摘要
目的:探讨长链非编码RNA(long non-coding RNA,lncRNA)肌联蛋白反义RNA1(titin antisense RNA 1,TTN-AS1)调节miR-107/肝癌衍生生长因子(hepatoma-derived growth factor,HDGF)轴对胃癌(gastric cancer,GC)细胞化疗耐药性的影响。方法:以不同浓度的顺铂(cisplatin,DDP)(0、3、6、12μg/mL)处理胃癌细胞HGC-27和顺铂耐药胃癌细胞HGC-27/DDP,MTT法测定细胞增殖;将HGC-27/DDP细胞分为si-TTN-AS1组、si-NC组、si-TTN-AS1+miR-107 inhibitor组、si-TTN-AS1+inhibitor-NC组、control组;利用qRT-PCR检测TTN-AS1、miR-107在HGC-27、HGC-27/DDP细胞中的表达水平;平板克隆形成实验测定细胞克隆能力;Transwell法测定细胞侵袭;流式细胞仪测定细胞凋亡;Western blot测定B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、耐药相关蛋白MDR-1、MRP1及HDGF的表达;双荧光素酶报告实验证实miR-107与TTN-AS1和HDGF的作用关系。结果:DDP以剂量依赖性的方式抑制HGC-27和HGC-27/DDP细胞增殖,并且与HGC-27比较,HGC-27/DDP细胞对DDP的敏感性降低;与HGC-27组比较,HGC-27/DDP组TTN-AS1水平上升,miR-107水平下降(P<0.05);相较于control组、si-NC组,si-TTN-AS1组TTN-AS1水平、侵袭数、克隆形成率及Bcl-2、MMP-2、PCNA、MRP1、MDR-1、HDGF表达下降,凋亡率、miR-107水平、Bax表达增加(P<0.05);相较于si-TTN-AS1组、si-TTN-AS1+inhibitor-NC组,si-TTN-AS1+miR-107 inhibitor组HGC-27/DDP细胞中克隆形成率、侵袭数及PCNA、Bcl-2、MMP-2、MDR-1、MRP1、HDGF蛋白表达水平升高,miR-107表达水平、凋亡率及Bax蛋白表达水平降低(P<0.05);TTN-AS1靶向调控miR-107表达,miR-107靶向负调控HDGF表达。结论:敲低TTN-AS1可能通过上调miR-107来降低HDGF表达,抑制HGC-27/DDP对DDP耐药性,进而诱导HGC-27/DDP细胞凋亡,阻滞增殖、侵袭。
Objective:To investigate the impact of long non-coding RNA(lncRNA)titin antisense RNA 1(TTN-AS1)on chemoresistance of gastric cancer(GC)cells by regulating miR-107/hepatoma-derived growth factor(HDGF)axis.Methods:Different concentrations of cisplatin(DDP)(0,3,6,12μg/mL)treated gastric cancer cells HGC-27 and cisplatin-resistant gastric cancer cells HGC-27/DDP,and the cell proliferation was detected by MTT method.HGC-27/DDP cells were grouped into si-TTN-AS1 group(transfected with si-TTN-AS1),si-NC group(transfected with si-NC),si-TTN-AS1+miR-107 inhibitor group(co-transfected with si-TTN-AS1 and miR-107 inhibitor),and si-TTN-AS1+inhibitor-NC group(co-transfected with si-TTN-AS1 and inhibitor-NC),control group(normal cultured cells).The expression levels of TTN-AS1 and miR-107 in HGC-27 and HGC-27/DDP cells were detected by qRT-PCR.Plate cloning assay was applied to detect the cell cloning ability.Transwell chamber was applied to detect cell invasion.Flow cytometry was used to detect apoptosis.The expression of B cell lymphocyte tumor 2(Bcl-2),matrix metalloproteinase-2(MMP-2),Bcl-2 associated X protein(Bax),proliferating cell nuclear antigen(PCNA),drug-resistance related protein MDR-1,MRP1 and HDGF was detected by Western blot,and the targeting relationship between TTN-AS1 and miR-107,miR-107 and HDGF was detected by double luciferase report experiment.Results:DDP inhibited the proliferation of HGC-27 and HGC-27/DDP cells in a dose-dependent manner,and compared with HGC-27,HGC-27/DDP cells were less sensitive to DDP.Compared with HGC-27 group,the expression level of TTN-AS1 mRNA in HGC-27/DDP group was higher and the expression level of miR-107 was lower(P<0.05).Compared with control group and si-NC group,the expression level of TTN-AS1 mRNA,invasion number,cloning rate and the expression levels of Bcl-2,MMP-2,PCNA,MRP1,MDR-1,HDGF protein in HGC-27/DDP cells in si-TTN-AS1 group were lower,the apoptosis rate,expression level of miR-107 and Bax protein expression were higher(P<0.05).Compared with si-TTN-AS1 group and si-TTN-AS1+inhibitor-NC group,the cloning rate,invasion number,and the expression levels of PCNA,Bcl-2,MMP-2,MDR-1,MRP1 and HDGF in HGC-27/DDP cells in si-TTN-AS1+miR-107 inhibitor group were higher,the expression level of miR-107,apoptosis rate and Bax protein expression were lower(P<0.05).TTN-AS1 targeted to regulate the expression of miR-107,and miR-107 targeted to negatively regulate the expression of HDGF.Conclusion:Knocking down TTN-AS1 may down-regulate the expression of HDGF and reduce the resistance of HGC-27/DDP to DDP by targeting miR-107,thus inhibiting the proliferation and invasion of HGC-27/DDP cells and promoting apoptosis.
作者
高正杰
张俏
孟涛
孙培胜
范龙鑫
刘亚飞
闫争强
朱绍辉
GAO Zhengjie;ZHANG Qiao;MENG Tao;SUN Peisheng;FAN Longxin;LIU Yafei;YAN Zhengqiang;ZHU Shaohui(Department of General Surgery,Ward 2,the First Affiliated Hospital of Xinxiang Medical College,Henan Xinxiang 453000,China)
出处
《现代肿瘤医学》
2025年第10期1676-1682,共7页
Journal of Modern Oncology
基金
河南省医学科技攻关计划项目(编号:LHGJ20210523)。
