tRNAHis来源小RNA的发现及其功能初步预测

Discovery and function prediction of tRNA-derived small RNA

目的证明肺癌细胞miRNA芯片中不存在高表达的miR-4454,而是来源于tRNAHis的同源序列16 nt小RNA,并对该16 nt小RNA生物学功能进行初步预测分析。方法利用生物素标记的anti-miR-4454成熟体探针Northern印迹(NB)检测肺癌H460细胞内miR-4454成熟体及其前体,利用该探针钓取与之特异结合的RNA,测序确定该RNA的性质。Dicer酶体外切割tRNAHis,检测其切割产物并对切割产生的小RNA进行测序鉴定。在H460细胞中过表达tRNAHis来源的16 nt小RNA,通过转录组测序预测其生物学功能。结果肺癌H460细胞中预测的miR-4454成熟体不存在,其前体序列与tRNAHis高度一致。Dicer酶体外切割tRNAHis能产生一条16 nt小RNA,序列鉴定与tRNAHis3′端完全匹配,是一条新的tRNAHis来源小RNA。H460细胞中过表达tRNAHis来源的16 nt小RNA,转录组测序结果分析显示,tRNAHis来源的16 nt小RNA可能参与有丝分裂、细胞周期调控及碱基错配修复。结论首次发现数据库预测的miR-4454在非小细胞肺癌细胞中不存在,证明它是来源于tRNAHis3′端由Dicer酶切割产生的一种16 nt小RNA。过表达16 nt小RNA的转录组测序结果提示其可能参与细胞有丝分裂、周期调控及碱基错配修复。

Objective To prove that miR-4454,which is highly expressed in the miRNA chip of lung cancer cells,does not exist and is a 16 nt small RNA derived from tRNAHis and to predict the biological function of the16 nt small RNA.Methods Mature miR-4454 and its precursor in H460 cells were identified with the biotin-labeled miR-4454 probe and confirmed by Northern blot(NB).The binding RNA was sequenced with the biotin-labeled miR-4454 probe.Dicer was used to cut tRNAHis in vitro,and the product was confirmed by sequencing.The tRNAHis derived 16 nt small RNA in H460 cells was overexpressed and its biological function was predicted via transcriptome sequencing analysis.Results It was found that mature miR-4454 did not exist in H460 cells,and the precursor sequence was proved to be highly consistent with tRNAHis.In vitro cleavage experiments confirmed that tRNAHis could produce a 16 nt small RNA after Dicer enzyme cleavage.Bioinformatic analysis of transcriptome sequencing of H460 cells that overexpressed 16 nt small RNA showed that the biological function of 16 nt small RNA might have been involved in mitosis,cell cycle regulation and base mismatch repair.Conclusion This is the first time that miR-4454 predicted by the database has been found to be non-existent in non-small cell lung cancer cells.It is a 16 nt small RNA derived from the 3′end of tRNAHis that may be involved in cell mitosis,cycle regulation and base mismatch repair.