Placenta-specific novel splice variants of Rho GDP dissociation inhibitor β are highly expressed in cancerous cells
- Keiichi Hatakeyama†1Email author,
- Yorikane Fukuda†1, 4,
- Keiichi Ohshima1,
- Masanori Terashima2,
- Ken Yamaguchi3 and
- Tohru Mochizuki1
© Hatakeyama et al.; licensee BioMed Central Ltd. 2012
Received: 28 September 2012
Accepted: 30 November 2012
Published: 3 December 2012
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.
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.
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.
KeywordsAlternative splicing ARHGDIB Biomarker Metastasis Splice variant
Alternative splicing of pre-mRNA transcripts is an important process by which genomic complexity is generated from the small number of genes currently estimated to be present within the human genome sequence. Alternative splicing not only plays a role in normal molecular processes but is also associated with cancer development [1–5]. While normal transcripts are ubiquitously expressed in normal tissues [6, 7], splice variants created through abnormal alternative splicing events are often expressed in cancer cells. When the different expression patterns of a splice variant and the normal transcript on the same gene can be detected, the variant is considered a potential biomarker.
The Rho GDP dissociation inhibitor β gene, commonly referred to as ARHGDIB but also known as LyGDI, GDI-D4, RhoGDI2, or RhoGDIβ, inhibits dissociation of GDP from Rho family GTPases. ARHGDIB has been reported to be involved in either the progression or suppression of metastasis [8–17] and is also ubiquitously expressed in normal tissues . Despite these recent findings, the presence of splice variants of ARHGDIB in cancerous cell lines has not yet been investigated. Employing validation analysis using RT-PCR and DNA sequencing, this study aimed to fill this research gap by investigating alternative splicing events in cancer cell lines and normal tissues to identify splice variants of ARHGDIB. The analysis showed the presence of 3 novel splice variants of the ARHGDIB gene, and their expression was observed only in cancer cell lines and normal placenta. However, no protein isoforms were translated from these variants since their protein-coding regions were identical.
Cell lines and culture conditions
The cell lines used in this study are listed in Additional file 1: Table S1. The MKN45P cell lines  were kindly provided by Dr. Yutaka Yonemura and Dr. Yoshio Endo. These cell lines were cultured and maintained in RPMI 1640, DMEM/F12, or DMEM medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 5–10% foetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), glutamine (0.3 mg/ml), penicillin (100 unit/ml), and streptomycin (0.1 mg/ml), in a humidified 5% CO2 incubator. Protein expression and mRNA analyses were performed using cells found to have 70–90% confluency and greater than 95% viability, as determined using trypan blue staining.
RNA extraction and cDNA synthesis
After total RNA had been extracted from each pellet using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), RNA concentration was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and total RNA quality was then confirmed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The purified total RNA from cancer cell lines and total RNA derived from normal tissues (Clontech, Mountain View, CA, USA) were then reverse-transcribed using ThermoScript Reverse Transcriptase (Invitrogen) and oligo(dT)20 primers in accordance with the manufacturer’s instructions. The normal tissues that were purchased are listed in Additional file 1: Table S2.
