储存红细胞ATP代谢circRNA筛选及功能预测  

The screening and function prediction of ATP-metabolism-associated circRNA in stored red blood cells

作　　者：张怡宇[1] 苑召虎[1] 黄慧瑛 李秋娴 李楠[1] 黄建云[1] 魏亚明[1] ZHANG Yiyu;YUAN Zhaohu;HUANG Huiying;Li Qiuxian;Li Nan;WEI Yaming(Department of Blood Transfusion, Guangzhou First People＇s Hospital, Guangzhou Medical University, Guangzhou 510180, China)

机构地区：[1]广州医科大学附属市一人民医院输血科,广东广州510180 [2]广州市番禺区中心医院

出　　处：《中国输血杂志》2018年第4期332-338,共7页Chinese Journal of Blood Transfusion

基　　金：广州市民生科技重大专项(201300000100)

摘　　要：目的筛选4℃储存前、后期红细胞差异表达的circRNA并初步预测其功能。方法采集健康无偿献血者5（人）份血样200 m L/份,制备成悬浮红细胞,滤除白细胞后（悬浮滤白红细胞）分为3组：4℃储存0、20及40 d组,对3组标本用第2代高通量测序技术检测,根据FDR〈0.05且｜log2FC｜〉1这2个条件,以火山图和表达模式聚类分析筛选出差异基因并用cytoscape作出mRNA—circRNA—miRNA网络图;通过KEGG和GO分析筛选出与ATP代谢相关的circRNA验证;最后通过生信软件预测hsacirc0007470—hsa-miR-155-5p—MRP4的分子结合具体区域及生物学功能。结果红细胞储存20 d较0 d时差异表达的circRNA有119个,40 d较20 d时差异表达的circRNA有110个,2个储存阶段都有差异表达的circRNA共有41个。KEGG和GO分析显示在差异表达的circRNA中hsacirc0007470的亲本基因MRP4富集于c AMP代谢通路。mireap、miranda、targetscan、circBase、miRBase、mirTar Base软件分析及序列比对显示hsacirc00007470可与hsa-miR-155-5p结合;hsa-miR-155-5p可与MRP4结合。经PCR验证,红细胞储存在0、20、40 d时,hsacirc00007470、MRP4、hsa-miR-155-5p的变化倍数分别为1.00±0.02、1.36±0.09、0.53±0.16,1.00±0.01、1.87±0.48、0.45±0.20,1.00±0.02、0.43±0.20、0.98±0.29。红细胞储存0、20、40 d时,总ATPase（U/gHb）分别是40.87±5.883、18.300±2.847、10.21±1.636;ATP（U/gHb）分别是8.708±1.25、3.082±0.166、2.462±0.252。结论储存红细胞中存在circRNA,且含量会随储存时间发生改变,储存20 d后红细胞的hsacirc00007470与ATPase及ATP表达呈同向相关性,hsacirc00007470或许能解除hsa-miR-155-5p对MRP4的抑制作用,通过ATP代谢途径改善红细胞变形能力。Objective To screen out red blood cell（RBC） circRNAs with different expression status during early and late stage storage at 4℃,and to prediction their function associated with ATP metabolism was also made based on the results.Methods Blood samples of 200 ml blood were collected from 5 healthy blood donors and prepared as suspended RBCs. Leukocytes were removed by filtration. The samples were then divided into 3 groups： The 0 d group,the 20 d group and the 40 d group. Second-generation high-throughput sequencing technology was applied to screen genes by volcano plots and expression pattern cluster analysis. Cytoscape was used to construct the mRNA-circRNA-miRNA network Figure; circRNA validation associated with ATP metabolism was screened by KEGG and GO analysis; Finally,molecular binding specific regions and biological functions of hsacirc0007470--hsa-miR-155-5 p--MRP4 were predicted by Bioinformatics. Results There were 119 and 110 circRNAs differently expressed on Day 20 vs Day 0 and on Day 40 vs Day 20,respectively. Forty one circRNAs changed in both the stages. KEGG and GO analysis showed that one of the differently expressed circRNA named MRP4,the parental gene of hsacirc0007470,was enriched in the c AMP metabolic pathway. mireap,miranda,targetscan,circ Base,miRbase,mir Tar Base software analysis,and sequence alignment showed that hsacirc00007470 can bind hsa-miR-155-5 p and hsa-miR-155-5 p can bind MRP4. PCR validation showed that,at 0,20,and 40 days,the fold change of hsa circ 00007470,MRP4,and hsa-miR-155-5 p was： 1. 00±0. 02,1. 36± 0. 09,0. 53 ± 0. 16;1. 00 ± 0. 01,1. 87 ± 0. 48,0. 45 ± 0. 20;1. 00±0. 02,0. 43 ± 0. 20,0. 98 ± 0. 29,respectively. The total ATPase and ATP concentration was 40. 87 ± 5. 883,18. 300 ±2. 847 and 10. 21±1. 636（U/gHb）,respectively; ATP was 8. 708±1. 25,3. 082±0. 166,2. 462±0. 252（U/gHb）,respectively. Conclusion This study has revealed that circRNA level changes in RBC during storage. The expression of hsacirc00007470 has been co-directed to ATP

关 键 词：储存红细胞ATP酶 环状RNA 小分子RNA CAMP MRP4 生物信息学 

分 类 号：Q522[生物学—生物化学] Q524.3