Abstract
目的
非酒精性脂肪性肝病(nonalcoholic fatty liver disease，NAFLD)严重影响人们的健康，近来研究发现6-甲基腺嘌呤(N6-methyladenosine，m6A)甲基化参与机体脂质代谢过程，但仍缺乏针对NAFLD的m6A修饰水平的研究。本研究探讨NAFLD患者肝组织中m6A修饰水平的变化。
方法
采用液相色谱质谱(liquid chromatography-mass spectrometry，LC-MS)分析NAFLD患者肝组织中总m6A修饰水平的变化，甲基化RNA免疫共沉淀(methylated RNA immunoprecipitation，MeRIP)和微阵列(包含44 122个mRNA和12 496个lncRNA)分析整个转录组的m6A修饰水平的变化，进而通过基因本体(gene ontology，GO)功能富集分析、基因和基因组京都百科全书(Kyoto Encyclopedia of Genes and Genomes，KEGG)通路富集分析对差异修饰的mRNA进行功能注释，最后随机抽取4个mRNA和4个lncRNA，通过MeRIP及实时反转录聚合酶链反应(real-time RT-PCR)验证微阵列结果。
结果
与对照组相比，NAFLD组m6A总修饰水平明显下降，NAFLD组有176个差异修饰的mRNA和44个差异修饰的lncRNA。其中15个mRNA和7个lncRNA m6A修饰水平上调，而161个mRNA和37个lncRNA m6A修饰水平下调；GO功能富集分析和KEGG通路富集分析显示差异修饰的mRNA主要富集于能量代谢、转录调控、翻译调控等生物学过程。
结论
与正常肝组织相比，NAFLD患者肝中m6A甲基化修饰发生了显著改变，可能在NAFLD的发生、发展过程中发挥重要作用。
Keywords: 非酒精性脂肪性肝病, m6A修饰, 脂质代谢
Abstract
Objective
Non-alcoholic fatty liver disease has seriously affected people’s health. Recent studies have found that N6-methyladenosine (m6A) methylation is involved in the lipid metabolism process of the body, but the study on the level of m6A modification in NAFLD is still not available. This study aims to explore the changes in the level of RNA m6A methylation modification in NAFLD liver tissues, and to provide experimental and theoretical basis for in-depth study on the role of RNA m6A methylation in the occurrence and development of NAFLD.
Methods
Changes in the m6A level in NAFLD liver tissues were measured by liquid chromatography-mass spectrometry (LC-MS). Total RNA was extracted from liver tissues of NAFLD patients or normal control individuals and subjected to methylated RNA immunoprecipitation (MeRIP) with microarray analysis (including 44 122 mRNAs and 12 496 lncRNAs) to determine the changes in m6A modification levels across the entire transcriptome. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to annotate the differentially modified mRNAs. Finally, 4 mRNAs and 4 lncRNAs were randomly selected to verify the microarray results by MeRIP and real-time transcription polymerase chain reaction.
Results
A total of 176 mRNAs and 44 lncRNAs were found to be differentially m6A-modified in the NAFLD group compared with the control group. Among them, 15 mRNAs and 7 lncRNAs were hypermethylated in NAFLD, while 161 mRNAs and 37 lncRNAs were hypomethylated in NAFLD. GO and pathway analysis showed that the differentially modified mRNAs were enriched mainly in biological processes such as carboxylic acid metabolism and transcriptional regulation.
Conclusion
The m6A modification profile is changed in NAFLD liver tissues compared with normal liver tissues, which may functionally impact the pathophysiological progress in NAFLD.
