Preliminary Study On The Mechanism Of "Metabolic Memory" Induced By High Glucose In Vascular Endothelial Cells Based On RNA-seq

Part I Study on the differential expression of mRNA in"metabolic memory" induced by high glucose in vascular endothelial cellsObjective:We try to screen the differentially expressed genes（DEGs）in the HUVECs of MM at the transcriptome level and explore their functional relationship in order to provide target molecules for further research in the future.Methods:1.The HUVECs cells of the 3rd to 10th passage were divided into the following three groups:（1）cells under the low glucose（LG）condition were cultured in the medium containing 5 mM glucose+20 mM mannitol for 6 days,（2）cells under the high glucose（HG）condition were cultured in the medium containing 25mM glucose for 6 days,（3）cells under the MM condition were cultured in the medium containing 25mM glucose for 3 days,and then changed to medium containing 5mM glucose+20 mM mannitol for 3 days.At the end of 6 days,the cell morphology was observed and the cell activity was detected by MTT.2.The total RNA was extracted by miRNA Isolation Kit.After conducted purifying and quality testing,we took 5 ug total RNA of each HUVEC under LG,HG and MM to construct cDNA library and conducted quality inspection of cDNA library.Finally,Cluster generation and sequencing were completed on Illumina 2500 sequencer.Data were processed to prepare for the subsequent differentially expressed gene analysis.3.The differentially expressed genes（DEGs）were analyzed by edgeR to get the high glucose-induced MM-involved DEGs（HGMMGs）:（1）The differentially expressed genes were compared between HG group and LG group（|FC| ≥ 1.2、p<0.05）,between MM group and LG group（|FC|≥1.2、p<0.05）.Then the differentially expressed genes were obtained.（2）Analysis of genes with no significant difference between MM and HG（p≥0.05）.The intersection of（1）and（2）represents the maladjusted genes associated with MM.4.Identified the up-regulating genes in MM induced by HG（up-HGMMGs）and the down-regulating genes in MM induced by HG（down-HGMMGs）.The biological function and signal transduction pathway of genes were analyzed by gene ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）.Protein-protein interaction network was constructed and clustering analysis of protein interaction network based on MCODE algorithm was carried out.5.The total RNAs were extracted with TRIzol reagent and the HGMMGs selected randomly from RNA sequencing were verified by real-time quantitative PCR.Results:1.Cell activity in HG group and MM group were lower than that in LG group.The concentration,purity and integrity of total RNA extracted from each group were excellent,and the results of this sequencing were qualified to meet the requirements of follow-up analysis.2.A total of 726 HGMMGs were identified,including 210 down-HGMMGs and 516 up-HGMMG;which were enriched in cell cycle,oocyte meiotic division,p53 signal pathwayand oxidative phosphorylation.The protein interaction（PPI）network consists of 462 nodes and 2656 connections.MCODE identified four main modules,including 49,12,11 and 8 interacting proteins,respectively.3.The real-time PCR results of differentially expressed genes CDK1,CCNB1,CCNA2,CDK6,RPL11 and CREB1 were consistent with those of RNA-seq,which proved that the RNA-seq results were credible.Conclusion:We identified for first time by RNA-seq that the abnormal expression of mRNAs related to endothelial cell dysfunction in MM after the conversion of environmental glucose concentration to normal level.Cell cycle,oocyte meiosis,p53 signaling pathway and oxidative phosphorylation may be potential therapeutic targets for high glucose induced endothelial cell MM.Further development of the role of these genes in MM may lead to new strategies for the treatment of diabetic vascular complications.Part Ⅱ Study on the differential expression of miRNA in"metabolic memory induced by high glucose in vascular