Expression Profile Analysis of the Cell Cycle in Diploid and Tetraploid Carassius auratus red var

Abstract

Polyploidization often leads to "transcriptome shock," and is considered an important factor in evolution of species. Analysis of the cell cycle, which is associated with survival in polyploidy, has proved useful in investigating polyploidization. Here, we used mRNA sequencing to investigate global expression in vitro (in cultured cells) and in vivo (in fin and liver tissues) in both the diploid and tetraploid Carassius auratus red var.. Differential expression (DE) of genes in diploid (7482, 36.0%) and tetraploid (3787, 18.2%) states suggested that in vitro and in vivo conditions dramatically change mRNA expression levels. However, of the 20,771 total shared expressed genes, 18,050 (87.0%), including 17,905 (86.2%) non-differentially expressed genes (DEGs) and 145 (0.7%) DEGs between diploids and tetraploids, showed the same expression trends in both cultured cells and liver tissues. Of the DEGs, four of seven genes in the cell cycle pathway had the same expression trends (upregulated in diploids and tetraploids) in both cultured cells and liver tissues. Quantitative PCR analysis confirmed the same expression trends in the nine DEGs associated with regulation of the cell cycle. This research on common characteristics between diploids and tetraploids provides insights into the potential molecular regulatory mechanisms of polyploidization. The steady changes that occur between diploids and tetraploids in vitro and in vivo show the potential value of studying polyploidy processes using cultured cell lines, especially with respect to cell cycle regulation.

Keywords: cell cycle; in vitro; in vivo; mRNA expression; polyploidy.

FIGURE 1

Strategy of the expression analysis and expression cluster in all samples. (A) mRNA-seq and qPCR methods were used to determine the expression levels in cultured cells, caudal fin tissues, and liver tissues. The comparison of “vs. 1” and “vs. 2” was used to assess the DE of in vivo and in vitro between diploids and tetraploids. The comparison of “vs. 3” and “vs. 4” was used to assess the DE of diploids and tetraploids between in vivo and in vitro. (B) Overall clustering of 12 samples including diploid and tetraploid liver tissues, and diploid (2N) and tetraploid (4N) cultured cells, using normalized count data calculated by Cufflinks. The heatmap drawn from all gene count data for the reference genome depicts the relationships of all transcriptomes.

FIGURE 2

Differentially expressed genes (DEGs) between diploid and tetraploid states in cultured cells and liver tissues. (A) The distribution of DEGs in cultured cells. (B) The distribution of DEGs in liver tissues. (C) Shared genes with no DE in cultured cell and liver samples. Log₂ counts per million (CPM). (D) Shared upregulated genes in diploid cultured cells and liver samples. (E) Shared upregulated genes in tetraploid cultured cells and liver samples.

FIGURE 3

Shared DEGs between diploids and tetraploids in cultured cells and liver tissues. The 145 shared DEGs, including 38 that were upregulated in 2N and 107 that were upregulated in 4N, were detected from the comparison between diploid and tetraploid states.

FIGURE 4

Distribution of differentially expressed genes (DEGs) of the cell cycle pathway (dre04110) in diploid and tetraploid states in vitro and in vivo. Green symbols represent upregulated expression in 2N cultured cells, yellow symbols represent upregulated expression in 4N cultured cells, red symbols represent upregulated expression in 2N liver, and blue symbols represent upregulated expression in 4N liver. Three genes (ep300a, myc, and gadd45) show the same DE trends between diploids and tetraploids. However, three other genes (smad4a, cul1a, and tp53) exhibited the opposite DE trends.

FIGURE 5

Expression levels of 11 genes detected by mRNA-seq and qPCR. (A) Heatmap of the expression distribution of 11 as detected by mRNA-seq in cultured cells and liver tissues. (B–L) The expression levels of 11 genes detected by qPCR in cultured cells and caudal fin.