Keywords: non-alcoholic fatty liver disease, m6A modification, lipid metabolism
非酒精性脂肪性肝病(non-alcoholic fatty liver disease，NAFLD)是我国最常见的慢性肝病之一，目前全球NAFLD患病率近30%[1]。随着肥胖及代谢综合征的全球化流行，NAFLD的发生率仍在逐年攀升。NAFLD可直接进展为肝硬化、肝癌，也与血管粥样硬化及2型糖尿病的发病密切相关，其不仅严重影响了患者的生活质量，也给家庭和社会带来了沉重的经济负担[2-3]。至今，NAFLD的发病机制尚不明确，临床上缺乏有效的治疗药物。因此，迫切需要进一步了解NAFLD的发病机制，以探索新的治疗方法。
研究[4-5]表明：RNA修饰参与各类疾病的发生发展过程，这些修饰被定义为转录组学变化，是一种新层次的基因表达调控方式，包括6-甲基腺嘌呤(N6-methyladenosine，m6A)、1-甲基腺嘌呤(N1-methyl-adenosine，m1A)、5-甲基胞嘧啶(5-methylcytidine，m5C)、5-羟甲基胞嘧啶(5-hydroxylmethylcytidine，hm5C)、假尿嘧啶(pseudouridine，Ψ)等；其中，N6位置的腺苷甲基化导致m6A的形成，m6A修饰是mRNA和lncRNA上含量最丰富的可逆性修饰，在转录后各个水平影响mRNA和lncRNA的代谢和功能。m6A修饰主要发生在RRACH(R是嘌呤基，A是腺嘌呤，C是胞嘧啶，H是非鸟嘌呤基)基序上[5-6]。在哺乳动物细胞中，N6位置的腺苷在甲基化转移酶(methyltransferase-like，METTL)和肾母细胞瘤1相关蛋白(Wilms’ tumor 1-asscociating protein，WTAP)等的作用下发生特异性甲基化修饰，即m6A。另外，RNA在脂质肥胖相关蛋白(fat mass and obesity-associated protein，FTO)和α-酮戊二酸/铁依赖的双加氧酶同源蛋白(α-ketoglutarate dependent dioxygenase homologue，ALKBH)2种去甲基化酶的作用下发生去甲基化，使得m6A修饰成为一种可逆反应。这些发生甲基化修饰的RNA碱基位点，需要特定的m6A结合蛋白YTH结构域蛋白家族(YT521-B homology，YTHDF)来识别，这些m6A结合蛋白能够识别发生了m6A修饰的碱基，从而参与下游翻译、mRNA降解、mRNA加速出核等过程。
近来研究[7]发现m6A甲基化参与机体脂质代谢并与异常脂质代谢相关，但缺乏针对NAFLD的m6A修饰水平的研究。因此，本研究旨在探讨NAFLD的m6A甲基化修饰水平的变化，以期更好地了解m6A修饰在NAFLD发生发展中的作用。
1. 对象与方法
1.1. 对象
根据2017年美国《非酒精性脂肪性肝病诊断与管理实践指南》，NAFLD诊断依据：1)无饮酒史或饮酒含乙醇量每周少于140 g(女性<70 g)；2)排除病毒性肝炎、药物性肝病、全胃肠外营养、肝豆状核变性等可导致脂肪肝的特定疾病；3)肝活检组织学改变符合脂肪性肝病的病理学诊断标准。收集2018年1月至12月在中南大学湘雅三医院住院并行胆囊切除手术的10例单纯性胆囊结石患者的肝组织标本，其中5例伴有NAFLD的标本为实验组，5例不伴NAFLD的标本为对照组。术前充分告知患者研究目的并获得书面知情同意书。术中于右肝第V段边缘用手术剪或手术刀片(避免使用电刀或超声刀) 切取一大小约1.0 cm×0.5 cm×1.0 cm的肝组织(50~100 mg) ，并立即置于液氮中，然后转至-80 ℃冰箱备用。纳入标准：1)25~55岁中青年患者；2)无其他代谢并发症；3)术后通过2名资深病理学家独立进行病理阅片证实疾病诊断。本研究经中南大学湘雅三医院医学伦理委员会批准(批准号：2016-S090)，并根据赫尔辛基宣言中的原则进行。
1.2. 材料
NEBNext Poly(A) mRNA磁性分离模块购自美国NEB公司，SYNBR Green PCR Master Mix试剂盒和Super RNA标记试剂盒购自美国Arraystar公司，RNeasy Mini试剂盒购自德国QIAGEN公司，RNase抑制剂购自美国Enzymatics公司，SuperScriptTM III反转录酶试剂盒购自美国赛默飞公司。
