Sequencing Analysis Of The CircRNAs Expression Profile In Human Banked Erythrocyte Suspension With Leucocyte Removal During Storage

BackgroundThe physiological function of red blood cells is to transport oxygen and carbon dioxide through hemoglobin.Clinically,infusion of red blood cell suspension can improve hemodynamics,increase oxygen carrying capacity,and maintain the body’s oxidation reaction.In the process of vitro storage of red blood cells,the metabolism,function,and morphology of the cells are changed one after the other.This is called storage damage.In English,it is specifically described as“storage lesion”,such as:ATP,3-DPG concentration,NO and its derivatives,etc.Continue to decrease.In vitro,stored red blood cells were usually using CPDA blood preservation solution placed in a constant temperature refrigerator at 4°C.The shelf life was 35 days.According to international practice,red blood cell input into the patient’s body,at least 70% of the red blood cells have function can be defined as effective blood transfusion.The reason of it is that deformation and senescence have occurred in vitro and are rapidly lysed and hemolyzed in the spleen,bone marrow or liver.Finding out factors related to red blood cell energy metabolism,morphological changes,etc.,to inhibit the rate of erythrocyte senescence is the only way to solve the problem of blood source tension and improve the efficacy of transfusion of red blood cells.RNA can be divided into messenger RNA(mRNA),small molecule RNA(miRNA)and circular RNA(circRNA).Preliminary studies have shown that mature red blood cells also have apoptotic signaling pathway or programmed cell death(PCD).This pathway involves miRNA regulated mRNA.In mammals,miRNAs usually bind to the target mRNA through 6-7 bases,inhibiting the translation(ie,post-transcriptional regulation)of the target gene protein to control the cell biology process,which makes it more conducive to better understanding of red blood cell storage lesion.The events that occurred because most of the red blood cells are cell-free nucleus,so lost the transcriptional regulation mechanism of nucleated cells,and they need survive for relying on the presence of post-transcriptional regulatory mechanisms in the cytoplasm.circRNA(circular RNA,circRNA)is a type of closed circular molecule formed by back splicing of mature linear RNA.More than 10% of human genes can be transcribed to produce circular RNA,which is equivalent to the linear RNA produced by its transcription.Circular RNA is enriched in a large number non-nucleated cells.Recently studies have shown that a part of circRNAs have the function of encoding proteins,but most circRNAs have miRNA sponge functions as non-coding RNAs,which can relieve miRNAs from their target gene inhibitors and promote the expression of target genes of miRNAs,thereby play an important role in cells physiological processes.Circ RNA has been widely studied in the leukemia field.Changes and functions in normal red blood cell storage have not been studied.On the background of this study,we used circRNA high-throughput sequencing of mature red blood cells stored at different time points,discovered a large number of unreported circRNAs,and used biological information from several known pathways to understanding differentially expressed circRNAs.In addition,we found circRNA related to ATP metabolism and predicted its target miRNA and miRNA-associated mRNA,in order to better understand the possible roles and molecular mechanisms of differentially expressed circRNA in the storaged red blood cells.ObjectiveTo investigate the circular RNAs expression profiles in human red blood cell during the storage,predict the potential biological function of circular RNAs and its target miRNA,and reveal the molecular mechanism of circular RNAs on target gene expression.MethodsRed blood cell were collected from 5 healthy men with O blood type group.The age of these people is about 25 years old.150 ml leukocyte removed from blood and mixed with CPDA-1 preservative fluid to prepare 5 suspension red blood cells,then stored in a special blood-storage refrigerator at(4±2)°C.Erythrocytes sample were extracted at 0,20,and 40 days,and total RNA was extracted from each sample and stored at-80℃.The circRNA high-throughput sequencing was performed at 40 days.hsa＿circ＿0004619,hsa＿circ＿0000446,hsa＿circ＿0000619,hsa＿circ＿0000722,hsa＿circ＿0000754,hsa＿circ＿0006717,hsa＿circ＿0008554,has＿circ＿0007127,hsa＿circ＿0007112,Novel＿circ＿0000311,hsa＿circ＿0000033,hsa＿circ＿0049462,hsa＿circ＿0000739,has＿circ＿0000847,has＿circ＿0005546,has＿circ＿0031038,has＿circ＿0004724,hsa＿circ＿0007470,MRP4,hsa-mi R-155-5p were chosen to validate by RT-PCR.Expression of each circRNA in high-throughput sequencing data Quantitative statistics were performed and all of differentially expressed genes were annotated to satisfy the condition of FDR<0.05(FDR is the calibrated P value)and |log2FC|>1.using volcano plot,KEGG enrichment and GO Analysis to selected circular RNAs which parental gene screened in c AMP signaling pathway,ubiquitin-mediated proteolysis pathway,apoptosis pathway,adhesion pathway,MAPK signaling pathway,cysteine methionine metabolism pathway,RNA degradation pathway,RNA transport pathway,TGF-beta pathway,and actin-regulated