DNA sequences of RT-PCR, quantitative RT-PCR, and DNA sequencing primers
Sense primer (location)
Antisense primer (location)
RT-PCR and DNA sequencing
ACTCAGAAGTCAGAGTTGAGAGAC (exon 1)
AGAGAATTCTTCCAGGTGGCAAGG (exon 6/6x)
variant 6 series
ACTGGGCTTTTGCTTCATTTGTTCAG (exon 1x/y/z)
AGAGAATTCTTCCAGGTGGCAAGG (exon 6/6x)
CGACTGGAGCACGAGGACACTGA (5′ RACE)
ACGAGTGAAGCAACATGGCACAAAG (exon 1x/y/z)
ACTGGGCTTTTGCTTCATTTGTTCAG (exon 1x/y/z)
GCTGTCAACGATACGCTACGTAACG (3′ RACE)
AGGAGTCTTTGTGCCATGTTGCTTCAC (exon 1x/y/z)
CGCTACGTAACGGCATGACAGTG (3′ RACE)
GGACACTGACATGGACTGAAGGAGTA (5′ RACE)
ACTCCTGAACAAATGAAGCAAAAGCCCC (exon 1x/y/z)
ATTCCTATGTGGGCGACGAGGC (exon 3)
TGGATAGCAACGTACATGGCTGG (exon 4)
GGACAGAGACGTGAAGCACTGA (exon 1)
GCTTGCTGTCCAGCTCATCA (exon 2)
GTCTTTGTGCCATGTTGCTTCA (exon 1x/y/z)
AGCTTGCTGTCCAGCTCATCA (exon 2)
TGGCACCCAGCACAATGA (exon 5)
CCGATCCACACGGAGTACTTG (exon 6/6x)
Standard curves in quantitative RT-PCR
ACTCAGAAGTCAGAGTTGAGAGAC (exon 1)
AGAGAATTCTTCCAGGTGGCAAGG (exon 6/6x)
ACTGGGCTTTTGCTTCATTTGTTCAG (exon 1x/y/z)
ACTCTCACAAACCAGGGTGAGC (exon 3)
Rapid amplification of cDNA ends and DNA sequencing analysis
Quantitative real-time PCR
Quantitative real-time PCR (qRT-PCR) was performed using the SYBR Green dye technique and the ABI PRISM 7900HT Fast Real Time PCR System (Applied Biosystems). The target fragment among the synthesized cDNA was amplified using specific primers (Table 1) according to the following protocol: preheating at 95°C for 20 sec; 40 cycles at 95°C for 1 sec and 60°C for 30 sec; and dissociation at 95°C for 15 sec, 60°C for 15 sec, and 95°C for 15 sec. The threshold cycle (Ct) values were converted into absolute copy numbers using a standard curve. This standard curve was constructed using data for purified PCR amplicons that were generated from the plasmids containing the sequences of the target transcripts. These conversions for quantification were performed for data obtained at high PCR amplification efficiencies (95–105%). Nonspecific amplification in qRT-PCR was checked by dissociation curve analysis and gel electrophoresis, which showed the amplicon size.
SW948, Colo741, and DLD-1 cells were harvested using a trypsin–EDTA solution (Invitrogen) and washed twice with phosphate-buffered solution (PBS, pH 7.4). After removal of the supernatant, cellular pellets were ultrasonicated in PBS with a protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Centrifugation of the lysates at 15,000 g for 10 min was followed by collection of the supernatant for determination of protein concentration using the Quick Start Bradford Protein Assay Kit (Bio-Rad, CA, USA). The quantified lysates were then subjected to one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using 5 μg of lysate protein/well on 12.5% polyacrylamide gel. After electroblotting of the lysate onto polyvinylidene difluoride membranes (pore size, 0.45 μm; Millipore, Bedford, MA, USA), the blots were probed with a mouse monoclonal anti-ARHGDIB/D4-GDI antibody (1:500 dilution; Acris Antibodies GmbH, Herford, Germany) and a rabbit polyclonal anti-TUBA4A antibody (1:5,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The bound antibodies were incubated with the corresponding anti-IgG secondary antibodies conjugated with horseradish peroxidase (1:50,000 dilution; Jackson Laboratories, Bar Harbor, ME, USA). Specific proteins were visualized using the ECL Plus Western Blotting Detection System (GE Healthcare, Little Chalfont Buckinghamshire, England) and the FUJIFILM Luminescent Image Analyzer LAS3000 (Fuji Film, Tokyo, Japan).
mRNA stability assay
To assay mRNA stability, actinomycin D was added to the culture medium when the SW948 cells reached 80–90% confluence. During the following 24 h, cells were harvested at 0, 2, 4, 6, 8, 10, or 24 h, and total RNA was extracted as described above. The amount of target transcripts (known form of ARHGDIB and variant 6a) and actin β (ACTB) at each time point was quantified using qRT-PCR.
