1. Genome Information Laboratory
Molecular characterization of the DYX1C1 gene and its application as a colorectal cancer biomarker
Yun-Ji Kim1*, Jae-Won Huh1,2*, Dae-Soo Kim3, Min-In Bae1, Tae-Oh Kim4, Ja-Rang Lee1, Hong-Seok Ha1, Kung Ahn1, Yu-Na Noh1, Sang-Je Park1, Jin-Han Bae1, Yi-Deun Jung1 and Heui-Soo Kim1,3§
1 Division of Biological Sciences, College of Natural Sciences, Pusan National University, Busan , Republic of Korea
2 National Primate Research Center (NPRC), KRIBB, Ochang, Chungbuk , Republic of Korea
3 PBBRC, Interdisciplinary Research Program of Bioinformatics, College of Natural Sciences, Pusan National University, Busan , Republic of Korea
4Division of Gastroenterology Department of Internal Medicine, College of Medicine Pusan National University, Pusan National University Hospital, Busan , Republic of Korea
Abstract
DYX1C1 has three alternatively spliced transcripts. So we expect that alternative transcripts of DYX1C1 are used as a biomarker to detect specific cancer. RT-PCR analysis is conducted in order to detect expression of the DYX1C1 gene and the PCR products were analyzed using the Image J program to compare the expression levels of each transcript. We found one of the transcripts was directly associated with an HERV-H LTR element that could be translated into protein sequence. Four new alternative transcripts were identified by RT-PCR analysis with various human tissue samples including 5 normal and adjacent tumor tissue sets. Semi-quantitative RT-PCR analysis showed the transcriptional activity of V3 and V2 was higher in normal than in tumor tissue samples, especially in the colorectal tissue samples. Our results indicated that alternatively spliced transcript variants of the
DYX1C1 gene could be used as cancer biomarkers to detect colorectal cancer ……………………………………………………………………………………………………………….
Introduction
Materials & Methods
Biomarker
Bioinformatics
( in silico analysis DYX1C1 gene )
The identification of cancer biomarkers has been a major focus of investigation in the field of cancer research. However, effective and reproducible cancer biomarker developments are still far off and enormous efforts are needed in this area (Brinkman et al. 2004). In cancerous cells, splicing mechanisms are significantly altered by defects in splice sites caused by frequent point mutations (Venables et al. 2004; Skotheim et al. 2007). Aberrant neuronal-specific splicing of amphiphysin 2 in non-neuronal cells may contribute to the progression human melanoma (Ge et al. 1999). Expression levels of alternatively spliced variants of survivin are not equal to each other in malignant breast tissues. In the case of breast cancer, different survivin transcripts show different functions in the progression of apoptosis (Ryan et al. 2005; Pajares et al. 2007). Our study focused on the application of cancer biomarkers using transcription variants.
PCR and RT – PCR in human tissues
and various normal/tumor sets
Alignment & Image J analysis
Application to colorectal patients
transformation
Plasmid isolation
ligation
Electrophoresis
PCR, RT-PCR
Human genomic DNA
inoculation
DNA
vector
Bioinformatics
Sequencing analysis
DYX1C1
DYX1C1 is a recently identified candidate gene for dyslexia. Disruption of the gene by a translocation was detected in dyslexia patients (Taipale et al. 2003; McGrath et al. 2006). The DYX1C1 gene maps to chromosome 15q21 and consists of 10 exons dispersed over about 78 kb of genomic DNA. The protein sequence encoded by DYX1C1 is 420 aa in length and the protein sequences of the nonhuman primate homolog are % similar to that of humans (Taipale et al. 2003). DYX1C1 is expressed broadly in several adult tissues including lung, kidney, and brain (Taipale et al. 2003; Wang et al. 2006), where its protein is localized in white matter including the nuclei of cortical neurons and glial cells (Taipale et al. 2003; Fisher and Francks 2006). Eight single nucleotide polymorphisms (SNPs) in the DYXICI genes of 20 patients with reading disabilities were detected (Taipale et al. 2003; McGrath et al. 2006). The 1249G-to-T and -3G-to-A mutations were among the SNPs found more frequently in patients with dyslexia than in controls (Fisher and Francks 2006). Developmental dyslexia is associated with migration anomalies in the neocortex as well genetic susceptibility (Pennington and Smith 1983; Galaburda et al. 1985; Smith et al. 1998; Chang et al. 2005; Sokol et al. 2006). DYX1C1 plays a role in the migration of neocortical neurons. Specifically it is required for the transition of the multipolar stage of migration (Wang et al. 2006).
Kim et al. Molecular characterization of the DYX1C1 gene and its application as a cancer biomarker.
J Cancer Res Clin Oncol in press
Results and Discussion
Fig.1 Spliced variants of the DYX1C1 gene and their structural analysis
Fig.2 Expression patterns of HERV-H LTR-associated (A) and -unassociated (B) DYX1C1 transcripts in normal human tissues. G3PDH indicates the positive control.
Fig.4 Comparative analysis of the other transcripts (V2, V3) in normal (N) and tumor (T) samples. G3PDH indicates the positive control 10
EXON1 2 3 4 5 6 7 8 9
NM_
NM_ V2 V3
NM_
NEW TRANSCRIPTS V1
V1-1
V1-2
V1-3
V1-4 7’ 8’ S1
AS1 S2
AS2
Fig. 3,4 - (b) RT-PCR was conducted three times and G3PDH was used as a control. PCR products were analyzed quantitatively using the Image-J program. The X-axis of the bar graph indicates normal (N)/tumor (T) tissue samples. Lanes: M, size marker; 1, colon (N); 2, colon (T); 3, liver (N); 4, liver (T); 5, uterus (N); 6, uterus (T); 7, breast (N); 8, breast (T); 9, stomach (N); 10, stomach (C) and the Y-axis of the bar graph indicates the relative expression levels of 2 transcripts (V2 and V3). P < 0.05 in
Student’s t-test is indicated by *.
Fig.3 Comparative analysis of HERV-H LTR-associated DYX1C1 transcripts (V1, V1-1, V1-2, V1-3, V1-4) in normal and tumor tissue samples. G3PDH indicates the positive control.
Fig.5 Comparative analysis of the other transcripts (V2, V3) in normal (N) and tumor (T) samples of colorectal cancer patients. G3PDH indicates the positive control
The DYX1C1 gene is shown to have three alternatively spliced transcripts in the GenBank database. V1, V2, and V3 are the originally reported transcripts, and V1-1, V1-2, V1-3, and V1-4 are newly identified alternative transcripts.
References
1. Adran A, Yoshida A, Ishikawa K, Goi T, Yamaguchi A, Ueda T, Inuzuka M. (2004) Identification of a novel splice variant of the human anti-apoptopsis gene survivin. Biochem Biophys Res
Commun. 314:
2. Brinkman BM. (2004) Splice variants as cancer biomarkers. Clin Biochem. 37:
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