靶向SOX9调控弥漫性大B细胞淋巴瘤代谢重编程的研究

doi:10.3969/j.issn.1674-8115.2023.10.003

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (10): 1236-1244.doi: 10.3969/j.issn.1674-8115.2023.10.003

• 论著 · 基础研究 • 上一篇

靶向SOX9调控弥漫性大B细胞淋巴瘤代谢重编程的研究

张漪蓉¹^,²(), 魏玮庆², 马皎²(), 张雪¹()

^1.上海中医药大学交叉科学研究院，上海 201203
^2.上海交通大学基础医学院生物化学与分子细胞生物学系，上海 200025

收稿日期:2023-04-06 接受日期:2023-05-22 出版日期:2023-10-28 发布日期:2023-10-28
通讯作者: 马皎,张雪 E-mail:rongrongz@sjtu.edu.cn;drjiaoma@shsmu.edu.cn;xuezhang@shutcm.edu.cn
作者简介:张漪蓉（1996—），女，硕士生；电子信箱：rongrongz@sjtu.edu.cn。
基金资助:
上海市自然科学基金(23ZR1436100)

Research on the role of SOX9 in regulating metabolic reprogramming in diffuse large B cell lymphoma

ZHANG Yirong¹^,²(), WEI Weiqing², MA Jiao²(), ZHANG Xue¹()

^1.Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
^2.Department of Biochemistry and Molecular Cell Biology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China

Received:2023-04-06 Accepted:2023-05-22 Online:2023-10-28 Published:2023-10-28
Contact: MA Jiao,ZHANG Xue E-mail:rongrongz@sjtu.edu.cn;drjiaoma@shsmu.edu.cn;xuezhang@shutcm.edu.cn
Supported by:
Natural Science Foundation of Shanghai(23ZR1436100)

摘要/Abstract

摘要：

目的·探索弥漫性大B细胞淋巴瘤（diffuse large B cell lymphoma，DLBCL）中差异表达的性别决定区Y框转录因子9（SRY-box transcription factor 9，SOX9）基因所起到的作用，尤其是在生发中心B细胞（germinal center B-cell，GCB）来源亚型中对代谢重编程的调控作用。方法·选取NCICCR-DLBCL数据库中的481例DLBCL患者的临床信息和基因表达谱数据，使用R语言4.1.3版本进行数据分析与可视化，并基于RNA-seq测序表达量的细胞组织来源亚型（cell of origin subtype，COO）分类算法进行分类；使用ABC/GCB特征注释基因集，通过基因集富集分析（gene set enrichment analysis，GSEA）对分类进行验证。以SOX9的表达量高低将ABC和GCB亚组分别二分类。使用DEseq2包进行差异分析。使用KEGG（Kyoto Encyclopedia of Genes and Genomes）与Hallmark注释集分析SOX9与DLBCL的代谢的关系。采用Kaplan-Meier方法绘制生存曲线。采用GEPIA2进行泛癌分析。采用ESTIMATE包进行微环境评分分析。结果·481例DLBCL患者样本中，481例均有RNA-seq的表达量数据，421例有临床分期，335例有国际预后指数（international prognostic index，IPI）评分，234例有生存数据。分类得出ABC亚型232例（48.2%）、GCB亚型173例（36.0%）、未分类76例（15.8%），与数据库声明的比例相符，经富集分析验证符合ABC/GCB表达谱特征。SOX9低表达量组与SOX9高表达量组相比，总生存期更短，预后分数更差。泛癌分析示该现象亦可见于其他类型肿瘤。差异分析显示，在GCB亚型中，与SOX9高表达量组相比，SOX9低表达量组中有上调基因156个、下调基因1 826个。对于细胞代谢水平的变化，下调基因富集于糖酵解。结论·在ABC-DLBCL中，SOX9基因通过调控代谢重编程影响ABC-DLBCL的生物学特征。低表达SOX9的DLBCL，预示着肿瘤中糖酵解减少；其肿瘤基质细胞浸润程度更低，并且有着更差的预后。

关键词: 性别决定区Y框转录因子9, 弥漫性大B细胞淋巴瘤, 代谢重编程

Abstract:

Objective ·To explore the role played by the differentially expressed SRY-box transcription factor 9 (SOX9) gene in diffuse large B cell lymphoma (DLBCL), particularly in the regulation of metabolic reprogramming in the germinal center B-cell (GCB) like subtype. Methods ·The clinical information and gene expression profile data of 481 DLBCL patients retrieved from the NCICCR-DLBCL database were included. Data analysis and visualisation were performed by using R language version 4.1.3. The classification was performed by using a cell of origin subtype (COO) classification algorithm based on RNA-seq sequencing of expression. ABC/GCB features were used to annotate gene sets, and the classification was verified by gene set enrichment analysis. The ABC and GCB subgroup was dichotomised based on the mean expression of SOX9. Differential analysis was performed by using the DEseq2 package. The relationship between SOX9 and ABC-DLBCL metabolism was analysed by using KEGG (Kyoto Encyclopedia of Genes and Genomes) with the Hallmark annotation set. The survival curves were plotted by using the Kaplan-Meier method. The pan-cancer analysis was performed by using GEPIA2. The microenvironmental scoring analysis was performed by the ESTIMATE package. Results ·Of the 481 DLBCL patient samples, all the patients had RNA-seq expression data, 421 had clinical staging, 335 had international prognostic index (IPI) scores and 234 had survival data. The classification yielded 232 (48.2%) ABC subtypes, 173 (36.0%) GCB subtypes and 76 (15.8%) unclassified, consistent with the proportions declared in the database, and the enrichment analysis was verified to be consistent with the ABC/GCB expression profile. Compared to the high SOX9 expression group, the overall survival was shorter in the low SOX9 expression group and the prognostic score was worse. The pan-cancer analysis showed that this phenomenon was also seen in other tumor types. The differential analysis showed that there were 156 upregulated genes and 1 826 downregulated genes in the GCB subtype in the low SOX9 expression group, compared to the high SOX9 expression group. For metabolic processes, down-regulated genes were enriched in glycolysis. Conclusion ·In the ABC subtype of DLBCL, the SOX9 gene affects the biological features of ABC-DLBCL by regulating metabolic reprogramming, and low expression of SOX9 in DLBCL, possibly caused by high methylation, predicts decreased glycolysis in tumors. The proportion of tumor stromal cells decreases, showing a worse prognosis.

