顺铂诱导的急性肾损伤中肾脏组织m6A甲基化水平的变化

doi:10.3969/j.issn.1674-8115.2021.12.009

上海交通大学学报(医学版) ›› 2021, Vol. 41 ›› Issue (12): 1603-1611.doi: 10.3969/j.issn.1674-8115.2021.12.009

顺铂诱导的急性肾损伤中肾脏组织m⁶A甲基化水平的变化

沈剑箫¹(), 王万鹏², 邵兴华¹, 吴晶魁¹, 李舒¹, 车霞静¹, 倪兆慧¹()

^1.上海交通大学医学院附属仁济医院肾脏科，上海 200127
^2.江苏省涟水县人民医院肾脏科，涟水 223400

收稿日期:2021-08-12 出版日期:2021-12-28 发布日期:2021-01-28
通讯作者: 倪兆慧，电子信箱：profnizh@126.com。
作者简介:沈剑箫（1985—），男，主治医师，博士；电子信箱：shenjianxiao@aliyun.com。
基金资助:
国家自然科学基金(81700586);上海市核酸化学与纳米医学重点实验室资助计划(2020ZYB009);上海申康医院发展中心临床三年行动计划(SHDC2020CR3029B);上海市卫生健康委员会科研基金(ZHYY-ZXYJHZX-202014);上海市科学技术委员会基金(13401906100)

Changes of m⁶A methylation in renal tissue during cisplatin-induced acute injury

Jian-xiao SHEN¹(), Wan-peng WANG², Xing-hua SHAO¹, Jing-kui WU¹, Shu LI¹, Xia-jing CHE¹, Zhao-hui NI¹()

^1.Department of Nephrology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China
^2.Department of Nephrology, Lianshui People's Hospital, Jiangsu Province, Lianshui 223400, China

Received:2021-08-12 Online:2021-12-28 Published:2021-01-28
Contact: NI Zhao-hui, E-mail: profnizh@126.com.
Supported by:
National Natural Science Foundation of China(81700586);Key Shanghai Laboratory of Nucleic Acid Chemistry and Nanomedicine(2020ZYB009);Clinical Research Plan of Shenkang Hospital Development Center(SHDC2020CR3029B);Science and Research Fund of Shanghai Municipal Health Commission(ZHYY-ZXYJHZX-202014);Fund from Science and Technology Commission of Shanghai Municipality(13401906100)

摘要/Abstract

摘要：

目的·探讨N⁶-甲基腺嘌呤（N⁶-methyladenosine，m⁶A）甲基化修饰在顺铂诱导的小鼠急性肾损伤进程中的作用。方法·选择4只C57bL/6小鼠，尾静脉注射顺铂（20 mg/kg）诱导急性肾损伤（损伤组）；另取4只C57bL/6小鼠，尾静脉注射等量生理盐水（对照组）。检测2组小鼠血清肌酐及血尿素氮水平变化，观察小鼠肾脏组织切片中病理损伤情况，评估模型是否成功。进一步运用甲基化RNA免疫共沉淀技术（methylated RNA immunoprecipitation，MeRIP）与RNA测序技术分别检测2组小鼠肾脏组织中m⁶A甲基化水平与RNA表达变化。运用基因本体论及京都基因和基因组数据库进行结果可视化和综合研究，并将RNA测序技术所得转录组数据与MeRIP技术检测所得表观遗传数据联合分析，寻找参与顺铂诱导急性肾损伤病理变化过程的候选基因。结果·顺铂可诱导小鼠血清肌酐与血尿素氮水平显著升高。光学显微镜观察肾组织发现广泛的肾小管空泡变性，上皮细胞剥脱，肾小管坏死，提示造模成功。MeRIP检测发现损伤组与对照组小鼠肾脏中共有2 227个基因含有2 981个差异化表达的m⁶A甲基化位点（表达变化倍数≥2且P<0.05），这些基因主要富集于代谢及细胞死亡通路。表达差异化m⁶A甲基化位点的基因与RNA差异化表达基因的联合分析发现1 002个表达趋势相同的基因，如纤维蛋白原α链、溶质载体12家族成员1和甲肝病毒细胞受体1等。结论·顺铂可诱导肾脏组织中基因mRNA上m⁶A甲基化位点的甲基化水平变化，促进急性肾损伤进程。

