The goal of this thesis was to investigate the role of circRNAs in B-cell lymphoma and breast cancer. We focused on the characterization of their expression patterns, functional mechanisms, and regulatory roles. In the first part, we conducted RNA sequencing to characterize the circRNA landscape of three main B-cell lymphoma subtypes and normal B-cells. This was followed by more in-depth studies of the roles of circPVT1 and circZDHHC11 in B-cell lymphoma. CircPVT1 and the linear PVT1 transcripts showed opposite differential expression patterns and both supported cell growth. In contrast to other cancers, we showed that the effect on growth was independent of their ability to bind miRNAs. CircZDHHC11 was strongly enriched in the AGO2-IP fraction upon miR-150 overexpression and supported the growth of B-cell lymphoma. However, its growth-supporting effect was independent of its ability to bind miR-150. In the second part, we investigated the role of circ-NOL10 in breast cancer. Circ-NOL10 was selected based on its most pronounced downregulation in triple-negative breast cancer (TNBC) compared to control tissue. Upon binding of MTDH and CASC3, circ-NOL10 levels were strongly reduced, resulting in the release of miR-149-5p, miR-330-3p, and miR-452-5p. These miRNAs subsequently targeted PDCD4 and this promoted progression of breast cancer. Overall, our study extends current knowledge of circRNAs and highlights specific functions of selected circRNAs in B-cell lymphoma and breast cancer.

The study of this thesis is related to the previously established MYC/miR-150/MYB/ZDHHC11 network regulating Burkitt lymphoma (BL) growth. Our first aim was to study the role of this network in Hodgkin lymphoma (HL) and diffuse large B-cell lymphoma (DLBCL). We found a similar role of three of the network components, while miR-150 overexpression showed no or only very mild effects on HL and DLBCL growth. Therefore, we considered miR-150 as a BL-specific component. Our second aim was to study the specific roles of the protein coding and circular ZDHHC11 gene products. ZDHHC11 belongs to a family of 24 ZDHHC proteins that are characterized by a DHHC motif which mediates palmitoylation. Loss of palmitoylation induced apoptosis in three B-cell lymphoma subtypes indicating the overall importance of protein palmitoylation for B-cell lymphoma. Based on the observed expression patterns we pinpointed seven DHHC family members that might be relevant in B-cell lymphoma. Further studies to elucidate the role of the ZDHHC11 protein were hampered due to difficulties in detecting the protein but provided some evidence that this protein is not a critical factor in regulating BL growth. Lastly, we showed that circular ZDHHC11 RNA transcripts do play a critical role in growth of B cell lymphoma, but this effect is independent of binding miR-150, as deletion of the miR-150 binding site region did not alter the observed phenotype. In summary, we extended current knowledge on the role of the MYC/miR-150/MYB/ZDHHC11 network and addressed the function of ZDHHC11 transcripts and protein in B-cell lymphoma.

A large number of aberrantly expressed microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) have been reported in B-cell lymphomas. However, their role in the pathogenesis of B-cell lymphoma remains largely unknown. In this thesis, the functions of non-coding RNAs were explored in two distinct subtypes of germinal center B-cell (GC-B) derived lymphomas, i.e. classical Hodgkin lymphoma (cHL) and Burkitt lymphoma (BL).
In a loss-of-function screen in three cHL cell lines we identified four miRNAs with an effect on cell growth. Follow-up experiments for miR-21-5p showed that this miRNA was upregulated in cHL compared to GC-B cells and protects cHL cells from apoptosis possibly via targeting BTG2 and PELI1. Small RNA sequencing in BL and GC-B cells revealed a clearly aberrant expression profile. In subsequent miRNA loss- and gain-of-function screens we identified 18 miRNAs that affected growth of BL, including some previously reported oncogenic miRNAs. Functional follow-up studies revealed promising target genes for miR-378a-3p and miR-26b-5p. Focusing on MYC-induced lncRNAs revealed 18 candidates that were consistently higher expressed in BL cell lines compared to GC-B cells. Three of these lncRNAs showed effects on BL cell growth in a loss-of-function screen. Further validation experiments of MAFG-AS1 confirmed its effect on growth in BL cell lines.
In summary, we identified several aberrantly expressed and MYC-regulated non-coding RNAs, with clear effects on growth of cHL and BL cells, and unveiled their underlying mechanisms in cell growth regulation. Our findings add to the current knowledge about the roles of non-coding RNAs in B-cell lymphomas.

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.