Effect of miR-146a on biological behavior of esophageal squamous cell carcinoma cells and its mechanism
10.3760/cma.j.cn112152-20190308-00136
- VernacularTitle:miR-146a对食管鳞癌细胞生物学行为的影响及其机制
- Author:
Wenying XIE
1
;
Sheng YANG
Author Information
1. 福建医科大学附属协和医院肿瘤内科，福州 350000
- Keywords:
Esophageal squamous cell carcinoma;
MiR-146a;
Eca109 cell;
Biological behavior;
Interleukin-1 receptor-associated kinase 1
- From:
Chinese Journal of Oncology
2020;42(11):912-918
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effects and mechanisms of miR-146a on the proliferation, invasion, migration, apoptosis and cell cycle of esophageal squamous cell carcinoma cells.Methods:The expressions of miR-146a in 3 esophageal squamous carcinoma cell lines (Eca109, KYSE140, KYSE150) were detected by real-time quantitative polymerase chain reaction (RT-qPCR). MiR-146a mimics was transfected into Eca109 to up-regulate the expression of miR-146a. Effects of miR-146a on cell proliferation, invasion and migration were evaluated by cell counting kit 8 (CCK-8), Transwell assay and wound-healing assay, respectively. The cell apoptosis and cycle were assessed by flow cytometry (FCM). Finally, relevant bioinformatics techniques were used to predict the target gene of miR-146a. Dual luciferase reporter assay was used to identify the interaction of 3′ terminal untranslated region (3′ UTR) of miR-146a and its target gene, interleukin-1 receptor-associated kinase (IRAK1). RT-qPCR and western blot were used to detect the mRNA and protein expressions of IRAK1, respectively.Results:The relative expressions of miR-146a in 3 esophageal squamous cell carcinoma cells (Eca109, KYSE140, KYSE150) were 0.36±0.05, 0.16±0.06 and 0.09±0.02, respectively, all of which were significantly lower than 1±0.05 of normal esophageal epithelial cells (HEEC) ( P<0.01). The model of esophageal squamous cell carcinoma cells was constructed by transfecting miR-146a mimics into Eca109 cells. The results showed that the ability of absorbance value, the number of transmembrane cells (52±18), the reduced scratch distances at 48 hours and 72 hours after transfection [(25.29±0.77) μm, (30.66±0.91) μm] were significantly lower than those of the negative control group and blank control group (all P<0.01). The early apoptosis rate was (6.13±0.91)%, higher than (2.50±0.68)% of the negative control group ( P<0.01) and (1.70±0.20)% of blank control group ( P<0.01). The percentage of cells in G 1 phase [(44.74±6.76)%] was decreased while the G 2/M phase [(41.88±2.88)%] was increased when compared with the negative control group and the blank control group ( P<0.05 and P<0.01, respectively). The results of dual luciferase reporter gene assay showed that luciferase activity in the group co-transfected with IRAK1-wild-type and miR-146a mimics was significantly lower than that in the control groups ( P<0.01). The results of RT-qPCR and western blot showed that the mRNA and protein expressions of IRAK1 in the co-transfected group were 1.02±0.28 and 1.00±0.05, respectively, both lower than those in the negative control group and the blank control group ( P<0.01). Conclusions:The expressions of miR-146a are decreased in the esophageal squamous cell lines, which plays a role as tumor suppressor gene. MiR-146a can inhibit the proliferation, invasion and migration of esophageal squamous cell cells, promote apoptosis, and block the cell cycle at G 2/M stage. MiR-146a may mediate the malignant biological behavior of esophageal cancer cells through the regulation of IRAK1.