pathway.Their corresponding miRNAs and mRNA also used for target prediction to construct mRNA-circRNA-miRNA co-expression network.Result1.Through high-throughput sequencing analysis,a total of 2586 known and 6216 newly discovered circRNAs were identified.More than 100 circRNAs expression levels were shifted.39 up and 80 down on day 20 compared to day 0;47 up and52 down on day 40 compared to day 0;68 up and 42 down on day 40 compared to day20.On this data basis,Sceening 18 circRNAs with high abundance and different expression patterns in the erythrocyte,then identified by RT-PCR.The RT-PCR verification results were generally consistent with the results of the microarray.The RT-PCR verification results are as follows:2.At 0,20,and40 days,the fold change of hsa＿circ＿0004619,hsa＿circ＿0000446,hsa＿circ＿0000619,hsa＿circ＿0000722,hsa＿circ＿0000754,hsa＿circ＿0006717 and hsa＿circ＿0008554 have same trend was 1.00±0.06,0.30±0.12,0.43±0.17;1±0.06,0.38±0.15,0.5±0.23;1±0.06,0.32±0.06,0.44±0.28;1±0.06,0.37±0.17,0.31±0.27;1±0.04,0.30±0.09,0.37±0.19;1.37±0,0.35±0.19,0.47±0.19;1±0.06,0.33±0.19,0.43±0.1,respectively.The fold change of has＿circ＿0007127,hsa＿circ＿0007112,Novel＿circ＿0000311,hsa＿circ＿0000033 and hsa＿circ＿0049462 was 1.00±0.06,0.35±0.06,1.00±0.46;1.00±0.06,0.384±0.22,0.74±0.19;1±0.06,0.29±0.17,0.68±0.17;1±0.06,0.40±0.18,1±0.30;1±0.06,0.40±0.06,0.90±0.08,respectively.The fold change of hsa＿circ＿0000739 was 1±0.06,1.36±0.36,0.41±0.13,respectively.The fold change of has＿circ＿0000847 was1±0.06,4.48±2.43,4.22±2.38,respectively.The fold change of has＿circ＿0005546 was 1±0.06,0.34±0.06,0.55±0.16.The fold change of has＿circ＿0031038 was 1±0.06,1.16±0.43,2.0±0.7.The fold change of has＿circ＿0004724 was 1±0.06,0.83±0.25,0.57±0.18.The fold change of hsa＿circ＿00007470,MRP4 and hsa-mi R-155-5p was1.00±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.3.mireap,miranda,targetscan,and mir Tar Base predicted that 20 days compared to0 days,there were 196 miRNAs that could be targeted with 86 circRNAs,and mRNAs that could bind to miRNAs were 246;40 days compared to 0 days,there were 171 miRNAs that can be targeted with 71 circRNAs,and 194 mRNAs that can bind to miRNAs.There are 160 miRNAs that can target 69 circRNAs at 40 days and 20 days,and 190 mRNAs can bind to miRNAs.GO analysis and KEGG analysis showed that these parental gene of changed circRNAs are mainly related to ubiquitin-mediated proteolysis,RNA degradation,RNA transport,c AMP signaling pathway,erythrocyte adhesion,TGF-βsignaling,AMPK signaling,apoptosis in the cell cycle.4.The target miRNAs of hsa＿circ＿0007470 were hsa-mi R-155-5p and hsami R-6827-5p predicted by mireap,miranda,targetscan and mir Tar Base.The target mRNA of these miRNAs was MRP4,which is the same gene as patental gene of hsa＿circ＿0007470.Calculation of total ATPase and ATP concentration:At 0,20,and 40 days,total ATPase(U/g Hb)was 40.87±5.883,18.300±2.847,and 10.21±1.636,respectively;ATP(U/g Hb)was 8.708±1.25,3.082±0.166,2.462±0.252.respectively.We are interested in the parental gene of hsa＿circ＿0007470 that plays a major role in the c AMP signaling pathway.Through comprehensive screening and software predictions,we found that hsa＿circ＿0007470 target miRNAs are hsa-mi R-155-5p and hsa-mi R-6827-5p,their target proteins is MRP4,which is also the parental gene of hsa＿circ＿0007470.According to erythrocyte ATPase and ATP test results,the expression of hsa＿circ＿00007470 in erythrocytes of 20-day storage fluid is in the same trend with ATPase and ATP expression,suggesting that hsa＿circ＿00007470 may indirectly regulate ATP metabolism by eliminating the inhibitory effect of miRNA on MRP4.This results needs further experimental verification.ConclusionStudies have shown that there are more than 100 circRNAs have different expression mode in stored red blood cells,among of which the number of down-regulated circRNAs is more and greater than the increase.During the entire red blood cell storage period,the ubiquitin-mediated proteolytic pathway keeped top1,as mean while the names and rankings of the remaining pathways change during storage.It shows that the differential expression of circRNA has sensitivity and correlation to a certain extent with the changes of erythrocyte physiological state.ATP content is an important indicator for judging the viability of red blood cells.According to the present study,the expression of hsa＿circ＿00007470 after 20 days was co-directed to ATPase and ATP expression,hsa＿circ＿00007470 might indirectly regulate ATP metabolism by releasing the inhibitory effect of miRNA on MRP4 and improve red blood cell deformability in ATP metabolism.In conclusion,this study aimed to reveal the relationship between the expression and function of target miRNA and its downstream target genes mRNA and the regulation pathways of these circRNAs to better understanding red blood cell energy metabolism,morphological changes,apoptosis and other red blood cell storage damage.It may be lay a theoretical foundation for reducing the metabolism of cryopreserved red blood cells in vitro,reducing their deformability,inhibiting the degradation of intracellular proteins,increasing the oxygen carrying capacity of red blood cells after transfusion,and improving the efficacy of single red blood cell transfusion.