高脂饮食诱导的小鼠NAFLD模型肝组织中m6A甲基化修饰表达谱分析

doi:10.3969/j.issn.1674-8115.2023.10.002

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (10): 1227-1235.doi: 10.3969/j.issn.1674-8115.2023.10.002

• 论著 · 基础研究 • 上一篇

高脂饮食诱导的小鼠NAFLD模型肝组织中m⁶A甲基化修饰表达谱分析

刘君君¹^,²(), 逯素梅², 张炳杨², 李永清², 马万山¹^,²()

^1.山东大学临床医学院，济南 250014
^2.山东第一医科大学第一附属医院（山东省千佛山医院）检验医学科，山东省医药卫生临床检验诊断学重点实验室，济南 250014

收稿日期:2023-04-14 接受日期:2023-09-12 出版日期:2023-10-28 发布日期:2023-10-28
通讯作者: 马万山 E-mail:liujing920927@163.com;mwsqianyi@163.com
作者简介:刘君君（1992—）女，主管技师，硕士生；电子信箱：liujing920927@163.com。
基金资助:
山东省自然科学基金(ZR2021MH187);山东省千佛山医院国家自然科学基金培育基金(QYPY2020NSFC1004)

Analysis of m⁶A methylation expression profiles in liver tissue of high-fat diet-induced mouse models of NAFLD

LIU Junjun¹^,²(), LU Sumei², ZHANG Bingyang², LI Yongqing², MA Wanshan¹^,²()

^1.School of Clinical Medicine, Shandong University, Jinan 250014, China
^2.Department of Clinical Laboratory Medicine, The First Affiliated Hospital of Shandong First Medical University/Shandong Provincial Qianfoshan Hospital, Shandong Medicine and Health Key Laboratory of Laboratory Medicine, Jinan 250014, China

Received:2023-04-14 Accepted:2023-09-12 Online:2023-10-28 Published:2023-10-28
Contact: MA Wanshan E-mail:liujing920927@163.com;mwsqianyi@163.com
Supported by:
Natural Science Foundation of Shandong Province(ZR2021MH187);Shandong Provincial Qianfoshan Hospital, National Natural Science Foundation of China Cultivation Fund(QYPY2020NSFC1004)

摘要/Abstract

摘要：

目的·利用微阵列芯片技术检测高脂饮食诱导的小鼠非酒精性脂肪性肝病（non-alcoholic fatty liver disease，NAFLD）模型中肝组织mRNA的m⁶A甲基化修饰和基因表达的变化。方法·以6~8周龄雄性C57BL/6J小鼠为实验动物，高脂饲料喂养16周诱导建立NAFLD模型（高脂组，n=10）；另设基础组（n=10）为对照，给予含10%脂肪的基础饲料喂养。苏木精-伊红（hematoxylin-eosin，H-E）染色评估小鼠肝组织病理改变，判断NAFLD模型是否构建成功。运用甲基化RNA免疫共沉淀技术（methylated RNA immunoprecipitation，MeRIP）和微阵列测序技术（microarray表达谱分析）检测2组小鼠肝组织中mRNA的m⁶A甲基化和表达水平变化。结果·基础组小鼠肝脏呈鲜红色，少见脂肪沉积；高脂组小鼠肝脏边界黄润，H-E染色可见肝细胞中脂滴弥漫浸润且相互融合，提示高脂饲料诱导的NAFLD模型构建成功。MeRIP-微阵列芯片检测结果显示，与基础组相比，高脂组小鼠肝脏中共有320个基因m⁶A甲基化修饰水平变化显著（P<0.05且变化倍数>1.5），其中有108个上调基因和212个下调基因。将组间m⁶A甲基化水平差异显著的基因与mRNA差异表达基因取交集，发现有163个基因m⁶A甲基化水平和mRNA表达水平均差异显著。结论·高脂饮食诱导的小鼠NAFLD模型中肝组织mRNA的m⁶A修饰变化显著，且该变化与mRNA的基因表达有关。

关键词: 非酒精性脂肪性肝病, m⁶A甲基化修饰, mRNA, 表观转录组学

Abstract:

