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.
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OBJECTIVE@#To analyze the action of miRNA-326 on its target gene BCL-XL and the molecular mechanism of platelet apoptosis regulated by miRNAs.@*METHODS@#Dual-luciferase vectors containing respectively the wild-type and mutant 3'-untranslated region (3'UTR) fragments of the BCL-XL gene were constructed with firefly and renilla luciferases and transfected into 293T cells. Relative fluorescence intensities of the transfected cells were measured.@*RESULTS@#Dual-luciferase reporter gene vectors for PsiCHECK- BCL-XL -3'UTR-WT (wild-type) and PsiCHECK- BCL-XL -3' UTR-MT (variant) were respectively constructed. Relative fluorescence intensities of the 293T cells co-transfected by miRNA-326 and PsiCHECK- BCL-XL -3'UTR-WT plasmid were significantly lower compared with the control group (co-transfected by a miRNA-326 negative sequence and PsiCHECK- BCL-XL -3' UTR-WT plasmid) ( P = 0.034). The relative fluorescence intensity was also significantly reduced in cells co-transfected by miRNA-326 and PsiCHECK- BCL-XL -3' UTR-WT plasmid compared with the mutant control group co-transfected by miRNA-326 and PsiCHECK- BCL-XL -3'UTR-MT plasmid (P = 0.022).@*CONCLUSION@#miRNA-326 may participate in the regulation of platelet apoptosis by acting on the 3'-UTR of the BCL-XL gene.
ABSTRACT
Objective To compare the clinical characteristics of community-acquired acute kidney injury (CA-AKI) and hospital-acquired acute kidney injury (HA-AKI) patients.Methods Hospital network system was employed to screen the clinical data of adult patients in the First Affiliated Hospital in Xinjiang Medical University in January to July 2013.A total of 19 528 patients were screened,and 544 AKI patients were identified based on KIDGO (Kidney Disease:Improving Global Outcomes) AKI guidelines.Three hundred and thirty patients were included in HA-AKI group and 214 patients in CA-AKI group.Clinical variables including mortality were analyzed retrospectively.Results The incidence of AKI in hospitalized patients was 2.8％ (544/19 528):1.7％ in CA-AKI group and 1.1％ in HA-AKI group.The mean age in CA-AKI group was significantly older than that in HA-AKI group [(62.9 ± 16.8) years vs (56.6± 15.9) years].Medical patients in CA-AKI group accounted for 62.4％,and surgical patients in HA-AKI group accounted for 64.1％.The co-morbid diseases were cardiac disease,hypertension,diabetes and chronic liver disease.Majority of AKI was caused by pre-renal etiologies.The length of hospitalization was significantly shorter in CA-AKI group compared to that in HA-AKI group [12(8,20) days vs 19 (12,27) days,P ＜ 0.01].Compared to that in HA-AKI group,all-cause mortality was significantly lower in CA-AKI group (11.5％ vs 20.1％,P=0.005).Results by multivariate logistic regression analysis demonstrated that the common independent risk factors of AKI in both groups were ICU hospitalization and shock.The independent risk factor of AKI in CA-AKI group was diabetes (OR=3.019).In contrary,the independent risk factors of AKI in HA-AKI group were elderly (≥65 years) (OR=3.303),oliguria (24 h urine volume ＜ 400 ml) (OR=6.906),use of antiinflammatory drugs (OR=13.079) and multiple organ dysfunction syndrome (OR=17.778).Conclusions The incidence of AKI in hospitalized patients is not rare,among which both communityacquired and hospital-acquired AKI are mainly caused by pre-renal etiologies.All-cause mortality is lower in community-acquired AKI compared to that in hospital-acquired AKI and the independent risk factors are different between CA-AKI and HA-AKI.