Identification of novel ARHGDIB exons
ARHGDIB is known to be involved in the progression of metastasis [10, 12, 14–17] in colon, pancreas, stomach, and breast cancer. Therefore, systematic RT-PCR screening of 46 cancer cell lines and of normal colon, pancreas, stomach, breast, and placental tissue was conducted using newly designed RT-PCR primers for several exons on the target gene and a primer pair spanning the region from exon 1 to exon 6 on the known ARHGDIB transcript registered in GenBank (NM_001175; Figure 1A). RT-PCR screening revealed positive signals (i) in 36 cell lines (78%) and all 4 types of normal tissue whose lengths were identical to those of the known transcript. The RT-PCR subsequently performed using another primer pair designed for annealing within exon 1 of the variant ARHGDIB-006 (ENST00000541644), which is predicted in the Ensembl and Havana models (http://www.ensemb.org), did not result in detection of the expected band. Likewise, other splice forms whose detection had been anticipated were not observed. Although other bands (ii–iv) were observed in 35 cell lines (76%), these bands were not detected in all the normal tissues. These results indicate that the unknown ARHGDIB transcripts are expressed along with the known ubiquitously expressed ARHGDIB transcripts only in cancer cells.
To determine the exon structures of the unknown transcripts (ii-iv), designated as variants 6a, 6b, and 6c, respectively (Figure 1B), DNA sequencing was performed using RACE. Although the spliced forms were found to consist of 6 exons, as seen in the predicted variant (ARHGDIB-006), their first and last exons differed from those of the predicted variant (Figure 1B). Specifically, the first exons (exon 1x–z) were derived from the intronic region in the known transcript and the sixth exons (exon 6x) from a part of exon 6 identical to the known transcript, while exons 2 to 5 were common in all the novel transcripts. Although the exon structures of these novel and known variants, along with the known transcript, were found to vary, no differences were observed in their protein-coding regions.
ARHGDIB splice variants in normal tissues
Validation of ARHGDIB mRNA and protein expression levels
Stability of variant 6a mRNA
Alternative splicing, a phenomenon by which a single gene generates multiple forms of mRNA that can be translated into diverse proteins , is responsible for an estimated 10–20% of all cancer-related gene alterations [5, 22, 23]. Both the ubiquitous normal transcripts and the unique splice variants generated from these alteration events are frequently expressed simultaneously in cancerous cells and tissues [6, 7, 24]. As such, those variants expressed as cancer-specific transcripts are possible candidate biomarkers [6, 7]. This study revealed that while the splice variants 6a, 6b, and 6c of the ARHGDIB gene are expressed in pancreas, stomach, colon, and breast cancer cell lines and in normal placental tissue but not in other types of normal tissue, the known form of ARHGDIB is ubiquitously expressed in many types of normal tissue and cancer cells. A similarly restricted profile has also been observed for PLAC1. PLAC1, reported to be the first member of a new class of antigens that specifically relates placentation to cancer [25, 26], has been observed to be ectopically activated in many human cancers and is essentially involved in malignant cell processes . These ARHGDIB variants can be considered potential biomarkers based on the similarity between ARHGDIB and PLAC1 reported in previous studies, as well as the observation of high levels of expression of the variants 6a, 6b, and 6c in placental and cancer cells and their relatively restricted expression in normal tissues in this study.
Splice variants are often translated into structurally defective proteins in cancer cells, affecting the functioning of the original protein [7, 27]. In a previous study, the authors of this study focused on examining splice variants and variant-derived protein isoforms to identify candidate biomarkers . However, the current study revealed that the variants 6a, 6b, and 6c and the normal transcript share a common protein-coding region and that these variants are slightly translated into the intact ARHGDIB protein. These results suggest that the structurally defective protein isoforms of ARHGDIB derived from the variant 6 series are not expressed in cancer cells and the placenta, thereby indicating that the mRNA of the cancer-specific variant 6 series may be a more suitable biomarker than the ARHGDIB protein.
