Study and expression analysis of novel alternative transcript variants of the human baculoviral IAP repeat containing 5 (BIRC5) gene in cancer cells using Next Generation Sequencing

Postgraduate Thesis uoadl:2933072 117 Read counter

Unit:
Κατεύθυνση Κλινική Βιοχημεία - Μοριακή Διαγνωστική
Library of the School of Science
Deposit date:
2021-01-20
Year:
2021
Author:
Adam Eleni
Supervisors info:
Σκορίλας Ανδρέας, Καθηγητής Κλινικής Βιοχημείας, ΕΚΠΑ
Original Title:
Ανάλυση και μελέτη της έκφρασης σε καρκινικά κύτταρα, νέων εναλλακτικών μεταγράφων του γονιδίου BIRC5 με χρήση μεθοδολογίας αλληλούχησης επόμενης γενιάς.
Languages:
Greek
Translated title:
Study and expression analysis of novel alternative transcript variants of the human baculoviral IAP repeat containing 5 (BIRC5) gene in cancer cells using Next Generation Sequencing
Summary:
The human baculoviral IAP repeat containing 5 (BIRC5), also known as survivin, is a conserved member of the inhibitor of apoptosis protein (IAPs) family. IAP proteins exert their regulatory action by binding to caspases 3, 7 and 9, thereby suppressing their action. Although, the expression levels of survivin are low in terminally differentiated cells and/or tissues, they can be found significantly increased in certain pathological conditions as well as in malignant tumors. Numerous studies have demonstrated the importance of the BIRC5 gene and its alternative transcripts in the development of cancer and the course and outcome of the disease. Conventional cloning and sequencing techniques have already confirmed that alternative splicing events within the survivin pre-mRNA result in five distinct alternative transcript variants of the gene. In the present study, however, we implemented an innovative, in-house developed, targeted DNA-seq assay to identify novel survivin alternative transcript variants with significantly increased depth of coverage that high-throughput sequencing approaches offer.
Next-generation sequencing technologies (NGS) are a key tool for researchers to discover new alternative splicing events but also to understand the role of alternative splicing in major cellular processes such as carcinogenesis. In the present study, next-generation NGS sequencing technology was used in order to identify new alternative copies of the BIRC5 gene in a wide range of human cancer cell lines with many different origins. For this purpose, total RNA was extracted from 17 human cancer cell lines and synthesized cDNA by the reverse transcription method, using an oligo-dT-adaptor as a primer to selectively reverse transcribe the BIRC5 mRNAs. After cDNA was produced, touchdown PCR was performed for the molecular cloning of all the BIRC5 transcripts. All PCR products were purified and library preparation and quantitation took place. At the next step NGS based on the semi-conductor sequencing technology was carried out in the Ion Torrent Personal Genome Machine™.
After collecting the data we received from the sequencing followed the bioinformatics analysis of the results which revealed several novel splice junctions between annotated exons of survivin gene as well as the existence of a novel exon of 117 nt, spanning between the annotated exons 3 and 3b. Validation of the NGS findings with PCR-based assays, using variant-specific primers, led to the identification of fourteen novel survivin alternative splice variants (BIRC5 v.4 - v.17), which demonstrate universal expression profiles in a wide established panel of human cell lines, originating from sixteen human malignancies.
Although our study provides a crystal-clear overview of the survivin mRNAs that are actually generated from the pre-mRNA, the biological function of all novel alternative transcript variants as well as the putative protein isoforms should be investigated in the near future. Future studies could also focus in the possible interactions of these isoforms with the main transcript of survivin as well as with each-other. Such studies will provide informations concerning the role of the balance between survivin isoforms in malignant cell proliferation and apoptosis, providing novel diagnostic, prognostic and predictive biomarkers as well as therapeutic targets. In addition, the study of the expression of alternative survivin transcripts in cancer and normal tissues, which will allow to clarify the actual processes of regulation of their transcription, as well as the possible interactions of these molecules in real cell tissues is a key part of future studies.
Main subject category:
Science
Keywords:
alternative splicing, next-generation sequencing (NGS), survivin,
Index:
No
Number of index pages:
0
Contains images:
Yes
Number of references:
174
Number of pages:
101
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