山姜素调控miR-654-3p/HMGB1轴影响肺癌细胞A549增殖、迁移和凋亡  

Alpinetin affects proliferation,migration and apoptosis of lung cancer cell A549 through regulating miR-654-3p/HMGB1 axis

作　　者：刘磊 孙京海 林家茂 LIU Lei;SUN Jing-hai;LIN Jia-mao(College of Integrated Traditional Chinese and Medicine,Jining Medical University,Jining Shandong 272076,China;Dept of Pharmacy,the Affiliated Cancer Hospital of Shandong First Medical University,Jinan 250117,China;Dept of Oncology,the Affiliated Cancer Hospital of Shandong First Medical University,Jinan 250117,China)

机构地区：[1]济宁医学院中西医结合学院,山东济宁272067 [2]山东第一医科大学附属肿瘤医院药剂科,山东济南250117 [3]山东第一医科大学附属肿瘤医院肿瘤内科,山东济南250117

出　　处：《中国药理学通报》2024年第10期1892-1898,共7页Chinese Pharmacological Bulletin

基　　金：山东省自然科学基金创新发展联合基金项目(No ZR2023LZY007);山东省中医药重点学科建设项目(鲁卫中医药科教字[2022]4号)。

摘　　要：目的探讨山姜素对肺癌细胞生物学行为的影响,并分析其抗肿瘤机制是否与调控miR-654-3p/高迁移率族蛋白B1(high mobility group box-1 protein,HMGB1)轴有关。方法将肺癌A549细胞分为0μmol·L^(-1)组、山姜素(50、100、200μmol·L^(-1))组、miR-NC组、miR-654-3p mimic组、si-NC组、si-HMGB1组、山姜素+miR-654-3p inhibitor组、山姜素+pcDNA-HMGB1组。细胞计数试剂盒(cell counting kit-8,CCK-8)法、流式细胞术分别检测抑制率和凋亡率。平板克隆实验、Transwell小室法分别检测细胞克隆形成数和迁移数。实时荧光定量聚合酶链式反应(quantitative reverse transcription polymerase chain reaction,RT-qPCR)分析miR-654-3p和HMGB1 mRNA表达。双荧光素酶报告实验确定miR-654-3p对HMGB1的靶向作用。蛋白免疫印记实验分析蛋白表达。结果与0μmol·L^(-1)组比较,山姜素(50、100、200μmol·L^(-1))组A549细胞抑制率、凋亡率、miR-654-3p表达上调,而克隆形成数、迁移数、HMGB1表达下调(P<0.01)。与miR-NC组比较,miR-654-3p mimic组A549细胞抑制率、凋亡率上升,而克隆形成数、迁移数、HMGB1表达下降(P<0.01)。与si-NC组比较,si-HMGB1组A549细胞抑制率、凋亡率增加,而克隆形成数、迁移数降低(P<0.01)。miR-654-3p与HMGB1直接特异性结合。与山姜素组比较,山姜素+miR-654-3p Inhibitor组、山姜素+pcDNA-HMGB1组A549细胞抑制率、凋亡率、Bcl-2相关x蛋白(Bcl-2-associated x protein,Bax)及E-cadherin蛋白表达下调,而克隆形成数、迁移数、B细胞淋巴瘤/白血病-2基因(B-cell lymphoma-2,Bcl-2)和N-cadherin蛋白表达上调(P<0.01)。结论山姜素在肺癌中具有抗增殖、抗迁移和促凋亡作用,其机制与上调miR-654-3p/HMGB1轴有关。Aim To investigate the effect of alpinetin on the biological behavior of lung cancer cells,and further to analyze the relation between its anti-tumor mechanism and the regulation of miR-654-3p/high mobility group protein B1(HMGB1)axis.Methods The lung cancer A549 cells were divided into 0μmol·L^(-1)group,alpinetin(50,100,200μmol·L^(-1))group,miR-NC group,miR-654-3p mimic group,si-NC group,si-HMGB1 Group,alpinetin+miR-654-3p inhibitor group,alpinetin+pcDNA-HMGB1 group.Cell counting kit-8(CCK-8)method and flow cytometry were performed to detect the inhibition rate and apoptosis rate respectively.Plate cloning assay and Transwell chamber method were applied to detect the numbers of clone formation and migration.Quantitative reverse transcription polymerase chain reaction(RT-qPCR)was employed to analyze the expression of miR-654-3p and HMGB1 mRNA.Dual luciferase reporter assay was used to confirm the targeting effect of miR-654-3p on HMGB1.Western blotting was performed to detect protein expression.Results Compared with the 0μmol·L^(-1)group,the inhibition rate,apoptosis rate and miR-654-3p expression of A549 cells in the alpinetin group(50,100,200μmol·L^(-1))increased,while clone formation numbers,migration numbers and HMGB1 expression were reduced(P<0.01).Compared with the miR-NC group,the inhibition rate and apoptosis rate of A549 cells in the miR-654-3p mimic group increased,while clone formation numbers,migration number and HMGB1 expression decreased(P<0.01).Compared with the si-NC group,the inhibition rate and apoptosis rate of A549 cells in the si-HMGB1 group increased,while clone formation numbers and migration were reduced(P<0.01).MiR-654-3p directly and specifically bound to HMGB1.Compared with the alpinism group,the inhibition rate and apoptosis rate of A549 cell and BCL2-associated x protein(Bax)and E-cadherin protein expression in alpinetin+miR-654-3p inhibitor group,alpinetin+pcDNA-HMGB1 group were reduced,while clone formation numbers,migration and B-cell lymphoma-2(Bcl-2)and N-cadherin protein

关 键 词：肺癌 山姜素 miR-654-3p HMGB1 增殖 迁移 凋亡 

分 类 号：R329.25[医药卫生—人体解剖和组织胚胎学] R329.28[医药卫生—基础医学] R342.2R734.2R916.4