Study On MiRNA Expression Profile Of Platelet With Trehalose Cryopreservation

The structure and function of platelets change over time during storage,which i s called platelet storage lesion.Now the platelet concentrate is stored at 22?C with gentle shake for 5 days,as a routine platelet preservation condition.In order to redu ce the platelets storage damage,people tried to use low temperature preservation me thods,because cryopreservation can extend the vitality of platelets in storage.But lo w temperature also can promote platelet activation,which makes its failure after inf usion into recipients and clearance rapidly from the body.Study shows that trehalose can inhibit platelet storage in the process of damage and reduce the occurrence of apoptosis,meanwhile its specific mechanism remains to be clarified.MicroRNA(miR NA)is a kind of noncoding single-stranded RNA molecules encoded by the endogen ous gene,by the length of 21 ~ 22 nucleotides and pertain to Small non-coding R NA molecules(Small non-coding RNAs,snc RNAs).Main function of micro RNA i s targeted inhibition to specific messenger RNA(m RNA)as well as participating in the process of intracellular regulation,various biological processes involved in varie ty of body biological processes.Platelet contains an abundant of miRNA,which play s important roles in the process of platelet storage lesion.Studies on platelet miRN As relative to storage damage were based on PCR or chip way.In this study we us e the second generation of high-throughput sequencing technologies,for the first tim e to study the expression changes of platelet miRNA with trehalose in cryopreservati on,and to try to further explore the possible molecular mechanism of trehalose prot ecting platelet at low temperature.Objective To explore the changes of miRNAs in platelet with trehalose cryopres ervation.Methods Three bags of apheresis platelets from three healthy volunteer blood donors of same blood type were mixed well,and subsequently distributed into the control group(22?C,with 100% plasma for 2 hours)and the experimental group(10?C,with 70% trehalose preservation solution(TPS)for 5 days)for the first experiment.The second control group: adding 70% without adding trehalose platelets fluid add 10 ? shock save 5 days;The experimental group: adding 70% trehalose save liquid 10 ? shock save five days.The second control group: adding 70% without adding trehalose platelets fluid add 10 ? shock save 5 days;The experimental group: adding 70% trehalose save liquid 10 ? shock save five days.Next generation small RNA sequencing was applied to the two groups,and then bioinformatics exploration was underway,including known miRNAs identification,differential expression analysis and KEGG pathway analysis.Results Ten million clean reads data for each sample were obtained by small RNA high-throughput sequencing.In the known miRNAs,a total of 460 miRNAs was differentially expressed.Statistical differences in expression of known miRNAs,a total of 44,among which 16 miRNAs were up-regulated and 28 miRNAs down-regulated,was respectively enriched in the relative signaling pathways of platelets storage lesion such as Calcium signaling pathway,MAPK pathway,apoptosis pathway and axon guidance.Comparing to the control group,the expression of has-miR-4449 and has-miR-1296 in experiment group were up-regulated significantly(p?0.05 and |log2Ratio|?1),meanwhile the expression of has-miR-665 was down-regulated.With trehalose platelets preservation process,multiple storage damage related signaling pathway changed,including MAPK signaling pathway,phototransduction,p53 signaling pathway,VEGF signaling pathways,apoptosis pathway and so on.A total of 92 miRNAs were differentially expressed significantly,among which 55 miRNAs were up-regulated and 37 miRNAs down-regulated.Including a variety of relevant miRNA expression appears to change,such as hsa-miR-2277-5 p increasing,hsa-miR-4707-3p,hsa-miR-136-3p and hsa-miR-431-3p rising significantly,as well as reduction of hsa-miR-188-5p,hsa-miR-28-5p expression significantly declined.Conclusion It was no doubt that there was a distinctive profile of differences expression in miRNAs of platelets after low temperature storage in TPS,which prompted trehalose regulated platelets storage lesion at low temperature by hsa-miR-2277-5p,hsa-miR-4707-3p,hsa-miR-188-5p,hsa-miR-28-5p regulation of protein kinase B(Akt)expression increases,the Bcl-2,IAP,MKP increasing significantly,Bax decreasing,FasL and protein kinase(PKC)declining significantly.The protective effects of p38 MAPK is closely associated with trehalose protect platelets in low temperature.