Dataset Information

TMEM16A alternative splicing coordination in breast cancer.

ABSTRACT:

Background

TMEM16A, also known as Anoctamin-1, is a calcium-activated chloride channel gene overexpressed in many tumors. The role of TMEM16A in cancer is not completely understood and no data are available regarding the potential tumorigenic properties of the multiple isoforms generated by alternative splicing (AS).

Methods

We evaluated TMEM16A AS pattern, isoforms distribution and Splicing Coordination (SC), in normal tissues and breast cancers, through a semi-quantitative PCR-assay that amplifies transcripts across three AS exons, 6b, 13 and 15.

Results

In breast cancer, we did not observe an association either to AS of individual exons or to specific TMEM16A isoforms, and induced expression of the most common isoforms present in tumors in the HEK293 Flp-In Tet-ON system had no effect on cellular proliferation and migration. The analysis of splicing coordination, a mechanism that regulates AS of distant exons, showed a preferential association of exon 6b and 15 in several normal tissues and tumors: isoforms that predominantly include exon 6b tend to exclude exon 15 and vice versa. Interestingly, we found an increase in SC in breast tumors compared to matched normal tissues.

Conclusions

As the different TMEM16A isoforms do not affect proliferation or migration and do not associate with tumors, our results suggest that the resulting channel activities are not directly involved in cell growth and motility. Conversely, the observed increase in SC in breast tumors suggests that the maintenance of the regulatory mechanism that coordinates distant alternative spliced exons in multiple genes other than TMEM16A is necessary for cancer cell viability.

SUBMITTER: Ubby I

PROVIDER: S-EPMC3728142 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Publications

TMEM16A alternative splicing coordination in breast cancer.

Ubby Ifeoma I Bussani Erica E Colonna Antonio A Stacul Giuseppe G Locatelli Martina M Scudieri Paolo P Galietta Luis L Pagani Franco F

Molecular cancer 20130716

<h4>Background</h4>TMEM16A, also known as Anoctamin-1, is a calcium-activated chloride channel gene overexpressed in many tumors. The role of TMEM16A in cancer is not completely understood and no data are available regarding the potential tumorigenic properties of the multiple isoforms generated by alternative splicing (AS).<h4>Methods</h4>We evaluated TMEM16A AS pattern, isoforms distribution and Splicing Coordination (SC), in normal tissues and breast cancers, through a semi-quantitative PCR-a ...[more]

PMID: 23866066

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Project description:BackgroundAlternative splicing (AS) is a central mechanism in the generation of genomic complexity and is a major contributor to transcriptome and proteome diversity. Alterations of the splicing process can lead to deregulation of crucial cellular processes and have been associated with a large spectrum of human diseases. Cancer-associated transcripts are potential molecular markers and may contribute to the development of more accurate diagnostic and prognostic methods and also serve as therapeutic targets. Alternative splicing-enriched cDNA libraries have been used to explore the variability generated by alternative splicing. In this study, by combining the use of trapping heteroduplexes and RNA amplification, we developed a powerful approach that enables transcriptome-wide exploration of the AS repertoire for identifying AS variants associated with breast tumor cells modulated by ERBB2 (HER-2/neu) oncogene expression.ResultsThe human breast cell line (C5.2) and a pool of 5 ERBB2 over-expressing breast tumor samples were used independently for the construction of two AS-enriched libraries. In total, 2,048 partial cDNA sequences were obtained, revealing 214 alternative splicing sequence-enriched tags (ASSETs). A subset with 79 multiple exon ASSETs was compared to public databases and reported 138 different AS events. A high success rate of RT-PCR validation (94.5%) was obtained, and 2 novel AS events were identified. The influence of ERBB2-mediated expression on AS regulation was evaluated by capillary electrophoresis and probe-ligation approaches in two mammary cell lines (Hb4a and C5.2) expressing different levels of ERBB2. The relative expression balance between AS variants from 3 genes was differentially modulated by ERBB2 in this model system.ConclusionsIn this study, we presented a method for exploring AS from any RNA source in a transcriptome-wide format, which can be directly easily adapted to next generation sequencers. We identified AS transcripts that were differently modulated by ERBB2-mediated expression and that can be tested as molecular markers for breast cancer. Such a methodology will be useful for completely deciphering the cancer cell transcriptome diversity resulting from AS and for finding more precise molecular markers.

Dataset Information

TMEM16A alternative splicing coordination in breast cancer.

Background

Methods

Results

Conclusions

Publications

TMEM16A alternative splicing coordination in breast cancer.

Similar Datasets

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