Objective ·To detect the differences in m⁶A methylation modification and gene expression of liver tissue mRNA in high-fat diet-induced mouse models of non-alcoholic fatty liver disease (NAFLD) using microarray technology. Methods ·The NAFLD models were established in 6-8 weeks old male C57BL/6J mice fed with high-fat chow for 16 weeks (high-fat group, n=10). The basal group (n=10) was given 10% fat diet. Hematoxylin-eosin (H-E) staining was used to assess the histopathological changes in liver tissue and to determine the success of the NAFLD models. The changes of mRNA m⁶A methylation and expression levels in the liver tissues of the two groups were detected by using methylated RNA immunoprecipitation (MeRIP) and microarray expression profiling. Results ·The livers of the mice in the basal group were bright red with few fat deposits, while the livers of the mice in the high-fat group were yellowish with diffuse infiltration and fusion of lipid droplets in the hepatocytes by H-E staining, suggesting that the high-fat diet-induced NAFLD models were successfully constructed. The results of the MeRIP-microarray showed that the m⁶A methylation levels of 320 genes in the livers of mice in the high-fat group were significantly altered compared with those in the basal group (P<0.05 and fold change>1.5), of which 108 genes were up-regulated and 212 genes were down-regulated. Genes with significant differences in m⁶A methylation levels between the two groups were intersected with those with differentially expressed mRNAs, and 163 genes were found to have significant differences in both m⁶A methylation level and mRNA expression level. Conclusion ·The change in m⁶A modification of liver tissue mRNA in the high-fat diet-induced mouse models of NAFLD is significant and the change is associated with the gene expression of mRNA.

Key words: non-alcoholic fatty liver disease (NAFLD), m⁶A methylation modification, mRNA, epitranscriptomics

中图分类号:

R446

刘君君, 逯素梅, 张炳杨, 李永清, 马万山. 高脂饮食诱导的小鼠NAFLD模型肝组织中m⁶A甲基化修饰表达谱分析[J]. 上海交通大学学报（医学版）, 2023, 43(10): 1227-1235.

LIU Junjun, LU Sumei, ZHANG Bingyang, LI Yongqing, MA Wanshan. Analysis of m⁶A methylation expression profiles in liver tissue of high-fat diet-induced mouse models of NAFLD[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(10): 1227-1235.

