Abstract:

Objective　To investigate the effects of aloe-emodin (AE) combined with siRNA-FOXC1 targeted silencing of the FOXC1 gene on regulating the Wnt/β-catenin pathway on the proliferation, apoptosis, and migration of glioma U251 cells. Methods　The study was conducted at the Medical Experimental Center of Binzhou Medical University Hospital from June 2022 to December 2023. The siRNA sequence (FOXC1-siRNA-2102) with the highest efficiency in targeting silencing of FOXC1 gene was selected, and the cells were transfected by liposome method. MTT method was used to detect the effects of different concentrations of AE on the proliferation of glioma U251 cells, and the IC50 value was calculated. CCK8 method was used to detect the changes of cell proliferation ability at 24 h, 48 h, 72 h, and 96 h after transfection. Cells were divided into a control group, a AE group, a siRNA-FOXC1 group, and a siRNA-FOXC1+AE group, and the effect on apoptosis in each group was detected by Annexin V-PE/7-AAD staining-flow cytometry. Transwell assay was used to detect the migration ability of cells in each group, qPCR assay was used to detect the mRNA expression level of the FOXC1 gene in each group, and Western blotting method was used to detect the protein expression levels of FOXC1, β-catenin, and C-myc in each group. F-test was used for statistical analysis. Results　With the increase of AE dose, the proliferation inhibition rate of glioma U251 cells was significantly increased [(0.04±12.86)% in the control group, (24.45±6.96)% in the 30 μmol/L group, (48.41±5.67)% in the 60 μmol/L group, (60.63±9.96)% in the 90 μmol/L group, and (78.23±13.34)% in the 120 μmol/L group], and the IC50 of AE in U251 cells was 61.43 μmol/L. The cell proliferation of the siRNA-FOXC1 group, AE group, and siRNA-FOXC1+AE group was higher than that of the control group over time, and the proliferation of the siRNA-FOXC1+AE group was the most significantly decreased; the apoptosis rates of the siRNA-FOXC1 group, AE group, and siRNA-FOXC1+AE group were higher than that of the control group over time, and the cell proliferation of siRNA-FOXC1+AE group was the most obvious; the mRNA expression levels of FOXC1 and protein expression levels of β-catenin, C-myc, and FOXC1 of the siRNA-FOXC1 group, AE group, and siRNA-FOXC1+AE group were lower than those of the control group over time, and those of the siRNA-FOXC1+AE group were the most significantly decreased(all P<0.05). The cell migration abilities of the siRNA-FOXC1 group, AE group, and siRNA-FOXC1+AE group were lower than that of the control group over time, and that of the siRNA-FOXC1+AE group was the most significantly decreased [the number of migrating cells was (193.00±14.17) in the control group was, (84.00±14.22) in the AE group, (80.67±5.69) in the siRNA-FOXC1 group, and (36.33±11.59) in the siRNA-FOXC1+AE group, with a statistically significant difference (F=93.55, P<0.05)]. Conclusions　AE can inhibit the proliferation and migration of glioma U251 cells and promote cell apoptosis. This mechanism is associated with FOXC1 gene regulation through Wnt/β-catenin pathway.

Key words:

Glioma, FOXC1, Aloe-emodin, Wnt/β-catenin pathway, C-myc

摘要：

目的　探讨芦荟大黄素（AE）联合siRNA-FOXC1靶向沉默FOXC1基因调控Wnt/β-catenin通路对胶质瘤细胞U251增殖、凋亡、迁移的影响。方法　研究时间为2022年6月至2023年12月，地点为滨州医学院附属医院医学实验中心。选择靶向沉默FOXC1基因效率最高的siRNA序列（FOXC1-siRNA-2102），采用脂质体法转染细胞。采用噻唑蓝（MTT）法检测不同浓度AE对胶质瘤细胞U251增殖的影响，并计算半数抑制浓度值（IC50）。CCK8法检测转染后24 h、48 h、72 h、96 h细胞增殖能力变化。将细胞分为对照组、AE组、siRNA-FOXC1组、AE+siRNA-FOXC1组，采用Annexin V-PE/7-AAD染色-流式细胞仪检测各组细胞凋亡的影响，Transwell小室检测各组细胞的迁移能力，qPCR法检测各组FOXC1基因的mRNA表达水平，蛋白质印迹法检测各组FOXC1、β-catenin、C-myc蛋白表达水平。统计学方法采用F检验。结果　随着AE剂量的增加，胶质瘤细胞U251增殖抑制率增高［对照组为（0.04±12.86）%，30 μmol/L组为（24.45±6.96）%、60 μmol/L组为（48.41±5.67）%、90 μmol/L组为（60.63±9.96）%、120 μmol/L组为（78.23±13.34）%］，AE在U251细胞的IC50值为61.43 μmol/L。随着时间延长，siRNA-FOXC1组、60 μmol/L AE组、AE+siRNA-FOXC1组细胞增殖抑制率均高于对照组，其中AE+siRNA-FOXC1组最高；siRNA-FOXC1组、AE组、AE+siRNA-FOXC1组的凋亡率均高于对照组，其中AE+siRNA-FOXC1组的细胞凋亡率最高；siRNA-FOXC1组、AE组、AE+siRNA-FOXC1组的FOXC1 mRNA表达水平及β-catenin、C-myc、FOXC1蛋白表达均低于对照组，其中AE+siRNA-FOXC1组最低（均P<0.05）。siRNA-FOXC1组、AE组、AE+siRNA-FOXC1组细胞迁移能力均低于对照组，其中AE+siRNA-FOXC1组最低［对照组迁移细胞数为（193.00±14.17）个，AE组为（84.00±14.22）个，siRNA-FOXC1组为（80.67±5.69）个，AE+siRNA-FOXC1组为（36.33±11.59）个，差异有统计学意义（F=93.55，P<0.05）］。结论　AE能抑制胶质瘤U251细胞增殖、迁移，促进细胞凋亡，其机制可能与FOXC1基因、Wnt/β-catenin通路有关。

关键词:

胶质瘤, FOXC1, 芦荟大黄素, Wnt/β-catenin通路, C-myc