Volume: 16, Issue: 3

Review Article

Reversible RNA Modification N1-methyladenosine (m1A) in mRNA and tRNA

Chi Zhang, Guifang Jia

More than 100 modifications have been found in RNA. Analogous to epigenetic DNA methylation, epitranscriptomic modifications can be written, read, and erased by a complex network of proteins. Apart from N6-methyladenosine (m6A), N1-methyladenosine (m1A) has been found as a reversible modification in tRNA and mRNA. m1A occurs at positions 9, 14, and 58 of tRNA, with m1A58 being critical for tRNA stability. Other than the hundreds of m1A sites in mRNA and long non-coding RNA transcripts, transcriptome-wide mapping of m1A also identifies >20 m1A sites in mitochondrial genes. m1A in the coding region of mitochondrial transcripts can inhibit the translation of the corresponding proteins. In this review, we summarize the current understanding of m1A in mRNA and tRNA, covering high-throughput sequencing methods developed for m1A methylome, m1A-related enzymes (writers and erasers), as well as its functions in mRNA and tRNA.
迄今为止人们已经在RNA上发现了100多种化学修饰。与表观遗传学中的DNA甲基化相似，表观转录组上的修饰也能够由一系列的蛋白引入、识别和去除。N1-甲基腺苷（m1A）是一种存在于tRNA和mRNA上的可逆修饰。它在tRNA上主要存在于9,14和58位，其中m1A58对tRNA的稳定性起到至关重要的作用。人们利用对转录组进行高通量测序的方法，在mRNA和lncRNA上鉴定出了超过100个m1A位点，同时在线粒体基因中也发现了20多个m1A位点，并发现线粒体基因中的m1A会抑制相关蛋白的翻译。在这篇综述中，我们总结了目前在mRNA和tRNA上对m1A的研究，介绍了针对m1A的高通量测序方法以及与m1A相关的蛋白（甲基转移酶和去甲基酶），并阐述了m1A在mRNA和tRNA上的功能。

Page 155-161

Original Research

Genome-wide MicroRNA Expression Profiles in COPD: Early Predictors for Cancer Development

Andreas Keller, Tobias Fehlmann, Nicole Ludwig, Mustafa Kahraman, Thomas Laufer, Christina Backes, Claus Vogelmeier, Caroline Diener, Frank Biertz, Christian Herr, Rudolf A. Jörres, Hans-Peter Lenhof, Eckart Meese, Robert Bals for the COSYCONET Study Group

Chronic obstructive pulmonary disease (COPD) significantly increases the risk of developing cancer. Biomarker studies frequently follow a case-control set-up in which patients diagnosed with a disease are compared to controls. Longitudinal cohort studies such as the COPD-centered German COPD and SYstemic consequences-COmorbidities NETwork (COSYCONET) study provide the patient and biomaterial base for discovering predictive molecular markers. We asked whether microRNA (miRNA) profiles in blood collected from COPD patients prior to a tumor diagnosis could support an early diagnosis of tumor development independent of the tumor type. From 2741 participants of COSYCONET diagnosed with COPD, we selected 534 individuals including 33 patients who developed cancer during the follow-up period of 54 months and 501 patients who did not develop cancer, but had similar age, gender and smoking history. Genome-wide miRNA profiles were generated and evaluated using machine learning techniques. For patients developing cancer we identified nine miRNAs with significantly decreased abundance (two-tailed unpaired t-test adjusted for multiple testing P < 0.05), including members of the miR-320 family. The identified miRNAs regulate different cancer-related pathways including the MAPK pathway (P = 2.3 × 10−5). We also observed the impact of confounding factors on the generated miRNA profiles, underlining the value of our matched analysis. For selected miRNAs, qRT-PCR analysis was applied to validate the results. In conclusion, we identified several miRNAs in blood of COPD patients, which could serve as candidates for biomarkers to help identify COPD patients at risk of developing cancer.

Page 162-171

Original Research

Tet2 Regulates Osteoclast Differentiation by Interacting with Runx1 and Maintaining Genomic 5-Hydroxymethylcytosine (5hmC)

Yajing Chu, Zhigang Zhao, David Wayne Sant, Ganqian Zhu, Sarah M. Greenblatt, Lin Liu, Jinhuan Wang, Zeng Cao, Jeanette Cheng Tho, Shi Chen, Xiaochen Liu, Peng Zhang, Jaroslaw P. Maciejewski, Stephen Nimer, Gaofeng Wang, Weiping Yuan, Feng-Chun Yang, Mingjiang Xu

