ESRP1 is a novel marker of germ cell development and gonadal cancer
AuthorSaeidi, Seyedeh Shaghayegh
AffiliationAnatomy and Neuroscience
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2020-08-06.
© 2018 Dr. Seyedeh Shaghayegh Saeidi
Alternative splicing (AS) plays critical roles in controlling developmental programs. To date, there is evident that many genes splice differently during gametogenesis. Since disruption in AS may result in various reproductive disorders such as cancer, regulation of this event would be crucial during gametogenesis. The regulation of alternative splicing occurs through a network of highly combinatorial molecular interactions and numerous RNA binding proteins (RBPs) and transcription factors have been implicated in this process. Among them, ESRP1 (Epithelial Splicing Regulatory Protein) is an important cell-type specific regulator that affects the splicing of various developmental genes. Given the high level of alternative splicing during gametogenesis and the association of ESRP1 with AS, I was interested in examining the expression of ESRP1 during germ cell development and gonadal cancer. In this study by droplet digital PCR (ddPCR), I identified that Esrp1 is expressed in developing murine male and female germ cells but not somatic cells. Esrp1 also showed a high level of expression in adult mouse spermatogonia. In addition, the result of immunofluorescence experiments showed that ESRP1 protein is most highly expressed in nuclei of pre-meiotic germ cells in adult testes and co-labeled with PLZF and c-KIT. However, it did not co-localized with SOX9 in somatic cells, indicating it is germ cell specific. Furthermore, no colocalization was detected between ESRP1 and SC35 (post-transcriptional splicing marker), which suggests a probable role for ESRP1 in co-transcriptional splicing. In addition, my studies on the expression of Esrp1 in gonadal cancers showed upregulation of Esrp1 in both serous and mucinous ovarian carcinomas, its correlation with the levels of E-cadherin (CDH1) expression and coincides with switches from mesenchymal to epithelial isoforms of CD44 and FGFR2. In testicular cancer, my data showed that Esrp1 was also up-regulated in both seminoma and mixed-germ cell testicular cancers. However, it did not regulate splicing of FGFR2 and CD44 in testicular cancers. RNA interference-mediated knockdown of ESRP1 expression in the seminoma-derived TCam-2 cell line, followed by RNA sequencing resulted in the identification of 576 novel potential splicing targets for ESRP1 in germ cell tumor cell line. IPA analyses of the data demonstrated ESRP1 regulates alternative splicing of genes that are involved in directing critical pathways involved in cell migration, morphology, and behaviors that occur during EMT. My data also showed that four mitochondrial complexes of oxidative phosphorylation are affected by differential gene expression after silencing of ESRP1. Overall, these data suggest that ESRP1 is required for germ cell development and raises the possibility that ESRP1 plays a role in splicing of mitotic and premeiotic transcripts during spermatogenesis, particularly in spermatogonia. Furthermore, my findings reveal that ESRP1 plays an important role in gonadal cancer progression by regulation of AS of numerous genes that are related to EMT. Furthermore, differential gene expression after silencing of ESRP1 suggesting that ESRP1 expression in testicular germ cells may alter ATP production and thus affect energy metabolism of these cells.
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