摘要
目的探讨姜黄素对膀胱癌化疗增敏作用机制。方法对经过姜黄素和放射处理的T24细胞进行microRNA表达(miRNA芯片测序)、细胞活力(细胞增殖试验试剂盒)、集落形成、凋亡(AnnexinV-F)分析、ITC/7-AAD流式细胞计数(ITC/7-AAD)、miR-1246和p53mRNA(实时PCR)和蛋白质(Westernblot)表达的分析。结果芯片测序分析鉴定出17个差异表达的miRNA,在姜黄素处理的细胞中,与对照组相比,姜黄素显著降低T24细胞活力,并呈现浓度依赖性。miR-1246在T24细胞中的表达显著高于SV-HUC-1细胞,且高浓度的姜黄素(10或20μg·mL^(-1))可显著下调T24细胞miR-1246的表达。20μg·mL^(-1)浓度的姜黄素组和放疗处理联合应用对抑制miR-1246的表达、细胞活力和集落形成的作用优于姜黄素或者进行单独放疗处理组。通过与对照组相比,抑制miR-1246显著降低T24细胞的存活率。转染antagomiR-1246可显著提高T24细胞处于G0/G1期细胞比例,而转染antagomiR-NC细胞则诱导细胞凋亡。荧光素酶试验显示,miR-1246的过表达会抑制p53 3‘-UTR基因的荧光素酶活性。结论 miR-1246通过靶向抑制p53基因在膀胱癌细胞中的表达,参与到姜黄素和放疗的抗肿瘤治疗中。
Objective Radiotherapy is the primary option for bladder cancer patients that with less obvious curativeeffects. This study was to investigate how to increase radiosensitivity in bladder cancer. Methods The curcumin andirradiation treated T24 cells were used for analysis of microRNA expression(miRNA microarray), cell viability(Cell Proliferation Assay Kit),colony formation,apoptosis(Annexin V-F),ITC/7-AAD flow cytometry(ITC/7-AAD), miR-1246 and p53 mRNAs(real-time PCR) and proteins(Western Blot) expression. Results Microarray assay identified 17 differentially expressed miRNAs(2-fold change) in curcumin treated cells compared to control cells that miR-1246 with the largest change. Curcumin significantly decreased T24 cells viability andcolony formation in a concentration-dependent manner compared to control cells. miR-1246 expression wassignificantly higher in T24 cells than in SV-HUC-1 cells and the higher concentrations(10 or 20 μg·mL^-1) ofcarcumin significantly down-regulated miR-1246 expression in T24 cells. Combination of 10 μg·mL^-1 curcumin andirradiation showed more effective in decreasing miR-1246 expression, cell viability and colony formation thancurcumin or irradiation alone. Inhibition of miR-1246 significantly decreased cell viability and colony formation inT24 cells. Transfection with antagomiR-1246 significantly increased the G0/G1-phase of T24 cells, and cellstransfected with antagomiR-NC induced apoptosis. Luciferase reporter assay showed that overexpression of miR-1246 suppressed the activity of luciferase of the p53 3’-UTR reporter genes. Conclusion miR-1246 is involved in the anti-cancer effects of curcumin and irradiation through targeted inhibition of p53 gene translation in bladder cancer cells.
作者
吴雅莉
吴文强
李华兵
徐冉
原海燕
屈健
唐甜甜
鲁琼
WU Yali;WU Wenqiang;LI Huabing;XU Ran;YUAN Haiyan;QU Jian;TANG Tiantian;LU Qiong(Department of Science and Education, Hunan Provincial Maternal cind Child Health Care Hospital, Changsha 410008 Hunan, China;Guangzhou University of Chinese Medicine, Guangzhou 510006 Guangdong, China;Department of Radiology, The Second Xiangya Hospital of Central South University, Changsha 410011 Hunan,China;Department of Urology, The Second Xiangya Hospital of Central South University, Changsha 410011 Hunan, China;Department of Pheinnacy, The Second Xiangya Hospital of Central South University, Changsha 410011 Hunan, China)
出处
《中药新药与临床药理》
CAS
CSCD
北大核心
2019年第3期319-326,共8页
Traditional Chinese Drug Research and Clinical Pharmacology
基金
湖南省自然科学基金(2016JJ2072)