As a dioxygenase, Ten-Eleven Translocation 2 (TET2) catalyzes subsequent steps of 5-methylcytosine (5mC) oxidation. TET2 plays a critical role in the self-renewal, proliferation, and differentiation of hematopoietic stem cells, but its impact on mature hematopoietic cells is not well-characterized. Here we show that Tet2 plays an essential role in osteoclastogenesis. Deletion of Tet2 impairs the differentiation of osteoclast precursor cells (macrophages) and their maturation into bone-resorbing osteoclasts in vitro. Furthermore, Tet2−/− mice exhibit mild osteopetrosis, accompanied by decreased number of osteoclasts in vivo. Tet2 loss in macrophages results in the altered expression of a set of genes implicated in osteoclast differentiation, such as Cebpa, Mafb, and Nfkbiz. Tet2 deletion also leads to a genome-wide alteration in the level of 5-hydroxymethylcytosine (5hmC) and altered expression of a specific subset of macrophage genes associated with osteoclast differentiation. Furthermore, Tet2 interacts with Runx1 and negatively modulates its transcriptional activity. Our studies demonstrate a novel molecular mechanism controlling osteoclast differentiation and function by Tet2, that is, through interactions with Runx1 and the maintenance of genomic 5hmC. Targeting Tet2 and its pathway could be a potential therapeutic strategy for the prevention and treatment of abnormal bone mass caused by the deregulation of osteoclast activities.
Tet2作为双加氧酶，负责催化5-甲基胞嘧啶（5mC）氧化的后续步骤。Tet2在造血干细胞的自我更新、增殖和分化中发挥着关键作用，但其对成熟造血细胞的影响尚未得到很好的阐释。在这里，我们的工作表明Tet2在破骨细胞发生过程中起着至关重要的作用。Tet2的缺失破坏破骨细胞前体细胞（巨噬细胞）的体外分化及其成熟为具有骨吸收功能破骨细胞的能力。此外，Tet2-/-小鼠表现出轻度骨硬化，伴随着体内破骨细胞数量的减少。Tet2在巨噬细胞中的缺失导致一系列破骨细胞分化相关基因表达的紊乱，如Cebpa、Mafb和Nfkbiz。Tet2缺失同时引起巨噬细胞内与破骨细胞分化相关的特定基因集的5-羟甲基胞嘧啶（5hmC）水平和表达水平的改变。此外，Tet2与Runx1发生相互作用，并负调控其转录活性。我们的研究揭示了Tet2通过与Runx1相互作用以及维持基因组5hmC水平来调控破骨细胞分化和功能的新型分子机制。靶向TET2及其通路可能是防治由破骨细胞活性失调引起的骨密度异常的潜在治疗策略。

Page 172-186

Original Research

5-Hydroxymethylome in Circulating Cell-free DNA as A Potential Biomarker for Non-small-cell Lung Cancer

Ji Zhang, Xiao Han, Chunchun Gao, Yurong Xing, Zheng Qi, Ruijuan Liu, Yueqin Wang, Xiaojian Zhang, Yun-Gui Yang, Xiangnan Li, Baofa Sun, Xin Tian

Non-small-cell lung cancer (NSCLC), the most common type of lung cancer accounting for 85% of the cases, is often diagnosed at advanced stages owing to the lack of efficient early diagnostic tools. 5-Hydroxymethylcytosine (5hmC) signatures in circulating cell-free DNA (cfDNA) that carries the cancer-specific epigenetic patterns may represent the valuable biomarkers for discriminating tumor and healthy individuals, and thus could be potentially useful for NSCLC diagnosis. Here, we employed a sensitive and reliable method to map genome-wide 5hmC in the cfDNA of Chinese NSCLC patients and detected a significant 5hmC gain in both the gene bodies and promoter regions in the blood samples from tumor patients compared with healthy controls. Specifically, we identified six potential biomarkers from 66 patients and 67 healthy controls (mean decrease accuracy >3.2, P < 3.68E−19) using machine-learning-based tumor classifiers with high accuracy. Thus, the unique signature of 5hmC in tumor patient’s cfDNA identified in our study may provide valuable information in facilitating the development of new diagnostic and therapeutic modalities for NSCLC.
肺癌是全球发病率和死亡率最高的恶性肿瘤之一，其中约85%为非小细胞肺癌（NSCLC）。由于缺乏高灵敏度、准确度和可靠性的早期诊断方法，NSCLC患者被确诊时往往已经到了中晚期，其预后较差。因此，探索NSCLC早期诊断和预后评估的分子标志物成为当前肺癌研究的热点领域。近年来研究表明，血浆游离DNA（cell-free DNA，cfDNA）携带丰富的肿瘤细胞基因组突变及表观改变的相关信息，在肿瘤早期诊断、预后评估等方面具有重要的应用价值。5-羟甲基胞嘧啶(5-hydroxymethylcytosine, 5hmC)作为DNA去甲基化过程中的稳定中间产物，在基因表达和肿瘤发生发展调控中具有重要作用。本研究中，我们采用一种高灵敏度、特异性强的测序技术对66例NSCLC患者和67例健康志愿者cfDNA中微量5hmC修饰进行测序，并发现在NSCLC患者基因体区和启动子区域5hmC富集特异性增加。我们进一步鉴定到2459个差异修饰基因，并发现这些基因可有效地区分NSCLC患者和健康对照样本，同时揭示了这些基因主要富集于与癌症发生、发展密切相关的通路上，如cGMP-PKG、Rap及PI3K-Akt信号通路等。为进一步筛选cfDNA 5hmC候选肿瘤生物标志物，我们通过随机森林的机器学习方法鉴定出6个在NSCLC患者中5hmC修饰水平上升的潜在分子标志物。相比现有的临床肿瘤标志物，这6个基因具有更高的灵敏度和准确性。总之，本研究表明cfDNA的5hmC有望成为NSCLC的新型生物标志物，用于NSCLC的早期诊断、预后评估、耐药监测等，具有十分重要的临床应用价值。