图/表 5

参考文献 26

1	祥蔚. IL-17A基因敲除减轻LPS诱导的脂肪肝小鼠炎症性肝损伤及其机制研究[D].重庆: 第三军医大学, 2017.
	XIANG W. IL-17A deficiency alleviates LPS-induced steatotic liver injury in mice and the underlying mechanism[D]. Chongqing: Third Military Medical University of Chinese P.L.A., 2017.
2	ESTES C, ANSTEE Q M, ARIAS-LOSTE M T, et al. Modeling NAFLD disease burden in China, France, Germany, Italy, Japan, Spain, United Kingdom, and United States for the period 2016‒2030[J]. J Hepatol, 2018, 69(4): 896-904.
3	CHENG Y, HOU T, PING J, et al. Quantitative succinylome analysis in the liver of non-alcoholic fatty liver disease rat model[J]. Proteome Sci, 2016, 14: 3.
4	XU Z J, SHI J P, YU D R, et al. Evaluating the relationship between metabolic syndrome and liver biopsy-proven non-alcoholic steatohepatitis in China: a multicenter cross-sectional study design[J]. Adv Ther, 2016, 33(11): 2069-2081.
5	YOUNOSSI Z M, KOENIG A B, ABDELATIF D, et al. Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes[J]. Hepatology, 2016, 64(1): 73-84.
6	YOUNOSSI Z M, GOLABI P, DE AVILA L, et al. The global epidemiology of NAFLD and NASH in patients with type 2 diabetes: a systematic review and meta-analysis[J]. J Hepatol, 2019, 71(4): 793-801.
7	PASCHOS P, PALETAS K. Non alcoholic fatty liver disease and metabolic syndrome[J]. Hippokratia, 2009, 13(1): 9-19.
8	STEFAN N, HÄRING H U, CUSI K. Non-alcoholic fatty liver disease: causes, diagnosis, cardiometabolic consequences, and treatment strategies[J]. Lancet Diabetes Endocrinol, 2019, 7(4): 313-324.
9	ALEXANDER M, LOOMIS A K, VAN DER LEI J, et al. Risks and clinical predictors of cirrhosis and hepatocellular carcinoma diagnoses in adults with diagnosed NAFLD: real-world study of 18 million patients in four European cohorts[J]. BMC Med, 2019, 17(1): 95.
10	FRIEDMAN S L, NEUSCHWANDER-TETRI B A, RINELLA M, et al. Mechanisms of NAFLD development and therapeutic strategies[J]. Nat Med, 2018, 24(7): 908-922.
11	DIEHL A M, DAY C. Cause, pathogenesis, and treatment of nonalcoholic steatohepatitis[J]. N Engl J Med, 2017, 377(21): 2063-2072.
12	王园园, 蒋建萍. 非酒精性脂肪肝与代谢综合征各组分关系的研究进展[J]. 中国当代医药, 2022, 29(15): 36-39, 43.
	WANG Y Y, JIANG J P. Research progress of the relationship between nonalcoholic fatty liver disease and components of metabolic syndrome[J]. China Modern Medicine, 2022, 29(15): 36-39, 43.
13	ZHOU J, ZHOU F, WANG W, et al. Epidemiological features of NAFLD from 1999 to 2018 in China[J]. Hepatology, 2020, 71(5): 1851-1864.
14	李永清. METTL3介导的CYP2B6 m6A甲基化修饰在非酒精性脂肪性肝病肝细胞胰岛素敏感性中的作用和机制研究[D]. 济南: 山东大学, 2022.
	LI Y Q. Role and mechanism of METTL3-mediated CYP2B6 m⁶A methylation modification in insulin sensitivity in hepatocytes with non-alcoholic fatty liver disease[D]. Jinan: Shandong University, 2022.
15	叶棣文, 马万山, 逯素梅. RNAm⁶A甲基化修饰在代谢综合征中的研究进展[J]. 临床检验杂志, 2022, 40(9): 691-694.
	YE D W, MA W S, LU S M. Advances in the study of RNA m⁶A methylation modification in the metabolic syndrome[J].Chinese Journal of Clinical Laboratory Science, 2022,40(9): 691-694.
16	SUMIDA Y, YONEDA M. Current and future pharmacological therapies for NAFLD/NASH[J]. J Gastroenterol, 2018, 53(3): 362-376.
17	陈子扬, 蒲锐, 邓爽, 等. m⁶A甲基化与代谢性疾病调控[J]. 中国组织工程研究, 2022, 26(20): 3250-3255.
	CHEN Z Y, PU R, DENG S, et al. N⁶-methyladenosine methylation and its regulation in metabolic diseases[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(20):3250-3255.
18	PENG Z, GONG Y, WANG X, et al. METTL3-m⁶A-Rubicon axis inhibits autophagy in nonalcoholic fatty liver disease[J]. Mol Ther, 2022, 30(2): 932-946.
19	韦冷云. Hacd2在脂质代谢中的功能及其作用机制[D]. 无锡: 江南大学, 2022.
	WEI L Y. Function and mechanism of Hacd2 in lipid metabolism[D]. Wuxi: Jiangnan University, 2022.
20	曾晗. CD36在肝细胞脂质从头合成中的作用及机制研究[D]. 重庆: 重庆医科大学, 2022.
	ZENG H. The role of CD36 in regulation of hepatocyte de novo lipogenesis[D]. Chongqing: ChongQing Medical University, 2022.
21	仝雪薇, 冶学燕, 刘春燕, 等. 2型糖尿病合并肥胖患者外周血PPARG、CIDEA、ECHDC3、CGN mRNA表达与血脂水平的关系研究[J]. 深圳中西医结合杂志, 2021, 31(6): 4-8, 199.
	TONG X W, YE X Y, LIU C Y, et al. Relationship between mRNA expression of PPARG, CIDEA, ECHDC3, CGN and lipid levels in peripheral blood of patients with type 2 diabetes mellitus complicated with obesity[J].Shenzhen Journal of Integrated Traditional Chinese and Western Medicine, 2021, 31(6): 4-8, 199.
22	SANS A, BONNAFOUS S, ROUSSEAU D, et al. The differential expression of CIDE family members is associated with NAFLD progression from steatosis to steatohepatitis[J]. Sci Rep, 2019, 9(1): 7501.
23	TANG J, ZHAO X, WEI W, et al. METTL16-mediated translation of CIDEA promotes non-alcoholic fatty liver disease progression via m6A-dependent manner[J]. PeerJ, 2022, 10: e14379.
24	QUIROGA A D, LI L, TRÖTZMÜLLER M, et al. Deficiency of carboxylesterase 1/esterase-x results in obesity, hepatic steatosis, and hyperlipidemia[J]. Hepatology, 2012, 56(6): 2188-2198.
25	钱胜南. ATP在胰岛素抵抗机制中对线粒体MECR蛋白的影响[D]. 上海: 上海海洋大学, 2020.
	QIAN S N. Effect of ATP on mitochondrial MECR protein in the mechanism of insulin resistance[D].Shanghai: Shanghai Ocean University, 2020.
26	潘晓燕. FOXO转录因子在饮食诱导的脂肪性肝病中的作用机制研究[D]. 苏州: 苏州大学, 2018.
	PAN X Y. Study on the mechanisms of action of FOXO transcriptional factors in diet-induced fatty liver disease[D].Suzhou: Soochow University, 2018.

