赖氨酸去甲基化酶5C对肾癌转移的作用研究

doi:10.3969/j.issn.1674-8115.2023.05.007

摘要/Abstract

摘要：

目的·研究失活的蛋白赖氨酸去甲基化酶5C（lysine demethylase 5C，KDM5C）对肾透明细胞癌迁移、侵袭的作用，分析它调控的基因，探索其失活后促癌的机制。方法·使用慢病毒构建KDM5C稳定敲除的人肾透明细胞癌786-O、Caki-1细胞株，通过Transwell小室观察肾癌细胞KDM5C敲除后迁移、侵袭的变化，蛋白质印迹法检测上皮-间充质转化（epithelial-mesenchymal transition，EMT）相关蛋白表达的变化；用786-O Control-sg、KDM5C-sg 2组细胞RNA测序数据和癌症基因组图谱（The Cancer Genome Atlas，TCGA）公共数据库做差异基因分析、功能通路富集分析，进一步探索KDM5C缺失后的促癌机制。根据EMT基因集筛选出测序数据中上调的EMT相关基因，并通过生存分析、单因素COX分析对基因进行初步筛选，最后通过LASSO回归分析、风险森林模型进一步筛选与KDM5C敲除后表型高度相关的基因。结果·KDM5C敲除后786-O和Caki-1细胞较对照组细胞表现出较为明显的迁移和侵袭表型（P=0.000）。TCGA数据库分析表明肾癌患者KDM5C突变会导致患者的预后情况变差（P=0.042）。RNA测序分析表明KDM5C敲除后可能通过影响黏附相关分子、上调上皮-间充质转化相关基因。通过蛋白质印迹法检测到2组细胞在敲除KDM5C后β-catenin、Vimentin、Snail蛋白表达水平均发生上调。最后将生存分析、单因素COX分析中得到的10个上调EMT基因进行LASSO回归分析、风险森林模型预测，结果表明KDM5C可能对PLAUR基因具有调控作用。结论·KDM5C在肾癌中突变可通过增强肿瘤迁移和侵袭促进肾癌的发生发展。

关键词: 赖氨酸去甲基化酶5C, 肾透明细胞癌, 迁移, 上皮-间充质转化

Abstract:

Objective ·To investigate the effects of lysine demethylase 5C (KDM5C) on the migration and invasion of renal clear cell carcinoma, analyze the genes regulated by KDM5C and explore the mechanisms of promoting renal cell carcinoma after its inactivation. Methods ·Lentivirus was used to construct human renal clear cell carcinoma 786-O and Caki-1 cell lines with KDM5C stable knockout. The changes of migration and invasion abilities of renal carcinoma cells were observed by Transwell assay. Epithelial-mesenchymal transition (EMT) protein expression was detected by Western blotting. Differential gene analysis and functional pathway enrichment analysis were performed by using 786-O Control-sg, KDM5C-sg 2 group cell RNA sequencing data and The Cancer Genome Atlas (TCGA) public database to further explore the cancer-promoting mechanisms after KDM5C deletion. The up-regulated EMT-related genes in sequencing data were screened out according to the EMT gene set, and the genes were screened by survival analysis and univariate COX analysis. Finally, LASSO regression analysis and risk forest model were used to further screen genes highly related to the phenotype after KDM5C knockout. Results ·After the deletion of KDM5C, both 786-O and Caki-1 cells showed significant migration and invasion phenotypes compared with control groups. Analysis of TCGA database indicated that mutation of KDM5C in renal cancer patients led to poor prognosis (P=0.042). RNA sequencing analysis showed that KDM5C knockout may affect cell adhesion molecules and upregulate epithelial-mesenchymal transition-related genes. Western blotting detected the increased expression levels of β-catenin, Vimentin and Snail proteins in two kinds of cells after KDM5C knockout. Finally, ten up-regulated EMT genes were obtained by survival analysis and univariate COX analysis for LASSO regression analysis and risk forest model prediction. The results showed that KDM5C may have a regulatory effect on PLAUR gene. Conclusion ·The mutation of KDM5C can promote the development of renal carcinoma by enhancing migration and invasion.

Key words: lysine demethylase 5C (KDM5C), renal clear cell carcinoma, migration, epithelial-mesenchymal transition

中图分类号:

R737.11

陈宁黛, 周冰倩, 陈哲逸, 陈诗宇, 郑英霞. 赖氨酸去甲基化酶5C对肾癌转移的作用研究[J]. 上海交通大学学报（医学版）, 2023, 43(5): 571-579.

CHEN Ningdai, ZHOU Bingqian, CHEN Zheyi, CHEN Shiyu, ZHEN Yingxia. Effect of lysine demethylase 5C on renal carcinoma metastasis[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(5): 571-579.

图/表 5

图1 KDM5C 在不同癌症中的表达情况Note： A. Expression of KDM5C mRNA in tumor samples and controls in the TCGA database. B. The mutation ratio of KDM5C in different cancers in the TCGA database. Red boxes indicate KDM5C.

Fig 1 Expression of KDM5C in various cancer cells

图2 KDM5C 敲除对肾透明细胞癌产生的影响Note： A. Transfection efficiency of KDM5C-sgRNA in 786-O and Caki-1 verified by Western blotting. B. Effect of transfection of KDM5C-sgRNA plasmid on migration of 786-O and Caki-1 cells. C. Effect of transfection of KDM5C-sgRNA plasmid on invasion of 786-O and Caki-1 cells. ①P=0.000, compared with Control-sg.

Fig 2 Effect of KDM5C knockout on renal clear cell carcinoma cell

图3 KDM5C 敲除后肾癌细胞的RNA测序Note： A. PCA plot and heatmap of the two groups. B. Venn plots of upregulated and downregulated genes in 786-O_KDM5C-sg cells compared to 786-O_-control-sg cells. C/D. Go analysis (C) and KEGG analysis (D) on the sequencing data. E. KM plot of the prognosis of KIRC patients with and without KDM5C mutation.

Fig 3 RNA sequencing analysis of renal carcinoma cells with KDM5C knockout

图4 KDM5C 敲除对EMT相关蛋白产生的影响

Fig 4 Effects of KDM5C knockout on EMT-related proteins expression

图5 KDM5C 敲除后上调的EMT相关基因对预后的影响Note： A. Venn plot showed the common genes of survival analysis (P<0.01) and cox proportional-hazards model (P<0.01). B. KM plots showed the prognosis of 10 common genes in the TCGA database. C. LASSO coefficient profiles of genes significantly associated with OS. D. The forest plots showed the univariate COX analysis of the four genes significantly associated with OS.

Fig 5 Effect of up-regulated EMT-related gene on prognosis after KDM5C knockout

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