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基于二代测序的不同分化阶段晶状体转录组分析
张丽芳
秦祯蔚
卢冰
吕丹旎
李嘉永
闫晨曦
宋帆
唐俏梅
尹厚发
傅秋黎
作者及单位信息
·
DOI： 10.3760/cma.j.cn112142-20200222-00095
Transcriptome profiling of differentiated lenses through RNA sequencing
Zhang Lifang
Qin Zhenwei
Lu Bing
Lyu Danni
Li Jiayong
Yan Chenxi
Song Fan
Tang Qiaomei
Yin Houfa
Fu Qiuli
Authors Info & Affiliations
Zhang Lifang
Eye Center, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China
Qin Zhenwei
Eye Center, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China
Lu Bing
Eye Center, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China
Lyu Danni
Eye Center, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China
Li Jiayong
Eye Center, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China
Yan Chenxi
Eye Center, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China
Song Fan
Eye Center, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China
Tang Qiaomei
Eye Center, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China
Yin Houfa
Eye Center, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China
Fu Qiuli
Eye Center, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China
·
DOI： 10.3760/cma.j.cn112142-20200222-00095
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摘要

目的了解分化中晶状体的转录谱，包括mRNA、长链非编码RNA（lncRNA）和环状RNA（circRNA）。

方法实验研究。提取人源性分化晶状体RNA并纯化。然后使用Illumina HiSeq 2500对16、23、25周晶状体总RNA样本进行测序，并使用生物信息学工具进行分析，筛选出最高表达和差异表达的mRNA和lncRNA；用维恩图分析3个时期晶状体的重叠基因表达情况；分析晶状体特异性基因的表达趋势，并用实时荧光定量PCR验证。

结果在16、23、25周晶状体中分别获得67 518 311、99 440 160和67 262 320条映射序列。基因重叠表达分析显示，前1 000个高表达mRNA、前300个高表达lncRNA和前100个高表达circRNA中分别有740、170、69个在16、23、25周晶状体中同时表达。晶状体特异性基因表达分析显示，晶状体蛋白（CRY）AA、CRYGA、CRYGB、CRYGC、CRYGD、CRYGEP和CRYGS基因表达随晶状体分化时间上调，缝隙连接（GJ）蛋白GJA3和GJA8基因表达随晶状体分化时间下调。

结论晶状体转录组表达谱显示不同分化阶段晶状体的高表达mRNA、lncRNA和circRNA中重叠表达者占半数以上，且均高表达晶状体特异性蛋白基因，如CRY、GJ蛋白基因等。（中华眼科杂志， 2020， 56： 356- 363）

晶体;高通量核苷酸序列分析;RNA，信使;RNA，长链非编码;RNA，环状;基因表达谱
ABSTRACT

ObjectiveTo gain insight into the transcriptional landscape including mRNA, long non-coding RNA (lncRNA), and circular RNA (circRNA) of the differentiated lens.

MethodsExperiment research. The total RNAs of the differentiated lenses were extracted and purified. Total RNAs of 16-week, 23-week, and 25-week differentiated lenses were then sequenced using Illumina HiSeq 2500, and analyzed using bioinformatics tools. The top expressed and differentially expressed mRNAs and lncRNAs were screened. The expressions of overlap genes among the 16-week, 23-week, and 25-week lenses were analyzed by Venn diagram. The expression tendency of lens-specific genes was obtained and verified with real-time polymerase chain reaction.

ResultsA total of 67 518 311 mapped reads were obtained from differentiated lenses at 16 weeks, 99 440 160 at 23 weeks, and 67 262 320 at 25 weeks. The gene overlap expression analysis showed 740 of the top 1 000 highly expressed mRNAs, 170 of the top 300 highly expressed lncRNAs, and 69 of the top 100 highly expressed circRNAs overlapping expressed in lenses at 16, 23, and 25 weeks, respectively. Lens specific gene expression analysis revealed that the expression of crystallin (CRY) AA, CRYGA, CRYGB, CRYGC, CRYGD, CRYGEP, and CRYGS was upregulated, while the expression of gap junction (GJ) A3 and GJA8 was downregulated with the differentiation of lenses.

ConclusionThe lens transcriptome profile shows that more than half of the high expressed mRNA, lncRNA and circRNA at different differentiation stages are overlapping expressed, and all of them have high expression of lens specific protein genes, such as CRY, GJ etc. (Chin J Ophthalmol, 2020, 56: 356- 363)

Lens, crystalline;High-throughput nucleotide sequencing;RNA, messenger;RNA, long noncoding;RNA, circular;Gene expression profiling
Fu Qiuli, Email: nc.defudabe.ujz9003132
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引用本文

张丽芳,秦祯蔚,卢冰,等. 基于二代测序的不同分化阶段晶状体转录组分析[J]. 中华眼科杂志,2020,56(05)：356-363.