微量分光光度计(NanoDrop)为美国赛默飞公司产品，Agilent 6460 QQQ质谱仪、Agilent扫描器G2505C为美国安捷伦公司产品，10 000-Da MWCO旋转过滤器为美国Satorius公司产品。
1.3. m6A水平的量化
采用琼脂糖凝胶电泳从肝组织中提取总RNA，并使用微量分光光度计对RNA进行定量。通过NEBNext Poly(A) mRNA磁性分离模块将mRNA从总RNA中分离出来，然后通过酶促反应将mRNA水解为单个脱磷酸核苷，使用Satorius 10 000-Da MWCO旋转过滤器对预处理的核苷进行脱蛋白质处理，最后使用多反应监测模式(MRM)在Agilent 6460 QQQ质谱仪上进行液相色谱质谱(liquid chromatography-mass spectrometry，LC-MS)分析[8]。
1.4. 甲基化RNA免疫共沉淀
在4 ℃下，将总RNA(2 μg)和带有m6A的RNA探针混合物悬浮在IP缓冲液(含50 mmol/L Tris-HCl，pH 7.4，150 mmol/L NaCl，0.1% NP-40和40 U/μL RNase抑制剂)中孵育 2 h，再与2 μg抗m6A抗体洗涤，然后与羊抗兔IgG悬浮液在4 ℃下用新鲜制备的0.5% BSA孵育2 h，用300 μL IP缓冲液洗涤3次，再重新悬浮于总RNA和上面制备的抗体混合物中。结合m6A-抗体的RNA在4 ℃下旋转2 h。然后用500 μL IP缓冲液洗涤3次，并用500 μL洗涤缓冲液(50 mmol/L Tris-HCl，pH7.4，50 mmol/L NaCl，0.1% NP40，40 U/μL RNase抑制剂)洗涤2次。富集的RNA在50 ℃下用200 μL洗脱缓冲液(10 mmol/L Tris-HCl，pH7.4， 1 mmol/L EDTA，0.05% SDS，40 U蛋白酶K)洗脱1 h。用酸性苯酚-氯仿提取RNA，并沉淀出乙醇。
1.5. 标记和杂交
使用Super RNA标记试剂盒中的Cy5荧光染料标记来自对照组和实验组(n=5)的免疫共沉淀的RNA样品，并使用RNeasy Mini试剂盒纯化。将Cy5标记的cRNA片段化并杂交到人类m6A外转录微阵列(8× 60 000)中，该微阵列包含44 122 个mRNAs和12 496个lncRNAs探针。然后使用Agilent扫描器G2505C扫描杂交阵列，2组各使用5个微阵列。
1.6. 微阵列数据分析
使用安捷伦特征提取软件(版本11.0.1.1)分析采集的阵列图像，IP(发生修饰的RNA，免疫沉淀，用Cy5标记)的原始强度和Sup(未修饰的RNA，上清液，用Cy3标记)强度用log2(Spike-in RNA)的平均值进行标准化。根据IP和Sup的归一化强度计算m6A甲基化水平。用倍数(倍数>1.5)和统计学显著性(P<0.05)阈值进行过滤，鉴定出两组差异化的m6A，并对差异修饰的RNA进行基因本体(gene ontology，GO)功能富集分析、基因和基因组京都百科全书(Kyoto Encyclopedia of Genes and Genomes，KEGG)通路富集分析以确定这些基因主要富集的生物学途径[9]。
1.7. Real-time RT-PCR验证
使用SuperScriptTM III反转录酶试剂盒，按照说明书对大约1 μg的总RNA进行反转录。实时PCR(real-time PCR)使用SYNBR Green PCR Master Mix试剂盒，按照说明书进行操作。在每个反应结束时，进行熔解曲线分析以确认不存在引物二聚体。甘油醛3-磷酸脱氢酶(GAPDH)基因用作标准化的内部对照。PCR的总反应体积为20 µL，其中包括10 µL SYBR Premix Ex Taq(2x)，2 µL PCR引物(10 mmol/L)，1 µL cDNA和7 µL蒸馏水。使用2-ΔΔCt方法对目标mRNA进行定量。对比GAPDH将所有样品标准化。部分具体序列见表1。
表1.
