Methylation degree of miRNA-4729 in renal cancer tissues and its effect on proliferation and migration abilities of renal cancer cells
10.3760/cma.j.cn115355-20230901-00077
- VernacularTitle:肾癌组织中miRNA-4729的甲基化程度及其对肾癌细胞增殖和迁移能力的影响
- Author:
Lei WANG
1
;
Geng HUANG
;
Weidong JIANG
;
Fei LIU
;
Ni KE
Author Information
1. 黄石市中心医院（湖北理工学院附属医院）超声医学科，黄石 435000
- Keywords:
Kidney neoplasms;
MicroRNAs;
Methylation;
Cell proliferation;
Cell migration assay
- From:
Cancer Research and Clinic
2024;36(9):646-651
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the methylation degree of miRNA-4729 (miR-4729) in renal cancer tissues and its impact on the proliferation and migration abilities of renal cancer cell lines.Methods:Data from the SurvivalMeth database (updated in October 2022) was used to analyze the methylation degree of miR-4729 in 178 renal cancer tissues. Target gene with complementary binding sites to miR-4729 was predicted by miRNApath software. The normal renal tubular epithelial cell line HK-2 and the renal cancer cell lines Caki-1, A-498, ACHN, and 786-O were selected. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the relative expression of miR-4729 in each cell line and the effect of methylation inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) on the expression of miR-4729 in renal cancer cells. A-498 cells with the lowest relative expression of miR-4729 were transfected with miR-4729 mimic (miR-4729 group) and miRNA-NC (NC group), and colony formation assay and scratch assay were used to detect the effect of overexpression of miR-4729 on the proliferation and migration abilities of A-498 cells. Dual-luciferase reporter gene assay was used to verify the targeting relationship between miR-4729 and DEAD box peptide 5 (DDX5). Western blotting was used to detect the effect of miR-4729 overexpression on the expression of DDX5 protein and AKT signaling pathway-related proteins (p-AKT, p-IKKα, p-Tpl2 and AS160) in A-498 cells.Results:The analysis results of data from the SurvivalMeth database showed that the methylation degree of miR-4729 in renal cancer tissues was higher than that in paracancerous tissues ( P < 0.01). The relative expressions of miR-4729 in renal cancer Caki-1, A-498, ACHN, 786-O cells and normal renal tubular epithelial HK-2 cells were 0.62±0.05, 0.16±0.04, 0.53±0.02, 0.69±0.03, and 0.99±0.07, respectively, and the difference was statistically significant ( F = 47.39, P < 0.01). Compared with various cell groups cultured with dimethyl sulfoxide (DMSO), the relative expressions of miR-4729 in renal cancer Caki-1, A-498, ACHN and 786-O cells cultured with 5-Aza-CdR were higher (all P < 0.01). The results of colony formation assay showed that the number of colonies formed in A-498 cells of the miR-4729 group and NC group were 53±6 and 102±10, respectively, and the difference was statistically significant ( t = 4.25, P < 0.01). The results of scratch assay showed that the scratch healing rates of A-498 cells in the miR-4729 group and NC group were (42.3±2.7)% and (67.6±4.8)%, respectively, and the difference was statistically significant ( t = 4.58, P < 0.01). The results of dual-luciferase reporter gene assay showed that miR-4729 directly targeted and bound to DDX5. The relative expressions of DDX5 mRNA in A-498 cells of the miR-4729 group and NC group were 0.93±0.25 and 5.29±0.74, respectively, and the difference was statistically significant ( t = 5.60, P < 0.01). The results of Western blotting showed that compared with the NC group, the expression of DDX5 protein in A-498 cells of the miR-4729 group was lower, and the expressions of AKT signaling pathway-related proteins p-AKT, p-IKKα, p-Tpl2 and AS160 were also lower. Conclusions:Overexpression of miR-4729 decreases the activation level of AKT signaling pathway by targeting and inhibiting the expression of DDX5 gene, thereby inhibiting the proliferation and migration of renal cancer cells.
