Effects of µ-Conotoxin GIIIB on the cellular activity of mouse skeletal musculoblast: combined transcriptome and proteome analysis

Abstract

µ-Conotoxin GIIIB (µ-CTX GIIIB) is a polypeptide containing three disulfide bridges, produced by the sea snail Conus geographus. This study was aimed to explored the cytotoxic effects of µ-CTX GIIIB on mouse skeletal musculoblast (Sol8). Sol8 cells were exposed to ouabain and veratridine to establish the cell injury model, and then treated with µ-CTX GIIIB. CCK-8 was adopted to evaluate the cytotoxicity of µ-CTX GIIIB. Then, proteomics and transcriptome were conducted, and the explore the differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) affected by µ-CTX GIIIB were found. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was used to investigate the affected signaling pathways. µ-CTX GIIIB increased the cell survival rate of injured Sol8 cells. We found and identified 1,663 DEGs and 444 DEPs influenced by µ-CTX GIIIB. 106 pairs of correlated DEGs and DEPs were selected by combining transcriptome and proteome data. The results of KEGG and GO analysis showed that µ-CTX GIIB affected the cell cycle, apoptosis, DNA damage and repair, lipid metabolism and other biological processes of Sol8 cells. µ-CTX GIIIB could affected cell cycle regulation, DNA damage repair, and activation of tumor factors, with potential carcinogenic effects. Our results provide an important basis for the study of in vitro toxicity, the mechanism of toxicity and injury prevention by µ-CTX GIIIB.

Keywords: Conotoxins; Proteomics; Transcriptomics; µ-Conotoxin GIIIB.

Fig. 1

µ-CTX GIIIB protects the Sol8 cells from the cell injury induced by ouabain and veratrine (O/V). a The effects of ouabain and veratrine on cell survival rate of Sol8 cells was detected by CCK-8. *P < 0.05 vs. 0 group; (b) The effects of µ-CTX GIIIB on cell survival rate was detected by CCK-8. *P < 0.05 vs. Control group, ^#P < 0.05 vs. O/V group

Fig. 2

The DEGs and DEPs were found and identified by transcriptome and proteome analysis. a The Difference statistics of the DEGs. Red indicates upregulated genes and green indicates downregulated genes. b The Volcano plot of the DEGs. Compared with the control group, the X-axis represents the fold change between groups, and the Y-axis represents the p value difference of DEGs. The gray dots represent genes with no significant change, the red dots represent genes that are up-regulated, and the green dots represent genes that are down-regulated. c Scatter diagram of the DEPs, in which the x-axis and y-axis respectively represent the protein expression levels in two samples, and each point represents a specific protein. The closer a point is to 0, the lower the expression level is. The farther a point is to 0, the higher the expression level is. The gray dots represent proteins with no significant change, the red dots represent proteins that are up-regulated, and the green dots represent proteins that are down-regulated. d Volcano plot of the DEPs. The X-axis represents the fold change between two groups, and the Y-axis represents the p value difference of DEPs. The gray dots represent proteins with no significant change, the red dots represent proteins that are up-regulated, and the green dots represent proteins that are down-regulated

Fig. 3

The comprehensive analysis of transcriptome and proteome. a Venn diagram of DEGs and DEPs. The green part represents DEGs, red represents DEPs, and blue represents associated genes and proteins. b Associated data Venn diagram. The diagram contains the up-regulated and down-regulated genes and proteins in associated data

Fig. 4

GO annotation analysis of DEGs and DEPs. Proteins and genes were divided into three major categories: biological processes, cellular components, and molecular functions. Green bars represent proteins and purple bars represent genes. The Y-axis (left) indicates the percentage of proteins or genes contained in the secondary classification to the total number of proteins or genes in the secondary classification, and the Y-axis (right) indicates the number of proteins or genes annotated to the secondary classification

Fig. 5

KEGG pathway analysis of DEGs and DEPs. a The Signal pathways affected by DEGs and DEPs. The borderless bars represent proteins and the borderless bars represent genes. The x axis indicates the number of genes and proteins that are matched, and the y-axis shows pathways in the KEGG classification. b The p53 signaling pathway influenced by DEGs and DEPs. In the figure, all green background boxes represent matched proteins of the candidate species, gray background boxes represent matched genes, and orange background boxes represent matched genes or proteins at the same time. The blue border represents the protein set and the red border represents the gene set. The red and blue border indicates the set of genes and proteins that are matched simultaneously

Fig. 6

qRT-PCR validation of nine DEGs. The mRNA expression of CDK2, CCNB1, CCNB2, TSP1, Plk1, BRCA1, Taok2, Acot2, Vegfa, Tk1, Plin2, Hmgcs1, Rrm2b, Aurkb, Ero1l, Ctnnal1, and Kif20a were detected by qRT-PCR. Red bars represent the data of qRT-PCR and black bars represent the RNAseq data. Cyclin dependent kinase 2 (CDK2); cyclin B1 (CCNB1); cyclin B2 (CCNB2); thrombospondin 1 (TSP1); polo like kinase 1 (Plk1); breast cancer type 1 susceptibility protein (BRCA1); TAO kinase 2 (Taok2); acyl-CoA thioesterase 2 (Acot2); vascular endothelial growth factor A (Vegfa); thymidine kinase 1 (Tk1); perilipin 2 (Plin2); 3-hydroxy-3-methylglutaryl -coenzyme A synthase 1 (Hmgcs1); ribonucleotide reductase M2 B (Rrm2b); aurkb aurora kinase B (Aurkb); endoplasmic reticulum oxidoreductase 1 alpha (Ero1l); catenin (cadherin associated protein)alpha-like 1 (Ctnnal1); kinesin family member 20 A (Kif20a)

Fig. 7

Validation of DEPs by PRM. The protein expression was detected by PRM analysis. Red bars represent the data of PRM and black bars represent the TMT data. ENSMUSP00000026416 (CDK2); ENSMUSP00000034742 (CCNB1); ENSMUSP00000071989 (CCNB2); ENSMUSP00000153208 (TSP1); ENSMUSP00000033154 (PLK1); ENSMUSP00000017290 (BRCA1); ENSMUSP00000112963 (Taok2); ENSMUSP00000021649 (Acot2); ENSMUSP00000115883 (Vegfa); ENSMUSP00000026661 (Tk1); ENSMUSP00000000466 (perilipin 2); ENSMUSP00000040694 (recombination signal binding protein for immunoglobulin kappa J region); ENSMUSP00000044903 (thrombospondin 1); ENSMUSP00000136944 (3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1); ENSMUSP00000026221 (stearoyl-Coenzyme A desaturase 2); ENSMUSP00000033283 (ribonucleotide reductase M1); ENSMUSP00000020980 (ribonucleotide reductase M2)