基于二代测序的不同分化阶段晶状体转录组分析

Transcriptome profiling of differentiated lenses through RNA sequencing

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67518311、99440160和67262320条映射序列。基因重叠表达分析显示,前1000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。

Objective To gain insight into the transcriptional landscape including mRNA, long non-coding RNA (lncRNA), and circular RNA (circRNA) of the differentiated lens.Methods Experiment 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.Results A 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.Conclusion The 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.