Biomarker Screening And Validation Of Plasma Derived Extracellular Vesicle Small RNA In Ischemic Stroke

Background and PurposeIschemic stroke(IS)is an acute cerebrovascular disease caused by a decrease in cerebral blood perfusion due to severe stenosis or obstruction of cerebral blood vessels.It usually leads to ischemia and hypoxia of the brain in the blood supply area and eventually brain tissue death.Stroke is the second leading cause of death worldwide according to the statistics of the World Health Organization,which is characterized by high rate of incidence,high mortality and disability.In recent years,the incidence of stroke showed a trend of younger age.Stroke is the leading cause of death in China,with first rank fatality rate of 142 deaths per 100,000 people,placing a great burden on health care.The current treatment strategy for ischemic stroke is based on thrombolytic therapy and endovascular intervention,but with narrow time window and low thrombolytic rate.At present,the clinical diagnosis of ischemic stroke mainly relies on CT and MRI imaging and laboratory tests such as rapid blood glucose,blood routine examination,blood electrolytes and coagulation function.Simple,rapid,and cost-effective plasma molecular diagnostic biomarkers screening strategy with highly sensitive and specific for ischemic stroke still needs further exploration.According to the comprehensive prevention and control policy for major diseases in the outline of the “Healthy China 2030” initiative,there is an urgent need to promote conditional screening for stroke and to incorporate appropriate technologies for early diagnosis and treatment into the clinical routine work.In recent years,with the development of precision medicine and molecular diagnosis,liquid biopsy based on extracellular vesicles(EVs)can better reflect the dynamic changes of the disease which shows an extensive application prospect.Extracellular vesicles are lipid bilayer membrane packets that naturally secreted from all eukaryotic cells.EVs are widely discovered in blood,urine,cerebrospinal fluid and other body fluids carrying functional "cargos" such as nucleic acids,proteins,lipids and metabolites,which is considered as important vehicle for mediating intercellular communication.When ischemic stroke occurs,brain cells interact with microenvironment to maintain homeostasis in the brain by releasing extracellular vesicles,which can pass cross the blood-brain barrier and enter the blood circulation.Alterations in the extracellular vesicles and their contents in the peripheral blood help indicate the pathological state of the brain.Studies have shown that a variety of noncoding small RNAs contained in extracellular vesicles are clinically important in the diagnosis,treatment and prognosis of ischemic stroke.They are also involved in the progression of ischemic stroke through various regulatory mechanisms.In this study,we applied next generation sequencing to screen potential plasma extracellular vesicle small RNAs(EV-sRNA)as molecular diagnostic markers for ischemic stroke.Next,we investigated the changes of cell viability at vascular endothelial cells and neuronal cells by up-take assay,and further validated the selected markers in both cell lines and plasma sample sets.By fitting a combined diagnostic model with multiple indicators,we found combinations of a set of EV-sRNA with high diagnostic efficacy,providing a theoretical basis for early diagnosis of ischemic stroke.Methods(1)Isolation and characterization of extracellular vesiclesPlasma derived extracellular vesicles were isolated from healthy controls and IS patients and characterized by transmission electron microscopy,nanoparticle tracking analysis and western blot for morphological characteristics,particle size and concentration,and EV membrane protein markers,respectively.(2)Extracellular vesicle small RNA library preparation,sequencing and bioinformatics analysisRNA was extracted from extracellular vesicles and constructed small RNA libraries from 3 IS patients and 3 matched healthy controls.The library size was estimated by Fragment Analyzer,while library was quantified by Qubit 4 fluorometer and qPCR method.Next generation sequencing was performed using Illumina Novaseq 6000.Fast QC was applied to raw data quality control,BBDuk was used to remove adaptor sequences and filter out low quality reads.Then,BBMap was used for sequences alignment.Data normalization and differential expression analysis were carried out using DESeq2.Heatmap was created by using pheatmap package and volcano plot was created by ggplot2 package.(3)In vitro experiment validation of EVs and EV-sRNA differential expressionOxygen glucose deprivation/re-oxygenation(OGD/R)models were constructed by using HUVEC and SH-SY5 Y cell lines.HUVEC derived EVs were labeled with the fluorescent dye PKH67 staining to illustrate that the EV internalization process by SH-SY5 Y cells.In addition,the influence of HUVEC-derived EVs on SH-SY5 Y cells was elucidated by hypoxic pretreatment.Then,EV-sRNA relative expression levels were calculated by comparing EVs derived from the cell culture medium of HUVEC and SH-SY5 Y cells under normoxia and OGD/R conditions.(4)Molecular diagnostic marker screening and population validation of plasma derived EVs for ISPlasma samples were collected from IS patients(n=32)and healthy controls(n=32).Differences of relative expression levels of plasma EV-sRNA between the two groups were detected,and the diagnostic efficacy of individual plasma EV-sRNA was assessed using ROC curve analysis.Multiple combinations of plasma EV-sRNA molecules were fitted using logistic regression analysis,and the diagnostic efficacy of the molecular combinations was assessed by ROC analysis to find a group of plasma EVs diagnostic markers with high diagnostic efficacy for IS.Target gene prediction of differentially expressed EV-miRNAs was performed by using Target Scan,miRDB and miRTar Base.And the miRNA-m RNA regulatory network was created by Cytoscape.Results(1)The typical EVs were observed in a cup-like structure under transmission electron microscopy.The results of nanoparticle tracking analysis showed that the particle size of the EVs suspension was around 100 nm.Western blotting showed that the clear EVs marked proteins of CD9,CD63,CD81 and Alix bands while negative marker bands of TOMM20 and Histone H3 were invisible,and Albumin bands were faint.(2)The fragment size of the small RNA library was 140-170 bp.The concentration of library quantitated by Qubit 4 and qPCR met the requirements of the NGS sequencer.Quality control showed good quality of sequencing data.Based on absolute value of Fold Change ≥1.5 and adjusted p value<0.05,we selected a batch of plasma derived EV-sRNA for subsequent validation and functional study.(3)The OGD/R models of HUVEC and SH-SY5 Y cells were successfully constructed.Uptake assay showed that OGD treatment of SH-SY5 Y increased the uptake of hypoxia-pretreated HUVEC cell-derived EVs and improved cell viability of SH-SY5 Y cells.Altered expression levels of partial EV-sRNAs after OGD/R treatment in HUVEC and SH-SY5 Y cells(p<0.05).(4)Population validation showed that hsa-miR-21-5p,hsa-miR-30a-3p,hsamiR-126-3p,hsa-miR-3074-5p and t RF-29 were differential expression(p<0.05)in plasma derived EVs between healthy controls and the IS patient group.By evaluating the diagnostic efficacy of several molecular combinations of plasma EVs and the consideration with clinical appliance,the molecular combination consisting of hsamiR-21-5p,hsa-miR-30a-3p,hsa-miR-126-3p and hsa-miR-3074-5p from plasma derived EVs have effective potential as a novel diagnostic marker for early diagnosis of ischemic stroke with a good diagnostic efficacy,with the AUC of 0.921,the sensitivity of 83.87% and the specificity of 93.1%(p<0.001).ConclusionPlasma derived EV-sRNA markers have potential value for early diagnosis of ischemic stroke.The molecular combination consisting of hsa-miR-21-5p,hsa-miR-30a-3p,hsa-miR-126-3p and hsa-miR-3074-5p from plasma derived EVs is a novel diagnostic candidate marker for early diagnosis of ischemic stroke.Moreover,hypoxia-pretreated HUVEC cell-derived EVs uptaken by SH-SY5 Y cells improved SH-SY5 Y cell viability after OGD.