Breast cancer is a significant global health issue, impacting millions of women worldwide. ER-positive breast cancer, the most common subtype, accounts for over 70% of cases. While hormone-based therapies like tamoxifen are effective, resistance develops in many patients, posing clinical challenges. Recent research has focused on the molecular mechanisms behind this resistance, aiming to identify new therapeutic targets.
This thesis investigates the regulatory role of microRNAs (miRNAs) in ER-positive breast cancer, particularly their impact on ER expression, cell proliferation, and tamoxifen sensitivity. Through a genome-wide miRNA high-throughput screen, we analysed regulatory networks involving miRNAs to understand their mechanisms better.
Two miRNAs affected ER expression in MCF7 cells, including miR-130a and miR-18b/miR-106a. MiR-18b was highlighted as a key regulator, influencing ER expression and cell proliferation. Analysis of potential targets of miR-18b identified amongst others target genes like ESR1, HMGCS1, and SON, supporting a role of miR-18b in these processes.
Additionally, we identified five miRNAs associated with tamoxifen resistance and validated their role in response to tamoxifen for miR-130a, let-7g, and miR-15b/miR-16-2. For miR-130a, 20 genes, including ESR1 and AIB1, were identified as predicted targets influencing tamoxifen sensitivity. Downregulation of ESR1 and AIB1 by miR-130a was confirmed via Western blotting.
These findings underscore the importance of miRNA regulation in ER-positive breast cancer and pave the way for potential miRNA-based therapeutic strategies to improve treatment outcomes. The research also highlights the complexity of miRNA-mediated regulation and the need for further studies to validate these findings across different breast cancer models.

Classic Hodgkin lymphoma (cHL) is a B-cell malignancy marked by Hodgkin and Reed/Sternberg (HRS) cells that highly express CD30. We explored the roles of microRNAs (miRNAs) in cHL pathogenesis and CD30-targeting therapy using Brentuximab Vedotin (BV) through high-throughput screening techniques. Review of miRNA-related studies on known and potential miRNA-target gene interactions identified multiple miRNA-gene pairs with potential relevance for cHL pathogenesis. This highlighted the relevance of miRNAs in cHL, warranting further studies to deepen our understanding.
A microRNA overexpression library screen revealed 11 miRNA-target gene pairs implicated in cHL pathogenesis and 66 miRNA constructs that regulate BV treatment response. Among these, miR-590 was found to inhibit CD30 expression and induce BV resistance. Additionally, miR-191-5p not only conferred BV resistance but also promoted cell growth. By using AGO2-immunoprecipitation, we identified a network of involved target genes, including PTEN.
To investigate endogenous miRNAs involved in BV resistance mechanisms, we established two BV-resistant cHL cell lines and observed dynamic changes in CD30 expression. Moreover, we utilized a miRNA-focused CRISPR-Cas9 screen to identify endogenous miRNAs that regulate CD30 expression and/or BV sensitivity. To this end, we generated two inducible Cas9 monoclonal cell lines and optimized the entire screening process. These screens await further data analysis.
In summary, this thesis highlights the functional importance of specific miRNAs in cHL and provides detailed procedures of the miRNA-focused screening approaches, from initial setup to optimization and validation of results.

In this thesis, we hypothesized that microRNAs (miRNAs) with deregulated expression in lung fibroblasts, are crucial players in the impaired lung tissue repair and remodelling as observed in chronic obstructive pulmonary disease (COPD). To explore this, we focused on miRNA expression changes in the lung, and in particular in fibroblasts in relation to the effects of TGF-β, and current smoking and associations with COPD and ageing. We identified 106 TGF-β-regulated miRNAs in control and/or in COPD lung fibroblasts. Of these, three miRNAs responded differently to TGF-β in COPD compared to control lung fibroblasts. Only one miRNA was higher expressed in COPD compared to control lung fibroblasts. Furthermore, we identified one miRNA that was lower expressed in lung fibroblasts from current compared to ex-smokers. We identified >960 genes that are actively regulated by miRNAs in lung fibroblasts, which were used to identify fibroblast-specific targets of the differentially expressed miRNAs. Our studies indicate that the identified miRNAs may affect the function of lung fibroblasts through these genes, and affect tissue repair and remodelling, and thus are implicated in COPD pathogenesis. In bronchial biopsies of healthy control subjects, 285 age-related genes and 27 age-related miRNAs were identified. Genes with lower expression with increasing age included several hallmarks of ageing whereas genes with higher expression with increasing age were amongst others involved in synapse-related processes. These studies provide a good stepping stone for further studies aiming to clarify the complex role of these miRNAs in relation to abnormal tissue repair in COPD and ageing.

In this thesis, we studied the role of microRNAs (miRNAs) in the pathogenesis of Hodgkin lymphoma (HL). Small RNA sequencing revealed 84 significantly differentially expressed miRNAs between HL cell lines and normal germinal center B cells. Inhibition of the in HL significantly overexpressed miR-24-3p resulted in decreased growth which was at least in part caused by an increase in apoptotic cells. MiRNA target gene identification using Ago2-IP in HL cell lines revealed 1,142 miRNA target genes of which 52 were predicted to be targeted by miR-24-3p. Western blotting analysis confirmed increased CDKN1B/P27kip1 upon miR-24-3p inhibition, possibly explaining the effect on cell growth. We next set up a next generation sequencing based high throughput loss- and gain-of-function screening approach to identify miRNAs that influence HL cell growth. The overexpression screen revealed that miR-19b-1 may enhance while miR-141 may repress HL cell growth. The inhibition screen revealed that inhibition of miR-449a-5p, miR-625-5p, let-7f-2-3p and miR-21-5p has a negative effect on HL cell growth. The highly abundant miR-21-5p showed significantly higher expression levels in HL. We confirmed the negative effects of miR-21-5p inhibition on cell growth and observed a concomitant significant increase in apoptotic cells. Among the in HL Ago2-IP enriched target genes, we identified 36 predicted miR-21-5p targets. We confirmed targeting of BTG2 and PELI1 by miR-21-5p using reporter assays and Western blot. Overall, we set up the technology for functional miRNA studies in HL and identified two miRNAs and their target genes relevant for the pathogenesis of HL.

While RNA was long thought to act as a mere intermediary between DNA and protein, we now know RNA transcripts can have diverse cellular functions themselves. The major class of long noncoding (lncRNAs) thus represent a missing piece in the puzzle of human cell biology. A variety of transcriptional as well as posttranscriptional regulatory mechanisms have been described for lncRNAs. This thesis addresses the relevance of lncRNAs in normal B cell development and B cell malignancies. Specifically, we study lncRNA expression changes in normal B cells before, during and after the maturation process using naive, germinal center [GC] and memory B cells, respectively. Vast lncRNA expression changes are observed in the highly proliferative GC B cells, indicating significant lncRNA involvement in the B cell maturation process. Furthermore we identify multiple lncRNAs overexpressed in Hodgkin lymphoma cell lines compared to their cell-of-origin, which show cancer cell specific expression in primary patient tissue. We defined lncRNAs regulated by the oncogenic transcription factor Myc and putatively involved in its proliferation-supportive effects. We observe deregulation of hundreds of lncRNAs in response to Myc, thus identifying lncRNAs as a major component of the Myc transcriptional network. Myc-induced lncRNA KTN1-AS1 is studied in more detail and shown to affect Burkitt lymphoma cell growth by reinforcing high Myc expression. In summary, our studies provide novel insights into the role of lncRNAs in normal B cells and B cell malignancies. The individual lncRNAs we identified can potentially serve as biomarkers or therapeutic targets for lymphoma detection and treatment.