The transcriptome of wild-type and immortalized corneal epithelial cells

Abstract

Cellular immortalization enables indefinite expansion of cultured cells. However, the process of cell immortalization sometimes changes the original nature of primary cells. In this study, we performed expression profiling of poly A-tailed RNA from primary and immortalized corneal epithelial cells expressing Simian virus 40 large T antigen (SV40) or the combination of mutant cyclin-dependent kinase 4 (CDK4), cyclin D1, and telomere reverse transcriptase (TERT). Furthermore, we studied the expression profile of SV40 cells cultured in medium with or without serum. The profiling of whole expression pattern revealed that immortalized corneal epithelial cells with SV40 showed a distinct expression pattern from wild-type cells regardless of the presence or absence of serum, while corneal epithelial cells with combinatorial expression showed an expression pattern relatively closer to that of wild-type cells.

Fig. 1

The workflow of RNA-seq of wild-type, K4DT, and SV40 immortalized corneal epithelial cells. (a), The workflow of RNA-seq. (b) Mapping ratio and total number of reads for each sample. (c) Correlation matrix plots of all samples. (d) Three-dimensional PCA of wild-type, K4DT, and SV40 cells.

Fig. 2

Heat map of cell cycle-related genes in the KEGG map and pathways related to cancer.

Fig. 3

Mapping of upregulated or downregulated genes related to the cell cycle pathway. In bar plots, 2× upregulated or 1/2 downregulated genes were mapped in the KEGG pathway. Upper arrows indicate upregulated genes and lower arrows indicate downregulated genes in the corresponding sample.

Fig. 4

Mapping of upregulated or downregulated genes in the cell cycle pathway. In bar plots, 2× upregulated or 1/2 downregulated genes were mapped in the KEGG pathway. Upper arrows indicate upregulated genes and lower arrows indicate downregulated genes in the corresponding sample.

Fig. 5

Morphologies of wild-type, K4DT, and SV40 immortalized corneal epithelial cells. (a) Wild-type (left panel) and K4DT immortalized corneal epithelial cells (right panel). (b) Morphology of SV40 immortalized corneal epithelial cells with serum (left panels) and without serum (right panels).

Fig. 6

PCR genotyping and detection of gene products by western blotting. (a) PCR products amplified from CDK4, cyclin D1, SV40, TERT, and endogenous TSC2 genes are listed. (b) Western blot analysis of anti-CDK4, SV40, cyclin D1.

Fig. 7

Analysis of cycle progression in wild-type, K4DT, and SV40 immortalized corneal epithelial cells. Representative results were shown.