微小RNA-218通过靶向调控LASP1影响食管癌细胞的生物学行为
MicroRNA-218 affects the biological behavior of esophageal cancer cells by targeting LASP1
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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si-LASP1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。
Abstract: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-1A) 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 (si-LASP1), 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-1A 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.
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