Placenta-specific novel splice variants of Rho GDP dissociation inhibitor β are highly expressed in cancerous cells

Abstract

Background: Alternative splicing of pre-mRNA transcripts not only plays a role in normal molecular processes but is also associated with cancer development. While normal transcripts are ubiquitously expressed in normal tissues, splice variants created through abnormal alternative splicing events are often expressed in cancer cells. Although the Rho GDP dissociation inhibitor β (ARHGDIB) gene has been found to be ubiquitously expressed in normal tissues and involved in cancer development, the presence of splice variants of ARHGDIB has not yet been investigated.

Results: Validation analysis for the presence of and exon structures of splice variants of ARHGDIB, performed using reverse-transcriptase polymerase chain reaction and DNA sequencing, successfully identified novel splice variants of ARHGDIB, that is, 6a, 6b, and 6c, in colon, pancreas, stomach, and breast cancer cell lines. Quantitative real-time polymerase chain reaction analysis showed that these variants were also highly expressed in normal placental tissue but not in other types of normal tissue.

Conclusions: Expression of ARHGDIB variants 6a, 6b, and 6c appears to be restricted to cancer cells and normal placental tissue, suggesting that these variants possess cancer-specific functions and, as such, are potential cancer-related biomarkers.

Figure 1

ARHGDIB expression pattern in cell lines and normal tissues. (A) ARHGDIB expression profile in colon, pancreas, stomach, and breast cancer cell lines. Amplicons (i), (ii), (iii), and (iv) correspond to the 4 types of transcripts shown in Panel B. (B) Exon structures on the ARHGDIB gene. The 5 forms shown include the known transcript NM_001175 (i), 3 novel variants (ii–iv), and the known variant ARHGDIB-006 predicted in Ensembl (v). The grey boxes show novel alternative splicing forms in variant 6a (ii), 6b (iii), and 6c (iv). The † and * symbols indicate the locations of the start codon and the stop codon, respectively. The number of exons is shown on the white and grey boxes. Primer positions for RT-PCR and qRT-PCR are indicated by black and grey arrows, respectively.

Figure 2

Comparison of splicing events in normal tissues. The copy number of transcripts was estimated by determining the mean of the values obtained by qRT-PCR analysis performed in duplicate. The left and right panels show the expression patterns of normal ARHGDIB and variant 6a, respectively.

Figure 3

Expression analysis of ARHGDIB at the mRNA and protein levels in colon cancer cell lines. (A) The expression levels of known ARHGDIB transcripts and variant 6a transcripts were analysed by qRT-PCR. (B) Protein expression was confirmed by immunoblotting with the ARHGDIB-specific monoclonal antibody. Immunoblotting of tubulin, α 4a (TUBA4A) protein, was conducted as a positive control experiment. The colon cancer cell lines Colo741, DLD-1, and SW948 were examined as representative cancer cell lines whose expression profiles of variant 6a differs from that of the normal transcript. Data are expressed as mean ± SD values calculated from information obtained from an experiment repeated 4 times. Statistical significance (*p < 0.05 and **p < 0.01) was evaluated by Student’s t-test.

Figure 4

Stability of ARHGDIB variant 6a mRNA and normal ARHGDIB mRNA. SW948 cells were cultured without (A) or with (B) actinomycin D (ActD). Amplification of actin β (ACTB) was conducted as a positive control experiment. The expression levels of transcripts before addition of ActD are shown as 100%. Data are expressed as mean ± SD values calculated from information obtained from an experiment repeated 4 times. Statistical significance (**p < 0.01) was evaluated by Student’s t-test.