endothelial cellsObjective:The purpose of this study was to analyze the miRNA expression profile of HUVECs in MM by small RNA sequencing,and to find out the significant MM-related differentially expressed miRNA（MMmis）,and to analyze the function of the target gene.Methods:1.The 3rd to 10th passage HUVECs cells were divided into three groups:（1）low glucose control group（LG）,（2）high glucose maintenance group（HG）,（3）metabolic memory group（MM）.The culture conditions of each group were the same as " Part I Study on the differential expression of mRNA in ’ Metabolic memory ’ induced by High glucose in Vascular Endothelial cells".2.Total RNA extraction,purification and quality inspection,cDNA library construction and library quality inspection were the same as " Part Ⅰ Study on the differential expression of mRNA in ’Metabolic memory induced by High glucose in Vascular Endothelial cells”.Single-read was selected for sequencing.The sequencing data were preprocessed by Fastx,Bowtie and other software to prepared for the analysis of differential expression in the later stage3.The differentially expressed genes were analyzed by edgeR,and the differential expression of MMmis induced by high glucose was obtained.（1）The differentially expressed genes were obtained through comparison between HG group and LG group,MM group and LG group（|FC|≥2.0、p<0.05）.（2）The miRNA with no significant difference between MM group and HG group（p≥0.05）.The intersection of（1）and（2）represents the differential expression of MMmis related to MM4.The target genes of differential miRNA were predicted by 3’-terminal UTR with mirwalk and mirtarbase database.The target genes of differential expressed MMmis were analyzed by GO and KEGG with clusterProfiler software package to explore the relationship between MMmis function and MM5.The sequencing results of randomly selected MMmis were verified by qPCR.Results:1.The gene mapping of the sequencing reads was satisfactory.Atotal of 1413 known miRNA and 101 unknown miRNA were identified.The sequencing quantity is sufficient to meet the requirements of subsequent analysis2.A total of 15 MM-related up-regulated miRNA（up-MMmis）and 9 down-regulated miRNA（down-MMmis）were identified.It is predicted that there are 12979 target genes of these MMmis.GO enrichment analysis shows that the functions of these genes are related to regulation of nitrogen compound metabolic process,regulation of metabolic process,protein binding and signal pathway regulation,all these are important biological processes.KEGG analysis showed that the main biological functional pathways enriched in cell adhesion molecule,Wnt signaling pathway,adhesion junction,ErbB signaling pathway and MAPK signaling pathway affecting inflammation and cell proliferation and so on.The abnormal expression of miR-181b-5p,miR-150-5p,miR-370-3p,miR-365a-5p,miR-769-5p,miR-143-3p,hsa-miR-205-5p and hsa-miR-1260b may be involved in the abnormal activity of PI3K pathway,endoplasmic reticulum stress injury and atherosclerosis（AS）formation induced by MM.3.The real-time PCR results of hsa-miR-143-3p,hsa-miR-590-3p,hsa-miR-1307-5p,hsa-miR-205-5p,hsa-miR-375-5p and hsa-miR-192-5p are consistent with those of RNA-seq.Conclusion:We identified the persistent abnormal expression of miRNAs in MM.Some meaningful pathways were condensed:（1）PIK3R3,a common target gene of miR-181b-5p,miR-150-5p,miR-370-3p,miR-365a-5p and miR-769-5p,may be the key target molecule for these abnormally expressed miRNA to participate in the formation of MM.（2）The up-regulated expression of miR-181b-5p and miR-143-3p in MM may be an important role of endoplasmic reticulum stress injury.