| Part Ⅰ REGULATION OF STROKE INJURY BY ADSCS EXOSOMES MIR-21 AND MIR-342 THROUGH CASPASE-3BackgroundCerebral stroke currently accounts for about 35%of the total incidence of cerebrovascular diseases,with a fatality rate of more than 50%.40%of survivors are left with severe disability,which is one of the major diseases threatening the health of the elderly and one of the leading causes of death and disability worldwide.It is an important public health problem.In recent years,comprehensive treatment methods such as surgical removal of hematoma,decompression of bone-flap,endarteriotomy,dehydration and reduction of intracranial pressure,neuronutrition and hyperbaric oxygen have been adopted in clinical practice.Although they can save the lives of some patients,the survivors of stroke still have a high disability rate.Mechanical compression and destruction of hematoma and brain tissue edema after stroke can have a direct killing effect on nervous system cells,while ischemia and hypoxia secondary to peripheral brain tissue after stroke will further lead to the necrosis and apoptosis of neurons,resulting in severe neurological function loss.A large number of studies have been conducted to elucidate the pathological mechanism of neurological injury in stroke and the therapeutic target of stroke,and to find better and faster treatment measures that can directly avoid ischemic stroke injury.How to reduce the neuronal cell necrosis and apoptosis after stroke is the key to improve the prognosis of such patients.Between ADSCs is a kind of extract Stem Cells from adult fat,because of its no immune rejection,autologous transplantation,has the advantages of multi-directional differentiation potential,a Stem cell transplant research hot spot in recent years.The transplantation of ADSCs for the treatment of neurological defects caused by various diseases has made valuable progress.Himself in the early stage of the group has successfully built a SD rat model of stroke and isolated ADSCs in vitro amplification,and induced into neuronal cells,and injected into the rat brain tissue damage local,observed damaged brain tissue increased local immature neurons and synapses regeneration obviously,increased mitotic activity,reduce the local neural tissue loss area,can promote the neural functional recovery of rats.On the basis of a large number of animal experiments,the research group carried out a clinical trial study on local injection of autologous ADSCs for the treatment of stroke.The results of the study confirmed that local injection of ADSCs into the damaged brain tissues around the hematoma space in stroke patients can effectively improve the neurological loss in the near term(6 months),and the effect is better than intravenous injection and subarachnoid injection.However,we also found that there was no significant statistical difference in neurological function score of patients treated with ADSCs after 12 months compared with the control group,indicating that the long-term effect of ADSCs on nerve injury after stroke was poor and still unsatisfactory.How to improve the nerve repair effect of ADSCs after transplantation is one of the problems faced by researchers.This suggests that we should conduct in-depth research on the mechanism of action of ADSCs in the treatment of stroke,and it is the key to improve the clinical therapeutic effect of ADSCs to find the key molecules and conduct targeted regulation.In the past,mesenchymal stem cells were thought to differentiate into different types of cells,but more research is now focused on the interactions between mesenchymal stem cells and other cells and their effects on other cells,in particular the exchange of information with other cells.Communication through extracellular vesicles has recently attracted more interest.There are many different types of secretory extracellular vesicles that vary in size,cell origin,biosynthesis,contents,and the physiological environment in which they are released.Exosomes(exosomes)are polyvesicles with a diameter of 40-100nm that are formed by intracellular lysosomal particles.It contains proteins,mRNAs,miRNAs and other genetic materials,ACTS on target cells and regulates the expression of related target genes,has the function of realizing inter-cell material exchange and information transmission,and participates in a variety of in vivo pathophysiological processes.Exosomes can be isolated in a variety of ways,characterized and labeled by electron microscopy.At present,it is believed that in the treatment of nerve injury diseases by ADSCs transplantation.Exosomes derived from stem cells can provide growth factors and other cell protective factors,which can play a variety of roles such as