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Oncogenic microRNA in T prolymphocytic leukemia
cells and the lowest expression in naïve CD4 T cells (Figure 3E). Furthermore, we confirmed that the expression of ZEB2 in T-PLL is downregulated compared to effector CD4 T cells (Figure 3E). However, TGFbR3 expression is more homogeneously expressed in normal T-cell fractions and significantly downregulated in T-PLL compared to average of all normal T-cell fractions (Figure 3E). In addition, down- regulation of ZEB2 and TGFbR3 (>80%) was found in a set of three additional CD4-positive T-PLL samples (T-PLL49, 44, 46) with high miR-200c and miR-141 levels compared to normal PBMC (Online Supplementary Figure S3E). ZEB2 mRNA contains four predicted well-conserved 8-mer sites, one 7-mer-m8 and one 7mer-A1 site for miR-200c-3p, three well-conserved 8-mer sites for miR-141-3p and is a validat- ed target of miR-200c and miR-141.30 ZEB2, has been iden- tified as a SMAD-interacting protein and regulates the tran- scriptional activities of the TGFb network.31 According to TargetScan, TGFbR3 contains one less conserved miR-200c site in the 3’-UTR. In order to further investigate the func- tions of miR-200c/141 in regulation of the TGFb network, we transduced Jurkat cells with pLX301-miR-200c/141 (co-expressing EGFP, miR-141 and miR-200c) or empty vec- tor (EV) pLX301 expressing EGFP only. We noted that Jurkat-miR-200c/141 cells showed a 10-fold overexpression of miR-141 and a 5-fold overexpression of miR-200c com- pared to EV-transduced control Jurkat cells. We also observed a downregulation of both ZEB2 (P=0.013) and TGFbR3 mRNA (P<0.1) in Jurkat-miR-200c/141 cells com- pared to EV-transduced control Jurkat cells (Figure 3F).
Ligand stimulated TGFb type II receptor (TGFbRII) interacts with and phosphorylates TGFbRI, which results in activation of its kinase domain. This allows the TGFbR1 to phosphorylate SMAD2 and SMAD3 transcrip- tion factors. TGFbRIII act as a regulator of TGFb signaling and SMAD pathway activation, which is highly context- dependent.32-34 In order to further investigate the role of miR-200c/141 in the regulation of TGFb-signaling, we transduced HeLa cells with either miR-200c/141 or EGFP only (EV) lentiviruses. HeLa-miR-200c/141 cells had a 397- fold miR-200c and a 624-fold miR-141 overexpression compared to HeLa-EV control cells (Figure 4A). Downregulation of ZEB2 and TGFbR3 mRNA levels in HeLa-miR-200c/141 cells was confirmed by quantitative polymerase chain reaction (qPCR) (Figure 4B). Interestingly, hu-TGFb1 stimulation of HeLa-miR- 200c/141 cells, resulted in increased p-SMAD3 levels, whereas p-SMAD2 was reduced and SMAD2, SMAD3 and TGFbR1 levels remained similar to their expression in control HeLa cells (Figure 4C). Next, we investigated hu- TGFb1-induced SMAD2 and SMAD3 phosphorylation in T-PLL. Notably, the expression of SMAD3 relative to SMAD2 was higher in T-PLL compared to PBMC and HeLa (Figure 4C and D). In addition, we observed a decreased p-SMAD2 and p-SMAD3 induction after hu- TGFb1 stimulation compared to the unstimulated condi- tion in T-PLL with high miR-200c/141 compared to T-PLL with normal levels of miR-200c/141 and healthy PBMC (Figure 4D and E). Together, our results indicate that the miR-200c/141-ZEB2-TGFb- axis is aberrant in T-PLL.
Discussion
In this study, we characterized a cohort of T-PLL patients both phenotypically and molecularly. In full
agreement with recently published data,2 we found multi- ple oncogenic abnormalities in our T-PLL cohort, such as aberrations in ATM, TP53 and high TCL1 overexpression. We did not observe the clinical and morphological hetero- geneity of T-PLL due to differences in protein-encoding gene expression profiles as reported in previous publica- tions.2,35 This discrepancy can be largely explained by the relatively small cohort of T-PLL patients used for this study. Furthermore, we found that T-PLL generally dis- played immunological characteristics mostly comparable to memory T cells, without evidence for identical TCR usage pointing towards a common antigen-driven leuke- mogenesis. Based on miRNA expression, T-PLL cells were most similar to effector T cells.
Our study reports an abnormal expression of miRNA in T-PLL. In total, we identified a set of 35 aberrantly expressed miRNA, that were upregulated compared to normal effector CD4 T cells. Although most T-PLL cases showed a relatively homogenous gene expression profile, T-PLL samples exhibited differential miRNA expression that correlated with blood cell counts and survival. The observation that differentially expressed miRNA are most- ly upregulated in T-PLL is striking, since global miRNA depletion is more commonly found in human cancer.14 However, we cannot rule out that some miRNA are actu- ally downregulated but with high degree of variation, such that they are not picked up in this small cohort of T-PLL cases. On the other hand, involvement of enhanced miRNA expression was already postulated based on ele- vated AGO2 expression as a result of chromosome 8 gains in a subset of T-PLL patients.2 Increased AGO2 expression has been detected in some human cancer types and it is well-established that this results in enhanced miRNA sta- bility and expression levels.36 As we did not find a general AGO2 upregulation in our cohort of T-PLL cases, it remains to be determined why miRNA are upregulated in T-PLL. Some upregulated miRNA in T-PLL concern well- known oncogenic miRNA, including the oncogenic driver miR-19b, which represses expression of the tumor sup- pressor gene PTEN37 and miR-125a, which strongly represses the expression of genes involved in apoptosis, such as TP53, PUMA and BAK1.38
Expression of miR-200c/141 is strongly upregulated in a subset of T-PLL. The miR-200c/141 cluster is frequently co-expressed with PTPN6 through transcriptional read- through and complex 3D chromatin interactions between PTPN6 and miR-200c/141 promoters.28 Accordingly, we found a significant upregulation of PTPN6 in T-PLL sam- ples with enhanced miR-200c/141 expression. It is well- established that the expression of miR-200c/141 is strong- ly induced by oxidative stress.39 As reactive oxygen species (ROS) levels are dramatically increased in T-PLL cells,2 this may therefore largely explain the elevated expression lev- els of miR-200c/141. Furthermore, MIR200C/141 is known to be transcriptionally upregulated by MYC,40,41 an oncogenic transcription factor that is frequently overex- pressed in T-PLL, including our cohort.2,42
In our search for the biological implications of miR- 200c/141 upregulation, we identified a potential role for the miR-200c/141-ZEB2-TGFbR axis in the pathogenesis of T-PLL. The TGFb-signaling pathway regulates critical cellular processes in hematopoietic cells such as prolifera- tion, differentiation, apoptosis and cell migration. Leukemia cells are commonly resistant to TGFb-con- trolled mechanisms due to mutations and deletion of mol-
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