miRNA-5089-5p inhibits proliferation and migration of esophageal cancer cells by regulating the expression of cathepsin B in vitro
10.3760/cma.j.cn115355-20200908-00513
- VernacularTitle:miRNA-5089-5p体外通过调节组织蛋白酶B表达抑制食管癌细胞增殖和迁移
- Author:
Cheng CHANG
;
Yuan GU
;
Pinfa WANG
;
Xiaojun TENG
- From:
Cancer Research and Clinic
2021;33(5):328-333
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effect of miRNA-5089-5p (miR-5089-5p) on proliferation and migration ability of esophageal cancer in vitro and its relationship with the expression of cathepsin B (CTSB) gene.Methods:Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of miR-5089-5p in 31 tissue samples from patients who underwent esophageal cancer resection and the corresponding pericarcinomatous tissues between in March 2017 and in December 2019 at Huangshi Central Hospital of Edong Healthcare Group, and TE-13, EC9706, Eca109, KYSE30 cell lines and normal esophageal mucosal epithelial HET-1A cells. The esophageal cancer cells with the lowest expression level of miR-5089-5p were divided into 2 groups: miR-5089-5p group transfected with miR-5089-5p mimics and the negative control group with negative control sequence. qRT-PCR was used to detect the expression level of miR-5089-5p after transfection for 48 h. CCK-8 method and scratch healing test were used to detect the proliferation and migration ability of cells in the two groups. The online tools microRNA.org and Deepbase v2.0 were used to predict the target genes of miR-5089-5p. The dual luciferase reporter gene assay was used to verify the target gene of miR-5089-5p. qRT-PCR and Western blot were used to detect the expression level of target genes in the two groups. The expressions of cell proliferation-related protein (PCNA and Ki-67) and migration-related protein (N-Cadherin and Twist) were detected by using Western blot.Results:The relative expression level of miR-5089-5p in esophageal cancer and pericarcinomatous tissues was 1.54±0.53 and 7.07±1.25, respectively ( t = 24.06, P < 0.01). The relative expression level of miR-5089-5p in the esophageal cancer cell lines was lower than that of normal esophageal mucosal epithelial HET-1A cells (all P < 0.05), and the cell line with the lowest relative expression was Eca109 cells (0.12±0.03). Compared with the negative control group, the proliferation ability of Eca109 cells in miR-5089-5p group was gradually reduced with the transfection time extension, and the difference was statistically significant between the two groups since 48 h (all P < 0.05), and the migration ability was also reduced [scratch healing rate: (29±5)% vs.(64±8)%, t=3.91, P < 0.01]. The online tool predicted that the target gene of miR-5089-5p might be CTSB, and the dual luciferase reporter gene assay confirmed that miR-5089-5p complemented CTSB 3'UTR. qRT-PCR results showed that compared with the negative control group, the relative expression level of CTSB mRNA in Eca109 cells of miR-5089-5p group was reduced (0.23±0.04 vs.1.01±0.09, t = 8.27, P < 0.01). Western blot results showed that the expression level of CTSB protein was reduced, and the expression levels of cell proliferation-related protein PCNA, Ki-67 and cell migration-related protein N-Cadherin, Twist were also reduced. Conclusions:The expression level of miR-5089-5p in esophageal cancer tissues and cell lines is low. miR-5089-5p can inhibit proliferation and migration of esophageal cancer Eca109 cells. The mechanism may be achieved by down-regulating CTSB gene expression.
