摘要
目的探讨紫草素介导Hippo信号通路对鼻咽癌(NPC)细胞株CNE2生物学功能的影响。方法于2021年1—12月使用含不同浓度紫草素培养液(0、2.0、4.0、8.0、16.0 mg/L)培养对数生长期人鼻咽癌细胞(CNE2细胞)48 h,细胞计数试剂盒-8(CCK-8)法检测细胞存活率;流式细胞术、平板克隆、伤口愈合及Transwell实验分别检测CNE2细胞凋亡、集落形成、迁移及侵袭情况;实时荧光定量逆转录聚合酶链式反应(RT-qPCR)技术检测细胞yes相关蛋白1(YAP1)、具有PDZ结合基序的转录共激活子(TAZ)mRNA相对表达情况;蛋白质印迹法检测YAP1、yes相关蛋白1(p-YAP1)、TAZ、磷酸化具有PDZ结合基序的转录共激活子(p-TAZ)、N-钙黏蛋白(N-cad)、波形蛋白(Vim)及E-钙黏蛋白(E-cad)蛋白表达情况。结果与0 mg/L浓度紫草素CNE2细胞存活率(100%)、集落形成数(514.67±25.81)个、划痕愈合率(88.58±3.40)%、侵袭细胞数(233.67±15.01)个、YAP1与TAZ mRNA及蛋白(1.01±0.02、1.00±0.01、0.68±0.04、0.51±0.03)比较,2 mg/L浓度紫草素[(92.70±5.92)%、(452.33±22.72)个、(69.91±3.03)%、(195.33±18.15)个、0.93±0.02、0.91±0.05、0.56±0.03、0.44±0.02],4 mg/L浓度紫草素[(81.75±3.83)%、(308.33±22.12)个、(53.61±3.21)%、(153.33±10.02)个、0.81±0.03、0.76±0.04、0.45±0.03、0.30±0.03],8 mg/L浓度紫草素[(54.93±3.89)%、(173.67±13.65)个、(30.32±1.68)%、(92.67±6.66)个、0.65±0.03、0.54±0.04、0.31±0.03、0.24±0.02],16 mg/L浓度紫草素[(33.89±2.14)%、(85.33±13.05)个、(18.31±1.42)%、(52.33±6.03)个、0.41±0.02、0.30±0.02、0.18±0.02、0.16±0.02]降低(P<0.05),CNE2细胞凋亡率、p-YAP1与p-TAZ及E-cad蛋白相对表达水平均随紫草素作用浓度增大而升高(P<0.05);紫草素作用效果呈剂量依赖性(P<0.05)。结论紫草素可抑制NPC细胞株CNE2细胞恶性生物学功能,其作用机制可能与抑制Hippo信号通路核心下游信号因子YAP1、TAZ蛋白激活,阻止上皮间质转化(EMT)有关。
Objective To investigate the effect of shikonin on the biological function of nasopharyngeal carcinoma(NPC)cell line CNE2 by regulating Hippo signaling pathway.Methods From January 2021 to December 2021,CNE2 cells in logarithmic growth phase were cultured by culture medium containing different concentrations of shikonin(0,2.0,4.0,8.0,16.0 mg/L)for 48 hours,the cell survival rate was detected by cell counting kit-8(CCK-8)method.The apoptosis,colony formation,migration and invasion of CNE2 cells were detected by flow cytometry,plate cloning,wound healing and Transwell assay.Real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)was used to detect the relative expression of cell yes associated protein 1(YAP1)and transcrip⁃tional coactivator with PDZ-binding motif(TAZ)with PDZ binding motif mRNA.The expression levels of YAP1,phosphorylated yes as⁃sociated protein 1(p-YAP1),TAZ,phosphorylated transcriptional coactivator with PDZ-binding motif(p-TAZ),N-cadherin(N-cad),vi⁃mentin(Vim)and E-cadherin(E-cad)protein were detected by western blotting.Results Compared with 0 mg/L shikonin CNE2 cell survival rate(100%),colony formation number(514.67±25.81)cells,scratch healing rate(88.58±3.40)%,invasive cell number[(233.67±15.01)cells],YAP1 and TAZ mRNA and protein(1.01±0.02,1.00±0.01,0.68±0.04,0.51±0.03),2 mg/L shikonin(514.67±25.81)cells,scratch healing rate(88.58±3.40)%,invasive cell number[(233.67±15.01)cells],YAP1 and TAZ mRNA and protein(1.01±0.02,1.00±0.01,0.68±0.04,0.51±0.03),2 mg/L shikonin[(92.70±5.92)%,(452.33±22.72)cells,(69.91±3.03)%,(195.33±18.15)cells,(0.93±0.02),(0.91±0.05),(0.56±0.03),(0.44±0.02),4 mg/L shikonin[(81.75±3.83)%,(308.33±22.12)cells,(53.61±3.21)%,(153.33±10.02)cells,(0.81±0.03),(0.76±0.04),(0.45±0.03),(0.30±0.03),8 mg/L shikonin[(54.93±3.89)%,(173.67±13.65)cells,(30.32±1.68)%,(92.67±6.66)cells,(0.65±0.03),(0.54±0.04),(0.31±0.03),(0.24±0.02),16 mg/L shikonin[(33.89±2.14)%,(85.33±13.05)cells,(18.31±1.42)%,(52.33±6.03)cells,(0.41±0.02),(0.30±0.02),(0.18±0.02),(0.16±0.02)decreased(P<0.05),and the apopto⁃sis rate of CNE2 cells,the relative expression levels of p-YAP1,p-TAZ and E-cad protein increased with the increase of shikonin con⁃centration(P<0.05).The effects of shikonin were dose-dependent(P<0.05).Conclusion Shikonin can inhibit the malignant biological function of NPC cell line CNE2 cells,and its mechanism may be related to inhibiting the activation of the core downstream signaling fac⁃tors of the Hippo signaling pathway including YAP1 and TAZ proteins,and preventing epithelial mesenchymal transformation(EMT).
作者
曹华琳
尹昕
梁天嵩
CAO Hualin;YIN Xin;LIANG Tiansong(Department of Otolaryngology,Nanyang Central Hospital,Nanyang,He'nan 473000,China;Department of Radiotherapy,The First Affiliated Hospital of Zhengzhou University,Zhengzhou,He'nan 450000,China)
出处
《安徽医药》
CAS
2024年第1期31-36,共6页
Anhui Medical and Pharmaceutical Journal
基金
2019年度河南省医学科技攻关计划项目(LHGJ20190046)。
