Role And Mechanism Of Exosomal LncRNA HCG15 In Acute Myocardial Infarction

Research backgroundAcute myocardial infarction?AMI?is a kind of myocardial necrosis caused by acute and persistent ischemia and hypoxia of coronary artery.The incidence is increasing year by year and tends to be younger.Therefore,it is of great significance to find new targets for the treatment of myocardial infarction.During the occurrence of AMI,myocardial cell apoptosis cascade reactions were activated and myocardial cell necrosis was caused by persistent hypoxia and ATP deficiency.Necrotic cardiomyocytes activate the immune system and produce severe inflammatory reactions.Appropriate inflammation is conducive to the repair of myocardial infarction,while excessive inflammation results in secondary myocardial injury.Tumor necrosis factor?TNF?and other pro-inflammatory factors often have both damage and protection effects.Recognition of biomarkers that overreact to inflammation and activate pro-inflammatory pathways may be helpful in treatment.Previous studies have found that exosomes participate in the inflammatory process of AMI^[1-3].The exosomes produced by AMI are rapidly absorbed by infiltrating monocytes in ischemic myocardium,which aggravates local inflammation.Exosomes also transmit information to nearby myocardial cells,even to fibroblasts,endothelial cells and peripheral circulation,leading to increased inflammation.Large-scale genomic studies have found multiple susceptible sites and segments of myocardial infarction^[4-6],suggesting that the occurrence and development of AMI is the result of environmental and genetic factors.Long non-coding RNA?lncRNA?is widely involved in signal transduction,affecting cell metabolism,growth,differentiation,apoptosis and death.It is of great significance in the occurrence and development of cardiovascular diseases.Experiments at home and abroad have confirmed that a variety of lncRNAs play an important role in myocardial infarction and participate in the regulation of the occurrence and development of AMI^7-9.However,the study of exosome lncRNA in AMI is rare,and its expression profile and role in myocardial infarction are not clear.Therefore,we screened the circulatory exosome lncRNA of patients with AMI by RNA sequencing in order to find the differentially expressed lncRNA in AMI.By constructing an in vitro myocardial infarction model and using small interfering RNA?siRNA?to down-regulate the expression of lncRNA in target exosome,we observed the effects of inflammation,cell growth and apoptosis of myocardial cells,and preliminarily studied its possible mechanism.This research will help to reveal the role of exosomal lncRNAs in AMI and its possible mechanism,discover new markers for the diagnosis of myocardial infarction,and provide new targets for the treatment of myocardial infarction.Part I Screening and verification of exosomal lncRNAs related to acute myocardial infarctionObjective:To identify differential expression of lncRNA in circulating exosomes in patients with AMI.Methods:Peripheral blood was collected from 6 patients with AMI and 6normal controls.Exosomes were extracted and differential expression profiles of lncRNA s were established by RNA sequencing.Then enlarge the sample size,verify the differential expression of interesting lncRNA by qRT-PCR,and analyze the correlation with troponin.Results:1)Exosomes were extracted and identified by transmission electron microscopy,most of which were approximately 40-200nm in diameter.2)The expression of exosome-specific biomarkers?CD63,CD9,and TSG101?were analyzed by western blot method3)RNA was extracted,then 65 differentially expressed lncRNAs were identified in AMI by RNA sequencing,comparing to the normal group.29 of which were up-regulated?fold change>2,padj-value<0.05?in AMI and 36 were down-regulated?fold change<-2,padj-value<0.05?.4)The expression of five selected lncRNAs?STX18-AS1,HCG15,LINC00265,NPH3-AS1,ENTPD1-AS1?were tested in 45 patients with AMI by qRT-PCR.All the 5 lncRNAs were significantly upregulated in AMI group,consistent with the RNA-Seq results.5)Simple correlation analysis of 5 lncRNAs and cTnT showed that the expression level of HCG15 was correlated with the level of cTnT,a marker of myocardial infarction?R2=0.5872,P<0.001?.In the multiple linear regression analysis,only HCG15 entered the regression equation at the level of?