摘要
目的观察miR-505对阿霉素(ADM)耐药肝癌细胞耐药性及HMGB1表达的影响并探讨其机制。方法采用药物浓度递增法建立肝癌细胞MHCC97阿霉素耐药细胞株MHCC97/ADM即为耐药组,培养48 h肝癌亲本细胞为MHCC97亲本细胞组;以MTT法检测MHCC97亲本细胞组及耐药细胞组(MHCC97/ADM细胞)的存活率及半数抑制浓度(IC_(50)),检测MHCC97亲本细胞及耐药细胞miR-505、HMGB1表达差异。对耐药细胞(MHCC97/ADM细胞)给予miR-505拟似剂及miR-505抑制剂转染,以上调或下调miR-505表达,qRT-PCR检测转染效率;与阴性对照组(不进行转染的细胞)比较,MTT法及流式细胞术观察miR-505表达水平对ADM耐药MHCC97细胞生存、IC_(50)及凋亡的影响,以双荧光素酶实验检测miR-505与HMGB1靶向调控关系。结果随着ADM浓度的升高,亲本细胞株细胞存活率降低,抑制率升高,MHCC97细胞株的IC_(50)值为(1.25±0.13)mM,耐药细胞株的IC_(50)值为(41.78±5.41)mM,耐药指数(RI)为33.25。与MHCC97亲本细胞比较,MHCC97耐药细胞miR-505表达下调,HMGB1 mRNA及蛋白表达上调(P<0.05);上调miR-505表达,MHCC97/ADM存活率降低,与阴性对照组比较,IC_(50)降至(23.05±3.88)mM,RI降至17.24。下调miR-505表达,MHCC97/ADM存活率升高,IC_(50)升至(80.28±11.34)mM,RI升至64.22(P<0.05);在MHCC97/ADM细胞组,与阴性对照组比较,上调miR-505表达,HMGB1mRNA及蛋白表达下降,下调miR-505表达,HMGB1mRNA及蛋白表达上调(P<0.05)。上调miR-505表达逆转MHCC97/ADM的耐药性,增加耐药细胞株凋亡率。在HMGB1的3′端存在miR-505的结合位点。结论上调miR-505表达通过负向调控HMGB1表达部分逆转肝癌细胞ADM耐药。
Objective To observe the effect of miR-505 on hepatocellular carcinoma cell adriamycin resistance and HMGB1 expression,and discuss the mechanism.Methods Adriamycin(ADM)resistance hepatocellular carcinoma cell lines(MHCC97/ADM,resistance group)were constructed by ADM concentration step increasing method.The 48 h hepatocellular carcinoma parent cells were cultured as the MHCC97 parent cells group.IC_(50) and cell viability in hepatocellular carcinoma parent cells and ADM resistance cells(MHCC97/ADM cells)were detected by MTT.miR-505 and HMGB1 expression were detected in MHCC97 and MHCC97/ADM cells.MHCC97/ADM cells were transfected with miR-505 mimics or inhibitor,qRT-PCR was used to identify the transfection efficiency,cornpared with negative control group(the cell without transfection),MTT and flow cytometry assay were used to observe the effect of miR-505 on MHCC97/ADM cells survival,IC_(50) and apoptosis.Dual luciferase reporter assay was used to identify the target relation between miR-505 and HMGB1.Results The viability of hepatocellular carcinoma parent cells was decreased with ascending ADM concentration;inhibition rate was increased;IC_(50) of MHCC97 cell lines was(1.25±0.13)mM;IC_(50) of MHCC97/ADM was(41.78±5.41)mM;RI was 33.25.Compared with MHCC97 parent cells,miR-505 in MHCC97/ADM was down-regulated,while HMGB1 mRNA and protein were up-regulated(P<0.05).Up-regulating miR-505 decreased MHCC97/ADM viability;compared with negative group,IC_(50) decreased to(23.05±3.88)mM,and RI decreased to 17.24.Down-regulating miR-505 increased MHCC97/ADM viability;IC_(50) increased to(80.28±11.34)mM,and RI increased to 64.22(P<0.05).In MHCC97/ADM cells,compared with negative group,up-regulating miR-505 decreased HMGB1 mRNA and protein expression,and down-regulating miR-505 increased HMGB1 mRNA and protein expression(P<0.05).Up-regulating miR-505 reversed the MHCC97/ADM resistance and increased apoptosis.There was a combining site at HMGB13′UTR end with miR-505.Conclusion Up-regulating miR-505 expression partially
作者
陆林
张冬辉
徐宇
杨景旭
LU Lin;ZHANG Dong-hui;XU Yu;YANG Jing-xu(Department of General Surgery,First Affiliated Hospital of Jinzhou Medical University,Jinzhou 121001,China;Department of Stomatology,Jinzhou Central Hospital,Jinzhou 121001,China)
出处
《实用药物与临床》
CAS
2022年第3期193-199,共7页
Practical Pharmacy and Clinical Remedies
基金
辽宁省自然科学基金项目(20180550440)。