Gene	mRNA_ID	Locus	RNA length/nt	Fold change	P value
Up-regulated gene
Micalc1	ENSMUST00000033033	chr7: 112374345-112395355: +	2 259	2.840	0.004
Cyp2b10	ENSMUST00000005477	chr7: 25897676-25926559: +	1 822	2.721	0.011
Ksr2	ENSMUST00000180430	chr5: 117414000-117775003: +	13 125	2.484	0.001
Ybx1	ENSMUST00000079644	chr4: 119277981-119294604: -	1 628	2.359	0.023
Paqr7	ENSMUST00000095074	chr4: 134497004-134510235: +	3 550	2.274	0.002
Casc5	ENSMUST00000099542	chr2: 119047119-119104121: +	6 525	2.270	0.000
Ubr3	ENSMUST00000131553	chr2: 69897303-69938659: +	1 801	2.113	0.005
Dpm3	ENSMUST00000107462	chr3: 89259358-89267077: +	827	1.978	0.025
Cd36	ENSMUST00000082367	chr5: 17782016-17835696: -	3 016	1.965	0.021
Tsc22d1	ENSMUST00000142683	chr14: 76487759-76506998: +	797	1.928	0.028
Down-regulated gene
Esd	ENSMUST00000176957	chr14: 74732384-74749848: +	1 117	0.356	0.010
Slc22a26	ENSMUST00000120522	chr19: 7781041-7802578: -	2 854	0.421	0.010
Folr1	NM_001252553	chr7: 101858331-101870788: -	1 337	0.437	0.000
Slc46a3	ENSMUST00000138244	chr5: 147893881-147894815: -	388	0.437	0.001
Inpp5d	ENSMUST00000167032	chr1: 87676239-87720502: +	4 125	0.462	0.010
Hoxd9	ENSMUST00000059272	chr2: 74697727-74700208: +	2 136	0.471	0.012
Romo1	ENSMUST00000088610	chr2: 156144039-156145797: +	569	0.484	0.005
Litaf	ENSMUST00000117360	chr16: 10960824-10975579: -	763	0.492	0.014
Mdh2	ENSMUST00000019323	chr5: 135778480-135790391: +	1 456	0.509	0.032
Sycp3	ENSMUST00000125612	chr10: 88459664-88473236: +	1 169	0.513	0.044

Gene	mRNA_ID	Locus	RNA length/nt	Fold change	P value
Up-regulated gene
Micalc1	ENSMUST00000033033	chr7: 112374345-112395355: +	2 259	2.840	0.004
Cyp2b10	ENSMUST00000005477	chr7: 25897676-25926559: +	1 822	2.721	0.011
Ksr2	ENSMUST00000180430	chr5: 117414000-117775003: +	13 125	2.484	0.001
Ybx1	ENSMUST00000079644	chr4: 119277981-119294604: -	1 628	2.359	0.023
Paqr7	ENSMUST00000095074	chr4: 134497004-134510235: +	3 550	2.274	0.002
Casc5	ENSMUST00000099542	chr2: 119047119-119104121: +	6 525	2.270	0.000
Ubr3	ENSMUST00000131553	chr2: 69897303-69938659: +	1 801	2.113	0.005
Dpm3	ENSMUST00000107462	chr3: 89259358-89267077: +	827	1.978	0.025
Cd36	ENSMUST00000082367	chr5: 17782016-17835696: -	3 016	1.965	0.021
Tsc22d1	ENSMUST00000142683	chr14: 76487759-76506998: +	797	1.928	0.028
Down-regulated gene
Esd	ENSMUST00000176957	chr14: 74732384-74749848: +	1 117	0.356	0.010
Slc22a26	ENSMUST00000120522	chr19: 7781041-7802578: -	2 854	0.421	0.010
Folr1	NM_001252553	chr7: 101858331-101870788: -	1 337	0.437	0.000
Slc46a3	ENSMUST00000138244	chr5: 147893881-147894815: -	388	0.437	0.001
Inpp5d	ENSMUST00000167032	chr1: 87676239-87720502: +	4 125	0.462	0.010
Hoxd9	ENSMUST00000059272	chr2: 74697727-74700208: +	2 136	0.471	0.012
Romo1	ENSMUST00000088610	chr2: 156144039-156145797: +	569	0.484	0.005
Litaf	ENSMUST00000117360	chr16: 10960824-10975579: -	763	0.492	0.014
Mdh2	ENSMUST00000019323	chr5: 135778480-135790391: +	1 456	0.509	0.032
Sycp3	ENSMUST00000125612	chr10: 88459664-88473236: +	1 169	0.513	0.044

