摘要
目的探讨微小RNA-124(miR-124)在肾癌细胞自噬和顺铂耐药中可能的作用靶点。方法以实时荧光定量逆转录聚合酶链反应(RT-qPCR)法检测34例肾癌组织和癌旁组织,人肾小管上皮细胞HK-2以及4种人肾癌细胞A498、SN12C、RXF393、769P中miR-124表达。以蛋白质印迹法检测用30μg·mL^(-1)顺铂处理对肾癌细胞A498自噬相关蛋白LC3表达的影响。将A498肾癌细胞分为4组:对照组、顺铂组、第1转染组(DMSO+miR-124)、第2转染组(顺铂+miR-124组),在转染组细胞中添加miR-124 mimics。以细胞计数试剂盒8(CCK8)检测过表达miR-124对顺铂半数致死量(IC50)的影响。以荧光素酶报告实验和蛋白质印迹法验证miR-124对组蛋白去乙酰化酶4(HDAC4)的靶向调控作用。结果肾癌组织和癌旁组织miR-124表达分别为0.48±0.21和1.56±0.46,差异有统计学意义(P<0.05);人肾小管上皮细胞HK-2和人肾癌细胞A498、SN12C、RXF393、769P中miR-124表达分别为1.09±0.23,0.38±0.08,0.46±0.10,0.58±0.09,0.44±0.12,差异有统计学意义(P<0.05)。A498细胞用顺铂分别处理0,6,12,24 h,LC3表达分别为1.01±0.03,1.35±0.35,1.68±0.46,2.21±0.53,随着时间的延长,LC3蛋白表达显著增加(P<0.05)。第1转染组和第2转染组IC50分别为(7.81±1.14),(22.26±1.14)μg·mL^(-1),差异有统计学意义(P<0.05)。生物信息学网站预测发现HDAC4是miR-124的靶基因,荧光素酶报告实验显示,miR-124过表达可以降低野生型HDAC43'UTR荧光强度(P<0.05),而对突变型无明显影响(P>0.05)。对照组、顺铂组、第1转染组、第2转染组HDAC4蛋白表达分别为0.48±0.08,0.96±0.11,0.22±0.07,0.21±0.08,第1转染组、第2转染组分别与顺铂组比较,差异均有统计学意义(均P<0.05)。结论肾癌中miR-124表达降低,顺铂化疗可以诱导肾癌细胞自噬,而过表达miR-124可以降低肾癌细胞自噬,增强顺铂敏感性,其作用的靶标可能为HDAC4。
Objective To explore the possible target of microRNA-124(miR-124) in renal cell autophagy and cisplatin resistance. Methods Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) was used to detect the expression of miR-124 in 34 cases of renal cell carcinoma and adjacent tissues, human renal tubular epithelial cells HK-2 and four kinds of human renal cell carcinoma cells A498, SN12 C, RXF393 and 769 P. Western blot was used to detect the effect of 30 μg·mL-1 cisplatin on the expression of autophagy related protein LC3. A498 cells were divided into four groups: control group,cisplatin group,transfection-1 group,transfection-2 group,miR-124 mimics were added into transfected cells. The effects of miR-124 overexpression on cisplatin IC50 were detected by cell counting kit 8( CCK8). Luciferase report test and Western blotting test were used to verify the targeting effect of miR-124 on histone deacetylase 4( HDAC4). Results The expression of miR-124 in kidney cancer tissue and adjacent tissues were 0. 48 ± 0. 21 and 1. 56 ± 0. 46,the difference was statistically significant( P < 0. 05). The expression of miR-124 in HK-2 and A498,SN12 C,RXF393 and 769 P cells were 1. 09 ± 0. 23,0. 38 ± 0. 08,0. 46 ± 0. 10,0. 58 ± 0. 09,0. 44 ± 0. 12,with statistical significance( P < 0. 05). When A498 cells were treated with cisplatin for 0,6,12 and 24 h,the expression of LC3 were 1. 01 ± 0. 03,1. 35 ± 0. 35,1. 68 ± 0. 46 and 2. 21 ± 0. 53,with the extension of time,the expression of LC3 protein increased significantly( P < 0. 05). The IC50 of transfection-1 group and transfection-2 group were( 7. 81 ± 1. 14) and( 22. 26 ± 1. 14) μg·mL-1,with significant difference( P < 0. 05). Bioinformatics website predicted that HDAC4 was the target gene of miR-124.Luciferase assay showed that overexpression of miR-124 could reduce the fluorescence intensity of 3’UTR of wild-type HDAC4( P < 0. 05),but had no effect on mutant type( P > 0. 05). The expression of HDAC4 protein in control group,cisplatin group,transfe
作者
江典存
李晓治
倪良诚
黄玉钿
谢辉
任新
JIANG Dian-cun;LI Xiao-zhi;NI Liang-cheng;HUANG Yu-dian;XIE Hui;REN Xin(Department of Urology,Fuzhou First Hospital,Fuzhou 350009,Fujian Province,China;Department of Pathology,Fuzhou First Hospital,Fuzhou 350009,Fujian Province,China)
出处
《中国临床药理学杂志》
CAS
CSCD
北大核心
2021年第20期2766-2769,共4页
The Chinese Journal of Clinical Pharmacology
基金
福建省自然科学基金资助项目(2019J01535)
