br profile Although some of
profile. Although some of the altered miRNAs such as miR-1, miR-21, miR-29a, and miR-208 were common between BIBR1532 and other inhibitors of TERT (Drevytska et al., 2014), some others were specific to BIBR1532 and to the best of our knowledge, none of these miRNAs have been previously reported in BIBR1532-treated cells. In order to provide a better interpretation of our findings, we searched the validated targets of each relevant miRNA in diﬀerent databases. Noteworthy, in agree-ment with qRT-PCR results and the decreased mRNA expression level of BCL2, validated target analysis revealed that the expression of this molecule is potentially regulated by more than 10 de-regulated miRNAs. Intriguingly, considerable number of proteins involved in NF-κB-mediated apoptosis was also found as validated target genes for 28 de-regulated miRNAs with at least 10-fold diﬀerence in their expres-sion. Multiple signaling networks, including pathways in cancer, hippo signaling pathway, regulating stem cell pluripotency, WNT, FoxO, and MAPK signaling pathways were enriched according to heatmap and cluster analysis.
In the list of BIBR1532-altered miRNAs, miR-125a-5p was the most de-regulated one, which its critical impact on diﬀerent signaling path-ways has been reported in several studies. It has also been demon-strated that miR-125a-5p serves as a potent tumor suppressor by in-hibiting cancer cell growth, at least partly, through induction of cyclin-dependent kinase inhibitor 1A (CDKN1A) (Potenza et al., 2017). In addition, Kim et al. indicated that there is a regulatory loop between miR-125a-5p expression and cyclin D1-dependnet G1 RNase Inhibitor arrest
(Kim et al., 2013). Given these, it was reasonable to speculate that BIBR1532 eﬀect is mediated through modulation of CDKN1A. Inter-estingly, our data showed that BIBR1532 not only increased the mRNA expression level of CDKN1A, but also halted transition from G1 phase of cell cycle in NB4 cells. This finding was in accordance with several studies which suggested that treatment of diﬀerent cancer cells in-cluding pre B ALL (Bashash et al., 2017b), Breast cancer (Shi et al., 2015), osteosarcoma (Chen et al., 2017), and glioma (Wang et al., 2012) with telomerase inhibitors induced G1 cell cycle arrest. It is well-established that upon various signaling, foremost NF-κB pathway, c-Myc is activated and subsequently drives cell cycle progression through suppression of CDKN1A (Denis et al., 1991). In agreement with the eﬀect on CDKN1A, we found that treatment of the cells with BIBR1532 decreased the mRNA expression level of c-MYC, which also serves as the most predominant positive regulator of TERT transcription (Cerni, 2000). Of note, our bioinformatics investigations delineated c-MYC as an important validated target for several de-regulated miRNAs, in-cluding miR-9 (Table 4). Collaborating with our results, a recent study showed that miR-9 could regulate both c-MYC and NF-κB pathway in diﬀused large B cell lymphoma (Lenze et al., 2011). Of great interest, a recent study delineated that blockage of PI3K resulted in APL cell apoptosis through NF-κB-mediated suppression of telomerase via down-regulation of c-MYC expression (Bashash et al., 2017a). Although it is early to hazard a conjecture on the mechanism that telomerase in-hibition employed to reduce NB4 cell survival, a possible candidate
A. Pourbagheri-Sigaroodi et al.
would be NF-κB, a key nuclear transcription factor that engages a cross-talk with c-Myc. To sum up with, a straightforward interpretation of our bioinformatics and mechanistic studies is that telomerase inhibition using BIBR1532 not only induced CDKN1A-mediated G1 arrest in NB4, but also resulted in a caspase-3-dependent apoptotic cell death mostly through suppression of NF-κB axis (Fig. 7). European Journal of Pharmacology 846 (2019) 49–62
Fig. 5. miRNA-validated target gene interac-tions network by using Cytoscape software. Interactions between miRNAs (≥10-Fold change) and their validated target genes re-lated to apoptosis and cell cycle pathways are represented as a network. As represented in this figure, the expression of BCL2 is poten-tially regulated by 11 miRNAs, which indicate its crucial role in BIBR1532-induced cytotoxi-city.
Fig. 6. Suppressive eﬀect of BIBR1532 on NF-κB activa-tion and its anti-apoptotic target genes expression. A) After treatment NB4 cells with 60 µM BIBR1532, the NF-κB phosphorylation level was determined by using a cell-based ELISA assay. B) The impact of the compound on the mRNA expression levels of the anti-apoptotic target genes of NF-κB were measured in BIBR1532-treated cells after normalizing the cycle thresholds (Ct) of each triplicate against their corresponding HPRT1. As depicted, 24 h treatment of the cells resulted in a significant down-reg-ulation of the mRNA levels of MCL1, BCL2, BIRC5, and XIAP. Values are given as mean ± S.D. of three in-dependent experiments. Statistical significance was cal-culated using paired two-tailed Student's t-tests (*, re-presents P ≤0.05).