12-O-tetradecanoyl phorbol-13-acetate br positive cells after h of treatment Our finding was
positive 12-O-tetradecanoyl phorbol-13-acetate after 48 h of treatment. Our finding was further sub-stantiated by the data obtained from caspase-3 assay and qRT-PCR analysis, which showed a concentration-dependent elevation in both enzymatic and transcriptional activity of caspase-3 (Fig. 2F). Overall, our results suggest that the anti-leukemic eﬀect of BIBR1532 in NB4 is probably induced through transcriptional suppression of c-MYC cou-pled with induction of CDKN1A-mediated G1 arrest and apoptosis.
3.3. Treatment of NB4 cells with BIBR1532 altered the expression levels of cancer-related miRNAs
The putative role of miRNAs in most, if not all, human malignancies have revealed by recent investigations showing that the deregulated miRNome not only is responsible for tumor progression (Iorio and Croce, 2012), but also aﬀects the sensitivity of cancer cells to che-motherapeutic drugs (Kovalchuk et al., 2008; Yang et al., 2008; Zhu et al., 2008). Moreover, it has been suggested that the over-expression of TERT in malignant cells could aﬀect the regulation of several miRNAs and thereby potentiates cancer progression (Lassmann et al., 2015). To gain more insight into the molecular mechanisms by which telomerase inhibition resulted in apoptotic cell death in APL, the eﬀects of BIBR1532 on alteration of 88 cancer-related miRNAs were in-vestigated in NB4 leukemic cells. Analysis of the diﬀerential expression showed that among all the studied miRNAs, 68 (77.27%) were up-regulated in response to drug treatment, while 20 miRNAs (22.73%) displayed a down-regulated expression pattern (Fig. 3A). As depicted, exposure to BIBR1532 deregulated the expression of miRNAs with change levels ranged from 38.29-fold down-regulation to 345.22-fold up-regulation for miR-29b and miR-125a-5p, respectively. To de-termine the ranking of significant dysregulated miRNAs based on
Fig. 2. Restriction of telomerase activity resulted in induction of G1 arrest and activation of a caspase-dependent apoptotic cell death in NB4. A) Average telomere length of NB4 cells. B) BIBR1532 hindered telomerase activity in a dose-dependent manner. C) After treatment of the cells with the drug for 24 h and harvesting the RNA, the expression levels of TERT and c-MYC were measured using qRT-PCR after normalizing Ct of each triplicate against their corresponding HPRT1. D) 48 h Treatment of NB4 cells with BIBR1532 (60 µM) elevated the mRNA expression level of CDKN1A. Moreover, BIBR1532 induced accumulation of the cells in G1, and decreased the cells population in S phase. Sub-G1, which regarded as dead cells, was also increased in inhibitor-treated cells. E) The percentage of both annexin-V and annexin-V/PI double positive cells were increased in response to drug treatments after 48 h. F) BIBR1532-induced caspase-3 activity and transcription. Values are given as mean ± S.D. of three independent experiments (*, represents P ≤ 0.05).
magnitude change of their expression, mean log2 ratio of the fold change between the BIBR1532-treated/untreated cells was calculated and plotted against the corresponding t-test P values to produce volcano plot. By considering the threshold of ≥2-fold change in the gene ex-pression (vertical dashed lines) and P value < 0.05 (horizontal blue line), we found that the expression levels of a substantial number of miRNAs were significantly altered, showing either an up- or down-regulated pattern (Fig. 3B). The scatter plot represented in Fig. 3C also indicated that out of 20 down-regulated miRNAs, 10 were significantly decreased upon exposure to 60 µM of BIBR1532 (P value < 0.05). No-teworthy, alterations in the majority of the up-regulated miRNAs were
statistically significant (91.1%).
3.4. Validated target analysis and bioinformatics studies of the diﬀerentially expressed miRNAs
To elucidate the contributory role of dysregulated miRNAs with at least 2-fold diﬀerence in their expression levels, validated target ana-lysis was performed by using miRTarBase computational programs. To reduce the number of false positives, only target genes validated by three methods (reporter assay, Western blot, and qRT-PCR) were ac-cepted for further bioinformatics investigations. By using GO analysis
Fig. 3. Fold change in the expression levels of 88 cancer-related miRNAs in response to BIBR1532. A) Fold change of dysregulated miRNAs with at least 2-fold diﬀerence in their expression levels. B) The volcano plot depicts the positions of significant dysregulated miRNAs expression. The x-axis indicates a diﬀerence in the expression level on a log2 scale, whereas the y-axis represents corresponding P values on a negative log scale. 2-fold change in miRNAs expression threshold shows by dashed lines, and the horizontal line indicates the significant level of P < 0.05. C) The scatter plot represents the alterations in the normalized expression level of each miRNA (2− Ct) between untreated (x-axis) and inhibitor-treated (y-axis). The dashed lines correspond to 2-fold up- and down-regulation.