Page 187-199

Original Research

Identification and Analysis of Human Sex-biased MicroRNAs

Chunmei Cui, Weili Yang, Jiangcheng Shi, Yong Zhou, Jichun Yang, Qinghua Cui, Yuan Zhou

Sex differences are widely observed under various circumstances ranging from physiological processes to therapeutic responses, and a myriad of sex-biased genes have been identified. In recent years, transcriptomic datasets of microRNAs (miRNAs), an important class of non-coding RNAs, become increasingly accessible. However, comprehensive analysis of sex difference in miRNA expression has not been performed. Here, we identified the differentially-expressed miRNAs between males and females by examining the transcriptomic datasets available in public databases and conducted a systemic analysis of their biological characteristics. Consequently, we identified 73 female-biased miRNAs (FmiRs) and 163 male-biased miRNAs (MmiRs) across four tissues including brain, colorectal mucosa, peripheral blood, and cord blood. Our results suggest that compared to FmiRs, MmiRs tend to be clustered in the human genome and exhibit higher evolutionary rate, higher expression tissue specificity, and lower disease spectrum width. In addition, functional enrichment analysis of miRNAs show that FmiR genes are significantly associated with metabolism process and cell cycle process, whereas MmiR genes tend to be enriched for functions like histone modification and circadian rhythm. In all, the identification and analysis of sex-biased miRNAs together could provide new insights into the biological differences between females and males and facilitate the exploration of sex-biased disease susceptibility and therapy.
男性与女性不仅在身体结构上，还在生理功能、治疗反应等方面都有所差异。为研究性别差异相关的分子机制，大量的性别差异表达基因已被鉴定出来。而microRNA作为近年来非编码RNA的一个研究热点，已有大量的转录组数据集将其囊括，但目前还没有研究对 microRNA性别差异表达情况进行全面分析。本研究基于公共数据库中符合条件的microRNA转录组数据集，从脑、结肠直肠粘膜、外周血和脐带血四种组织中鉴定出73个女性高表达的microRNA和163个男性高表达microRNA，并对其生物学特性进行了系统分析。结果表明，与女性高表达的microRNA相比，男性高表达microRNA在基因组上更倾向于成簇分布，具有更高的进化速率、表达组织特异性和更低的疾病谱宽度。此外，microRNA的功能富集分析结果显示女性高表达的microRNA显著富集代谢和细胞周期相关功能，而男性高表达microRNA倾向于富集组蛋白修饰和昼夜节律等功能。鉴定和分析具有性别差异的microRNA可以为理解男女间生物学差异提供新的视角，并将有助于性别差异治疗方案的研究。

Page 200-211

Letter

A Systems Biology Approach for Studying Heterotopic Ossification: Proteomic Analysis of Clinical Serum and Tissue Samples

Erin L. Crowgey, Jennifer T. Wyffels, Patrick M. Osborn, Thomas T. Wood, Laura E. Edsberg

Heterotopic ossification (HO) refers to the abnormal formation of bone in soft tissue. Although some of the underlying processes of HO have been described, there are currently no clinical tests using validated biomarkers for predicting HO formation. As such, the diagnosis is made radiographically after HO has formed. To identify potential and novel biomarkers for HO, we used isobaric tags for relative and absolute quantitation (iTRAQ) and high-throughput antibody arrays to produce a semi-quantitative proteomics survey of serum and tissue from subjects with (HO+) and without (HO−) heterotopic ossification. The resulting data were then analyzed using a systems biology approach. We found that serum samples from subjects experiencing traumatic injuries with resulting HO have a different proteomic expression profile compared to those from the matched controls. Subsequent quantitative ELISA identified five blood serum proteins that were differentially regulated between the HO+ and HO− groups. Compared to HO− samples, the amount of insulin-like growth factor I (IGF1) was up-regulated in HO+ samples, whereas a lower amount of osteopontin (OPN), myeloperoxidase (MPO), runt-related transcription factor 2 (RUNX2), and growth differentiation factor 2 or bone morphogenetic protein 9 (BMP-9) was found in HO+ samples (Welch two sample t-test; P < 0.05). These proteins, in combination with potential serum biomarkers previously reported, are key candidates for a serum diagnostic panel that may enable early detection of HO prior to radiographic and clinical manifestations.

Page 212-220

Corrigendum

Corrigendum to “GoldCLIP: Gel-omitted Ligation-dependent CLIP” [Genomics Proteomics Bioinformatics 16 (2) (2018) 136–143]

Jiaqi Gu, Ming Wang, Yang Yang, Ding Qiu, Yiqun Zhang, Jinbiao Ma, Yu Zhou, Gregory J. Hannon, Yang Yu

Page 221-222