柚皮素通过p53-MDM2信号通路对人骨肉瘤SJSA-1细胞增殖及周期的影响

Effects of Naringin on Proliferation and Cycle of HUMAN Osteosarcoma SJSA-1 Cells through p53-MDM2 Signaling Pathway

目的采用网络药理学、分子对接及生物分子实验共同探讨柚皮素抑制人骨肉瘤(OS)细胞SJSA-1细胞增殖及周期的机制作用。方法通过CCK-8法确定柚皮素对SJSA-1细胞增殖的抑制作用,检索TCMSP和SwissTargetPredictation数据库获得柚皮素潜在靶点;通过GeneCards、OMIM和TTD数据库筛选骨肉瘤靶点,将柚皮素与OS作用的交集潜在靶点导入STRING绘制PPI网络。对交集靶蛋白进行GO功能富集分析和KEGG通路分析;对鼠双微体基因2(murine double minute 2,MDM2)靶点使用Schr9dinger软件进行分子对接。用Western blot法测定p53、MDM2及p21蛋白表达,并用流式细胞法分析细胞周期变化。结果共筛选出102个柚皮素潜在靶点与260个骨肉瘤靶点,GO富集分析和KEGG对12个交集潜在靶点进行更深入分析。对接结果显示柚皮素能够较好地和MDM2结合并与MDM2蛋白分子氨基酸残基产生多种作用力。生物实验结果显示柚皮素在SJSA-1细胞中IC为18.3μmol/L并诱使细胞周期G2期发生阻滞。结论基于网络药理学、分子对接及细胞生物实验,表明柚皮素通过p53-MDM2信号通路抑制人骨肉瘤细胞SJSA-1增殖和诱导细胞G2期阻滞。

Objective The mechanism of naringenin inhibiting the proliferation and cell cycle of osteosarcoma(OS)cells SJSA-1 was studied by network pharmacology,molecular docking and biomolecular experiments.Methods The inhibitory effect of naringenin on SJSA-1 cell proliferation was determined by CCK-8 method,and the potential targets of naringenin were obtained by searching TCMSP and SwissTargetPredictation database.Targets of osteosarcoma were screened by GeneCards,OMIM and TTD databases,and the intersection with potential targets of naringin and OS action were imported into STRING to draw PPI network.GO functional enrichment analysis and KEGG pathway analysis were performed for the intersection of target proteins.Schr9 dinger software was used for molecular docking with naringin and murine double mirute 2(MDM2).The expressions of p53,MDM2 and P21 were determined by Western blotting assay,and the cell cycle was analyzed by flow cytometry.Results A total of 102 potential targets of naringin and 260 targets of osteosarcoma were screened out.GO enrichment analysis and KEGG analysis were performed to further analyze the intersection of 12 potential targets.The docking results showed that naringin could bind to MDM2 and generate multiple interactions with amino acid residues of MDM2 protein.Bioassay results showed that the ICvalue of naringin in SJSA-1 cells was 18.3μmol/L and furthermore naringin induced G2 phase arrest.Conclusion Based on network pharmacology,molecular docking and cell biology experiments,it was demonstrated that naringin inhibited SJSA-1 proliferation and induced G2 phase arrest of human osteosarcoma cells through p53-MDM2 signaling pathway.