储存血小板差异性长链非编码RNA表达谱研究

Expression profile of the long non-coding RNA in human platelets during apheresis storage

目的探讨机采血小板储存过程中差异性lncRNA表达谱变化及其差异表达的lncRNA在血小板储存损伤中潜在的生物学功能。方法收集13（人）份O型健康男性机采血小板各50 m L,于（22±2）℃下震荡储存,应用lncRNA芯片技术检测储存2、5、8 d机采血小板中lncRNA和mRNA表达谱变化;运用GO分析及KEGG分析预测差异表达的lncRNA功能分布,通过顺式调控分析预测lncRNA调控邻近蛋白编码基因表达的分子机制;采用实时PCR（RT-PCR）技术,验证8条lncRNA（MARCH2-2∶1、SLC2A2-1∶1、WBSCR16-3∶6、WEE1-2∶1、ENTPD6-2∶1、NKD2-1∶1、FOXS1-2∶1、MAPK13-3∶1）和4条mRNA（BCL2L1、CAPN2、MAPK14、VAMP8）的表达。结果芯片检测：血小板储存5与2 d相比,有162种lncRNA的表达量发生明显变化,表达升高的4种、降低的158种;8与2 d相比,有691种lncRNA表达量发生明显变化,表达升高的20种、降低的671种;8与5 d相比,246种lncRNA表达量发生明显变化,升高的2种,降低的244种。GO和KEGG分析：差异表达的lncRNA可能参与的生理过程有血小板激活、血小板聚集、内吞、凋亡、肌动蛋白纤维聚集、肌动蛋白骨架的调节;顺式调控分析：lncRNA具有调控血小板相关mRNA表达的功能,USP39-1调控VAMP8的表达、TMEM86B-2调控GP6的表达、FOXS1-2调控BCL2L1的表达等。RT-PCR验证：与芯片检测结果一致,其中lncRNA MARCH2-2∶1、WEE1-2∶1和NKD2-1∶1表达量升高,WBSCR16-3∶6、SLC2A2-1∶1、ENTPD6-2∶1、FOXS1-2∶1和MAPK13-3∶1表达量下降,BCL2L1,CAPN2,MAPK14和VAMP8的表达量也下降。结论血小板中含有大量的lncRNA,其表达量随着血小板储存时间延长而呈不同的变化趋势,总体来讲下调的lncRNA数量较多,且下调的幅度较大。部分变化的lncRNA可能与血小板生理功能密切相关,并且在血小板储存损伤中发挥作用。

Objective To investigate the long noncoding RNA（lncRNA） expression profile andtheir potential biological function in human platelet during the apheresis storage period.Methods Thirteen platelet samples（50 ml/each） were collected from healthy men with O blood group. The LncRNA profiles were characterizedby Human lncRNA Array in human platelets after being stored for 2,5,and 8 days. Bioinformatics analysis was performed to construct the lncRNA and mRNA co-expression profile,where GO and KEGG analysis predicted the lncRNA＇s function and cis-regulation analysis revealed the potential molecular mechanism of lncRNA on the target gene expression.EightlncRNAs（MARCH2-2 ∶ 1,SLC2 A2-1 ∶ 1,WBSCR16-3 ∶ 6,WEE1-2 ∶ 1,ENTPD6-2 ∶ 1,NKD2-1 ∶ 1,FOXS1-2 ∶ 1,MAPK13-3 ∶1） and four mRNAs（BCL2 L1,CAPN2,MAPK14,VAMP8） were further confirmed by RT-PCR.Results 162 and 691 lncRNAsshowedexpression levelshifts on d5 and d8 compared to d2,respectively. As for the d5 IncRNA expression profiles： 4 went upand 158 dropped down while at d8,20 went up and 671 dropped down. Comparing the profiles at d8 and d5,246 lncRNAs are differentially expressed： 2 increased and 244 decreased. The GO and KEGG analysis showed that lncRNAs co-expressed mRNAs may beinvolved in platelet activation,platelet aggregation,endocytosis,apoptosis,actinfilament polymerization and the regulation of actin cytoskeleton. Cis-analysis showed that lncRNAspossessedthe potential to cis-regulate the expression of protein coding gene associated with platelet physiological function： USP39-1 regulating VAMP8,TMEM86 B-2 regulating GP6,FOXS1-2 regulating BCL2 L1 and so on.The result of RT-PCR was consistent with the microarray： MARCH2-2 ∶1,WEE1-2 ∶1 and NKD2-1 ∶ 1 expression level increased; WBSCR16-3 ∶ 6,SLC2 A2-1 ∶ 1,ENTPD6-2 ∶ 1,FOXS1-2 ∶1 and MAPK13-3 ∶1 reduced; 4 possible targeted mRNAs BCL2 L1,CAPN2,MAPK14,VAMP8 were down-regulated during platelet storage.Conclusion A large number of lncRNAs weredetectedin apheresis platelets. The expression level of lncRNAs changedasthe platelet storage time extended.Overall,the number of down-regulated lncRNAswas much greater than that of the up-regulated ones.Some of theselncRNAs might be involved in the regulation of platelet physiological function and play a role in the occurrence of platelet storage lesion.