Transcriptome Sequencing-based Investigation Of The Effect Of Vitamin C On Senescence In Caenorhabditis Elegans Through Insulin Signaling Pathway

Objective Aging is a common characteristic of organisms and a high-risk factor for a variety of diseases,so studying aging is particularly important for overcoming diseases.In order to explore the influence of vitamin C on the aging of Caenorhabditis elegans,a model of vitamin C-treated Caenorhabditis elegans was constructed,and genes related to the possible regulation of life pathway were found.The concentration gradients of vitamin C in the blank control group(0 mg/mL vitamin C),0.4 mg/mL,0.8 mg/mL and1.6 mg/mL were set to detect the relationship between vitamin C and the life span of Caenorhabditis elegans.The blank control group and the treatment group used Illumina Hi Seq to sequence the gene transcriptome,and the differential gene expression analysis was excavated by the Gene Ontology(GO)and the Kyoto Encyclopedia of Genes and Genomes(KEGG)to excavate the mtl-2,ctl-1,ctl-2 and sek-4 genes.They express significant upregulation and participate in the regulation of life-related pathways in Caenorhabditis elegans.The study found that the lifespan of Caenorhabditis elegans was significantly longer than that of other concentrations under 0.8 mg/mL vitamin C treatment,and the expression levels of genes and proteins related to the insulin signaling pathway of Caenorhabditis elegans were detected by qRT-PCR and Western Blotting,which provided a theoretical basis for delaying the aging process.Methods Firstly,NGM medium and OP50 E.coli broth were prepared.After the vitamin C mother liquor was mixed with OP50 bacterial solution at different concentrations,100μL was added dropwise into NGM medium and transferred into Caenorhabditis elegans in NGM medium and fed in a constant temperature incubator at 20 ℃.Under sterile conditions,the Caenorhabditis elegans in the oviposition period were synchronized,and the eggs were cultured in a constant temperature and humidity incubator at 20 ℃ for about 64 h to mature.The mature Caenorhabditis elegans were transferred into the blank control group(0 mg/mL vitamin C group),0.4 mg/mL vitamin C group,0.8 mg/mL vitamin C group,1.6 mg/mL vitamin C group NGM medium,and the optimal vitamin C concentration was selected by life observation experiment.Total RNA was extracted from Caenorhabditis elegans tissue samples,c DNA was reverse transcribed by RNA reverse transcription,adaptor was connected,and finally sequenced by Illumina.After sequencing,RSEM software was used to analyze the expression levels and differences among different samples,and the results were used to draw the volcano map of differential gene expression.After screening the differential gene expression,the GO database was used to annotate the differentially expressed genes,and the results were used to draw the GO annotation histogram.The KEGG database was used to analyze the function of differential genes,and the KEGG annotation histogram was drawn according to the classification of signal pathways.Four differentially expressed genes in lifespan of Caenorhabditis elegans screened between 0.8 mg/mL vitamin C group and control group were detected by qRT-PCR.The expression levels of related genes and proteins in the insulin signaling pathway of Caenorhabditis elegans were detected by qRT-PCR and Western Blotting.Results1.Vitamin C affects the lifespan of Caenorhabditis elegans.2.Under 0.8 mg/mL vitamin C treatment,the lifespan of Caenorhabditis elegans was significantly longer than that of other concentrations.3.Differential expression gene screening indicated that the presence of differentially expressed genes may be involved in the regulation of life expectancy of Caenorhabditis elegans.4.GO analysis suggests that differential genes may be involved in the regulation of oxidative aging in Caenorhabditis elegans.5.KEGG pathway analysis showed that four differentially expressed genes,mtl-2,ctl-1,ctl-2 and sek-4,were significantly up-regulated and involved in the regulation of lifespan in Caenorhabditis elegans.6.qRT-PCR detection showed that the expression of mtl-2,ctl-1 and ctl-2 genes in the 0.8mg/mL vitamin C treatment group was stable and significantly upregulated compared with the control group,which was located downstream of the signature gene daf-16 of aging in the insulin signaling pathway of Caenorhabditis elegans.7.In the insulin signaling pathway of Caenorhabditis elegans,qRT-PCR detection showed that daf-16 gene expression was upregulated;The age-1,daf-2 and akt-2 genes were located upstream of the daf-16 gene and their expression was down-regulated.The sod-3 gene is located downstream of the daf-16 gene and its expression is upregulated.8.In the insulin signaling pathway of Caenorhabditis elegans,Western Blotting detection showed that the expression of akt-2 and p-akt-2 proteins was down-regulated.The expression of ctl-1,ctl-2 and sod-3 proteins is upregulated.Conclusion By constructing a vitamin C treatment model of Caenorhabditis elegans,the concentration gradients of blank control group(0 mg/mL vitamin C),0.4 mg/mL,0.8mg/mL and 1.6 mg/mL vitamin C were set up,and the life observation experiment was carried out,and 0.8 mg/mL was selected as the optimal vitamin C concentration.Illumina Hi Seq was used to sequence the transcriptome of genes,and the four genes of mtl-2,ctl-1,ctl-2 and sek-4 were mined through the GO database and KEGG database,and their expression was significantly upregulated and participated in the regulation process of liferelated pathways in Caenorhabditis elegans.qRT-PCR detection showed that the expression of mtl-2,ctl-1 and ctl-2 genes in the 0.8 mg/mL vitamin C treatment group was stable and significantly upregulated compared with the control group,which was located downstream of the signature gene daf-16 of aging in the insulin signaling pathway of Caenorhabditis elegans.The expression levels of related genes and proteins in the insulin signaling pathway of Caenorhabditis elegans were detected,and qRT-PCR detection showed that daf-16 gene expression was up-regulated.The age-1,daf-2 and akt-2 genes were located upstream of the daf-16 gene and their expression was downregulated.The sod-3 gene was located downstream of the daf-16 gene and its expression was upregulated.Western Blotting detection showed that akt-2 and p-akt-2 gene expression were down-regulated.The expression of ctl-1,ctl-2 and sod-3 genes is upregulated.This study explores the effect of vitamin C on the aging of Caenorhabditis elegans,and provides a theoretical basis for delaying the aging.