长链非编码RNA LINC00261通过调控miR-324-3p/EST1促进胃癌进展
Long non-coding RNA LINC00261 promotes progression of gastric cancer by regulating miR-324-3p/EST1
谢锐 1尹源 1滕俊 1梁红亮1
作者信息
- 1. 三六三医院消化内科,成都 610041
- 折叠
摘要
目的:探讨长链非编码RNA LINC00261通过干扰miR-324-3p表达调控E26转录因子1(EST1)水平促进胃癌(GC)的进展.方法:收集2018年6月至2020年10月在三六三医院接受手术治疗的GC患者(n=80)的癌组织及其相应癌旁正常组织作为研究样本,qRT-PCR检测LINC00261和miR-324-3p表达水平.分析LINC00261与临床病理参数之间的相关性.将siRNA(si-LINC00261)、miRNA模拟物(miR-324-3p)和miRNA抑制剂(miR-324-3p in)及其相应对照转染至MGC-803和SGC-7901细胞;克隆形成试验评估细胞增殖能力;Transwell试验评估细胞迁移和侵袭能力;Western blot检测E-cadherin、N-cadherin和ETS1蛋白表达水平;肿瘤异种移植实验检测LINC00261在体内的肿瘤形成能力;荧光素酶报告、RNA沉淀分析LINC00261、miR-324-3p及EST1之间的作用关系.结果:与癌旁组织相比,GC组织中LINC00261表达明显上调,差异具有统计学意义(P<0.05).LINC00261表达与患者TNM分期、组织分化程度淋巴结转移及微血管侵犯有关(P<0.05);通过数据库预测、萤火虫荧光素酶实验及RNA免疫沉淀结果显示,LINC00261和miR-324-3p存在靶向作用关系;GC组织中miR-324-3p水平明显低于癌旁正常组织(P<0.05);miR-324-3p水平与LINC00261表达呈负相关(P<0.05);与in NC+si-NC组相比,in NC+si-LINC00261组细胞增殖、迁移和侵袭能力及N-cadherin表达明显被抑制(P<0.05),E-cadherin表达明显升高(P<0.05);与in NC+si-LINC00261组相比,siLINC00261+miR-324-3p in组细胞增殖、迁移和侵袭能力及N-cadherin表达明显提高(P<0.05),E-cadherin表达明显降低(P<0.05),Targetscan预测及萤火虫荧光素酶实验表明ETS1是miR-324-3p的下游结合位点;转染miR-324-3p后,ETS1 蛋白水平显著下调(P<0.05),但转染miR-324-3p in后,ETS1蛋白水平显著上调(P<0.05);在GC组织中ETS1表达明显高于癌旁正常组织(P<0.05);miR-324-3p水平与ETS1呈负相关(P<0.05);转染si-LINC00261的MGC-803细胞产生的肿瘤重量低于转染si-NC的MGC-803细胞产生的肿瘤重量(P<0.05),在si-LINC00261来源的肿瘤中,ETS1的蛋白水平低于si-NC来源的肿瘤(P<0.05).结论:LINC00261在GC组织中高表达,其可能通过调控miR-324-3p影响EST1表达促进GC进展.
Abstract
Objective:To investigate the role of long non-coding RNA LINC00261 in regulating E26 transcription factor 1(EST1)by interfering with the expression of miR-324-3p in promoting the development of gastric cancer(GC).Methods:Cancer tis-sues and corresponding adjacent normal tissues of GC patients(n=80)who underwent surgical treatment in 363 Hospital from June 2018 to October 2020 were collected as research samples,and the expression levels of LINC00261 and miR-324-3p were detected by qRT-PCR.The correlation between LINC00261 and clinicopathological parameters were analyzed.siRNA(si-LINC00261),miRNA mimic(miR-324-3p),miRNA inhibitor(miR-324-3p in)and their corresponding controls were transfected into MGC-803 and SGC-7901 cells.Clonogenesis assay was used to evaluate cell proliferation.The Transwell assay assessed cell migration and invasion.The protein expression levels of E-cadherin,N-cadherin and ETS1 were detected by Western blot.Tumor xenotransplantation assay was used to detect the tumorigenesis of LINC00261 in vivo.Luciferase report and RNA precipitation were used to analyze the interaction between LINC00261,miR-324-3p and EST1.Results:Compared with adjacent tissues,the expression of LINC00261 in GC tissues was significantly up-regulated with statistical significance(P<0.05).The expression of LINC00261 was correlated with TNM stage,tissue differentiation degree,lymph node metastasis and microvascular invasion(P<0.05).The database prediction,firefly luciferase assay and RNA immunoprecipitation results showed that LINC00261 had a targeted relationship with miR-324-3p.The level of miR-324-3p in GC tissues was significantly lower than that in paracellular normal tissues(P<0.05).There was a negative correlation between miR-324-3p level and LINC00261 expression(P<0.05).Compared with in NC+si-NC group,the cell proliferation,migration and invasion ability and the expression of N-cadherin in in NC+si-LINC00261 group were significantly inhibited(P<0.05),while the expression of E-cadherin was significantly increased(P<0.05).Compared with in NC+si-LINC00261 group,cell proliferation,migra-tion and invasion ability and expression of N-cadherin in si-LINC00261+miR-324-3p in group were significantly increased(P<0.05),while the expression of E-cadherin was significantly decreased(P<0.05).Targetscan prediction and firefly luciferase assay showed that ETS1 was the downstream binding site of miR-324-3p.After transfection with miR-324-3p,ETS1 protein level was significantly down-regulated(P<0.05),but after transfection with miR-324-3p in,ETS1 protein level was significantly up-regulated(P<0.05).The expression of ETS1 in GC tissue was significantly higher than that in adjacent normal tissue(P<0.05).The miR-324-3p level was negatively correlated with ETS1(P<0.05).The tumor weight of MGC-803 cells transfected with si-LINC00261 was lower than that of MGC-803 cells transfected with si-NC(P<0.05),and the protein level of ETS1 in si-LINC00261-derived tumors was lower than that of si-NC tumors(P<0.05).Conclusion:LINC00261 is highly expressed in GC tissue,which may affect EST1 expression and promote GC progress by regulating miR-324-3p.
关键词
胃癌/侵袭/增殖/长链非编码RNA/LINC00261/E26转录因子1Key words
Gastric cancer/Invasion/Proliferation/Long non-coding RNA LINC00261/E26 transcription factor 1引用本文复制引用
出版年
2024
国家科技期刊平台
NSTL
万方数据