（3）The down-regulation of mRNA atherosclerotic gene by hsa-miR-375,hsa-miR-205-5p,hsa-miR-1260b,hsa-miR-192-5p et al may play a role in MM injury.Part Ⅲ Study on the differential expression of circular RNA in "metabolic memory induced by high glucose in vascular endothelial cellsObjective:In order to explore the role of competing endogenous RNAs（ceRNA）,in MM.Methods:1.The grouping and culture conditions of HUVECs cells were the same as " Part Ⅰ Study on the differential expression of mRNA in ’ Metabolic memory’ induced by High glucose in Vascular Endothelial cells".2.The sequencing method is the same as " Part Ⅰ Study on the differential expression of mRNA in ’ Metabolic memory ’ induced by High glucose in Vascular Endothelial cells”.The sequencing data were processed with FastQC,BWA-MEM,circBase and other software to prepare for the next step analysis3.The differentially expressed genes were analyzed by edgeR.The MM related circRNA（MMcis）were obtained in accordance with the following criteria:（1）MMcis were significantly different between HG group and LG group,MM group and LG group（|FC|≥2.0、p<0.05）.（2）Meanwhile,get the MMcis with no significant difference between MM group and HG group（p≥0.05）.The intersection of（1）and（2）represents the differential expression of MMcis in MM.The characteristics of parental gene of differential expression MMcis and the predicted circRNA-miRNA interaction were analyzed4.CeRNA analysis based on the actual expression of various RNA.（1）Get differential MMcis（|FC|>2,p<0.05）as above.（2）Predicting the complementary miRNA of differential MMcis.The target gene of miRNA was predicted by mirwalk and mirtarbase database.（3）The correlation between the actual expression values of each RNA was calculated by R package HSMAC.The results of circRNA-mRNA,circRNA-miRNA and miRNA-mRNA were positive correlation,negative correlation and negative correlation,respectively（p<0.05）.（4）Obtained ceRNA pairs with binding potential and expression correlation by intersection of（2）and（3）.Conducted functional analysis of the important ceRNA pairs.5.The randomly selected MMcis sequencing results were verified by qPCR method.Results:1.Sequencing quality control data is the same as“Part I Study on the differential expression of mRNA in ’Metabolic memory ’ induced by High glucose in Vascular Endothelial cells ".9135 circRNAs were annotated in Circbase.Most of them were derived from exons of protein coding genes2.A total of 22 MM-related MMcis were identified,including up-regulated MMcis 12（up-MMmis）and 10 down-regulated MMcis（down-MMmis）Hsa circ 0002317 is an intergenic circRNAs,and the rest of the circRNAs are exon circRNA or intron circRNAs,derived from 21 parental genes.The analysis of parental gene characteristics of hsa_circ₀006137,hsa_circ 0025638 and hsa_circ₀087015 showed that these circRNA synthesis processes may affect the linear RNA expression of their parent genes.Thus,it will affect the formation of AS,apoptosis and senescence,autophagy and other functions of HUVECs in MM.Bioinformatics analysis showed that the 22 MMcis were rich in miRNA response elements（MREs）’So there were many potential ceRNA based on miRNA targeting regulatory genes.We have also established these predictive circRNA-miRNA interaction network diagrams.3.CeRNA mechanism analysis of circRNA,miRNA and mRNA.18 differentially expressed circRNA,15 differentially expressed miRNA and 351 differentially expressed mRNA in MM were obtained.Predictive analysis showed that 38 circRNA-miRNA pairs and 573 miRNA-mRNA pairs were formed.GO and KEGG analysis showed that the following ceRNA should be paid attention to:（1）Hsa circ₀002317,which is highly expressed in MM,indirectly regulates the expression of DEPDC1B/PRKD2/PRR11 and other genes by targeting miR-455-5p/miR-365a-5p/miR-522-3p.It may participate in the regulation of cell cycle and autophagy.（2）The high expression of hsa_circ 0000215 in MM may indirectly up-regulate the expression of CDK1/ID3/AURKA/MAPK1 and other cell cycle proteins or cell cycle regulated kinases by targeting miR-152-5p/miR-205-5p/miR-365a-5p/miR-769-5P.It can affect diabetic vascular complications through endothelial cell proliferation,apoptosis,autophagy and other functional disorders.