DOI:10.3760/cma.j.cn112142-20200222-00095

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晶状体是一种无血管的透明组织，其起源于表皮外胚层，逐步形成晶状体板、晶状体泡，后者由单层细胞组成，其前壁细胞分化为晶状体上皮细胞，而后不断增长形成次级晶状体纤维；后壁细胞形成初级晶状体纤维，并逐渐退化形成晶状体核 [ 1 ]。这个过程伴随着细胞伸长、晶状体蛋白合成和细胞器程序化降解 [ 1 , 2 , 3 ]。晶状体发育障碍可导致多种晶状体疾病，如球形晶状体、先天性无晶状体眼 [ 4 ]、先天性晶状体脱位 [ 5 ]、先天性白内障等 [ 6 , 7 ]。先天性白内障是全球儿童失明的首要原因，由胚胎期的基因突变、感染和代谢紊乱等因素引起 [ 8 ]。因此，了解晶状体发育的分子机制对于晶状体疾病的早期干预和治疗具有重要意义。
晶状体的发育是一个高度协调的过程，涉及细胞增殖和分化过程中基因表达的精确调控。目前对晶状体发育过程的基因表达调控仍缺乏全面了解。尽管在小鼠、鸡、青蛙等动物中已经进行了晶状体发育的分子机制研究 [ 2 , 9 ]，但关于晶状体发育机制的人源性数据仍然缺乏。目前，已经使用二代测序对晶状体发育进行了高通量的转录谱分析 [ 10 , 11 , 12 ]。Khan等 [ 10 ]研究者通过对小鼠晶状体转录组在不同分化阶段的测序分析，增加了对转录组在晶状体透明性维持和疾病表现中作用的理解。尽管动物晶状体可以模仿人晶状体，但小鼠和人类转录组并不完全相同。Wistow等 [ 13 ]在2002年研究了2名成人（40岁）晶状体的表达谱，提供了宝贵的人晶状体转录组信息。与人分化中晶状体相比，成年人晶状体相对稳定，其转录组与分化中晶状体转录组不尽相同。因此，仍然需要对不同分化阶段的晶状体组织转录组进行分析。
长链非编码RNA（long non-coding RNA，lncRNA）是一类长度大于200个核苷酸，通过顺式或反式作用在多种生物通路中发挥重要调控作用的非编码RNA [ 14 ]。一些研究发现lncRNA[如心肌梗死相关转录物（myocardial infarction associated transcript，MIAT）和钾电压门控通道Q亚家族成员1反义转录本1（potassium voltage-gated channel subfamily Q member 1 opposite strand/antisense transcript 1，KCNQ1OT1）]在透明和白内障晶状体中表达不同，提示其在晶状体病理过程中的作用 [ 15 , 16 , 17 ]。虽然lncRNA在生理病理调节过程中发挥重要作用，但仍缺乏其在晶状体分化中的表达谱和功能研究。迄今为止，仅有2项研究报道了分化中鼠晶状体的lncRNA表达谱 [ 11 , 14 ]。环状RNA（circular RNA，circRNA）是一类新型的具有连接3′和5′末端的共价闭环结构的非编码RNA [ 18 ]。越来越多的证据表明，circRNA的异常表达与眼病的发病密切相关 [ 19 , 20 ]。据报道，circHIPK3[由同源域相互作用蛋白激酶3（homeodomain interacting protein kinase 3，HIPK3）基因的2号外显子形成]在年龄相关性白内障中显著下调 [ 19 ]，而circKMT2E[由赖氨酸甲基转移酶2E（lysine methyltransferase 2E，KMT2E）基因的4~15号外显子形成]在糖尿病性白内障中上调 [ 20 ]。然而，对circRNA在晶状体中的作用认识仍不全面。本研究将16、23、25周分化晶状体组织，进行RNA测序和生物信息学分析，以探索每个分化阶段mRNA、lncRNA和circRNA的转录组表达谱，为先天性晶状体疾病的早期干预和治疗提供信息。
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备注信息
A
傅秋黎，Email： nc.defudabe.ujz9003132
B
所有作者均声明不存在利益冲突
C
国家自然科学基金 (81670833、81870641、81800806)
浙江省重点研发项目 (2019C03091)
中央高校基本科研业务费与专项资金资助基础研究基金 (2019QNA7026)
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