@phdthesis{Vardapour2022,
  author    = {Vardapour, Romina},
  title     = {Mutations in the DROSHA/DGCR8 microprocessor complex in high-risk blastemal Wilms tumor},
  doi       = {10.25972/OPUS-23140},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-231404},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Wilms tumor (WT) or nephroblastoma is the most common kidney tumor in childhood. Several genetic alterations have been identified in WT over the past years. However, a clear-cut underlying genetic defect has remained elusive. Growing evidence suggests that miRNA processing genes play a major role in the formation of pediatric tumors, including WT. We and others have identified the microprocessor genes DROSHA and DGCR8 as key players in Wilms tumorigenesis. Exome sequence analysis of a cohort of blastemal-type WTs revealed the recurrent hotspot mutations DROSHA E1147K and DGCR8 E518K mapping to regions important for catalyic activity and RNA-binding. These alterations were expected to affect processing of miRNA precursors, ultimately leading to altered miRNA expression. Indeed, mutated tumor samples were characterized by distinct miRNA patterns. Notably, these mutations have been observed almost exclusively in WT, suggesting that they play a specific role in WT formation. The aim of the present work was to first examine the mutation frequency of DROSHA E1147K and DGCR8 E518K in a larger cohort of WTs, and to further characterize these microprocessor gene mutations as potential oncogenic drivers for WT formation. Screening of additional 700 WT samples by allele-specific PCR revealed a high frequency of DROSHA E1147K and DGCR8 E518K mutations, with the highest incidence found in tumors of high-risk histology. DROSHA E1147K was heterozygously expressed in all cases, which strongly implies a dominant negative effect. In contrast, DGCR8 E518K exclusively exhibited homozygous expression, suggestive for the mutation to act recessive. To functionally assess the mutations of the microprocessor complex in vitro, I generated stable HEK293T cell lines with inducible overexpression of DROSHA E1147K, and stable mouse embryonic stem cell (mESC) lines with inducible overexpression of DGCR8 E518K. To mimic the homozygous expression observed in WT, DGCR8 mESC lines were generated on a DGCR8 knockout background. Inducible overexpression of wild-type or mutant DROSHA in HEK293T cells showed that DROSHA E1147K leads to a global downregulation of miRNA expression. It has previously been shown that the knockout of DGCR8 in mESCs also results in a significant downregulation of canonical miRNAs. Inducible overexpression of wild type DGCR8 rescued this processing defect. DGCR8 E518K on the other hand, only led to a partial rescue. Differentially expressed miRNAs comprised members of the ESC cell cycle (ESCC) and let-7 miRNA families whose antagonism is known to play a pivotal role in the regulation of stem cell properties. Along with altered miRNA expression, DGCR8-E518K mESCs exhibited alterations in target gene expression potentially affecting various biological processes. We could observe decreased proliferation rates, most likely due to reduced cell viability. DGCR8-E518K seemed to be able to overcome the block of G1-S transition and to rescue the cell cycle defect in DGCR8-KO mESCs, albeit not to the full extent like DGCR8-wild-type. Moreover, DGCR8-E518K appeared to be unable to completely block epithelial-to-mesenchymal transition (EMT). Embryoid bodies (EBs) with the E518K mutation, however, were still able to silence the self-renewal program rescuing the differentiation defect in DGCR8-KO mESCs. Taken together, I could show that DROSHA E1147K and DGCR8 E518K are frequent events in WT with the highest incidence in high-risk tumor entities. Either mutation led to altered miRNA expression in vitro confirming our previous findings in tumor samples. While the DROSHA E1147K mutation resulted in a global downregulation of canonical miRNAs, DGCR8 E518K was able to retain significant activity of the microprocessor complex, suggesting that partial reduction of activity or altered specificity may be critical in Wilms tumorigenesis. Despite the significant differences found in the miRNA and mRNA profiles of DGCR8 E518K and DGCR8-wild-type mESCs, functional analysis showed that DGCR8 E518K could mostly restore important cellular functions in the knockout and only slightly differed from the wild-type situation. Further studies in a rather physiological environment, such as in a WT blastemal model system, may additionally help to better assess the subtle differences between DGCR8 E518K and DGCR8 wild-type observed in our mESC lines. Together with our findings, these model systems may thus contribute to better understand the role of these microprocessor mutations in the formation of WT.},
