摘要
[目的 ]微小RNA-218(miR-218)作为一种肿瘤抑制因子,参与多种肿瘤的发生与进展。肌动蛋白骨架蛋白1(LASP1)是一种新的肌动蛋白结合蛋白,参与细胞骨架的重组调控及细胞迁移,与肿瘤的增殖、侵袭和转移密切相关。食管癌细胞中miR-218与LASP1基因间的调控关系尚未有报道,本研究对食管癌细胞中miR-218是否可以通过靶向调控LASP1而发挥抑癌作用进行初步探讨。[方法 ]应用实时定量逆转录聚合酶链反应(qRT-PCR)检测3株食管癌细胞(EC109、EC9706、KYSE510)和1株永生化食管上皮细胞(Het-1A)中miR-218的表达水平。以EC109为靶细胞,分别转染miR-218模拟物(miR-218 mimic)、miR-218 mimic的空载体对照(miR-NC)、LASP1的小干扰RNA(si-LASP1)、miR-218 mimic和si-LASP1(miR-218 mimic+si-LASP1)以及si-LASP1的空载体对照(si-NC)。采用qRT-PCR和Western blot法检测细胞的转染效果,采用细胞增殖与毒性检测试剂盒观察各组细胞生长曲线,采用细胞平板克隆形成实验、Transwell小室法和流式细胞术检测细胞增殖、迁移、侵袭和凋亡,通过生物信息学分析和双荧光素酶报告基因实验对miR-218的靶基因进行预测及验证。[结果] KYSE510、EC109和EC9706中miR-218表达水平分别为Het-1A的0.32%、1.81%和2.15%(均P <0.05)。生物信息学分析结果显示LASP1的3’非编码区有miR-218的结合位点,miR-218 mimic转染组EC109中LASP1 mRNA表达水平是miR-NC转染组的28.20%(P <0.05),Western blot结果显示过表达miR-218可下调LASP1蛋白表达水平。双荧光素酶报告基因实验结果显示LASP1-WT(野生型)+miR-218 mimic组的荧光素酶活性为0.31±0.02,低于LASP1-WT+miR-NC组(0.56±0.04)和LASP1-MUT(突变型)+miR-218 mimic组(0.49±0.07)(均P <0.05)。与miR-NC组相比,转染miR-218 mimic可抑制EC109的生长速率、迁移和侵袭能力(均P <0.05),促进细胞凋亡(P <0.05)。转染si-LASP1的EC109观察到相同的结果趋势。与siLASP1单独转染组比较,miR-218 mimic和si-LASP1共转染组细胞生长速率下降,平板克隆形成率降低(P <0.05),迁移和侵袭能力降低(P <0.05),凋亡增加(P <0.05)。[结论 ] miR-218在食管癌细胞中低表达;在EC109中miR-218可通过直接靶向调控LASP1基因的表达,抑制细胞的增殖、迁移和侵袭,促进凋亡,从而在食管癌的发展进程中发挥抑癌作用。
[Objective] As a tumor suppressor, microRNA-218(miR-218) is involved in the occurrence and progression of various tumors. Actin cytoskeleton protein 1(LIM and SH3 domain structure protein 1, LIM and SH3 protein 1, LASP1) is a newly identified actin binding protein, participating in cytoskeleton reorganization regulation and cell migration, and closely related to the proliferation, invasion, and metastasis of tumor. The regulatory relationship between miR-218 and LASP1 in esophageal cancer cells has not been reported. Therefore, this study aims to investigate whether miR-218 functions as a tumor suppressor by targeting LASP1.[Methods] The expression levels of miR-218 in three esophageal cancer cells(EC109, EC9706, and KYSE510) and one permanent esophageal epithelial cell(Het-1 A) were detected by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR). EC109 was separately transfected with miR-218 mimic, negative control of miR-218 mimic(miR-NC), small interfering RNA of LASP1(siLASP1), miR-218 mimic + si-LASP1, and negative control of si-LASP1(si-NC). The cell transfection efficiency was detected by qRT-PCR and Western blot;the cell growth curve of each group was observed by cell proliferation and toxicity assay kit(Cell Counting Kit-8, CCK-8);the cell proliferation, migration, invasion, and apoptosis were detected by plate clone formation experiment, Transwell assay, and flow cytometry;the target genes of miR-218 were predicted and verified by bioinformatics analysis and dual-luciferase reporter gene experiment.[Results] The expressions of miR-218 in KYSE510, EC109, and EC9706 were 0.32%, 1.81%, and 2.15% of that in Het-1 A respectively(Ps < 0.05). The results of bioinformatics analysis showed that 3’ untranslated regions(3’UTR) of LASP1 had binding sites of miR-218, and LASP1 mRNA expression level in EC109 transfected with miR-218 mimic was 28.20% of that in miR-NC transfection group(P < 0.05);the Western blot results showed that overexpression of miR-218 down-regulated LASP1 protein expression level. The dual-luciferase reporter gene experiment showed that the luciferase activity in the LASP1-WT(wild type)+ miR-218 mimic group was 0.31±0.02, lower than those in the LASP1-WT + miR-NC group(0.56±0.04) and the LASP1-MUT(mutant)+ miR-218 mimic group(0.49±0.07)(Ps < 0.05). Compared with the miR-NC group, transfection with miR-218 mimic inhibited the growth rate, cell plate clone formation rate, cell migration, and cell invasion(Ps < 0.05), as well as promoted cell apoptosis rate(P < 0.05). The same trend pattern was observed in EC109 transfected with siLASP1. Compared with the EC109 transfected with si-LASP1 alone, co-transfection with miR-218 mimic and si-LASP1 lowered the cell growth rate, plate clone formation rate(P < 0.05), migration(P < 0.05), and invasion(P < 0.05), as well as elevated cell apoptosis rate(P < 0.05).[Conclusion] The findings show low miR-218 expression in esophageal cancer cells. In addition, miR-218 may inhibit the proliferation, migration, and invasion and promote the apoptosis of esophageal cancer cells by directly targeting LASP1 gene expression in EC109, playing a cancer suppressive role in the development of esophageal cancer.
作者
郑雨虹
马月
张颖
赵超
刘冉
浦跃朴
尹立红
ZHENG Yu-hong;MA Yue;ZHANG Ying;ZHAO Chao;LIU Ran;PU Yue-pu;YIN Li-hong(Key Laboratory of Environmental Medicine Engineering of Ministry of Education, School of Public Health, Southeast University, Nanjing, Jiangsu 210009,China)
出处
《环境与职业医学》
CAS
CSCD
北大核心
2019年第3期217-225,共9页
Journal of Environmental and Occupational Medicine
基金
国家自然科学基金项目(81872588
81573191
81573108)