[1]	刘薇薇, 王龙. 女性绝经与非酒精性脂肪性肝病的关系及相关治疗的研究进展[J]. 上海交通大学学报（医学版）, 2023, 43(1): 125-131.
[2]	宗春燕, 何杰, 张哲, 贾仁兵, 沈键锋. APOBEC3B调控葡萄膜黑色素瘤复制应激的研究[J]. 上海交通大学学报（医学版）, 2022, 42(8): 1034-1044.
[3]	张晓文, 王祎, 张婵, 张迪, 贠航, 黄笛. Pcsk9基因干扰对高脂诱导的大鼠非酒精性脂肪性肝病合并动脉粥样硬化的影响[J]. 上海交通大学学报(医学版), 2022, 42(2): 150-157.
[4]	王海红, 高硕, 朱军, 周隽. 干扰素调节因子2结合蛋白2在斑马鱼胚胎早期发育过程中的表达谱分析[J]. 上海交通大学学报(医学版), 2021, 41(3): 314-319.
[5]	桑潮, 梁丹丹, 谢国祥, 贾伟, 陈天璐. 非酒精性脂肪性肝病血清学无创诊断的研究进展[J]. 上海交通大学学报(医学版), 2021, 41(1): 112-117.
[6]	刘永湄，谭明，雷鸣. 人源THOC1-THOC2-THOC3亚复合物的电镜结构研究[J]. 上海交通大学学报（医学版）, 2020, 40(1): 1-.
[7]	朱凡凡，仰礼真. 甲状腺功能正常的2型糖尿病患者血清甲状腺激素水平与非酒精性脂肪性肝病的相关性[J]. 上海交通大学学报（医学版）, 2018, 38(7): 763-.
[8]	菅朝慧，包玉倩. 自噬与非酒精性脂肪性肝病的研究进展[J]. 上海交通大学学报（医学版）, 2018, 38(6): 690-.
[9]	应晨 1，刘彩虹 2，胡家安 2，江石湖 3，徐志红 2，孙璟 2. 阻塞性睡眠呼吸暂停低通气综合征合并非酒精性脂肪性肝病患者血清脂肪代谢相关激素水平的研究[J]. 上海交通大学学报（医学版）, 2018, 38(10): 1203-.
[10]	侯亚楠，禤立平，彭魁，杜瑞，徐瑜，陈宇红，陆洁莉，毕宇芳，徐敏，王卫庆，宁光 . 社区中老年人群中上臂围与非酒精性脂肪性肝病及胰岛素抵抗的相关性研究[J]. 上海交通大学学报（医学版）, 2017, 37(9): 1232-.
[11]	孙雯雯，阮玉凤，连鹏，应晨，胡家安，徐志红，孙璟. 阻塞性睡眠呼吸暂停低通气综合征对非酒精性脂肪性肝病的影响[J]. 上海交通大学学报（医学版）, 2016, 36(5): 707-.
[12]	范梦夏，乔洁. 黄体生成素受体mRNA结合蛋白的研究进展[J]. 上海交通大学学报（医学版）, 2015, 35(6): 906-.
[13]	康冰，彭川，左的于，等. 非酒精性脂肪性肝病患者血清SFRP5水平与血脂相关性分析[J]. 上海交通大学学报（医学版）, 2015, 35(5): 694-.
[14]	朱超霞，仓桢，加孜热亚·再依拿提，等. 盐酸小檗碱对高脂饮食诱导的非酒精性脂肪性肝病大鼠肠道菌群的影响[J]. 上海交通大学学报（医学版）, 2015, 35(4): 483-.
[15]	张生枝，邵华江，马建婷. 人乳头瘤病毒E6/E7 mRNA与宫颈病变关系的研究进展[J]. 上海交通大学学报（医学版）, 2014, 34(5): 754-.

高脂饮食诱导的小鼠NAFLD模型肝组织中m⁶A甲基化修饰表达谱分析

Analysis of m⁶A methylation expression profiles in liver tissue of high-fat diet-induced mouse models of NAFLD

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 5

参考文献 26

相关文章 15

编辑推荐

Metrics