Real-time PCR引物序列
Table 1 Primers used for real-time PCR
| 基因名 | 序列 | 引物长度/bp |
|---|---|---|
| NR_110023(DKC1) |
F: 5'-AGTTGGTGGTCAGATGCAGG-3' R: 5'-AAGCAAGTGGAGTGCCATCA-3' |
133 |
| ENST00000269216(GATA6) |
F: 5'-CTCCAACTTCCACCTCTTCTAAC-3' R: 5'-GAGCCCATCTTGACCCGA-3' |
161 |
| ENST00000441492(AC004383.4) |
F: 5'-CACCCACAGGACAACGAAGAA-3' R: 5'-GTTTACCAGGAACCGCAGAG-3' |
145 |
| ENST00000397270(INS-IGF2) |
F: 5'-CGGGGAACGAGGCTTCTTCTA-3' R: 5'-TCTGCCGAAACTGCCTGGAC-3' |
197 |
| NR_136616(EFR3A) |
F: 5'-AGTGGAACTCTTGCTGCTTGA-3' R: 5'-AGACGTCATTCTCTGCACCTT-3' |
118 |
| ENST00000511705(SH3RF2) |
F: 5'-CAGAAGTTACATGCACAAGGCAA-3' R: 5'-GTAGGGTCAGTTGAGCCACG-3' |
128 |
| ENST00000409638(STK16) |
F: 5'-AAGGCTCATGATAGCGTAGTG-3' R: 5'-GCTGACGATGCTCAGACTGG-3' |
149 |
| ENST00000396679(CENPK) |
F: 5'-AACTCAGAGTAGCCAGATGAT-3' R: 5'-ATGTAGCAGGATCGGTGTAA-3' |
115 |
1.8. 统计学处理
采用SPSS 18.0统计学软件对数据进行处理。计量数据以均数±标准差( ±s)表示，两组比较采用t检验(双尾)。P<0.05为差异有统计学意义。
2. 结 果
2.1. NAFLD中总m6A修饰水平下降
采用LC-MS方法测定总m6A修饰水平，结果显示NAFLD组中总m6A修饰水平是对照组的0.45倍(图1)。
图1.
非酒精性脂肪性肝病组和正常对照组的峰值图
Figure 1 Peak values of the non-alcoholic fatty liver disease group and the normal group
A: Normal group; B: Non-alcoholic fatty liver disease group. The total methylation level in non-alcoholic fatty liver disease group is reduced.
2.2. 微阵列分析m6A修饰的改变
采用微阵列分析差异修饰的RNA，结果显示：NAFLD组有220个转录本(176个mRNA和44个lncRNA)发生差异化修饰(倍数变化>1.5，P<0.05；n=5)。与对照组相比，实验组中有10%的转录本(15个mRNA和7个lncRNA)呈高甲基化，90%的转录本(161个mRNA和37个lncRNAs)呈低甲基化(图2)。
图2.
火山图显示了实验组和对照组中m6A表达水平的变化
Figure 2 Volcano plot shows the changes of the expression level of m6A in the experimental group and the control group
A: mRNA; B: LncRNA. Green indicates hypomethylation and red indicates hypermethylation.
2.3. GO功能富集分析和KEGG通路富集分析
GO功能富集分析显示：低甲基化修饰的RNA富集于DNA转录、染色质组成、RNA代谢等主要的生物学过程，同时也富集在DNA结合、转录因子活性等主要分子功能上；而高甲基化修饰的RNA富集于DNA合成、翻译调控等生物学过程，同时也富集在核苷三磷酸二磷酸酶活性(提供能量使得mRNA向细胞质转移)、PPARs的结合、受体启动剂活性的调节等主要分子功能上(图3)。
图3.
m6A差异修饰的 mRNA 转录本中基因本体分析的分子功能和生物学过程
Figure 3 Molecular function and biological process terms of gene ontology (GO) analysis in m6A differentially modified mRNA transcripts
A: Differential modification downregulation group; B: Differential modification upregulation group; the X-axis is the gene count.
KEGG通路富集分析显示：低甲基化修饰的RNA主要与突触小泡循环过程(synaptic vesicle cycle)和百日咳(pertussis)相关(图4)，而高甲基化修饰的RNA无任何相关通路。
图4.
m6A差异修饰的mRNA转录本中低甲基化组的KEGG通路富集分析结果
Figure 4 KEGG pathway analysis of differentially m6A modified mRNA transcripts in the methylation downregulation group
The X-axis is the gene count. No pathway was related to the differential expression upregulation group.
2.4. 甲基化RNA免疫共沉淀及real-time RT-PCR验证
随机选取了4个差异修饰的lncRNAs(NR_110023，ENST00000441492，ENST00000511705，NR_ 136616)和4个差异修饰的mRNAs(ENST00000397270，ENST00000269216，ENST00000409638，ENST-00000396679)(P<0.01)，通过甲基化RNA免疫共沉淀取得RNA甲基化沉淀，然后行real-time RT-PCR。与微阵列数据一致，lncRNAs(NR_110023，NR_136616)和mRNAs(ENST00000269216，ENST00000409638)的m6A甲基化下调，而lncRNAs(ENST00000441492，ENST00000511705)和mRNAs(ENST00000397270，ENST00000396679)的m6A修饰上调(图5)。
图5.