In general, mRNA stability is related to UTR length as well as secondary structure , as both influence the regulation of protein expression . In this study, the 5′ and 3′ UTRs of the variant 6 series were observed to clearly differ from those of the normal ARHGDIB, resulting in the decreased mRNA stability of these variants, while the translation efficiency of the variants into intact ARHGDIB protein was observed to be distinctly low. These findings suggest that the mRNA stability of the novel variants identified in this study may be involved in the regulation of protein expression.
In summary, validation analysis using RT-PCR and DNA sequencing successfully identified variants 6a, 6b, and 6c—3 novel splice variants of the ARHGDIB gene—in colon, pancreas, stomach, and breast cancer cell lines; these variants were also found to be highly expressed in normal placental tissue. In contrast, the mRNA expression of known ARHGDIB was ubiquitous in many normal tissues and cancer cells. These findings suggest that these cancer and placenta-specific variants possess cancer-specific functions and, as such, are potential cancer-related biomarkers.
This research was supported by a Grant-in-Cooperation from the Regional Innovation Cluster Program 2010, Grant-in-Aid for Young Scientists (B) (No. 23701092, 2011), and Grant-in-Aid for Scientific Research (C) (No. 22591472, 2010) from the Ministry of Education, Culture, Sports, Science and Technology. The authors thank Dr. Miyuki Murata for her special advice and Yuko Watanabe, Tomomi Ide, and Kaori Kanto for their kind technical assistance.
- Becker KF, Atkinson MJ, Reich U, Becker I, Nekarda H, Siewert JR, Hofler H: E-cadherin gene mutations provide clues to diffuse type gastric carcinomas. Cancer Res. 1994, 54: 3845-3852.PubMedGoogle Scholar
- Gayther SA, Barski P, Batley SJ, Li L, de Foy KA, Cohen SN, Ponder BA, Caldas C: Aberrant splicing of the TSG101 and FHIT genes occurs frequently in multiple malignancies and in normal tissues and mimics alterations previously described in tumours. Oncogene. 1997, 15: 2119-2126. 10.1038/sj.onc.1201591.PubMedView ArticleGoogle Scholar
- Ge K, DuHadaway J, Du W, Herlyn M, Rodeck U, Prendergast GC: Mechanism for elimination of a tumor suppressor: aberrant splicing of a brain-specific exon causes loss of function of Bin1 in melanoma. Proc Natl Acad Sci U S A. 1999, 96: 9689-9694. 10.1073/pnas.96.17.9689.PubMedPubMed CentralView ArticleGoogle Scholar
- Barbour AP, Reeder JA, Walsh MD, Fawcett J, Antalis TM, Gotley DC: Expression of the CD44v2-10 isoform confers a metastatic phenotype: importance of the heparan sulfate attachment site CD44v3. Cancer Res. 2003, 63: 887-892.PubMedGoogle Scholar
- Faustino NA, Cooper TA: Pre-mRNA splicing and human disease. Genes Dev. 2003, 17: 419-437. 10.1101/gad.1048803.PubMedView ArticleGoogle Scholar
- Hatakeyama K, Ohshima K, Fukuda Y, Ogura S, Terashima M, Yamaguchi K, Mochizuki T: Identification of a novel protein isoform derived from cancer-related splicing variants using combined analysis of transcriptome and proteome. Proteomics. 2011, 11: 2275-2282. 10.1002/pmic.201100016.PubMedView ArticleGoogle Scholar