anti-apoptosis,promoting angiogenesis,enhancing the differentiation of nerve cells and repairing damaged tissues.The role of exosomes in nerve repair of ADSCs has become a hotspot in the study of its mechanism.A large number of studies have confirmed that fat-derived mesenchymal stem cells can secrete exosomes and play an important role in the physiological functions of stem cells.In studies on stroke related exosomes,Xin et al.reported that ADSCs can promote axonal growth by secreting exosome mir-133b which is absorbed by neurons and astrocytes.Moreover,the effect of ADSCs transplantation combined with mir-133b overexpression on axonal plasticity and synaptic remodeling was better than that of ADSCs alone.The team is also the first to use ADSCs-derived exosomes to replace ADSCs in the treatment of animal models of stroke,which can also improve the prognosis of stroke.However,the contents of exosomes play different roles in different pathological situations,and this difference may lead to the exact opposite fate of the target cell.Therefore,the study of the biological function of exosomes in certain pathological conditions such as hypoxia and ischemia will be of great benefit.Among the exosome contents,miRNAs have been shown to regulate important processes in pathophysiological outcomes of ischemic stroke.MiRNAs can not only promote and inhibit the apoptosis of glial cells and cerebrovascular endothelial blood cells,but also regulate acute ischemic stroke.However,it is not clear whether miRNA derived from adipose mesenchymal stem cells can participate in the regulation of apoptosis of glial cells and cerebrovascular endothelial cells in the case of cerebral hypoxia and ischemia.In this study,we investigated the protective effect of adipose-derived mesenchymal stem cells-derived exosomes on glial cells and cerebrovascular endothelial cells in ischemic and hypoxic models,suggesting that the mechanism is mainly through the regulation of caspase-3 proteins in these two types of cells by exosomes mir-21 and mir-342.At the same time,our extensive research work may also provide a potential cellular therapeutic strategy for ischemic stroke.Objective1.The expression levels of mir-21 and mir-342 in stem cell exosomes and in vitro models of stroke were detected.2.The relationship between the expression levels of mir-21 and mir-342 and the apoptosis of stroke cells was analyzed.3.To evaluate the regulatory effect of stem cell exosomes on stroke injury by interfering with the expression of mir-21 and mir-342.4.To study the regulatory mechanism and regulatory targets of mir-21 and mir-342 involved in stroke injury.Methods1.Human fat mesenchymal stem cells were cultured,exosomes were successfully extracted by ultra-centrifugation method and identified by electron microscopy.Human glial T98 cells and vascular endothelial cells HCMEC were cultured,and the ischemia and hypoxia models of T98 and HCMEC were successfully established with appropriate concentration of cobalt chloride solution.RNA-seq,MTT and qRT-PCR methods were used to detect and screen differentially expressed miRNAs,and miRNAwith high specificity(mir-21 and mirna-342)were selected from them.2.T98 and HCMEC cell apoptosis were observed under electron microscope,and the mimic and inhibitor of mir-21 and mir-342 were transfected with LP3000 transient transfection method,and the relationship between cell apoptosis and improvement and miRNA expression was observed.3.The differential expression of miRNAs in mesenchymal stem cells was verified by qRT-PCR,exosomes were co-cultured with the hypoxic-ischemic cell model to observe apoptosis and improvement.After interfering with the expression levels of mir-21 and mir-342,exosomes were isolated and co-cultured.Cell apoptosis and improvement were observed by qRT-PCR,Western Blot and flow cytometry.4.Through the luciferase detection method to detect miR-21 and the role of miR-342 in the stroke damage model protein targets and caspase 3,combined with biological information technology to test the effect of miRNAs and their target genes cell signaling pathways,and to observe the apoptosis of cerebral apoplexy and the correlation of caspase 3 expression,verify again miR-21 and miR-342 targets in stroke damage model,try for the regulation of stroke damage potential of cell therapy strategies.Results1.Differentially expressed miRNAs and mRNAs were analyzed by RNA sequencing.RNA sequencing was performed on normal carotid artery samples from 3 patients with atherosclerosis and 2 patients with accidental car accident.There were 141 differentially expressed miRNAs in atherosclerosis specimens,among which 68 were up-regulated and 73 were down-regulated.The apoptosis rates of