（3）In MM group,the low expression of hsa circ 0005263,targeting miR-150-5p/miR-504-5p/miR-590-3p/miR-346 can indirectly down-regulated ATP1B1/SAMD4A/GCLM/CDK6/ATP6V1E1/RPL14,etc.These ceRNA effects may affect the maintenance of redox homeostasis and antioxidant response of endothelial cells,resulting in subsequent MM-related damage.4.qPCR verification results confirm that RNA-seq results are reliable.Conclusion:We screened out the persistent abnormal expressed circRNA in MM.and proposed the potential ceRNA regulatory axis:（1）Hsa_circ 0002317,miR-455-5p/miR-365a-5p/miR-522-3p,DEPDC1B/PRKD2/PRR11.（2）Hsa_circ₀000215,miR-152-5p/miR-205-5p/miR-365a-5p/miR-769-5P,CDK1/ID3/AURKA.（3）Hsa circ 0005263,miR-150-5p/miR-504-5p/miR-590-3p/miR-346,ATP1B1/SAMD4A/GCLM/CDK6/ATP6V1E1/RPL14.Part Ⅳ Study on the function of circ-0000215 as miR-769-5p"sponge in metabolic memory of HUVECsObjective:Based on the previous results,it is preliminarily verified whether circ 0000215 in metabolic memory affects the expression of MAPK1 through miRNA and the activity of autophagy.Methods:1.The culture conditions were the same as "Part Ⅰ Study on the differential expression of mRNA in&Metabolic memory’ induced by High glucose in Vascular Endothelial cells".Circ 0000215,miR-769-5p and MAPK1 were verified by qPCR.HUVECs interfered with SiRNA were divided into HG and MM groups respectively for circRNA function analysis.2.The expression of circ 0000215,miR-769-5p and MAPK1 in HG,MM and LG groups were quantitatively detected by qPCR.Circ-0000215 siRNA interference experiments was used to observe the gene expression of circ 0000215,miR-769-5p and MAPK1 in HG group and MM group.Western blot was used to detect the expression of MAPK1,mTORC1 and LC3 protein,and MTT method was used to detect the growth activity of cells.3.According to the sequence of miR-769-5p binding site at the 3’ UTR of MAPK1 or circ 0000215 genes,wt and Mutant pmirGLO recombinant vector plasmids were designed respectively.PmirGLO vectors were used as control,and miR-769-5p mimics or miR-769-5p NC were transfected respectively.The relative luciferase expression of firefly in 293T cells was used to determine whether miR-769-5p was bound to the target gene site.Results:I.qPCR results showed that the expression of circ 0000215 and MAPK1 in HG and MM group were higher than that in LG group（p<0.001）,but the expression of miR-769-5p in LG group was lower than that in LG group（p<0.001）.2.siRNA interference experiments showed that after circ 0000215 siRNA silencing circ₀000215,the expression of circ₀000215 and MAPK1 decreased（p<0.001）,and the expression of miR-769-5p increased（p<0.001）in both HG and MM groups.In both HG and MM groups,after circ 0000215 siRNA interference,the expression of ERK1/2 and mTORC1 in HUVECs decreased,while the expression of LC3 II increased（P<0.01）.MTT test showed that both HG and MM groups could increase cell viability by about 40%after circRNA siRNA intervention（P<0.001）.3.The dual-luciferase reporter gene assay showed that the luciferase activity in the 293T transfected with miR-769-5p mimics+pmirGLO-MAPK1 was significantly lower than that in the group with miR-769-5p NC+pmirGLO-MAPK1（P<0.01）.The same results were found in the double fluorescein reporter gene experiment on whether miR-769-5p binds to the 3’ UTR sequence of circ 0000215.（P<0.01）.The results showed that miR-769-5p could bind directly to the 3 ’UTR binding site of circ 000215 and MAPK1.Conclusion:The change of HUVECs in MM group was the same as that in HG,hsa circ 0000215 and low expression of miR-769-5p in HUVECs and MAPK1.Silencing circ₀000215 can lead to low expression of autophagy pathway protein mTORC1 and high expression of LC3 Ⅱ,which may increase autophagy and promote cell survival.The double fluorescein reporte gene confirmed that miR-769-5p could act on the 3’ UTR binding sites of circ 0000215 and MAPK1 genes.Circ 0000215 and miR-769-5p may be potential targets for the treatment of autophagy in MM.