- Dorard C, de Thonel A, Collura A, Marisa L, Svrcek M, Lagrange A, Jego G, Wanherdrick K, Joly AL, Buhard O, Gobbo J, Penard-Lacronique V, Zouali H, Tubacher E, Kirzin S, Selves J, Milano G, Etienne-Grimaldi MC, Bengrine-Lefèvre L, Louvet C, Tournigand C, Lefèvre JH, Parc Y, Tiret E, Fléjou JF, Gaub MP, Garrido C, Duval A: Expression of a mutant HSP110 sensitizes colorectal cancer cells to chemotherapy and improves disease prognosis. Nat Med. 2011, 17: 1283-1289. 10.1038/nm.2457.PubMedView ArticleGoogle Scholar
- Gildea JJ, Seraj MJ, Oxford G, Harding MA, Hampton GM, Moskaluk CA, Frierson HF, Conaway MR, Theodorescu D: RhoGDI2 is an invasion and metastasis suppressor gene in human cancer. Cancer Res. 2002, 62: 6418-6423.PubMedGoogle Scholar
- Harding MA, Arden KC, Gildea JW, Gildea JJ, Perlman EJ, Viars C, Theodorescu D: Functional genomic comparison of lineage-related human bladder cancer cell lines with differing tumorigenic and metastatic potentials by spectral karyotyping, comparative genomic hybridization, and a novel method of positional expression profiling. Cancer Res. 2002, 62: 6981-6989.PubMedGoogle Scholar
- Ota T, Maeda M, Suto S, Tatsuka M: LyGDI functions in cancer metastasis by anchoring Rho proteins to the cell membrane. Mol Carcinog. 2004, 39: 206-220. 10.1002/mc.20006.PubMedView ArticleGoogle Scholar
- Theodorescu D, Sapinoso LM, Conaway MR, Oxford G, Hampton GM, Frierson HF: Reduced expression of metastasis suppressor RhoGDI2 is associated with decreased survival for patients with bladder cancer. Clin Cancer Res. 2004, 10: 3800-3806. 10.1158/1078-0432.CCR-03-0653.PubMedView ArticleGoogle Scholar
- Zhang Y, Zhang B: D4-GDI, a Rho GTPase regulator, promotes breast cancer cell invasiveness. Cancer Res. 2006, 66: 5592-5598. 10.1158/0008-5472.CAN-05-4004.PubMedView ArticleGoogle Scholar
- Ma L, Xu G, Sotnikova A, Szczepanowski M, Giefing M, Krause K, Krams M, Siebert R, Jin J, Klapper W: Loss of expression of LyGDI (ARHGDIB), a rho GDP-dissociation inhibitor, in Hodgkin lymphoma. Br J Haematol. 2007, 139: 217-223. 10.1111/j.1365-2141.2007.06782.x.PubMedView ArticleGoogle Scholar
- Abiatari I, DeOliveira T, Kerkadze V, Schwager C, Esposito I, Giese NA, Huber P, Bergman F, Abdollahi A, Friess H, et al: Consensus transcriptome signature of perineural invasion in pancreatic carcinoma. Mol Cancer Ther. 2009, 8: 1494-1504. 10.1158/1535-7163.MCT-08-0755.PubMedView ArticleGoogle Scholar
- Cho HJ, Baek KE, Park SM, Kim IK, Choi YL, Nam IK, Hwang EM, Park JY, Han JY, Kang SS, Kim DC, Lee WS, Lee MN, Oh GT, Kim JW, Lee CW, Yoo J: RhoGDI2 expression is associated with tumor growth and malignant progression of gastric cancer. Clin Cancer Res. 2009, 15: 2612-2619. 10.1158/1078-0432.CCR-08-2192.PubMedView ArticleGoogle Scholar
- Tan AC, Jimeno A, Lin SH, Wheelhouse J, Chan F, Solomon A, Rajeshkumar NV, Rubio-Viqueira B, Hidalgo M: Characterizing DNA methylation patterns in pancreatic cancer genome. Mol Oncol. 2009, 3: 425-438. 10.1016/j.molonc.2009.03.004.PubMedView ArticleGoogle Scholar