关键词: N⁶-甲基腺嘌呤甲基化, 顺铂诱导急性肾损伤, 纤维蛋白原α链, 溶质载体12家族成员1, 甲肝病毒细胞受体1

Abstract:

Objective·To investigate the role of N⁶-methyladenosine (m⁶A) methylation modification in the process of cisplatin-induced acute injury in mice.

Methods·Four C57BL/6 mice were injected with cisplatin (20 mg/kg) through tail vein (the injury group); Another 4 C57BL/6 mice were injected with the same amount of saline (the control group). The changes of serum creatinine and urea nitrogen levels in the mice and the pathological injury in the renal tissue sections of the mice were evaluated to judge the success of the model. Methylated RNA immunoprecipitation (MeRIP) and RNA sequencing were used to detect the changes of m⁶A methylation and RNA expression in the kidney tissue of the two groups of mice. Gene ontology and Kyoto Encyclopedia of genes and genomes were used for visualization and comprehensive research. Transcriptome data and epigenetic data were combined to find candidate genes for pathological changes of cisplatin-induced acute injury.

Results·Cisplatin could induce significant increase in the levels of serum creatinine and urea nitrogen compared with those in the baseline. Light microscope showed extensive tubular vacuolar degeneration, epithelial cell exfoliation and tubular necrosis, suggesting the success of modeling. MeRIP detection showed that a total of 2 277 genes contained 2 981 differentially expressed m⁶A methylation sites (expression multiple ≥2 and P<0.05) in the kidneys of mice in the injury group and the control group. These genes were mainly concentrated in the metabolic and cell death pathways. The joint analysis of genes expressing differential m⁶A methylation sites and RNA differential expression genes found 1 002 genes with the same expression trend, such as fibrinogen α chain, solute carrier family 12 member 1 and hepatitis A virus cellular receptor 1.

Conclusion·Cisplatin can induce the change of methylation level of m⁶A methylation site on mRNA in renal tissue, and promote the process of acute renal injury.

Key words: N⁶-methyladenosine (m⁶A) methylome, cisplatin-induced acute kidney injury (CI-AKI), fibrinogen α chain (FGA), solute carrier family 12 member 1 (Slc12a1), hepatitis A virus cellular receptor 1 (Harvc1)

中图分类号:

R692.5

沈剑箫, 王万鹏, 邵兴华, 吴晶魁, 李舒, 车霞静, 倪兆慧. 顺铂诱导的急性肾损伤中肾脏组织m⁶A甲基化水平的变化[J]. 上海交通大学学报(医学版), 2021, 41(12): 1603-1611.

Jian-xiao SHEN, Wan-peng WANG, Xing-hua SHAO, Jing-kui WU, Shu LI, Xia-jing CHE, Zhao-hui NI. Changes of m⁶A methylation in renal tissue during cisplatin-induced acute injury[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021,(12): 1603-1611.

图/表 6

图1 顺铂诱导的C57bL/6小鼠AKI模型建立Note： A. Analysis of serum creatinine level in mice following different treatments.①P=0.000. B. Analysis of urea nitrogen level in mice following different treatments. ②P=0.000. C. Image of hematoxylin and eosin staining in kidney (black arrows indicating the injury). D. Score for characteristic histologic signs of renal injury. ③P=0.000.

Fig 1 Establishment of CI-AKI model in C57bL/6 mice

图2 对照组和损伤组mRNAs中m6A变化情况Note： A. Venn diagram showing the overlap of m6A peaks within mRNAs between the injury group and the control group. B. The top motif enriched across m6A peaks was identified from both groups. C. Proportion of genes harboring different numbers of m6A peaks in both groups. D. Pie charts showing the percentage of m6A peaks in five segments of transcripts. m6A peaks were most enriched in the coding sequence segment.

