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.
DownLoad: