Comprehensive Analysis Of LncRNA Expression Profiles And Regulatory Mechanism Of Key LncRNA In ACS Patients Monocyte-derived Dendritic Cells

Background:As one of the most common fatal diseases,acute coronary syndromes（ACS）has a high morbidity and mortality.Therefore,in-depth understanding of the pathogenesis of ACS,reducing the mortality rate of patients and improving the quality of life has become a global social health issue that cannot be ignored.Although a large number of studies have reported a variety of complex pathogenesis of ACS,the pathological mechanisms of different types of ACS lead to different clinical treatment strategies and prognosis,and the understanding of the pathogenesis of each subtype of ACS is still in its infancy.The theory of immune inflammatory response is closely related to the occurrence,development and outcome of ACS.During the pathogenesis of ACS,the necrotic cardiomyocytes activate the immune system and produce a violent inflammatory response,which has repaired the necrotic tissue area.However,whether different subtypes of ACS have different degrees of inflammatory response,and whether the occurrence and development of inflammatory response are regulated by precise molecules,it is worthy of our further research.As a special antigen presenting cell,the function of dendritic cell（DC）in the pathogenesis of different types of ACS is essential for the balance of innate and adaptive immunity.Therefore,exploring the specific functional roles of dendritic cells in different subtypes of ACS and revealing their specific mechanisms in biological effects is the main content of this study.This study intends to use RNA sequencing to explore the differential expression profiles of lncRNAs and m RNAs derived from moDC in different types of ACS patients.Find the lncRNAs related to the differentiation and development of moDCs,and conduct in-depth discussions on the specific mechanisms of their regulation of DC functions to reveal the source of moDC The relationship between lncRNA in the activation of DC and the occurrence and development of ACS.Lnc RNAs related to the differentiation and development of moDC were identified,and the specific mechanism of its regulation of DC function was further explored,so as to reveal the correlation between the moDC-derived lncRNA in the activation of DC and the occurrence and development of ACS.Part Ⅰ The morphology and function of MODC in patients with different types of ACS are differentObjective: To cultivate moDC of patients with different types of ACS or healthy people,and to investigate the relationship between moDC and different types of ACS by detecting it’s morphology and function.Methods:（1）Fresh human peripheral blood was separated by Ficoll density gradient centrifugation to obtain peripheral blood mononuclear cells（PBMCs）.CD14+mononuclear cells were collected by immunomagnetic bead positive sorting method,and induced by complete medium,cell growth factor GM-CSF and IL-4 for 6 days,the moDC needed for the experiment could be cultured.Morphological characteristics of moDC were observed by inverted phase contrast microscope.DC-related surface molecules（CD11c,CD14,CD80,CD86,HLA-DR）were identified by flow cytometry（FCM）.6 patients with unstable angina（UA group）,8 patients with non-ST segment elevation myocardial infarction（NST group）,9 patients with ST segment elevation myocardial infarction（ST group）,and 8 healthy volunteers（CON group）were collected.The number of PBMC and moDC were calculated to obtain the MODC yield rate.In order to explore the functional differences of moDC between different types of ACS patients.The morphology of DC in each group was observed by scanning electron microscope（SEM）.FCM was used to detect the surface costimulatory molecules（CD80,HLA-DR）and ovalbumin（OVA）of moDC.The content of IL-6,IL-10 and IL-12p70 in the supernatant of moDC was detected by ELISA.Result:（1）FCM showed that CD14+ positive rate of PBMC was as high as 95% after MACS sorting.CD14+ mononuclear cells differentiated into DC after 6 days of induction by cytokines GM-CSF and IL-4.Microscopically,DC can be seen as suspension cell,with a small amount of dendritic pseudopods sticking out and gathering like grapes,which is a typical morphology of DC.FCM showed that DC-specific surface molecules CD11 c and HLA-DR were highly expressed,while CD80 and CD86 were weakly expressed without any antigen stimulation.（2）In the same culture system,the number of PBMC extracted from ACS patients and the rate of DC obtained after induction were higher than those from healthy volunteers（p <0.05）.SEM found that compared with the CON group,DCs in the UA group,NST group,and ST group had longer and denser pseudopod-like protrusions.Compared with the CON group and the UA group,the expression levels of CD80 and HLA-DR in the ST group and the NST group were increased,and the expression levels of OVA were decreased（p<0.05）.The expression levels of IL-6 and IL-12p70 in the moDC supernatants of the ST group and the NST group were significantly higher than those of the CON group and the UA group（p<0.05）,while there was no significant difference in the expression of IL-10 among the four groups.Conclusion:（1）Ficoll density gradient centrifugation combined with MACS can effectively cultivate dendritic cells with high purity and considerable quantity.