Expression Levels of BIRC5 Isoforms in Different Neuroblastoma Cell Lines




Haiquan Lu

Introduction

BIRC5 (also called Survivin) is a member of inhibitor of apoptosis (IAP) gene family that exhibits differential expression in nearly all human cancers but not in most normal tissues (Altieri, 2003). The human BIRC5 gene, spanning 14.7 kb on telomeric position of chromosome 17, produces a 16.5 kDa protein (Ambrosini et al., 1998). This protein has one N-terminal baculoviral IAP repeat (BIR) domain, a long C-terminal α-helix coiled region, and a dimeric arrangement (Verdecia et al., 2000).

BIRC5 exists in several isoforms as the result of alternatively spliced transcripts (Mahotka et al., 1999). These isoforms include full-length BIRC5, BIRC5-2B, and BIRC5-ΔEx-3. They may have differential localization, expression, and function in tumor cells (Kappler et al., 2001; Krieg et al., 2002). The BIRC5 gene has a three-intron, four-exon structure. The BIRC5-2B transcript results from the retention of a portion of intron 2, whereas BIRC5-ΔEx-3 transcript results from the removal of exon 3 (Fig 1).

The sequence alteration produced from the splice variants result in marked changes in the corresponding protein structure and differences in their ability to inhibit apoptosis (Mahotka et al., 2002). In this study, the ratio of different isoforms of BIRC5 is tested in SH-EP1 and SK-N-ER cell lines by RT-PCR.

Figures


Figure 1-Structure of different isoforms of BIRC5. The full-length BIRC5 has four exons. BIRC5-2B has an additional exon (exon 2B) inserted between the exons 2 and 3, and BIRC5-ΔEx-3, shows a loss of exon 3. (Adapted from Mahotka et al., 1999)


Figure 2-Primers designed specific to BIRC5. The forward and reverse primers were located in exon 2 and exon 4, respectively.


Figure 3-RT-PCR analysis of the different isoforms of BIRC5 expression in SH-EP1 and SK-N-ER.


Figure 4-Sequence alignment of the RT-PCR products, in reference to the reported NCBI reference sequence.


Neuroblastoma, a neuroendocrine tumor, is the most common extracranial solid cancer in infancy and childhood. BIRC5 is a member of inhibitor apoptosis gene family and its expression level may be related to neuroblastoma. There are several isoforms of BIRC5. In this study, mRNA forms of this gene were detected in two types of neuroblastoma cell lines, SH-EP1 and SK-N-ER.

There were two isoforms of BIRC5 found in SH-EP1 and SK-N-ER. Sequencing confirmed that they were full-length BIRC5 and BIRC5-ΔEx-3. Because full-length BIRC5 and BIRC5-ΔEx-3 are involved in protection against apoptosis, they were expected to be main isoforms of this gene in tumor cells. The result of this work was consistent with this expectation.

There was no significant difference in the ratio of these two isoforms in SH-EP1 and SK-N-ER. There are some possible explanations to it. Full-length BIRC5 and BIRC5-ΔEx-3 may have similar ability to suppress apoptosis, or neither of them is the critical factor to suppress apoptosis in neuroblastoma.

Further study in more types of cells may give a more detailed pattern of different isoforms of BIRC5 in different cell lines. In addition, immunological methods such as western blot can also be used in further studies to see the influence of posttranscriptional decoration on the regulation of the expression level of different BIRC5 isoforms.

Please refer to the attached full paper for more details.

Full Paper

Acknowledgments

I would like to thank Leleesha Samaraweera and Joseph Frezzo for their kind and patient guidance throughout the whole project. I also want to thank Jinsong Qiu, Bo Liu and all my classmates for invaluable help. Thanks for Dr. Berish Rubin to provide me such a chance to finish my first own project and his help in this project.


This document was last modified 05/09/2007.
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