- Xu SG, Yan PJ, Shao ZM: Differential proteomic analysis of a highly metastatic variant of human breast cancer cells using two-dimensional differential gel electrophoresis. J Cancer Res Clin Oncol. 2010, 136: 1545-1556. 10.1007/s00432-010-0812-0.PubMedView ArticleGoogle Scholar
- Iinuma H, Maruyama K, Okinaga K, Sasaki K, Sekine T, Ishida O, Ogiwara N, Johkura K, Yonemura Y: Intracellular targeting therapy of cisplatin-encapsulated transferrin-polyethylene glycol liposome on peritoneal dissemination of gastric cancer. Int J Cancer. 2002, 99: 130-137. 10.1002/ijc.10242.PubMedView ArticleGoogle Scholar
- Emani S, Zhang J, Guo L, Guo H, Kuo PC: RNA stability regulates differential expression of the metastasis protein, osteopontin, in hepatocellular cancer. Surgery. 2008, 143: 803-812. 10.1016/j.surg.2008.02.005.PubMedPubMed CentralView ArticleGoogle Scholar
- Pavithra L, Sreenath K, Singh S, Chattopadhyay S: Heat-shock protein 70 binds to a novel sequence in 5′ UTR of tumor suppressor SMAR1 and regulates its mRNA stability upon prostaglandin A2 treatment. FEBS Lett. 2010, 584: 1187-1192. 10.1016/j.febslet.2010.02.025.PubMedView ArticleGoogle Scholar
- Black DL: Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem. 2003, 72: 291-336. 10.1146/annurev.biochem.72.121801.161720.PubMedView ArticleGoogle Scholar
- Blencowe BJ: Alternative splicing: new insights from global analyses. Cell. 2006, 126: 37-47. 10.1016/j.cell.2006.06.023.PubMedView ArticleGoogle Scholar
- Greenman C, Stephens P, Smith R, Dalgliesh GL, Hunter C, Bignell G, Davies H, Teague J, Butler A, Stevens C, Edkins S, O’Meara S, Vastrik I, Schmidt EE, Avis T, Barthorpe S, Bhamra G, Buck G, Choudhury B, Clements J, Cole J, Dicks E, Forbes S, Gray K, Halliday K, Harrison R, Hills K, Hinton J, Jenkinson A, Jones D, et al: Patterns of somatic mutation in human cancer genomes. Nature. 2007, 446: 153-158. 10.1038/nature05610.PubMedPubMed CentralView ArticleGoogle Scholar
- Thorsen K, Sorensen KD, Brems-Eskildsen AS, Modin C, Gaustadnes M, Hein AM, Kruhoffer M, Laurberg S, Borre M, Wang K, Brunak S, Krainer AR, Tørring N, Dyrskjøt L, Andersen CL, Orntoft TF: Alternative splicing in colon, bladder, and prostate cancer identified by exon array analysis. Mol Cell Proteomics. 2008, 7: 1214-1224. 10.1074/mcp.M700590-MCP200.PubMedView ArticleGoogle Scholar
- Koslowski M, Sahin U, Mitnacht-Kraus R, Seitz G, Huber C, Tureci O: A placenta-specific gene ectopically activated in many human cancers is essentially involved in malignant cell processes. Cancer Res. 2007, 67: 9528-9534. 10.1158/0008-5472.CAN-07-1350.PubMedView ArticleGoogle Scholar
- Silva WA, Gnjatic S, Ritter E, Chua R, Cohen T, Hsu M, Jungbluth AA, Altorki NK, Chen YT, Old LJ, Simpson AJ, Caballero OL: PLAC1, a trophoblast-specific cell surface protein, is expressed in a range of human tumors and elicits spontaneous antibody responses. Cancer Immun. 2007, 7: 18-PubMedPubMed CentralGoogle Scholar
- Karni R, de Stanchina E, Lowe SW, Sinha R, Mu D, Krainer AR: The gene encoding the splicing factor SF2/ASF is a proto-oncogene. Nat Struct Mol Biol. 2007, 14: 185-193. 10.1038/nsmb1209.PubMedPubMed CentralView ArticleGoogle Scholar
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