Key words: SRY-box transcription factor 9 (SOX9), diffuse large B cell lymphoma (DLBCL), metabolic reprogramming

中图分类号:

R733

张漪蓉, 魏玮庆, 马皎, 张雪. 靶向SOX9调控弥漫性大B细胞淋巴瘤代谢重编程的研究[J]. 上海交通大学学报（医学版）, 2023, 43(10): 1236-1244.

ZHANG Yirong, WEI Weiqing, MA Jiao, ZHANG Xue. Research on the role of SOX9 in regulating metabolic reprogramming in diffuse large B cell lymphoma[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(10): 1236-1244.

图/表 6

表1 糖酵解相关引物序列

Tab 1 Primers for glycolysis

Gene	Forward primer (5'→3')	Reverse primer (5'→3')
NT5E	AAGGACTGATCGAGCCACTC	GGAAGTGTATCCAACGATTCCCA
EGFR	CCCACTCATGCTCTACAACCC	TCGCACTTCTTACACTTGCGG
VCAN	GTAACCCATGCGCTACATAAAGT	GGCAAAGTAGGCATCGTTGAAA
COL5A1	GCCCGGATGTCGCTTACAG	AAATGCAGACGCAGGGTACAG
GPC1	TGAAGCTGGTCTACTGTGCTC	CCCAGAACTTGTCGGTGATGA
CLDN3	AACACCATTATCCGGGACTTCT	GCGGAGTAGACGACCTTGG
SDC1	CTGCCGCAAATTGTGGCTAC	TGAGCCGGAGAAGTTGTCAGA
GPC3	ATTGGCAAGTTATGTGCCCAT	TTCGGCTGGATAAGGTTTCTTC
GPC4	GTGGGAAATGTGAACCTGGAA	CGAGGGACATCTCCGAAGG
PKP2	ATGACATGCTAAAGGCTGGCA	GGGAGCTGTACTGTGCTGTTC

图1 ABC/GCB亚型分类结果的验证Note： The classification for ABC/GCB subtypes was obtained by a classifier based on transcriptome. GSEA analysis was used for the validation.

Fig 1 Validation for the classification of ABC/GCB subtypes classification

图2 SOX9 表达对ABC亚型的DLBCL患者生存的影响Note： A. Kaplan-Meier survival curve comparing the overall survival time for different SOX9 expression groups (upper 50% vs lower 50%) in GCB-DLBCL from NCICCR-DLBCL patient cohort. B. Distribution of IPI scores between different SOX9 groups (upper 50% vs lower 50%) in GCB-DLBCL from the NCICCR-DLBCL patient cohort. C. Kaplan-Meier survival curve comparing the overall survival time for different SOX9 expression groups (upper 50% vs lower 50%) in ABC-DLBCL from NCICCR-DLBCL patient cohort. D. Distribution of IPI scores between different SOX9 groups (upper 50% vs lower 50%) in ABC-DLBCL from the NCICCR-DLBCL patient cohort. "Low risk" contains "Low risk" and "Low-intermediate risk" in original cohort, likewise the "High". Statistical significance was determined by Fisher's exact test.

Fig 2 SOX9's effect on DLBCL patients' survival

图3 SOX9 在肿瘤中的表达与预后价值Note： A. Changes of SOX9 expression between tumor samples and normal samples from testicular germ cells tumors and skin cutaneous melanoma based on TCGA database. B. Kaplan-Meier survival curve comparing the overall survival time for different SOX9 expression groups in follicular lymphoma, adrenocortical carcinoma and breast cancer. Statistical significance was determined by the Log-rank test. ①P=0.000.

Fig 3 Expression and prognosis value of SOX9 in various cancer types

图4 SOX9 表达差异对GCB亚型DLBCL代谢通路的调控Note： A. Volcano plot of differential expression analysis. B. Representative GESA plots with hallmark and KEGG as reference gene sets. C/D. Change of glycolysis was confirmed by qPCR analysis in Karpas-422 (C) and OCI-LY1 (D) cell line. ①P=0.000, ②P=0.006, ③P=0.016, ④P=0.002, ⑤P=0.003, ⑥P=0.254, ⑦P=0.036, ⑧P=0.716, ⑨P=0.496, ⑩P=0.129. E/F. Change of glycolysis was confirmed by Western blotting analysis in Karpas-422 (E) and OCI-LY1 (F) cell line.

Fig 4 Regulation of metabolic pathways by SOX9 in GCB-DLBCL

图5 表达差异 SOX9 的DLBCL的浸润情况Note： Scores of stromal content, immune cell infiltration and tumor purity derived from the ESTIMATE algorithm. ①P=0.006.

Fig 5 Infiltration of DLBCL with different expression of SOX9

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靶向SOX9调控弥漫性大B细胞淋巴瘤代谢重编程的研究

Research on the role of SOX9 in regulating metabolic reprogramming in diffuse large B cell lymphoma

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