=0.05.The results showed that there was a correlation between HCG15 expression level and cTnT level.6)The ROC curve analysis of the diagnostic value of five lncRNAs for AMI showed that the area under the ROC curve of HCG15 was 0.952[95%confidence interval,0.915-0.989].Part?Research on the role and mechanism of lncRNA HCG15Obective:To investigate the role and mechanism of exosomal lncRNA HCG15 in myocardial infarction model.Methods:Myocardial infarction model was constructed using AC16 human cardiomyocyte cell line.SiRNA technology was used to inhibit the expression of lncRNA HCG15,and the cell growth and apoptosis levels were detected.The expression levels of inflammatory factors?IL-6,IL-1?and TNF-??were detected.The level of activity of related signaling pathways?MAPK,NF-?B,etc.?was examined.Signal pathway inhibitors were used to further verify whether the above signaling pathways are involved in this process.Results:1 Exosomes mediate the transmission of lncRNA HCG151)Hemispherical exosomes of saucer type or side depression with diameter of 40-200nm were observed by transmission electron microscopy.Exosomal markers?CD63,CD9 and TSG101?were detected by Western Blot.2)The expression of HCG15 in cardiomyocytes and exosomes increased after hypoxia,especially in exosomes,and peaked at hours.3)After incubation with exosomes derived from hypoxic cardiomyocytes,the expression level of HCG15 in cardiomyocytes increased and peaked at 12 hours.4)Phagocytosis experiments showed that cardiomyocytes uptake PKH67-labeled exosomes.5)TUNEL assay showed that the apoptosis of AC16 cells increased when incubated with Exo-H.2 Research on the effect of exosomal HCG15 on cardiomyocyte function1)Transfected with HCG15 siRNA,the interference effect of siRNA on HCG15 of cardiomyocyte reached 70%.2)After incubation with Exo-H,the expression of HCG15 was significantly up-regulated in AC16 cells,while the up-regulation of HCG15 siRNA HCG15 was partially inhibited.3)MTT analysis showed that after incubation with Exo-H,the growth of AC16 cells was significantly inhibited,while the growth inhibition of HCG15 siRNA group was alleviated to some extent.4)TUNEL and flow cytometry showed that apoptosis of AC16 cells increased significantly after incubation with Exo-H,but the increase of apoptosis was antagonized in HCG15 siRNA transfection group.5)ELISA results showed that after incubation with Exo-H,the production of inflammatory cytokines IL-6,IL-1?and TNF-?in cardiac myocytes increased significantly,and the increase of inflammatory factors in HCG15 siRNA transfection group was antagonized.3 Preliminary research on mechanism underlying effect of exosomal HCG15on myocardial cell function1)Western blot was used to detect the expression of NF-?B pathway protein and MAPKs pathway such as p38 MAPK,JNK1/2 and ERK1/2.After incubation with Exo-H,the expression of phosphorylated NF-?B and p38was up-regulated in AC16 cell group,but the upregulation in HCG15siRNA transfection group was antagonized,and the expression of other signaling molecules did not change significantly.2)PDTC inhibited NF-kappa B signaling pathway and PD169316 inhibited p38 MAPK signaling pathway,respectively.When incubated with Exo-H,the growth of AC16 cells was inhibited,while PDTC,or PD169316,a pathway inhibitor,partially antagonized the growth inhibition.3)TUNEL and flow cytometry showed that apoptosis of cardiomyocytes induced by Exo-H was antagonized when AC16 cells were treated with PDTC or PD169316.4)The elevation of IL-6,IL-1?,TNF-?induced by Exo-H was detected by ELISA,while the elevation of IL-6,IL-1?,TNF-?was antagonized by blocking the pathways of NF-kappa B and p38.Conclusion:1.Through RNA sequencing,65 differentially expressed lncRNAs were found in circulating exosomes of AMI patients,of which 29 were up-regulated and 36 were down-regulated.The five lncRNAs verified by qRT-PCR were consistent with the sequencing results.2.The area under the ROC curve of HCG15 in diagnosing AMI was 0.952[95%confidence interval,0.915-0.989].Simple correlation analysis and multiple linear regression analysis also showed that there was a correlation between HCG15 expression level and cTnT level.HCG15 may be a specific marker of AMI and play a role in AMI.3.Hypoxic AC16 cardiomyocytes-derived exosomes overexpressed HCG15,while HCG15 was transported to normal cardiomyocytes through exosome pathway4.HCG15 promotes cardiomyocyte apoptosis,promotes the production of inflammatory cytokines IL-6,IL-1?,TNF-?,and inhibits cell growth.Inhibiting the expression of HCG15 in the exosomes released by hypoxic-treated cells can alleviate myocardial injury.5.HCG15 promotes cardiomyocyte apoptosis and inflammatory cytokine production through NF-?B and p38 pathways.When HCG15 was up-regulated,NF-kappa B and P38 were up-regulated,inflammatory factors and cell apoptosis were increased,and cell growth was inhibited.Inhibition of HCG15 or inhibition of NF-kappa B and P38 with pathway inhibitors could antagonize these effects.HCG15 derived from hypoxic cell-derived exosomes mediates myocardial cell damage via NF-?B and p38 pathways.In summary,our results showed that lncRNA HCG15was highly expressed in exosomes derived from AMI patients serum and hypoxia cells,and exsomal HCG15 released from hypoxia cells promoted cardiocyte apoptosis and inflammatory cytokines production via activation NF-?B and p38 pathway.This study not only provides the understaning of exosomal lncRNA function in AMI pathogenesis,but also present a promising novel treatment for AMI.