vascular endothelial cells at H2O2 0,1000,1500 and 2000 um were 2.6%,11.1%,20.5%and 41.1%,respectively.Meanwhile,with the increase of H2O2 concentration,the ratio of bcl-2 to BAX showed a decreasing trend(P<0.05,P<0.01).These results support the successful construction of vascular endothelial cell lesion model.2.Objective miRNA miR-342-5p was identified and promoted the apoptosis of vascular endothelial cells damaged by H2O2The top 30 up-regulated miRNAs were screened according to log2FC value sequencing,and their expression profile heat map was shown in figure 3A.Subsequently,10 miRNAs(i.e.Mir-147b-3p,mir-378d,mir-10399-3p,mir-146a-5p,mir-146b-5p,mir-2277-5p,mir-342-5p,mir-181a-3p,mir-378a-3p,and mir-142-3p)were considered candidates,which ranked first in RNA sequencing or were reported to be associated with atherosclerosis.In the vascular endothelial cell injury model,the relative expression of has-rna-342-5p was the highest among the ten miRNAs.As the concentration of H2O2(1000,1500 or 2000 um)gradually increased,the expression of mir-342-5p was detected.Obviously,with the increase of H2O2 concentration,the miRNA expression is gradually enhanced.The endothelial cells were transfected with mir-342-5p mimic,inhibitor and their corresponding NC,respectively,and then treated with 1500um H2O2.3.In the vascular endothelial cells damaged by H2O2,PPP1R12B was identified as the target of mir-342-5pCluster analysis can cluster genes with similar expression patterns,indicating that they may have common functions or participate in common metabolic and signaling pathways.Cluster analysis was conducted using differentially expressed mRNAs from RNA sequencing.In RNA sequencing,2694 mRNAs were down-regulated,and 240 genes were from three databases.Thus,25 genes were obtained,which were not only targets for mir-342-5p,but also downregulated in atherosclerosis.The 25 genes were then used for GO and KEGG enrichment analysis.The interactions of the 25 overlapping genes were first analyzed by STRING and then imported into Cytoscape.KEGG analysis showed that PPP1R12B,which scored the highest among these hub genes,was associated with both vasosmooth muscle contraction and oxytocin signaling.miR-342-5p inhibited the expression of PPP1R12B in vascular endothelial cell injury model.The above evidence indicates that PPP1R12B is a target of mir-342-5p in h2o2-impaired vascular endothelial cells.4.ADSCs derived exosomes can inhibit the expression of mir-342-5p in vascular endothelial cells damaged by H2O2.The ADSCs were isolated and cultured,and the exosomes purified from the supernatant of ADSCs were characterized by TEM.The results are shown in FIG.5B.The exosomes are circular membranous capsules with diameters ranging from 30 nm to 150nm.Western blot was used to detect the expression levels of CD9,CD63 and TSG101,which are commonly used as markers for exosomes.Exosomes were added to the vascular endothelial cells damaged by H2O2 at a concentration of 1500um.Subsequently,the expression of mir-342-5p was detected by q rt-PCR.The results showed that ADSCs-derived exosomes inhibited the expression of mir-342-5p in h2o2-impaired vascular endothelial cells.Conclusions1.The apoptosis progression of glial cells and vascular endothelial cells after stroke injury was significantly correlated with the expression of mir-21 and mir-342.2.Exosomes derived from mesenchymal stem cells can participate in the apoptosis of glial cells and vascular endothelial cells through mir-21 and mir-342.3.Mesenchymal stem cell exosomes mir-21 and mir-342 are involved in the regulation of stroke injury through caspase-3.SignificanceThis study makes it clear that we can draw a conclusion that exosomes mir-21,mir-342 and caspase-3 derived from adipose mesenchymal stem cells are promising potential targets for stroke treatment.Part Ⅱ ADIPOSE-DERIVED MESENCHYMAL STEM CELL DIRIVED EXOSOME MEDIATED MICRORNA-342-5P PROTECTS ENDOTHELIAL CELLS AGAINST ATHEROSCLEROSISBackgroundAtherosclerosis is the major underlying contributor to cardiovascular diseases and brings much morbidity and mortality across the globe.A series of hyperlipidemia-induced chronic inflammatory processes,covering complicated interactions among modified lipoproteins,monocytes and T lymphocytes with cellular components sourced from the vessel wall,facilitate the buildup of atherosclerosis lesion.To date,numerous diagnostic techniques have been employed to assess the cardiovascular disease risk and definitive therapies,and been generally classified into invasive and noninvasive kinds.The former kind is exemplified by invasive angiography and optical coherence tomography,the latter kind is by blood biomarkers,stress testing,CT and nuclear scanning.Of