猫爪草乙醇提取物调控结直肠癌细胞HCT116凋亡的分子机制研究

Study on the molecular mechanism of cat's claw ethanol extract in regulating apoptosis of colorectal cancer cell HCT116

目的探讨猫爪草乙醇提取物(CCEE)调控结直肠癌细胞HCT116凋亡的潜在分子机制。方法使用CCEE处理结直肠癌细胞HCT116,检测其凋亡水平。将CCEE处理后结直肠癌细胞HCT116进行半定量质谱分析。过表达差异蛋白后,检测结直肠癌细胞HCT116的凋亡水平。通过miRDB在线分析和荧光素酶报告系统检测差异蛋白的miRNA。结果CCEE处理24 h后,结直肠癌细胞HCT116的凋亡水平上升[(23.18±3.15)%vs.(68.21±12.05)%],差异有统计学意义(t=6.26,P<0.05)。CCEE处理结直肠癌细胞HCT11624 h后,半定量蛋白质组学发现BAX的蛋白表达水平上升(P<0.05)。当过表达BAX时,结直肠癌细胞HCT116的凋亡水平上升[(28.00±6.68)%vs.(85.08±4.97)%],差异有统计学意义(t=11.87,P<0.05)。CCEE处理结直肠癌细胞HCT116后,miR-761的表达水平显著下降[(0.82±0.10)vs.(0.11±0.02)],差异有统计学意义(t=12.00,P<0.05)。过表达miR-761后,BAX的蛋白和mRNA水平下降[(0.061±0.003)vs.(0.004±0.001),t=16.08,P<0.05],细胞凋亡水平下降[(21.59±5.66)%vs.(6.04±2.13)%,t=4.45,P<0.05],差异均有统计学意义。此外,miR-761靶向BAX mRNA的3端非编码区[(100.00±9.59)vs.(14.22±2.45),t=15.01,P<0.05]。结论CCEE抑制结直肠癌细胞HCT116中miR-761的表达水平,减少因miR-761所致的BAX mRNA降解,增强BAX的蛋白翻译,促进结直肠癌细胞HCT116的细胞凋亡。

Objective To explore the potential molecular mechanism of cat's claw ethanol extract(CCEE)regulating the apoptosis of colorectal cancer cell HCT116.Methods The ethanol extract of cat's claw was used to treat colorectal cancer cells HCT116,and the apoptosis level was detected.Colorectal cancer cells HCT116 were analyzed by semi-quantitative mass spectrometry after treatment with ethanol extract of cat's claw.After overexpressing the differential protein,the apoptosis level of colorectal cancer cell HCT116 was detected.miRNAs of differential proteins were detected through miRDB online analysis and luciferase reporter system.Results After CCEE treatment,the apoptosis level of colorectal cancer cell HCT116 was increased[(23.18±3.15)%vs.(68.21±12.05)%,t=6.26,P<0.05].After 24 h treatment with CCEE,the expression level of BAX protein in colorectal cancer cell HCT116 was increased by semi-quantitative proteomics(P<0.05).When BAX was overexpressed,the apoptosis level of colorectal cancer cell HCT116 was increased[(28.00±6.68)%vs.(85.08±4.97)%,t=11.87,P<0.05].The expression level of miR-761 was significantly decreased after CCEE treatment of colorectal cancer cells HCT116[(0.82±0.10)vs.(0.11±0.02),t=12.00,P<0.05].After overexpression of miR-761,the protein level and mRNA level of BAX were decreased[(0.061±0.003)vs.(0.004±0.001),t=16.08,P<0.05].The apoptosis level was decreased[(21.59±5.66)%vs.(6.04±2.13)%,t=4.45,P<0.05].In addition,miR-761 targeted the 3-terminal non-coding region of BAX mRNA[(100.00±9.59)vs.(14.22±2.45),t=15.01,P<0.05].Conclusion Cat's claw ethanol extract could inhibit the expression level of miR-761 in colorectal cancer cells HCT116,reduce the degradation of BAX mRNA due to miR-761,enhance the protein translation of BAX,and promote colorectal cancer cells HCT116 apoptosis.