The MIR23A cluster in BL and DLBCL patients

rates of 7-fold. Notably, the miR-27a overexpressing clones show a significant reduction of apoptosis rate (5-fold) compared to controls. The rate of double positive (Annexin V/7AAD) late apoptotic or dead cells is not affected by miRNA overexpression.

In conclusion, the overexpression of miR-23a and miR-27a reduces the ability of etoposide treated cells to undergo apoptosis.

3.4. The MIR23A cluster in BL and DLBCL patients

The role of the MIR23A cluster is discussed controversially in literature. Although intensively investigated in many different human diseases and cancers, the miRNAs encoded in this clus-ter can have diverse functions. MiR-23a was reported to act as a tumor suppressor or as an onco-miR, depending on the cellular context (Caoet al., 2012; Heet al., 2014). Nevertheless, several studies have shown altered expression of MIR23A cluster in different cancers, among these also some hematopoietic malignancies (APL (Saumetet al., 2009), ALL and AML (Mi et al., 2007)). For B-NHL it was shown by Lenzeet al. that miR-23a is differentially expressed between BL vs. DLBCL (Lenzeet al., 2011). Wang et al. already indicated that miR-23a is aberrantly expressed in DLBCL compared to healthy controls (Wanget al., 2014). However, the expression status of the whole MIR23A cluster in DLBCL compared to germinal center cells, which are the healthy progenitor cells from which DLBCL develops, was unknown so far.

Therefore, the publicly available data base of the International Cancer Genome Consortium (ICGC) Project “Determining Molecular Mechanisms in Malignant Lymphoma by Sequencing”

(ICGC-MMML, release v22, published in (Hezavehet al., 2016)) was retrieved to compare the MIR23A expression of BL and DLBCL patients to healthy GCBs (fig. 3.39). The differential expression of miR-23a in BLvs. DLBCL described by Lenzeet al. in 2011 could be verified.

Additionally, the comparison of the MIR23A cluster expression of BL and DLBCL patients to healthy GCBs reveals a significant overexpression of miR-23a, miR-27a and miR-24 in the patient samples (fig. 3.39).

Taken together, the patient sequencing data show that the whole MIR23A cluster is aberrantly activated in BL and in DLBCL compared to healthy controls (GCBs).

100 3| Results

Figure 3.39.: MIR23A cluster expression in B-NHL patients

MicroRNA sequencing data of 14 BL and 16 DLBCL patients compared to GCB isolated from three healthy controls (ICGC v22 published in Hezavehet al.2016). Mann-Whitney U-test *p≤0.05.

3.4.1. Expression of newly identified and validated miR-23a and miR-27a targets in BL and DLBCL patients

The observation that MIR23A levels are aberrantly high in BL and DLBCL compared to controls raises the question which consequences arise from this aberrant high expression of MIR23A cluster for the DLBCL patients. Wang et al. showed already in 2014 that high miR-23a lev-els were correlated with a worse overall survival rate of DLBCL patients. Thus, the MIR23A cluster might have an onco-miR function in DLBCL. Although a miRNA does not necessarily downregulate their targets on RNA level, the expression of the direct miR-23a/27a targets, identified in this study, were analyzed for BL and DLBCL patients on RNA sequencing data provided by the ICGC data base.

Although VRK3 protein could not be validated to be downregulated by miR-23a overexpres-sion under normal conditions in DLBCL cell line U2932 R1 (fig. 3.32), its mRNA levels are decreased in BL and DLBCL patients compared to healthy GCBs (fig. 3.40 a).

In the case of LIMK1, a validated target gene of miR-27a in this study (fig. 3.2.9.2), BL and DLBCL patients show a decrease inLIMK1mRNA levels (fig. 3.40 b).

3.4. THE MIR23A CLUSTER IN BL AND DLBCL PATIENTS 101

Figure 3.40.: MiR-23a and miR-27a target expression in BL and DLBCL patients

Expression of selected targets in BL and DLBCL patients and normal GCBs (ICGC v22 published in Hezavehet al.2016). (a) Expression of possible miR-23a target VRK3. (b) Expression of validated miR-27a target LIMK1. (c) Expression of validated miR-27a target PUMA. (d) Expression of ZNFs that are targeted by miR-23a. BL = Burkitt lymphoma, DLBCL = diffuse large B cell lymphoma, GCB = germinal center B cells, VRK3 = Vaccina related kinase 3, LIMK1 = LIM domain kinase 1, PUMA = p53 upregulated modulator of apoptosis, ZNF = zinc finger.

Mann-Whitney U-test *p≤0.05.

In contrast, the mRNA levels of validated miR-27a targetPUMA(fig. 3.34) are downregulated in BL, whereas the mean expression of DLBCL patients is higher compared to controls (fig.

3.40 c).

102 3| Results Interestingly, most ZNFs, that were identified as possible miR-23a targets in DLBCL model cell line U2932 R1, are downregulated in BL and DLBCL patients compared to healthy GCBs.

In general, miRNA regulation in mammalia must not result in downregulation of target mRNA levels. However, the mRNA transcripts of VRK3, LIMK1 and most ZNFs are decreased in B-NHL compared to healthy GCBs.

4. Discussion

This study had two major aims: the first one was to identify signaling pathways that regulate the expression of MIR23A cluster in DLBCL, in order to enlighten the processes that lead to aberrant high MIR23A cluster expression in DLBCL. The second aim was to identify the targetomes of miR-23a and miR-27a in DLBCL in order to predict the cellular functions in which miR-23a and miR-27a are involved and to test the onco-miR hypothesis for the MIR23A cluster.

DLBCL and BL patients show increased miR-23a levels compared to healthy controls (Wang et al., 2014) (fig. 3.39), indicating that the MIR23A cluster is de-regulated in aggressive B cell lymphoma. Furthermore, DLBCL patients with higher miR-23a levels show a worse overall survival rate than patients with low miR-23a levels, suggesting the MIR23A cluster to have a onco-miR function in DLBCL (Wanget al., 2014).

This thesis shows that the MIR23A cluster is activated by BCR signaling in BL and DLBCL cell lines. In detail, the BCR downstream MEK/ERK signaling cascade was identified to mediate the activation of MIR23A cluster in DLBCL cell lines. Furthermore the onco-miR hypothesis for the MIR23A cluster in DLBCL could be supported by identification and validation of the miR-23a and miR-27a specific targetome in a DLBCL model cell line.