槲皮素通过调控TP53基因抑制肾透明细胞癌的增殖和迁移

doi:10.12122/j.issn.1673-4254.2025.02.12

摘要/Abstract

摘要：

目的筛选槲皮素治疗肾透明细胞癌（ccRCC）的潜在分子靶点。方法运用网络药理学方法从多个数据库中筛选槲皮素的治疗靶点，运用孟德尔随机化方法从4907种血浆蛋白中筛选与ccRCC显著相关的靶点，构建药物-疾病网络模型，筛选出潜在的关键靶点，运用生物信息学技术评估这些靶点的作用，培养ccRCC细胞并用不用浓度槲皮素处理，行CCK-8实验、创伤愈合实验、RT-qPCR和Western blotting对筛选靶点进行验证。结果网络药理学和孟德尔随机化综合分析筛选出了TP53（OR=3.325，95% CI：1.805-6.124，P=0.0001）、ARF4（OR=0.173，95% CI：0.065-0.456，P=0.0003）和DPP4（OR=0.463，95% CI：0.302-0.711，P=0.0004）3个槲皮素治疗ccRCC的核心靶点。生物信息学分析表明TP53在肾透明细胞癌中表达增高（P<0.001）；生存分析显示高表达TP53的肾癌患者预后更差（P<0.001）；分子对接显示槲皮素与TP53的结合能为-5.83 kcal/mol；CCK-8实验和创伤愈合实验显示槲皮素在体外抑制786-O细胞的增殖和迁移（P<0.05）；RT-qPCR和Western blotting显示槲皮素在体外抑制TP53 mRNA和蛋白的表达（P<0.05）。结论 TP53可能是槲皮素治疗ccRCC的关键靶点，为槲皮素治疗肾透明细胞癌的分子机制研究奠定了基础。

关键词: 槲皮素, 肾透明细胞癌, 网络药理学, 孟德尔随机化, TP53基因, 分子靶点

Abstract:

Objective To identify potential molecular targets of quercetin in the treatment of clear cell renal carcinoma (ccRCC). Methods The therapeutic targets of quercetin were screened from multiple databases by network pharmacology analysis, and the targets significantly correlated with ccRCC were screened from 4907 plasma proteins using a Mendelian randomization method. The drug-disease network model was constructed to screen the potential key targets. The functions of these targets were evaluated via bioinformatics analysis, and the screened targets were verified in cultured ccRCC cells. Results Network pharmacology analysis combined with Mendelian randomization identified TP53 (OR=3.325, 95% CI: 1.805-6.124, P=0.0001), ARF4 (OR=0.173, 95% CI: 0.065-0.456, P=0.0003), and DPP4 (OR=0.463, 95% CI: 0.302-0.711, P=0.0004) as the core targets in quercetin treatment of ccRCC. Bioinformatics analysis showed that TP53 was highly expressed in ccRCC, and patients with high TP53 expressions had worse survival outcomes. Molecular docking studies showed that the binding energy between quercetin and TP53 was -5.83 kcal/mol. In cultured 786-O cells, CCK-8 assay and wound healing assay showed that treatment with quercetin significantly inhibited cell proliferation and migration. Quercetin treatment also strongly suppressed the expression of TP53 at both the mRNA and protein levels in 786-O cells as shown by RT-qPCR and Western blotting. Conclusion TP53 may be the key target of quercetin in the treatment of ccRCC, which sheds light on potential molecular mechanism that mediate the therapeutic effect of quercetin.

Key words: quercetin, renal clear cell carcinoma, network pharmacology, Mendelian randomization, TP53 gene, molecular targets

高俊杰, 叶开, 吴竞. 槲皮素通过调控TP53基因抑制肾透明细胞癌的增殖和迁移[J]. 南方医科大学学报, 2025, 45(2): 313-321.

Junjie GAO, Kai YE, Jing WU. Quercetin inhibits proliferation and migration of clear cell renal cell carcinoma cells by regulating TP53 gene[J]. Journal of Southern Medical University, 2025, 45(2): 313-321.

图/表 9

图1 获取槲皮素靶基因

Fig.1 Quercetin target information acquisition. A: Chemical structure of quercetin. B: Visualization of potential targets of quercetin.

