| Objective:In order to comprehensively explore the molecular toxicity regulation mechanism of fluoride exposure-induced myocardial injury at the transcriptional and protein levels,bioinformatics techniques were used to explore the changes of relevant signal transduction pathways and the interaction between genes.Methods:The rat model of subchronic fluorosis(0,30,60,90 mg/L)was established and the rat myocardial tissue samples were sequenced by RNA-Seq and Itraq technology.After passing quality control,bioinformatics was used for systematic and in-depth joint omics analysis.The specific analysis methods are as follows:(1)Identification and quantitative expression profile construction: Firstly,format conversion,data filtering and quality control of the original downloading sequence were carried out,and then the processed data were compared and identified with the reference database of specific organisms.Finally,quantification of transcripts/genes and proteins were completed by statistical model and abundance identification respectively.(2)Cluster analysis: R software was used for Euclidean distance calculation,Z-score standardization processing and image drawing of quantitative genes in each omics and each fluoride-treatment group,to complete cluster analysis of the relationship between similarity and difference of associated quantitative genes.(3)Principal component analysis: R software was used to project the quantitative values of potentially related genes to a new coordinate system without linear relationship for data dimension reduction,and the principal component analysis of similarity and difference between omics and treatment groups was completed.(4)Differential expression analysis: Based on the quantitative expression results of proteomics and transcriptomics,the threshold of the difference multiple and statistical index is defined.The genes / proteins meeting the above conditions is regarded as the genes / proteins with significant difference expression.(5)Enrichment analysis: DAVID analysis system combined with String database was used to annotate,enrich and analyze the function and pathway of differentially expressed molecules.(6)Gene interaction network analysis: STRING database and Cytoscape software were used to analyze the interaction between genes with joint differential expression.(7)Correlation analysis: Pearson correlation coefficients of inter-genes and inter-omics were calculated using R software to construct correlation coefficient heat maps.(8)Immunohistochemical analysis: The selected core differential proteins in the pathway were analyzed by immunohistochemical analysis to verify the reliability of sequencing results.Results:(1)Joint-omics analysis of the regulatory effect on differentially expressed molecules and the screening of co-enrichment pathways by fluoride exposure.Based on the joint-omics analysis,the three fluoride-treatment groups of low,Middle and high fluoride were matched to 900,903 and 898 associated quantitative genes,respectively.The results of clustering heat map and principal component analysis showed that the degree of gene expression between different fluoride-treatment groups of the same omic showed dose-dependence with fluoride concentration,however,there were significant differences in the expression of the same genes between different omics.The results of the differential analysis showed that 173,118 and 129 differentially expressed proteins(DEPs)were screened in the three fluoride-treatment groups of low,Middle and high,respectively,and 372,235 and 254 differentially expressed genes(DEGs)were obtained at the transcriptional level,respectively,of which 96 DEGs and 33 DEPs were co-expressed in the three fluoride-treatment groups.GO functional and KEGG pathway co-enrichment analysis were performed on the above differentially expressed molecules,and the GO functional analysis results showed that DEGs and DEPs were associated with 74,448 and 116 GO functional entries in cellular components,biological processes and molecular functional dimensions,while 40 co-enrichment pathways including oxidative phosphorylation and cardiac contraction pathways were successfully localized in the KEGG pathway co-enrichment analysis.(2)Joint-omics analysis of the regulatory effects of fluoride exposure on differential genes related to the oxidative phosphorylation pathway.Based on the screening results of the previous part,we extracted the expression profiles of oxidative phosphorylation pathway at protein and transcription levels respectively,and finally obtained 22 differentially expressed genes related to oxidative phosphorylation.In addition to COX7 c,other genes were significantly down regulated at protein level.At the functional level,GO annotation results showed that most of the above genes were located in mitochondria and respiratory chain complexes,involved in complex assembly,electron / proton transport and other oxidative phosphorylation processes,and regulated the activity of related proteins and the synthesis of various functional enzymes.The results of KEGG pathway showed that the above mitochondrial respiratory chain related genes were also differentially expressed in KEGG pathways with high energy demand,such as Parkinson’s disease and Alzheimer’s disease.At the gene level,the results of gene interaction network and correlation analysis further showed the close interaction between the genes of each complex,in which fluoride exposure had a more significant regulatory effect on the genes of respiratory chain complex I.Finally,the expression of Ndufa10 was verified by immunohistochemistry,and the results were the same as the sequencing results,that is,compared with the control group,the expression of Ndufa10 in the fluoride-treatment group was significantly down regulated.(3)Joint-omics analysis of the regulatory effects of fluoride exposure on differential genes related to the myocardial contraction pathway.Similarly to the second part,12 differentially expressed genes related to myocardial contraction were identified in this section.Among them,11 genes are jointly involved in oxidative phosphorylation,Alzheimer’s disease,Parkinson’s disease and metabolism related pathways,and 3 genes are jointly involved in cardiovascular disease related pathways related to abnormal myocardial contraction,such as dilated cardiomyopathy and hypertrophic cardiomyopathy.Most of the above 12 genes are located in cytoplasm,membrane and intracellular space through GO functional annotation,which can participate in calcium mediated signal transduction and myocardial contraction regulation through the action of various enzyme activities.The expression of Acta2 、 Ry R2 and Tnni3 in dilated cardiomyopathy,hypertrophic cardiomyopathy and other cardiovascular diseases were observed in KEGG pathway enrichment and gene interaction network,and their correlation coefficients were significantly positive.Finally,the central protein Ry R2 was selected for immunohistochemical verification.The results further verified the expression of differential genes of myocardial contraction,indicating the adverse effects of fluoride exposure on myocardial contraction.Conclusion:(1)Based on the results of high-throughput sequencing and combined omics analysis,it was found that long-term fluoride exposure can lead to different degrees of differential expression of genes at transcriptome and proteomic levels,which has a significant regulatory role in cardiac function related pathways,including oxidative phosphorylation and myocardial contraction.(2)Long-term fluoride exposure can interfere with the assembly and biological activity of mitochondrial complexes by down-regulating the expression of oxidative phosphorylation-related genes represented by Ndufa10,thereby affecting the energy transport during ATP synthesis.(3)Long term fluoride exposure can induce the differential expression of genes related to myocardial contractile signaling pathway,resulting in cardiac function damage and myocardial contractile dysfunction.Among them,Acta2 and Tnni3,which are involved in various heart disease pathways,and Ry R2 and ATP1a1,which regulate intracellular and extracellular ion balance,may be the key proteins of fluoride induced myocardial contractile dysfunction. |
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