甲基化RNA免疫沉淀结合实时RT-PCR验证NAFLD组和对照组肝组织中差异修饰的lncRNA和 mRNA，证实了微阵列数据的有效性
Figure 5 Methylated RNA immunoprecipitation-real-time RT-PCR verified the differentially modified lncRNAs and mRNAs in liver tissues in the NAFLD group and the control group, confirming the validity of the microarray data
3. 讨 论
RNA修饰是表观遗传学的重要组成部分，与胚胎发育、细胞分化、免疫应答等生理过程密切相关。m6A修饰是RNA修饰中最为常见的修饰方式之一，已有研究表明：m6A修饰参与胶质母细胞瘤[10]、宫颈癌[11]、乳腺癌[12]、牙周炎[13]、类风湿关节炎[14]、冠状动脉疾病[15]、缺血性脑卒中[16]等多种疾病的发生发展，同时也影响机体血压[17]、血脂[18]、骨密度[19]等指标。因此，m6A修饰在多种生物学过程和疾病的发生发展中起关键作用。
研究[20]表明：m6A修饰在代谢性疾病中起重要作用，如FTO能通过调节剪切位点周围的m6A水平来控制脂质合成因子(RUNX1 partner transcriptional co-repressor 1，RUNX1T1)的外显子剪接，从而调节肥胖。胎盘相关基因胰岛素受体(insulin receptor，INSR)和胰岛素受体底物1(insulin receptor substrate 1，IRS1)的m6A水平显著下降，参与妊娠期糖尿病的发生[21]。近来研究[22]发现：高脂饮食喂养8周后，小鼠肝组织的m6A修饰水平发生了显著改变，与对照组相比，高脂饮食组中小鼠的m6A修饰水平降低，其中高甲基化基因主要富集于脂质代谢过程，而低甲基化基因主要富集于代谢和翻译过程。在另一项研究[23]中，敲低小鼠甲基化转移酶METTL3，抑制了m6A修饰过程中的“编写器”，肝内脂质积聚明显减少。以上研究提示m6A修饰参与调控小鼠肝脂质代谢。为明确NAFLD患者肝组织中m6A修饰水平的变化，本研究收集了5例NAFLD患者的肝组织及5例正常肝组织标本，通过微阵列分析人肝组织中m6A修饰水平变化，结果显示NAFLD患者肝组织标本中m6A总甲基化水平降低，证实m6A修饰在脂质代谢中的重要性。基于微阵列数据，我们在NAFLD患者肝组织中发现了220个差异修饰基因，其中44个RNA呈高甲基化，176个RNA呈低甲基化，进一步对差异修饰基因行GO和KEGG分析发现，高甲基化基因主要富集于PPARs的结合、受体激动剂活性的调节等过程，低甲基化基因主要富集于转录因子活性调节、转录调控等过程。PPARs信号通路是重要的脂质代谢调控通路，提示PPARs信号通路因子可能通过改变m6A修饰水平参与NAFLD的发生发展。这为m6A调控肝脂质代谢提供了证据，有利于阐明NAFLD的发病机制。
本研究首次分析了NAFLD患者肝组织中m6A修饰水平的变化，发现44个lncRNA存在m6A甲基化修饰，且其中多个差异修饰的lncRNA参与调节脂质代谢。例如：组蛋白脱乙酰酶8(histone deacetylase 8，HDAC8)通过催化长链脂肪酰基赖氨酸的水解来调节脂质代谢[24]，G蛋白偶联受体78(G protein coupled receptor 78，GPR78)通过磷酸化参与内质网应激来调控脂质代谢[25]，酰基辅酶A合成酶家族成员3(acyl-CoA synthetase family member 3，ACSF3)作为脂肪酸合成的关键酶调控脂质代谢[26]，而丝苏氨酸蛋白激酶3(RIO kinase 3，RIOK3)通过激活炎症途径参与脂质代谢[27]。以上结果提示lncRNAs可能通过改变m6A修饰水平参与NAFLD的发生发展。
综上所述，本研究发现NAFLD患者肝组织中m6A修饰水平发生了显著变化，但差异化修饰的基因在NAFLD中的具体调控作用及分子机制有待进一步深入研究。
基金资助
国家自然科学基金(82070597)；湖南省自然科学基金(2019JJ40466，2019JJ40467)；湖南省卫生健康委员会课题(20180314)。
This work was supported by the National Natural Science Foundation (82070597), the Natural Science Foundation of Hunan Province (2019JJ40466, 2019JJ40467), and the Project of Hunan Provincial Health Commission (20180314), China.
利益冲突声明
作者声称无任何利益冲突。
原文网址
http://xbyxb.csu.edu.cn/xbwk/fileup/PDF/202108785.pdf
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