表1 血浆蛋白和肾透明细胞癌孟德尔随机化分析结果

Tab.1 Result of Mendelian randomization between plasma proteins and ccRCC

Exposure	Outcome	Gen	Method	Nsnp	b	Se	P
9816_37_ISOC1_ISOC1	KIRC	ISOC1	IVW	8	0.736746	0.171048	1.65E-05
6354_13_HEPHL1_HPHL1	KIRC	HEPHL1	IVW	5	-1.28851	0.310833	3.39E-05
17742_2_RRAS_RRAS	KIRC	RRAS	IVW	5	-1.33358	0.325935	4.29E-05
12621_55_PPP2R1A_PP2AAA	KIRC	PPP2R1A	IVW	4	-0.9648	0.243754	7.56E-05
5345_51_CKAP2_CKAP2	KIRC	CKAP2	IVW	15	-0.66094	0.169567	9.71E-05
12807_89_ARHGAP30_RHG30	KIRC	ARHGAP30	IVW	3	1.721247	0.446005	0.000114
6123_69_TP53_p53	KIRC	TP53	IVW	7	1.201512	0.311593	0.000115
9511_61_NXPH2_NXPH2	KIRC	NXPH2	IVW	18	-0.39638	0.103952	0.000137
6388_21_CCDC126_CC126	KIRC	CCDC126	IVW	18	-0.46547	0.124775	0.000191
13673_21_CCT7_TCPH	KIRC	CCT7	IVW	4	-1.0039	0.269598	0.000196
7083_74_MATN4_MATN4	KIRC	MATN4	IVW	7	0.634937	0.174364	0.000271
4568_17_SLITRK5_SLIK5	KIRC	SLITRK5	IVW	12	0.821988	0.226306	0.000281
15460_9_DPP4_CD26	KIRC	DPP4	IVW	3	-1.75547	0.495302	0.000394
18408_26_ARF4_ARF4	KIRC	ARF4	IVW	8	-0.76964	0.218311	0.000423
8040_9_LEMD1_LEMD1	KIRC	LEMD1	IVW	3	-2.26505	0.647584	0.000469
6578_29_TNMD_TNMD	KIRC	TNMD	IVW	3	-1.94913	0.558892	0.000488
9012_1_PRPF6_PRP6	KIRC	PRPF6	IVW	2	-2.88761	0.828756	0.000493
8535_102_DMKN_Dermokine	KIRC	DMKN	IVW	2	-1.63853	0.479425	0.000632
8748_45_CD74_HG2A	KIRC	CD74	IVW	3	-2.22233	0.652226	0.000656
10085_25_STAR_STAR	KIRC	STAR	IVW	5	-1.39378	0.418791	0.000874
18198_51_HBQ1_HBAT	KIRC	HBQ1	IVW	8	-0.86326	0.261832	0.000977
18380_78_ALB_Albumin	KIRC	ALB	IVW	11	-0.41249	0.13172	0.001739
12587_65_ARL2_ARL2	KIRC	ARL2	IVW	12	-0.47391	0.152486	0.001884
12041_33_HSPA1L_HS71L	KIRC	HSPA1L	IVW	11	-0.55241	0.178001	0.001913
10842_7_GCNT4_GCNT4	KIRC	GCNT4	IVW	4	-1.6224	0.541404	0.00273
11160_56_RNF122_RN122	KIRC	RNF122	IVW	6	-0.87475	0.299384	0.00348

图2 槲皮素和孟德尔随机化分析的核心靶基因

Fig. 2 Venn diagram of core target gene acquisition of quercetin and Mendelian randomization.

图3 肾透明细胞癌的孟德尔随机化结果

Fig.3 Mendelian randomization results on ccRCC. A-C: Forest plot showing the results of Mendelian randomization of the core genes. D-F: Scatter plots of Mendelian randomization results on core targets and ccRCC. G: Forest plot showing Mendelian randomization results of the core genes.

表2 血浆蛋白质与肾透明细胞癌孟德尔随机化的异质性分析

Tab. 2 Heterogeneity analysis of Mendelian randomization between plasma proteins and ccRCC

Id.exposure	Outcome	Exposure	Method	Q	Q_pval
18408_26_ARF4_ARF4	KIRC	ARF4	MR Egger	2.46079	0.782388313
18408_26_ARF4_ARF4	KIRC	ARF4	Inverse variance weighted	3.836625	0.698774273
15460_9_DPP4_CD26	KIRC	DPP4	MR Egger	0.27862	0.597607084
15460_9_DPP4_CD26	KIRC	DPP4	Inverse variance weighted	0.608893	0.737531351
6123_69_TP53_p53	KIRC	TP53	MR Egger	9.021383	0.060567227
6123_69_TP53_p53	KIRC	TP53	Inverse variance weighted	10.29088	0.06740046

图4 孟德尔随机化的敏感性分析

Fig.4 Sensitivity analysis of Mendelian randomization. A-C: MR funnel plot for core targets and ccRCC. D-F: Leave-one-out sensitivity analysis for potential core genes and ccRCC.

表3 血浆蛋白质和肾透明细胞癌之间孟德尔随机化的多效性分析

Tab.3 Pleiotropy analysis of Mendelian randomization between plasma proteins and ccRCC

Id.exposure	Outcome	Exposure	Egger_intercept	Se	P
18408_26_ARF4_ARF4	KIRC	ARF4	0.066322526	0.056543	0.293645
15460_9_DPP4_CD26	KIRC	DPP4	0.264971682	0.461066	0.667936
6123_69_TP53_p53	KIRC	TP53	0.08422687	0.112264	0.494821

图5 TP53在肾透明细胞癌中的表达

Fig.5 TP53 expression in ccRCC. A: TP53 mRNA evpression in different tumor samples. B: TP53 mRNA expression in ccRCC samples and normal tissues. C: TP53 mRNA expression in ccRCC and paired adjacent normal tissues. D,E: TP53 mRNA expression levels in different ccRCC clinical stages. F: Receiver operating characteristic analysis (ROC) of TP53 in patients with ccRCC (AUC=0.833). G: Immunohistochemical staining of TP53 in renal cancer tissues and normal renal tissues (Original magnification: ×20).

图6 细胞实验与分子对接验证

Fig.6 Cell experiments and molecular docking validation. A: Viability of 786-O cells measured using CCK-8 assay after 24 h of quercetin treatment. B: Wound healing assay for assessing migration capacity of 786-O cells after quercetin treatment for 12 h (scale bar=200 μm). C: Docking of quercetin with TP53 molecule. D: Effect of different concentrations of quercetin on TP53 mRNA expression in 786-O cells detected using RT-qPCR (*P<0.05,***P<0.001 vs 0 μmol/L). E: Effect of different concentrations of quercetin on TP53 protein expression in 786-O cells detected using Western blotting.

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