摘要
目的 探讨沉默环状RNA(circRNA)ciRS-7对胃癌细胞顺铂耐药性的影响及相关机制。方法 构建顺铂耐药胃癌细胞HGC-27/DDP,沉默ciRS-7并用顺铂处理HGC-27/DDP细胞后,用实时荧光定量PCR(qRT-PCR)检测ciRS-7、微小RNA-944(miR-944)表达水平,细胞计数试剂盒-8(CCK-8)检测细胞存活率、克隆形成实验检测克隆形成数,蛋白免疫印迹(Western blot)检测细胞周期蛋白D1(cyclinD1)、增殖细胞核抗原(PCNA)、B淋巴细胞瘤-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)、裂解的天冬氨酸特异性半胱氨酸蛋白酶3(cleaved-caspase-3)蛋白表达水平,流式细胞术检测细胞凋亡率,原位末端标记法(TUNEL)检测细胞凋亡指数,双荧光素酶实验验证ciRS-7与miR-944的靶向关系。结果 ciRS-7在顺铂耐药胃癌组织和细胞中高表达,miR-944在顺铂耐药胃癌组织和细胞中低表达,沉默ciRS-7明显降低顺铂处理的HGC-27/DDP细胞存活率、克隆形成数、cyclinD1、PCNA、Bcl-2蛋白表达水平,明显增加顺铂处理的HGC-27/DDP细胞ciRS-7、凋亡率、凋亡指数、Bax、cleaved-caspase-3蛋白表达水平,ciRS-7靶向抑制miR-944的表达。结论 沉默ciRS-7可逆转胃癌细胞对顺铂的耐药性,其机制可能与沉默ciRS-7可促进miR-944的表达有关。
Aim To investigate the effect of silencing circRNA ciRS-7 on cisplatin resistance of gastric cancer cells and its related mechanism.Methods Cisplatin-resistant gastric cancer cells HGC-27/DDP were constructed,ciRS-7 was silenced and HGC-27/DDP cells were treated with cisplatin.Real-time quantitative PCR(qRT-PCR)was used to detect the expression levels of ciRS-7 and microrNA-944(miR-944).Cell counting kit-8(CCK-8)was used to detect cell survival rate.Clone formation assay was used to detect the number of clones. Western blot was used to detect the protein expression levels of CyclinD1,proliferating cell nuclear antigen (PCNA),B-lymphocytoma-2-associated X protein (Bax),B-lymphocytoma-2 (Bcl-2) and cleaved aspartate-specific cysteine proteinase 3 (cleaved-caspase-3). Flow cytometry was used to detect cell apoptosis rate. In situ end labeling (TUNEL) was used to detect apoptosis index. Dual luciferase assay was used to verify the targeting relationship between ciRS-7 and miR-944. Results CiRS-7 was highly expressed in cisplatin-resistant gastric cancer tissues and cells,while miR-944 was low expressed in cisplatin-resistant gastric cancer tissues and cells. The survival rate,clonal formation number,protein expression levels of CyclinD1,PCNA and Bcl-2 in cisplatin-treated HGC-27/DDP cells were significantly reduced by silencing ciRS-7,while ciRS-7 expression level,apoptosis rate,apoptosis index,Bax,cleaved-caspase-3 protein expression levels of cisplatin-treated HGC-27/DDP cells were significantly raised. CiRS-7 targetedly inhibited the expression of miR-944. Conclusions Silencing ciRS-7 can reverse cisplatin resistance of gastric cancer cells,and the mechanism may be related to the promotion of miR-944 expression by silencing ciRS-7.
作者
魏文娥
郭翠萍
刁银卓
骆玉霜
WEI Wen-e;GUO Cui-ping;DIAO Yin-zhuo;LUO Yu-shuang(Dept of Oncology,Affiliated Hospital of Qinghai University,Dept of Oncology,Affiliated Cancer Hospital of Qinghai University,Xining 810001,China)
出处
《中国药理学通报》
CAS
CSCD
北大核心
2023年第7期1325-1332,共8页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No.81760730)。