note,considerable studies concerning the cell and molecular biology of atherogenesis have enhanced the understanding of the underlying risk factors involved in atheroma development and its clinical features.Therefore,continued research about the potential mechanism of atherogenesis is conductive to better combating this chronic disease.Conveniently,the pathogenesis of atherosclerosis goes through three phases,including initiation,progression and complications.Damage or dysfunction of endothelial cells is recognized as critical events in the initiation of atherosclerotic-plaque development.The adaptation of arterial endothelial cells to disturbed blood flow is critical for,or even decisive in the susceptibility at branching sites of arteries,where atherosclerosis preferentially occurs.It thus appears that seeking effective approaches to protect endothelial cells may provide fruitful strategies to prevent atherosclerosis progression.MicroRNAs(miRNAs),specific messenger RNAs(mRNAs).Recently,a considerable literature has recognized the dysregulated expression of several miRNAs associated with pro-atherosclerosis and anti-atherosclerosis as the critical mechanisms of atherosclerosis development.Numerous miRNAs are reported to exert essential regulated role in lipid metabolism,inflammation and atherogenesis,supportive by the cases of miR-19b and miR-144-3p involved in regulation of lipid metabolism,miR-92a and miR-155 as critical regulators of inflammation,miR-30c and miR-126-5p preventing atherosclerosis.By this token,targeting miRNAs may potentially alleviate the development of atherosclerosis.Despite bountiful atherosclerosis-associated miRNAs reported,much remains to be done in seeking novel or valuable miRNAs and exploring their potential biological functions in atherosclerosis.In this study,we aimed to screen the atherosclerosis-associated miRNAs and preliminarily investigate the potential regulatory mechanism of atherosclerosis.At first,RNA-sequencing was employed to comparatively analyze the differentially expressed miRNAs and mRNAs in patients with atherosclerosis and corresponding healthy controls.The lesion model for human umbilical vein endothelial cells(HUVECs)was constructed.Secondly,our aimed miRNA,miR-342-5p,was identified on basis of bioinformatics analysis and experimental verifications.The effect of miR-342-5p on the apoptosis of lesion model of HUVECs was also evaluated.According to a series of bioinformatics analysis and experimental confirmation,an atherosclerosis-associated target of miR-342-5p was identified.Finally,adipose-derived mesenchymal stem cells(ADSCs)-derived exosomes were characterized,and their effect on miR-342-5p expression in lesion model for HUVECs was investigated.Collectively,this work identified an atherosclerosis-associated miRNA(miR-342-5p)and unearthed a potential mechanism for this miRNA functioning in atherosclerosis.Objective1.The expressions of miRNA and mRNA in human carotid atherosclerosis specimens were detected compared with normal carotid arteries.2.An in vitro endothelial cell injury model was established,and the apoptosis-related genes and functions were detected by WB,Hoechst and FCM techniques,among which the effect of mir-342-5p on the endothelial cell injury model was also detected.3.Search for and validate downstream target genes of mir-342,and study the effects of inhibition and overexpression of mir-342 on the expression levels of downstream target genes and proteins.4.The downstream target genes of mir-342 were interfered with and overexpressed,respectively,to study the effect of mir-342 on the anti-atherosclerosis effect of vascular endothelial cells.5.To study the effect of stem cell derived exosomes on the expression of mir-342-5p.Methods1.The expressions of miRNA and mRNA in human carotid atherosclerosis specimens were detected compared with those in normal carotid arteries.Differentially expressed miRNAs and mRNAs in atherosclerotic samples were observed using RNA sequencing data using a range of appropriate analytical tools.In the Venn diagram,the Hub gene was found in the overlapping targets of mir-342-5p.2.Establish an in vitro endothelial cell injury model,detect apoptosis-related genes and functions,and detect the effect of mir-342-5p on the endothelial cell injury model.MTT method was used to screen the appropriate concentration of H2O2 to establish an in vitro endothelial cell injury model.3.To study the effect of stem cell derived exosomes on the expression of mir-342-5p.The exosomes of ADSCs were isolated and identified,the stem cell exosomes were extracted and co-cultured with the endothelial cell injury model,the effects of exosomes on the expression of mir-342 were detected by