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Alternative CD44 splicing identifies epithelial prostate cancer cells from the mesenchymal counterparts.

ABSTRACT: An epithelial to mesenchymal transition (EMT) has been shown to be a necessary precursor to prostate cancer metastasis. Additionally, the differential expression and splicing of mRNAs has been identified as a key means to distinguish epithelial from mesenchymal cells by qPCR, western blotting and immunohistochemistry. However, few markers exist to differentiate between these cells by flow cytometry. We previously developed two cell lines, PC3-Epi (epithelial) and PC3-EMT (mesenchymal). RNAseq was used to determine the differential expression of membrane proteins on PC3-Epi/EMT. We used western blotting, qPCR and flow cytometry to validate the RNAseq results. CD44 was one of six membrane proteins found to be differentially spliced between epithelial and mesenchymal PC3 cells. Although total CD44 was positive in all PC3-Epi/EMT cells, PC3-Epi cells had a higher level of CD44v6 (CD44 variant exon 6). CD44v6 was able to differentiate epithelial from mesenchymal prostate cancer cells using either flow cytometry, western blotting or qPCR.

SUBMITTER: Hernandez JR

PROVIDER: S-EPMC4391735 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:As a signaling molecule and an inhibitor of histone deacetylases (HDACs), butyrate exerts its impact on a broad range of biological processes, such as apoptosis and cell proliferation, in addition to its critical role in energy metabolism in ruminants. This study examined the effect of butyrate on alternative splicing in bovine epithelial cells using RNA-seq technology. Junction reads account for 11.28 and 12.32% of total mapped reads between the butyrate-treated (BT) and control (CT) groups. 201,326 potential splicing junctions detected were supported by ? 3 junction reads. Approximately 94% of these junctions conformed to the consensus sequence (GT/AG) while ~3% were GC/AG junctions. No AT/AC junctions were observed. A total of 2,834 exon skipping events, supported by a minimum of 3 junction reads, were detected. At least 7 genes, their mRNA expression significantly affected by butyrate, also had exon skipping events differentially regulated by butyrate. Furthermore, COL5A3, which was induced 310-fold by butyrate (FDR <0.001) at the gene level, had a significantly higher number of junction reads mapped to Exon#8 (Donor) and Exon#11 (Acceptor) in BT. This event had the potential to result in the formation of a COL5A3 mRNA isoform with 2 of the 69 exons missing. In addition, 216 differentially expressed transcript isoforms regulated by butyrate were detected. For example, Isoform 1 of ORC1 was strongly repressed by butyrate while Isoform 2 remained unchanged. Butyrate physically binds to and inhibits all zinc-dependent HDACs except HDAC6 and HDAC10. Our results provided evidence that butyrate also regulated deacetylase activities of classical HDACs via its transcriptional control. Moreover, thirteen gene fusion events differentially affected by butyrate were identified. Our results provided a snapshot into complex transcriptome dynamics regulated by butyrate, which will facilitate our understanding of the biological effects of butyrate and other HDAC inhibitors.

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Alternative CD44 splicing identifies epithelial prostate cancer cells from the mesenchymal counterparts.

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