（2）In the same culture system,compared with the CON group or the UA group,the moDC of AMI patients has stronger antigen presentation ability,pro-inflammatory secretory function and weaker phagocytic ability.Part Ⅱ Screening and bioinformatics analysis of differential expression lncRNA from Mo DC in patients with different types of ACSObjective: In order to explore the potential regulatory mechanism of DElncRNA in the pathogenesis of different types of acute coronary syndromes,lncRNAs with differential expression were screened and verified through moDC-derived lncRNA expression profiles and biochemistry analysis in patients with distinct subsets of ACS.Methods:（1）Collected 3 patients with UA（UA group）,3 patients with NSETMI（NST group）,3 patients with STEMI（ST group）,and 3 healthy volunteers（CON group）dendritic cells induced by peripheral blood mononuclear cells.Use RNA sequencing technology to establish differential expression profiles of m RNA and lncRNA.The candidate lncRNAs were screened out by GO analysis,KEGG analysis and co-expression network analysis.（2）RT-PCR was used to verify the candidate delncRNAs by expanding the sample size.The correlation between delncRNA and dendritic cell costimulatory molecules（CD80,CD86,HLA-DR）,white blood cells（WBC）,and AMI related diagnostic indicators（CKMB,TNT-HS）was analyzed.Result:（1）Pairwise comparison of m RNA and lncRNA expression in each group showed no significant changes between the CON group and the UA group,and between the ST group and the NST group.Samples from the STEMI group and the NSTEMI group were combined into a new experimental group,named the acute myocardial infarction group（AMI）,and conduct a significance test with the CON group.The results showed that 833 m RNAs（229 up-regulated,604 down-regulated）and 70 lncRNAs（21 up-regulated,49 down-regulated）were abnormally expressed in the CON group compared with the AMI group（fold change >2.0,p<0.05）.（2）GO analysis and KEGG pathway analysis were performed on the differential m RNA between the AMI group and the CON group.The results showed that most of the enriched signaling pathways and their biological effects were closely related to the differentiation,development and activation of DC.The lncRNA-m RNA co-expression network showed that the co-expression network consisted of 47 network nodes and 35 connections between 13 lncRNAs and 34 m RNAs.Among them,ENST00000418499.3 could be linked to up to 12 m RNAs.（3）lncRNAs（NR₀26793.1,NR₀27922.3,ENST00000446952.1,ENST00000457097.1,ENST00000590780.1）were selected based on the conservativeness score and differential expression multiples of lncRNAs,and were verified through RT-PCR in 14 AMI patients and 5 healthy volunteers.Consistent with RNA-sequencing results,the expressions of NR₀26793.1,NR₀27922.3,ENST00000446952.1,and ENST00000590780.1 were up-regulated.However,the expression level of ENST00000457097.1 was not statistically significant between the two groups.（4）RT-PCR showed that the costimulatory molecules（CD80,CD86,HLA-DR）of Mo DC in AMI patients were up-regulated at the m RNA level.In addition,the correlation analysis showed that the expression level of ENST00000590780.1（R=0.61,p<0.05）was positively correlated with CD80.The expression levels of NR₀27922.3（R=0.56,p<0.05）and ENST00000446952.1（R=0.61,p <0.05）were positively correlated with CD86.The expression levels of NR₀27922.3（R=0.47,p<0.05）and ENST00000446952.1（R=0.69,p<0.05）were positively correlated with WBC.The expression levels of NR₀27922.3（R=0.60,p<0.05）and ENST00000446952.1（R=0.63,p<0.05）were positively correlated with CK-MB.The expression levels of NR₀27922.3（R=0.66,p<0.05）and ENST00000446952.1（R=0.58,p<0.05）were positively correlated with TNT-hs.Conclusion:（1）moDC-derived m RNA and lncRNA in AMI patients are differentially expressed with normal healthy people,while the number of differentially expressed m RNA and lncRNA is not significant between UA patients and healthy normal people,ST patients and NST patients.（2）DElncRNAs derived from moDC in AMI patients have a potential connection with the pathogenesis of ACS,which may be related to the regulation of the biological functions of dendritic cells by these DElncRNAs.Part 3: The role and mechanism of delncRNA CCl15-CCl14 in MODCObjective: To explore the regulatory role and specific mechanisms of lncRNA CCL15-CCL14 in the biological functions of dendritic cellMethods:（1）Overexpression or silencing of lncRNA CCL15-CCL14 was used to detect the changes in the biological functions of moDC,including antigen presentation and cytokine secretion by flow cytometry and RT-PCR.The phosphorylation level of PI3K-Akt protein pathway in Mo DC was detected by Western Blot.（2）Fluorescence in situ hybridization was used to explore the sublocalization of lncRNA CCL15-CCL14 in Mo DC,and to predict its possible targets in combination with the lncRNA database.Result:（1）Using lentivirus as a vector,lncRNA CCL15-CCL14 can be successfully overexpressed into moDC.Using liposomes as vectors,CCL15-CCL14 Smart silencers were successfully transfected into moDC to silence lncRNA CCL15-CCL14.（2）Overexpression of CCL15-CCL14（OE CCL15-CCL14）can promote the activation of moDC,enhance the ability of antigen presentation,and promote the secretion of pro-inflammatory factors by cells.What’s more,silencing CCL15-CCL14（SS CCL15-CCL14）can inhibit the positive immune regulation of Mo DC.The results of Western Blot showed that changes in the expression level of lncRNA would affect the phosphorylation level of PI3K/AKT in moDC.（3）Fluorescence in situ hybridization technology and lncRNA database analysis results show that: CCL15-CCL14 is a non-coding RNA that spans the two sites of CCL14 and CCL15.It is mainly located in the nucleus,which may have a positive regulatory effect on the chemokine CCL14.Conclusion:lncRNA CCL15-CCL14 can regulate the expression level of CCL14 and cause changes in the phosphorylation level of PI3K/AKT protein pathway,thereby affecting the biological effects of moDC.