[Expression Analysis of miRNA Profiles in Apheresis Platelets during Storage]

To study the expression profiles changes of miRNA in apheresis platelets after 1, 3 and 5 days of storage.The apheresis platelets were collected from 20 volunteer blood donors. After mixing fully, the platelets were stored in a shaker with (22±2) ℃ horizontal oscillation. The samples were taken on the 1st, 3rd and 5th day, and used to sequence for miRNAs by DNA nanoball (DNB) sequencing technology, which were named as C_1, C_3 and C_5, respectively. The expression level of platelets miRNA was standardized by transcripts per kilobase million (TPM) algorithm. MiRNAs with P-value0.001 and the expression difference of more than two times were considered as significant difference between two groups. The expression of miRNAs was verified by real-time fluorescence quantitative PCR (RT-qPCR).By DNB sequencing, there were 688, 730, and 679 platelet miRNAs expressed in C_1, C_3 and C_5 group, respectively. Cluster analysis showed that the expression profile of miRNAs changed significantly. The expression level of the first 20 high abundance miRNAs was about 4/5 of the total amounts of expressed miRNAs in each group, which the top five miRNAs were miR-21-5p, miR-26a-5p, miR-199a-3p, miR-126-3p, and let-7f-5p. The correlation of high abundance platelet miRNAs among the three groups was high (R2=0.876, R2=0.979, R2=0.937, respectively) and the differences were not statistically significant (P0.05). Compared with the differential expression of platelet miRNAs with more than 1 000 TPM in the C_3 and C_1 group, there were 6 differentially expressed miRNAs, including 3 up-regulated (miR-146a-5p, miR-379-5p, and miR-486-5p) and 3 down-regulated (miR-652-3p, miR-142-5p, and miR-7-5p). While in the C_5 and C_1 group, there were 4 differentially expressed miRNAs, including 2 up-regulated (miR-146a-5p and let-7b-5p) and 2 down-regulated (miR-30d-5p and miR-142-5p). Compared with the differentially expression of platelet miRNAs between 1-1 000 TPM in the C_3 and C_1 group, there were 133 differentially expressed miRNAs, in which 99 were up-regulated and 34 were down-regulated. While in the C_5 and C_1 group, there were 77 differentially expressed miRNAs, in which 31 were up-regulated and 46 were down-regulated. The six selected differentially expressed miRNAs verified by RT-qPCR were consistent with those of sequencing.The expression profiles of platelets miRNAs change significantly among 1, 3, and 5 d of storage in vitro.血小板miRNA的表达谱分析.研究在体外常规储存1、3和5 d血小板微小RNA(miRNA)的表达谱，探讨miRNA在血小板储存期的变化.留取来自无偿献血捐献者单采血小板20份，摇匀后等量混合，贮存于（22±2）℃恒温血小板振荡保存箱中。分别在d 1、3和5取样（分别命名为C_1、C_3和C_5），应用DNA纳米球（DNB）测序技术对血小板miRNA组进行测序，采用每千百万碱基的转录本（TPM）算法标准化miRNA的表达水平，组间miRNA表达差异2倍（P0.001）认为表达差异具有统计学意义。采用实时荧光定量PCR方法验证miRNA的表达.通过DNB测序，分别获得C_1、C_3和C_5组688、730和679个表达的血小板miRNA，聚类分析表明其表达谱发生明显变化。在C_1、C_3和C_5组中前20个高表达miRNA的表达量约占各组miRNA总表达量的4/5，各组前5位的miRNA均为miR-21-5p、miR-26a-5p、miR-199a-3p、miR-126-3p和let-7f-5p。C_1、C_3和C_5组血小板高表达的55个miRNA（1 000 TPM）组间表达模式高度相似，其差异无统计学意义（P0.05）。通过对高表达miRNA（1 000 TPM）在各组中的差异表达分析发现，与C_1组相比，C_3组共有6个miRNA显著差异表达，其中表达上调的是miR-146a-5p、miR-379-5p和miR-486-5p，表达下调的是miR-652-3p、miR-142-5p和miR-7-5p；C_5组共有4个miRNA显著差异表达，表达上调的是miR-146a-5p和let-7b-5p，表达下调的是miR-30d-5p和miR-142-5p。对表达量为1-1000 TPM之间miRNA差异表达分析发现，与C_1组相比，C_3组有133个显著差异表达的miRNA，其中表达上调是99个，表达下调是34个；C_5组中有77个显著差异表达的miRNA，其中表达上调是31个，表达下调是46个。检测的6个差异表达miRNA的荧光定量PCR结果与测序结果基本一致.通过对储存1、3和5 d血小板miRNA组进行测序，发现随着血小板体外储存时间的延长，miRNA表达谱发生明显改变.