Study On The Mechanism Of NiSO₄ Exposure Induced Cytotoxicity In HUVECs Based On Transcriptome And Proteomics

Objective:A model of HUVECs treated with Ni SO₄ was established,transcriptional sequencing and proteomics analysis were conducted,and multi-component bioinformatics analysis was used to reveal the gene differential expression in HUVECs after exposure to Ni SO₄ to explore the possible molecular mechanism of NISO₄-induced cellular toxicity in HUVECs in order to provide experimental clues for further exploring the mechanism of Ni SO₄ cellular toxicity in cardiovascular system.Methods:（1）HUVECs cultured in vitro during 5-10 generations of logarithmic growth were selected,and 1000μmol/L Ni SO₄ of the final concentration in culture medium was choose to expose to HUVECs for 24 h;（2）Detection of the effect of Ni SO₄on the survival rate of HUVECs cells using CCK-8 reagent kit;（3）The cells were treated with Trizol and centrifuged in sterile,enzyme-free EP tubes to produce cell precipitates,which were frozen in liquid nitrogen and sent for full-length transcriptome sequencing and proteomic analysis by Biomaker Biotechnology Co.;（4）Differential transcript and protein analysis,KEGG,GO and GSEA analysis were performed on transcriptomic and proteomic data using R software,Web Gestalt database and Biomaker Bio Cloud platform;（5）Cross-linking analysis of transcriptomic and proteomic data using Biomaker Bio Cloud Platform,R software to find associated quantitative genes;（6）Using the String and mcode plugins in Cytoscape software to construct PPI for quantitative genes and screen out KEGG functional subnetworks with the top differentially expressed genes;（7）PPI construction of HIF-1 signaling pathway and endoplasmic reticulum protein processing KEGG pathway using Cytoscape software and cytohubba plug-in,and screening of the top five key genes;（8）Histological assay results were validated by PCR and WB:m RNA expression levels of TIMP-1,LDHA,DNAJC3,ERO1A,DDIT3,HSP90B1,ATF6B,BAK1 and PDHA1were detected by PCR;WB determined the protein expression levels of DNAJC3,HMOX1,HSP90B1 and TIMP-1 protein expression levels.Results:（1）The IC50 of Ni SO₄ on HUVECs cells was determined to be 931.4μM in CCK-8 assay,and the next highest cytotoxic dose of 1000μM was selected as the dose for this study;（2）According to the sample quality control results,the biological duplicate samples within the same group were highly similar,with significant differences between the control and contaminated group samples:（1）Transcriptome sequencing results showed that the full-length nucleic acid sequences of all samples were above 80%and the data quality was above Q12;（2）Principal component analysis of proteomic samples as well as spatial principal component analysis showed good reproducibility of samples within groups and significant differences between groups;（3）Transcriptome sequencing found 2736 differentially expressed transcripts between groups,1377 up-regulated transcripts,and 1359 down regulated transcripts,including783 newly annotated transcripts.GO functional analysis found that differentially expressed transcripts were mainly enriched in cell process,single organism process,metabolic process,biological regulation,response to stimulation and other items;On the cell group stratification plane,it is mainly concentrated in cells,cell components,organelle,molecular complexes,cell membranes and other items;At the molecular functional level,it is mainly enriched in terms of binding,catalytic activity,molecular function regulation,structural molecular interaction,nucleic acid binding transcription factor activity,and other items.Traditional KEGG and GSEA based KEGG pathway annotation analysis showed that differentially expressed transcripts were mainly enriched in oxidative phosphorylation,HIF-1 signaling pathway,endoplasmic reticulum protein processing,Parkinson’s disease and other functional pathways;（4）Proteome sequencing obtained 115 differentially expressed proteins,of which 44 were upregulated and 71 were downregulated.GO functional analysis found that the differentially expressed proteins mainly participated in cell process,single organism process,biological regulation,metabolic process and response to stimulation and other biological processes;It is enriched and expressed in cells,cell membranes,cell components,molecular complexes and organelle;Participated in cellular functions such as binding,catalytic activity,molecular functional regulation,structural molecular interactions,and transduction activity.Traditional KEGG pathway annotation analysis showed that differentially expressed proteins were mainly enriched in complement and coagulation cascades,endoplasmic reticulum protein processing,AGE-RAGE signaling pathway in diabetes complications,interaction of ECM receptors,HIF-1signaling pathway,amebiasis,systemic lupus erythematosus and other annotation items;（5）A total of 43 differentially expressed quantitative genes were found by multiomic analysis.Comprehensive KEGG pathway annotation analysis was carried out for transcriptome and proteome,and the main enrichment pathway HIF-1 signal pathway and endoplasmic reticulum protein processing were screened out;（6）Four proteins and nine transcripts in the HIF-1 signaling pathway and endoplasmic reticulum protein processing were randomly screened and subjected to WB and Rt-q PCR tests,which showed that the trend expression was consistent with the sequencing results;（7）PPI construction was performed on differentially expressed quantitative genes in the HIF-1signaling pathway,and five key genes HIF-1A,VEGFA,LDHA,PGK1,and SLC2A1were screened.Based on GO analysis of differentially expressed genes of HIF-1signaling pathway and literature search,the possible toxicity mechanism points to aerobic glycolytic process;（8）The quantitative genes of endoplasmic reticulum protein processing differential expression were constructed by PPI,and five key genes HSPA5,P4HB,DNAJC3,CANX and EDEM1 were screened out.Based on GO analysis of endoplasmic reticulum protein processing differentially expressed genes and literature search,the possible toxicity mechanism points to endoplasmic reticulum stress.Conclusion:（1）Validation experiments of the histological results showed that the results of transcriptome sequencing and proteome analysis were accurate and reliable;（2）Through database annotation and bioinformatics analysis,multi-omics cross-linking analysis finally revealed that differentially expressed quantitative genes were significantly enriched in HIF-1 signaling pathway and endoplasmic reticulum protein processing KEGG annotation pathway,and their associated aerobic glycolysis and endoplasmic reticulum stress mechanisms may play an important role in the toxicity mechanism of HUVECs caused by Ni SO₄,which can be used as a later stage in exploring the toxicity mechanism of Ni SO₄ in HUVECs toxicity mechanism,which could be an important direction in the later exploration of Ni SO₄ toxicity mechanism.