Using an exon microarray to identify a global profile of gene expression and alternative splicing in K562 cells exposed to sodium valproate

Abstract

To investigate the effect of valproate treatment on the K562 cell line, a model for chronic myelogenous leukaemia, the growth and survival of the K562 cell line were investigated using the Annexin-V/PI dual staining method, and global profiles of gene expression and alternative splicing in K562 cells were assessed using exon microarrays. A significant increase in cell apoptosis was observed in valproate-exposed K562 cells using flow cytometry. A total of 628 transcripts were identified as being significantly differentially expressed. The number of genes demonstrating increased expression levels was greater than the number of genes demonstrating decreased expression levels (445 genes vs. 183 genes, respectively). The significant enrichment analysis of GO terms for the differentially expressed genes revealed that these genes are involved in many important biological processes such as apoptosis. Six of the genes observed to be differentially expressed that might be involved in apoptosis were selected to undergo qRT-PCR validation. In total, 198 candidates of alternative splicing variants were identified. Among them, three alternative splicing events were selected for validation, and CBLC and TBX1 were confirmed to be alternatively spliced by semi-nested PCR. In conclusion, valproate exposure facilitated cell apoptosis, altered mRNA expression and alternative splicing events in the K562 cell line.

Figure 1

Flow cytometry analysis of apoptosis induced by exposure to 2 mM of VPA for 48 h in K562 cells using Annexin-V/PI.

Figure 2

Alterations in the expression levels identified by microarray were confirmed using qRT-PCR. Fold-changes between K562 cells treated with and without vaproate detected by microarray were compared with those measured by qRT-PCR. In the qRT-PCR assay, RNA was isolated from the K562 cell line treated with or without valproate exclusively for validation of differentially expressed genes and AS genes. mRNA levels were normalized with GADPH and fold-changes were calculated by dividing the mRNA levels of each K563 cell line treated with vaproate by mean mRNA levels from control samples in triplicate. Data are mean values ± standard deviations of the means from three independent experiments performed in triplicate.

Figure 3

Validation of alternative splicing using a semi-nested PCR assay. Total RNA, isolated from K562 cell line treated with or without valproate exclusively for validation of differentially expressed genes and AS genes, was reverse transcribed with random hexanucleotide primers using M-MLV reverse transcriptase. Following reverse transcription, two rounds of PCR were performed with reverse primers that target the predicted exon rather than the constitutive exon.