基于转录组的华法林作用后肝细胞miRNA表达谱的分析

Analysis of miRNA expression profile in hepatocytes treated with warfarin based on transcriptome

目的探究miRNA与华法林药物反应性的关系。方法用Illumina测序平台对华法林干预不同时间后的LO2肝细胞系进行全转录组测序,通过数据清理、质控、比对,与新miRNA预测,筛选组间差异基因,预测miRNA靶基因,从而发现新的华法林变异基因及其信号通路。结果运用生物信息学分析方法预测到359个新miRNA,3组组间差异miRNA共220个,其中168个差异miRNA为上调趋势,52个差异miRNA为下调趋势。差异miRNA通过韦恩图取交集,在3组对比分析中均存在差异的miRNA共有7个,分别是19_42692、5_15895、4_13863、2_6317、2_3682、X_46770和hsa-miR-3656。用实时荧光定量聚合酶链反应验证的表达趋势与测序结果均为上调表达,说明测序结果准确性较高,可以用于后续的功能分析。结论本研究基于全转录组测序方法经过鉴定及筛选出的miRNAs可能在华法林耐药机制中发挥显著作用,这些可能为华法林剂量的研究提供了新的见解和策略。

Objective To screen the differentially expressed miRNAs of warfarin drug intervention in liver cells to investigate their possible role in the mechanism of warfarin resistance.Methods Whole transcriptome sequencing was performed on LO2 liver cell lines after warfarin intervention for different periods using Illumina sequencing platform.Through data cleaning,quality control and comparison,prediction of new miRNA,screening and identification of differently expressed genes between groups,and then prediction of miRNA regulatory target genes,the new warfarin variant genes and their signaling pathways were explored.Results Bioinformatics analysis was applied to the sequencing results,and 359 new miRNAs were predicted,including a total of 220differentiated miRNAs among the three groups,among which 168differentiated miRNAs were up-regulated and 52 differentiated miRNAs were down-regulated.The intersection of differential miRNAs was taken by Venn diagram,and there were 7 differential miRNAs in the comparative analysis of the three groups,namely 19_42692,5_15895,4_13863,2_6317,2_3682,X_46770 and hsa-miR-3656.The expression trend was verified by quantitative real-time polymerase chain reaction and the sequencing results were up-regulated,indicating that the sequencing results were highly accurate and could be used for subsequent functional analysis.Conclusion In this study,key miRNAs identified and screened by whole-transcriptome sequencing may play a significant role in the mechanism of warfarin resistance,which may provide new insights and strategies for warfarin dose research.