriate concentration of mancoxanthin was screened according to the IC50 value.Hep-2 cells cultured were treated with,20μmol/L formononetin(20μmol/L),40μmol/L formononetin(40μmol/L),formononetin(40μmol/L)+negative control(transfected with miR-140-5p inhibitor negative control),formononetin(40μmol/L)+miR-140-5p inhibitor(transfected with miR-140-5p inhibitor).After transfection and drug treatment,the expression of miR-140-5p and T cell immunoglobulin and ITIM domain protein(TIGIT)in Hep-2 cells in each group was detected by real-time fluorescence quantitative PCR(qRT-PCR)experiment and western blot;the proliferation of Hep-2 cells in each group was detected by MTT assay and plate colony formation assay;the apoptosis of Hep-2 cells in each group was detected by flow cytometry;Western blot was used to detect the proliferation(PCNA,Cyclin D1)and apoptosis-related protein(Bax,cleaved caspase-3)expression of Hep-2 cells in each group.Nude mice were subcutaneously injected with Hep-2 cells on the back to establish a transplanted tumor model in nude mice,and they were randomly grouped into a control group,a low-dose formononetin(25 mg/kg)group,a high-dose formononetin(50 mg/kg)group,high-dose formononetin(50 mg/kg)+negative control(transfected with miR-140-5p antagomir negative control)group,high dose of forsythia(50 mg/kg)+miR-140-5p knockdown(transfected with miR-140-5p antagomir)group.After treatment,the quality and volume of transplanted tumors in nude mice of each group were detected;a dual-luciferase reporter assay was used to analyze the targeted regulation of TIGIT by miR-140-5p in Hep-2 cells.Results Compared with the contr

关 键 词：芒柄花黄素 miR-140-5p/TIGIT 喉鳞状细胞癌细胞 增殖 凋亡 

分 类 号：R739.65[医药卫生—肿瘤]