q rt-PCR and Western blot methods,and the effects of Western blot and other techniques on the transcription of target genes and protein expression levels were detected.Interference and overexpression of mir-342 and to detect the improvement of vascular endothelial cell apoptosis.Results1.Differentially expressed miRNAs and mRNAs were analyzed by RNA sequencingRNA sequencing was performed on normal carotid artery samples from 3 patients with atherosclerosis and 2 patients with accidental car accident.There were 141 differentially expressed miRNAs in atherosclerosis specimens,among which 68 were up-regulated and 73 were down-regulated.The apoptosis rates of vascular endothelial cells at H2O2 0,1000,1500 and 2000 um were 2.6%,11.1%,20.5%and 41.1%,respectively.Western blot analysis showed that the expression levels of cleaved-PARP/PARP,cleed-caspase3/caspase3,cytochrome C and p53 increased significantly with the increase of H2O2 concentration(P<0.05,P<0.01,P<0.001).Meanwhile,with the increase of H2O2 concentration,the ratio of bcl-2 to BAX showed a decreasing trend(P<0.05,P<0.01).These results support the successful construction of vascular endothelial cell lesion model.2.Objective miRNA mir-342-5p was identified and promoted the apoptosis of vascular endothelial cells damaged by H2O2The top 30 up-regulated miRNAs were screened according to log2FC value sequencing,and their expression profile heat map was shown in figure 3A.Subsequently,10 miRNAs(i.e.Mir-147b-3p,mir-378d,mir-10399-3p,mir-146a-5p,mir-146b-5p,mir-2277-5p,mir-342-5p,mir-181a-3p,mir-378a-3p,and mir-142-3p)were considered candidates,which ranked first in RNA sequencing or were reported to be associated with atherosclerosis.In the vascular endothelial cell injury model,the relative expression of has-ma-342-5p was the highest among the ten miRNAs.As the concentration of H2O2(1000,1500 or 2000 um)gradually increased,the expression of mir-342-5p was detected.Obviously,with the increase of H2O2 concentration,the miRNA expression is gradually enhanced.The endothelial cells were transfected with mir-342-5p mimic,inhibitor and their corresponding NC,respectively,and then treated with 1500um H2O2.3.In the vascular endothelial cells damaged by H2O2,PPP1R12B was identified as the target of mir-342-5pCluster analysis can cluster genes with similar expression patterns,indicating that they may have common functions or participate in common metabolic and signaling pathways.Cluster analysis was conducted using differentially expressed mRNAs from RNA sequencing.In RNA sequencing,2694 mRNAs were down-regulated,and 240 genes were from three databases.Thus,25 genes were obtained,which were not only targets for mir-342-5p,but also downregulated in atherosclerosis.The 25 genes were then used for GO and KEGG enrichment analysis.The interactions of the 25 overlapping genes were first analyzed by STRING and then imported into cytoscape.KEGG analysis showed that PPP1R12B,which scored the highest among these hub genes,was associated with both vasosmooth muscle contraction and oxytocin signaling.miR-342-5p inhibited the expression of PPP1R12B in vascular endothelial cell injury model.The above evidence indicates that PPP1R12B is a target of mir-342-5p in h2o2-impaired vascular endothelial cells.4.ADSCs derived exosomes can inhibit the expression of mir-342-5p in vascular endothelial cells damaged by H2O2.The ADSCs were isolated and cultured,and the exosomes purified from the supernatant of ADSCs were characterized by TEM.The results are shown in FIG.5B.The exosomes are circular membranous capsules with diameters ranging from 30 nm to 150nm.Western blot was used to detect the expression levels of CD9,CD63 and TSG101,which are commonly used as markers for exosomes.Exosomes were added to the vascular endothelial cells damaged by H2O2 at a concentration of 1500um.Subsequently,the expression of mir-342-5p was detected by q rt-PCR.The results showed that ADSCs-derived exosomes inhibited the expression of mir-342-5p in h2o2-impaired vascular endothelial cells.Conclusions1.Mesenchymal stem cell derived exosomes can inhibit the expression of mir-342-5p,protect endothelial cells against atherosclerosis,and mainly inhibit the signaling pathway of mitochondrial apoptosis induced by oxidative stress.2.In H2O2-impaired vascular endothelial cells,PPP1R12B is a target of mir-342-5p.3.miR-342-5p mediated by ADSCs derived exosomes is involved in the regulation of vascular endothelial cells against atherosclerosis.SignificanceOur study may provide a better understanding of the pathogenesis of atherosclerosis and valuable insights into potential therapeutic targets for atherosclerosis. |
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