  subject      = {Nephroblastom},
  language  = {en}
}
@article{VetrivelZhangEngeletal.2022,
  author    = {Vetrivel, Sharmilee and Zhang, Ru and Engel, Mareen and Oßwald, Andrea and Watts, Deepika and Chen, Alon and Wielockx, Ben and Sbiera, Silviu and Reincke, Martin and Riester, Anna},
  title     = {Characterization of adrenal miRNA-based dysregulations in Cushing's syndrome},
  series = {International Journal of Molecular Sciences},
  volume    = {23},
  journal   = {International Journal of Molecular Sciences},
  number    = {14},
  issn      = {1422-0067},
  doi       = {10.3390/ijms23147676},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284394},
  year      = {2022},
  abstract  = {MiRNAs are important epigenetic players with tissue- and disease-specific effects. In this study, our aim was to investigate the putative differential expression of miRNAs in adrenal tissues from different forms of Cushing's syndrome (CS). For this, miRNA-based next-generation sequencing was performed in adrenal tissues taken from patients with ACTH-independent cortisol-producing adrenocortical adenomas (CPA), from patients with ACTH-dependent pituitary Cushing's disease (CD) after bilateral adrenalectomy, and from control subjects. A confirmatory QPCR was also performed in adrenals from patients with other CS subtypes, such as primary bilateral macronodular hyperplasia and ectopic CS. Sequencing revealed significant differences in the miRNA profiles of CD and CPA. QPCR revealed the upregulated expression of miR-1247-5p in CPA and PBMAH (log2 fold change > 2.5, p < 0.05). MiR-379-5p was found to be upregulated in PBMAH and CD (log2 fold change > 1.8, p < 0.05). Analyses of miR-1247-5p and miR-379-5p expression in the adrenals of mice which had been exposed to short-term ACTH stimulation showed no influence on the adrenal miRNA expression profiles. For miRNA-specific target prediction, RNA-seq data from the adrenals of CPA, PBMAH, and control samples were analyzed with different bioinformatic platforms. The analyses revealed that both miR-1247-5p and miR-379-5p target specific genes in the WNT signaling pathway. In conclusion, this study identified distinct adrenal miRNAs as being associated with CS subtypes.},
  language  = {en}
}
@article{DetomasPivonelloPellegrinietal.2022,
  author    = {Detomas, Mario and Pivonello, Claudia and Pellegrini, Bianca and Landwehr, Laura-Sophie and Sbiera, Silviu and Pivonello, Rosario and Ronchi, Cristina L. and Colao, Annamaria and Altieri, Barbara and De Martino, Maria Cristina},
  title     = {MicroRNAs and long non-coding RNAs in adrenocortical carcinoma},
  series = {Cells},
  volume    = {11},
  journal   = {Cells},
  number    = {14},
  issn      = {2073-4409},
  doi       = {10.3390/cells11142234},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-281795},
  year      = {2022},
  abstract  = {Non-coding RNAs (ncRNAs) are a type of genetic material that do not encode proteins but regulate the gene expression at an epigenetic level, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). The role played by ncRNAs in many physiological and pathological processes has gained attention during the last few decades, as they might be useful in the diagnosis, treatment and management of several human disorders, including endocrine and oncological diseases. Adrenocortical carcinoma (ACC) is a rare and aggressive endocrine cancer, still characterized by high mortality and morbidity due to both endocrine and oncological complications. Despite the rarity of this disease, recently, the role of ncRNA has been quite extensively evaluated in ACC. In order to better explore the role of the ncRNA in human ACC, this review summarizes the current knowledge on ncRNA dysregulation in ACC and its potential role in the diagnosis, treatment, and management of this tumor.},
  language  = {en}
}