姜黄素抑制NF-κB核易位调控胰腺癌对吉西他滨的敏感性  

Curcumin regulates sensitivity of pancreatic cancer to gemcitabine by inhibiting nuclear translocation of NF-κB

作　　者：王艳丽 戈立秀 曾昭伟[1,2] 王睿 张爱民 WANG Yanli;GE Lixiu;ZENG Zhaowei;WANG Rui;ZHANG Aimin(Department of Clinical Laboratory,Tianjin Nankai Hospital,Tianjin 300100,China;Tianjin Institute of Integratied Traditional Chinese and Western Medicine for Acute Abdominal Disease,Tianjing 300100,China;Tianjin Jinyu Medical Laboratory Co.,Ltd,Tianjin 300100,China)

机构地区：[1]天津市南开医院检验科,天津300100 [2]天津市中西医结合急腹症研究所,天津300100 [3]天津金域医学检验实验室有限公司,天津300100

出　　处：《重庆医学》2024年第15期2247-2253,共7页Chongqing Medical Journal

基　　金：天津市卫生健康委员会中医药管理局中西医结合科研课题一般项目(2021097);天津市自然科学基金项目(18JCQNJC13400);天津市中西医结合医院防治关键技术及方案优化2022年度重点专项(NKYY-IIT-2022-009-3)。

摘　　要：目的探讨姜黄素调控胰腺癌细胞增强吉西他滨敏感性的机制。方法为验证姜黄素对胰腺癌PANC1细胞中p53及核因子-κB(NF-κB)p65核易位的影响,将细胞以0、10、20、30μmol/L姜黄素处理,得到空白组、10μmol/L姜黄素组、20μmol/L姜黄素组和30μmol/L姜黄素组;采用免疫荧光染色技术在荧光显微镜下确定NF-κB p65细胞内分布情况;Western blot技术确定细胞内p53蛋白的表达水平;逆转录-实时荧光定量PCR技术确定细胞内p53 mRNA的表达水平;联合采用miRstar和JASPAR软件预测寻找可能受NF-κB p65核转位调控的微RNA(miRNA);采用双荧光素酶报告实验分别验证miRNA与p53的关系。为验证姜黄素是否通过NF-κB p65/miR-26b-5p/p53轴影响吉西他滨对PANC1细胞的敏感性,以10μmol/L的吉西他滨处理细胞得到吉西他滨组,以20μmol/L姜黄素及10μmol/L吉西他滨处理细胞得到姜黄素联合吉西他滨组;经姜黄素(20μmol/L)和吉西他滨(10μmol/L)联合处理细胞后,分别将寡核苷酸对照(mimic NC)和miR-26b-5p模拟物(mimic)转染至细胞,得到mimic NC和mimic miR-26b-5p组;将pcDNA3.1空载质粒和pcDNA 3.1-p53过表达质粒分别转染至细胞,得到pcDNA3.1组和p53组;采用MTT实验检测细胞活力;采用膜联蛋白-V(Annexin V)/碘化丙啶(PI)双染色流式细胞术测定细胞凋亡水平。结果与空白组比较,各姜黄素处理组(10、20、30μmol/L姜黄素组)细胞内p53 mRNA及蛋白表达水平均升高;与空白组比较,20μmol/L姜黄素组细胞核内NF-κB p65表达水平降低;miRstar和JASPAR预测软件找到8个miRNA可能受NF-κB p65核易位调控,其中有3个具有靶向p53基因的潜力,尤其以miR-26b-5p效果最明显。双荧光素酶报告实验验证发现miR-26b-5p与p53确有相互作用。与mimic NC组比较,mimic miR-26b-5p组p53 mRNA和蛋白表达水平均下降;与吉西他滨组比较,姜黄素联合吉西他滨组细胞活力降低、细胞凋亡率增加;与mimic NC组比较,mimic miR-26b-5pObjective To explore the mechanism of curcumin in enhancing gemcitabine sensitivity by regulating pancreatic cancer cells.Methods In order to verify the effect of curcumin on p53 and NF-κB p65 nuclear translocation in pancreatic cancer cell PANC1,the pancreatic cancer PANC1 cells were treated with 0,10,20,30μmol/L curcumin to obtain the blank group,10μmol/L curcumin group,20μmol/L curcumin group and 30μmol/L curcumin group.The intracellular distribution of NF-κB p65 was determined by immunofluorescence staining under fluorescence microscope.The expression level of p53 protein in cells was determined by Western blot.The expression level of p53 mRNA was determined by reverse transcription-real-time fluorescence quantitative PCR.The combined use of miRstar and JASPAR softwares predicted to seek microRNA(miRNA)potentially regulated by NF-κB p65 nuclear translocation.The double luciferase reporting assay was used to respectively verify the relationship between miRNA and p53.In order to verify whether curcumin infect the sensitivity of PANC1 cell through NF-κB p65/miR-266-5p/p53 axis,the gemcitabine group was obtained by 10μmol/L gemcitabine treating cells,and the curcumin combined gemcitabine group was obtained by 20μmol/L curcumin and 10μmol/L gemcitabine treating cells.After the cells were treated with curcumin(20μmol/L)and gemcitabine(10μmol/L),oligonucleotides mimic NC and miR-26b-5p mimic were transfected into cells,and the mimic NC group and miR-26b-5p group were obtained.pcDNA3.1 no-load plasmid and pcDNA3.1-p53 overexpression plasmid were transfected into cells,respectively,and the pcDNA3.1 group and p53 group were obtained.The cell activity was detected by MTT assay.The cellular apoptosis level was determined by Annexin V/PI double staining.Results Compared with the blank group,the intracellular p53 mRNA and protein expression levels in the various curcumin treated groups(10,20,30μmol/L curcumin groups)all were increased.Compared with the blank group,the expression level of NF-κB p65 in the nuc

关 键 词：姜黄素 NF-κB p65/miR-26b-5p/p53轴 胰腺癌 吉西他滨敏感性 

分 类 号：R285[医药卫生—中药学]