ABSTRACT
【Objective】 To analyze the changes of microRNA (miRNA) expression profiles on day 1 and day 5 after storage with or without riboflavin and ultraviolet-B (UVB) light (VB2-PRT) treatment, and to explore the molecular mechanism of miRNAs involved in the regulation of platelet storage lesion (PSL) under VB2-PRT treatment. 【Methods】 20 apheresis platelet concentrates (5mL / sample), collected from voluntary donors, were split into two group after mixing and agitation. One was treated with riboflavin (final concentration 50 μmol/L) plus 6.24 J/mL UVB light(E group), and the other worked as a control group (C group) without any treatment. Both groups were subjected to agitated storage at (22±2) ℃ horizontally. The platelet concentrates were sampled on d1 and d5 (5mL) during storage, named as E1, E5, C1 and C5 groups, respectively, and sequenced by DNA nanoball (DNB) sequencing technology. The differentially expressed miRNAs between E and C groups were screened by using DEGseq and MA-plot analysis software, and GO function enrichment analysis and KEGG pathway enrichment analysis were further performed when the different expression between groups reached twofold and above. 【Results】 Compared with C1 group, 487 miRNAs with significantly different expression (P<0.01) were screened in E1 group, including 220 up-regulated miRNAs, such as miR-146a and let-7b, and 267 down regulated miRNAs, such as miR-7 and miR-1260. Compared with C5 group, 229 miRNAs with significantly different expression (P<0.01) were screened in E5, including 80 miRNAs with up-regulated expression, such as miR-423 and miR-378, and 149 down regulated miRNAs, such as miR-451 and miR-30.The target genes with differentially expressed miRNAs in E1 vs C1 groups and E5 vs C5 groups were similar in the numbers of enriched GO terms, including cell components, organelles, cell membrane and other cell structures, molecular functions such as adhesion, catalysis, molecular transformation, transportation, transcription factors and receptor activity, cell processing, metabolism, biological regulation, stress and other biological processes etc. Compared with E1 and C1 groups, E5 and C5 groups lacked of signal pathways related to environmental adaptation, translation and mucin synthesis, however, it increased inositol phosphate metabolism, phosphatidylinositol signaling system and chemokine signaling pathway. 【Conclusion】 The expression profiles of platelets miRNAs treated with VB2-PRT has changed significantly after storage for a period of time. Functional prediction suggests that these miRNAs might be involved in the regulation of platelet PSL induced by VB2-PRT.
ABSTRACT
【Objective】 To investigate the changes of platelet microRNA (miRNA) expression profiles of storage in vitro, and to explore the molecular mechanism of miRNAs involved in the regulation of platelet storage lesion (PSL). 【Methods】 20 platelet samples (5 mL / sample) were collected from apheresis platelet donors, fully mixed and stored in a shaker with (22±2) ℃ horizontal agitation, sampled on day 1 and day 5, and sequenced by DNA nanoball (DNB) sequencing technology. The miRNAs with more than 2 times expressions (P<0.01) were considered as significantly differences between d5 and d1 groups. The miRanda and TargetScan softwares were used to predict the target genes. Gene Ontology (GO) function enrichment analysis and Kyoto Encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed on the target genes of significant differentially expressed miRNAs. The expression of miRNAs was verified by real-time fluorescence quantitative PCR (qRT-PCR). 【Results】 Compared with d1 group, 315 miRNAs with significantly different expression (P<0.01) were screened in d5 group, including 146 up-regulated miRNAs (such as miR-146a, let-7b), and 169 down-regulated miRNAs (such as mir-30d, mir-142). Among 126 known miRNAs, 43 were up-regulated and 83 were down-regulated. There are 189 new miRNA sequences. The enriched GO terms of target genes of differentially expressed miRNAs in d5 and d1 groups included cell components, organelles, cell membrane and other cell structures, molecular functions such as adhesion, catalysis and activity, and biological processes such as cell processing, metabolism, biological regulation and stress. The corresponding pathways in the top 10 of KEGG enrichment were mainly signal transduction, secretion, membrane transport, amino acid metabolism, polysaccharide metabolism, protein synthesis and environmental adaptation. The 6 randomly selected differentially expressed miRNAs verified by qRT-PCR were consistent with those of DNB sequencing. 【Conclusion】 The expression profiles of platelets miRNAs have changed significantly between the d1 and d5 of storage in vitro. Functional prediction suggested that these miRNAs might be involved in the regulation of platelet PSL.
ABSTRACT
【Objective】 To investigate the changes of platelet microRNA (miRNA) expression profiles on d1 and d5 during storage with riboflavin and ultraviolet-B (UVB) light (VB2-PRT) treatment, and to explore the molecular mechanism of miRNAs involved in the regulation of platelet storage lesion (PSL) under VB2-PRT treatment. 【Methods】 20 apheresis platelet concentrates (5mL / sample) were collected from voluntary blood donors. After mixing and shaking, the samples was treated with riboflavin (final concentration 50 μmol/L) and 6.24J/mL UVB light for 8min, then split into two aliquots and agitated stored at (22±2) ℃. The concentrates were sampled (5mL) on d1 and d5, respectively, and sequenced by DNA nanoball (DNB) sequencing technology. The differentially expressed miRNAs between the two groups (at different storage periods) were screened by DEGseq and MA-plot analysis software. The miRNAs, reached more than 2 times different expression between groups, were considered significant different(P<0.01). The miRanda and TargetScan softwares were used to predict the target genes. Gene Ontology (GO) function enrichment analysis and Kyoto Encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed on the target genes of significant differentially expressed miRNAs. The expression of miRNAs was verified by real-time fluorescence quantitative PCR (qRT-PCR). 【Results】 miRNA expression profile: compared with d1 platelets, there were 590 miRNAs with significantly different expression (P< 0.01) in d5 group, including 255 up-regulated miRNAs (such as miR-99b, miR-7) and 335 down regulated miRNAs (such as miR-451a, miR-19b). Among the 272 known miRNAs, 112 were up-regulated and 160 were down regulated. There were 318 new miRNAs sequences. The enriched GO terms of target genes of differentially expressed miRNAs in d5 and d1 groups included cell components, organelles, cell membranes and other cellular structures, molecular functions such as adhesion, catalysis, molecular conversion, transportation, transcription factor and receptor activity, and biological processes such as cell processing, metabolism, biological regulation and stress. The corresponding pathways in the top 10 of KEGG enrichment were mainly secretion, glucose metabolism, signal transduction, membrane transport, translation, environmental adaptation and other signal pathways. The six randomly selected differentially expressed miRNAs verified by qRT-PCR was consistent with those of DNB sequencing. 【Conclusion】 The expression profiles of platelets miRNAs has changed significantly between d1- and d5-storage under VB2-PRT treatment. Functional prediction suggests that these miRNAs might be involved in the regulation of platelet PSL underVB2-PRT treatment.