Splice variants of zinc finger protein 695 mRNA associated to ovarian cancer

Abstract

Background: Studies of alternative mRNA splicing (AS) in health and disease have yet to yield the complete picture of protein diversity and its role in physiology and pathology. Some forms of cancer appear to be associated to certain alternative mRNA splice variants, but their role in the cancer development and outcome is unclear.

Methods: We examined AS profiles by means of whole genome exon expression microarrays (Affymetrix GeneChip 1.0) in ovarian tumors and ovarian cancer-derived cell lines, compared to healthy ovarian tissue. Alternatively spliced genes expressed predominantly in ovarian tumors and cell lines were confirmed by RT-PCR.

Results: Among several significantly overexpressed AS genes in malignant ovarian tumors and ovarian cancer cell lines, the most significant one was that of the zinc finger protein ZNF695, with two previously unknown mRNA splice variants identified in ovarian tumors and cell lines. The identity of ZNF695 AS variants was confirmed by cloning and sequencing of the amplicons obtained from ovarian cancer tissue and cell lines.

Conclusions: Alternative ZNF695 mRNA splicing could be a marker of ovarian cancer with possible implications on its pathogenesis.

Figure 1

Overlapping gene expression patterns in healthy ovary and benign tumors that differ from ovarian cancer. Principal component analysis (PCA) depicting gene expression profiles of healthy (HOT, green), benign ovarian tumors (BOT, red), tumor (MOT, purple) and ovarian cell lines (OCL, blue).

Figure 2

Hierarchical clustering of ovarian cancer gene. Heat maps depicting hierarchical clustering of gene expression in healthy ovarian tissue, benign ovarian tumours, ovarian cancer and ovarian cancer cell lines. Graphics were generated by means of Partek Genomics Suite™ software. Differentially expressed gene values were included only when fold-change was > 3 and false discovery ratio < 0.005 and only the number of genes clustering to over- expression (top), no significant changes (middle) and under-expression (bottom) are shown. Gene names, RefSeq, and other details are shown in Additional file 2.

Figure 3

Differential ZNF695 exon usage in ovarian cancer. A) All possible splice variants of zinc finger protein (ZNF) 655 mRNAs retrieved from UCSC Genome Browser. B) Graphic depicting levels of individual exon expression in healthy ovarian tissue (HOT, green), benign ovarian tissue (BOT, red), malignant ovarian tumours (MOT, purple) and ovarian cancer cell lines (OCL, blue). C) Heat map of each of the peaks containing the individual samples.

Figure 4

ZNF695 transcripts of healthy ovarian tissue, individual ovarian cancer samples, and cell lines. A) Agarose gel showing the raw RT-PCR products obtained from one HOT (n = 6) sample and MOT (n = 7) samples. Above, RPL4 protein (housekeeping), below, ZNF695. B) RT-PCR products run on a DNA Chip Agilent bioanalyzer. Top row: housekeeping gene RPL4. Middle and bottom rows: individual transcripts of each of the differentially spliced mRNAs in malignant ovarian cancer tissue (MOT, n = 10) and individual ovarian cancer cell lines (n = 4, shown by their names).

Figure 5

Cloning and sequencing of novel ZNF695 transcripts associated to ovarian cancer. A) PCR product of ZNF695 showing the three different RNA transcript sizes of ZNF695 expressed in malignant ovary tissue sample 6 (MOT6, right) and the individual amplicons used for cloning, with the size (in base pairs) shown on the left. Ladder (line 1), ZNF695 splice variants 1 and/or 2 (lane 2, 400 bp), ZNF695 splice variant 4 (lane 3, 360 bp), ZNF695 splice variant 5 (lane 4, 310 bp), and ZNF695 amplicons in MOT6 (line 5). B) Sanger sequence plot of ZNF695 splice variant 1 and 2 at the boundary of exons 1 and 2. C) Sanger sequence plot of ZNF695 splice variant 1 and 2 at the boundary of exons 2 and 3. D) Sanger sequence plot of ZNF695 splice 5. This transcript has alternative splicing in exon 1–2, with the arrow illustrating the alternative splicing site at the exon 1–2 boundary. E) Sanger sequence plot of ZNF695 splice variant 4. This sequence has alternative site splicing in exon 2–3 (arrows), this sequence are contain in ZNF695 variant 5 too. F) AS model of ZNF695 gene. Black lines represent primary splicing of exons 1-2-3 of ZNF695 gene containing the full coding sequence that corresponds to the largest amplicon. Green dotted line shows exons 2–3 AS, which corresponds to the intermediate amplicon. Red dotted line shows exon 1 AS together with exons 2–3 AS, which are contained in the smaller amplicon.