Fig 2 Overview of m6A methylation within mRNAs in the control group and the injury group

表1 甲基化水平变化差异最大的20个m6A甲基化位点

Tab 1 Top 20 differentially methylated m6A peaks

Gene name	Full name	Gene ID	Fold change	Regulation	Chromsome	Peak start	Peak end	Peak length
BC061237	cDNA sequence BC061237	385138	1 489.5	Up	Chr14	44504106	44504291	185
BC061237	cDNA sequence BC061237	385138	816.8	Up	Chr14	44500121	44500197	76
Krt20	Keratin 20	66809	498.7	Up	Chr11	99430752	99430920	168
Krt20	Keratin 20	66809	498.0	Up	Chr11	99429021	99429078	57
Krt20	Keratin 20	66809	483.5	Up	Chr11	99432181	99432343	162
1700001F09Rik	RIKEN cDNA 1700001F09 gene	71826	383.8	Up	Chr14	43346701	43346790	89
Ccdc85b	Coiled-coil domain containing 85B	240514	328.6	Up	Chr19	5454141	5454580	439
Gm3543	Predicted gene 3543	100041849	318.0	Up	Chr14	41982201	41982290	89
Serpina3n	Serinepeptidase inhibitor， clade A， member 3N	20716	316.3	Up	Chr12	104414261	104414329	68
Gm3543	Predicted gene 3543	100041849	254.0	Up	Chr14	41982133	41982180	47
Alms1	ALMS1 centrosome and basal body associated protein	236266	503.6	Down	Chr6	85694833	85694951	118
Tas2r119	Taste Receptor， type 2， member 119	57254	385.6	Down	Chr15	32177288	32177620	332
Ctnna2	Catenin α 2	12386	220.8	Down	Chr6	77600041	77600380	339
Afm	Afamin	280662	195.1	Down	Chr5	90518931	90519060	129
Slc5a4a	Solute carrier family 5， member 4a	64452	126.7	Down	Chr10	76163688	76163769	81
Nat1	N-acetyltransferase 1	17960	123.5	Down	Chr8	67490861	67491460	599
Pzp	PZP α-2-macroglobulin like	11287	111.1	Down	Chr6	128526621	128526720	99
Dpf3	Double PHD fingers 3	70127	99.2	Down	Chr12	83215461	83215800	339
Dgkg	Diacylglycerol Kinase γ	110197	95.3	Down	Chr16	22479365	22479426	61
Dpf3	Double Phd fingers 3	70127	94.3	Down	Chr12	83214541	83214840	299

图3 DMMS在染色体上分布情况Note： A. Chromosomal distribution of all DMMSs. Red lines indicate up regulation m6A methylation sites; Blue line indicate down regulation m6A methylation sites. B. Relative occupancy of DMMSs in each chromosome was normalized by length. C. Pie chart showing the percentage of DMMS peaks in five non-overlapping segments. D. Statistics of fold change of DMMS peaks in five segments. The histogram shows the mean of the fold change.

Fig 3 Distribution of DMMSs in chromosomes

图4 GO与KEGG分析包含DMMS的编码基因Note： A. Major GO terms were enriched for the genes containing up-regulated m6A sites. B. Major GO terms were enriched for the genes containing down-regulated methylated m6A sites. C. Major enriched pathways for the genes containing up-regulated methylated m6A sites. D. Major enriched pathways for the genes containing down-regulated methylated m6A sites.

Fig 4 GO and KEGG analyses of coding genes containing DMMSs

图5 差异甲基化基因和差异表达基因的联合分析Note： A. Four-quadrant graph exhibiting the differentially expressed genes containing DMMS peaks. B. Visualization of m6A-modified gene Fga. C. Visualization of m6A-modified gene Slc12a1. D. Visualization of m6A-modified gene Havcr1.

Fig 5 Conjoint analysis of differentially methylated genes and differentially expressed genes

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顺铂诱导的急性肾损伤中肾脏组织m⁶A甲基化水平的变化

Changes of m⁶A methylation in renal tissue during cisplatin-induced acute injury

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