Effect of miRNA-5193 on the sensitivity of cervical cancer Caski cells to cisplatin / 肿瘤研究与临床

ABSTRACT

Objective:To investigate the effect and mechanism of miRNA-5193 (miR-5193) on the sensitivity of cervical cancer Caski cells to cisplatin.Methods:The expression of miR-5193 in cervical cancer cell lines C33A, SiHa, Caski and normal cervical cell line Ect1/E6E7 were determined by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). Caski cells were divided into control group (no transfection, normally cultured), miR-5193-negative control (miR-NC) group (transfected with miR-NC mimic), miR-5193 group (transfected with miR-5193 mimic), miR-NC+cisplatin group (transfected with miR-NC mimic and treated with 10 μg/ml cisplatin), miR-5193+cisplatin group (transfected with miR-5193 mimic and treated with 10 μg/ml cisplatin), miR-5193+cisplatin+NC group (cotransfected with Foxp3-negative control vector and miR-5193 mimic, and treated with 10 μg/ml cisplatin), and miR-5193+cisplatin+Foxp3 group (cotransfected with Foxp3 overexpression vector and miR-5193 mimic, and treated with 10 μg/ml cisplatin). Proliferation was detected by methyl thiazolyl tetrazolium (MTT), cell cycle was detected by PI single staining method, cell apoptosis was detected by Annexin V-FITC/PI double staining method, and expressions of CDK2, p27 and C-caspase-3 proteins in cells were detected by Western blot. Bioinformatics software was used to predict miR-5193 target genes, and the luciferase reporting system was used to identify the targeting relationship.Results:The relative expression of miR-5193 in cervical cancer C33A, SiHa and Caski cells was lower than that in normal cervical Ect1/E6E7 cells (0.56±0.06, 0.41±0.03, 0.23±0.02 vs. 1.00±0.10, all P < 0.05). Compared with the control group and miR-NC group, the cell proliferation activity (absorbance value) in miR-5193, miR-NC+cisplatin and miR-5193+cisplatin groups decreased (0.58±0.06, 0.59±0.07 vs. 0.38±0.04, 0.40±0.05, 0.23±0.02, all P < 0.05), the cell apoptosis rate increased [(2.5±0.2)%, (2.7±0.3)% vs. (12.6±1.2)%, (11.9±1.5)% , (18.9±1.7)%, all P < 0.05], and the proportion of cells in G 0/G 1 phase increased [(50.4±4.2)%, (51.3±6.3)% vs. (62.3±3.2)%, (61.9±5.8)%, (71.4±5.4)%, all P < 0.05]. The expression levels of p27 and C-caspase-3 proteins increased, and the expression level of CDK2 protein decreased. The software predicted that the target gene of miR-5193 was Foxp3, which was confirmed by the luciferase reporting system. Compared with the miR-5193+cisplatin+NC group, the cell proliferation activity (absorbance value) in miR-5193+ cisplatin+Foxp3 group increased (0.24±0.03 vs. 0.65±0.05, t = 21.094, P < 0.01), the proportion of cells in G 0/G 1 phase decreased [(71.0±6.4)% vs. (60.3±4.1)%, t = 4.196, P < 0.01], the apoptosis rate of cells decreased [(19.6±1.6)% vs. (11.5±1.2)%, t = 11.880, P < 0.01], the expression levels of p27 and C-caspase-3 proteins in cells decreased, and the expression levels of CDK2 and Foxp3 proteins increased. Conclusion:The miR-5193 may increase the sensitivity of cervical cancer Caski cells to cisplatin in vitro by targeted inhibition of the Foxp3 gene.