MicroRNAs (miRNAs) are short non-coding RNAs that control gene expression at the post-transcriptional level by inducing mRNA decay or translation repression. A subclass of miRNAs, named epi-miRNAs, is known to exert anti-tumor activity by targeting effectors of the epigenetic machinery. We recently demonstrated a key role for the tumor suppressor miR-29b in reducing the global DNA methylation of multiple myeloma (MM) cells through the targeting of DNA-methyltransferases. In silico search of additional miR-29b targets contributing to clarify its role as epi-miRNA unveiled the class II histone deacetylase HDAC4. Since histone deacetylases represent promising molecular targets for cancer treatment, we sought to characterize HDAC4 expression and function and its regulation by miR-29b in MM cells. HDAC4 protein levels and enzymatic activity were found up-regulated in a panel of 11 MM cell lines as compared to peripheral blood mononuclear cells from healthy donors. Moreover, the analysis of HDAC4 mRNA levels in a microarray dataset consisting of 4 healthy controls, 55 MM and 29 plasma cell leukemia patients indicated significant over-expression in cancer samples, suggesting that high HDAC4 expression might be involved in MM pathogenesis. Notably, the analysis of miRNA/mRNA paired expression in the same microarray dataset revealed an inverse correlation between miR-29b and HDAC4 mRNA, strengthening the relevance of miR-29b as a key regulator of HDAC4. Synthetic miR-29b mimics transfected in MM cells (SKMM1 and NCI-H929) down-regulated HDAC4 mRNA and protein levels and inhibited the 3’UTR of HDAC4 cloned in a luciferase reporter vector, whereas failed to regulate a 3’UTR devoid of two predicted miR-29b target sites. On the other hand, lentiviral-mediated overexpression of HDAC4 strongly inhibited miR-29b expression. These results underscore a negative feedback loop occurring between miR-29b and its target HDAC4 in MM cells. Through loss of function experiments, we assessed the functional significance of HDAC4 in MM cells. Stable silencing of HDAC4 by shRNAs induced growth inhibition, caspase 3/7-dependent apoptosis and autophagy in U266 and KMS11 cells, which occurred together to miR-29b up-regulation. Interestingly, the pan-HDAC inhibitor vorinostat also triggered apoptosis and autophagy in MM cells, along with the induction of miR-29b and the down-regulation of HDAC4 and other miR-29b-canonical targets like CDK6, MCL-1 and Sp1. miR-29b itself was able to promote autophagy, as assessed by beclin-1 up-regulation and LC3A/B proteolytic cleavage in miR-29b mimics-transfected MM cells, which was abrogated by constitutive expression of HDAC4. Finally, we provided evidence that miR-29b over-expression potentiated, whereas its stable inhibition dampened, apoptosis and autophagy triggered by vorinostat. In conclusion, our findings shed light on the oncogenic role of HDAC4, which can be targeted through miR-29b-based therapeutic approaches, and identify miR-29b as a relevant effector of vorinostat activity in MM cells.
Tumor Suppressor MiR-29b Targets HDAC4 and Modulates Vorinostat Activity In Multiple Myeloma Cells
Nicola Amodio;Rossi M;
2013-01-01
Abstract
MicroRNAs (miRNAs) are short non-coding RNAs that control gene expression at the post-transcriptional level by inducing mRNA decay or translation repression. A subclass of miRNAs, named epi-miRNAs, is known to exert anti-tumor activity by targeting effectors of the epigenetic machinery. We recently demonstrated a key role for the tumor suppressor miR-29b in reducing the global DNA methylation of multiple myeloma (MM) cells through the targeting of DNA-methyltransferases. In silico search of additional miR-29b targets contributing to clarify its role as epi-miRNA unveiled the class II histone deacetylase HDAC4. Since histone deacetylases represent promising molecular targets for cancer treatment, we sought to characterize HDAC4 expression and function and its regulation by miR-29b in MM cells. HDAC4 protein levels and enzymatic activity were found up-regulated in a panel of 11 MM cell lines as compared to peripheral blood mononuclear cells from healthy donors. Moreover, the analysis of HDAC4 mRNA levels in a microarray dataset consisting of 4 healthy controls, 55 MM and 29 plasma cell leukemia patients indicated significant over-expression in cancer samples, suggesting that high HDAC4 expression might be involved in MM pathogenesis. Notably, the analysis of miRNA/mRNA paired expression in the same microarray dataset revealed an inverse correlation between miR-29b and HDAC4 mRNA, strengthening the relevance of miR-29b as a key regulator of HDAC4. Synthetic miR-29b mimics transfected in MM cells (SKMM1 and NCI-H929) down-regulated HDAC4 mRNA and protein levels and inhibited the 3’UTR of HDAC4 cloned in a luciferase reporter vector, whereas failed to regulate a 3’UTR devoid of two predicted miR-29b target sites. On the other hand, lentiviral-mediated overexpression of HDAC4 strongly inhibited miR-29b expression. These results underscore a negative feedback loop occurring between miR-29b and its target HDAC4 in MM cells. Through loss of function experiments, we assessed the functional significance of HDAC4 in MM cells. Stable silencing of HDAC4 by shRNAs induced growth inhibition, caspase 3/7-dependent apoptosis and autophagy in U266 and KMS11 cells, which occurred together to miR-29b up-regulation. Interestingly, the pan-HDAC inhibitor vorinostat also triggered apoptosis and autophagy in MM cells, along with the induction of miR-29b and the down-regulation of HDAC4 and other miR-29b-canonical targets like CDK6, MCL-1 and Sp1. miR-29b itself was able to promote autophagy, as assessed by beclin-1 up-regulation and LC3A/B proteolytic cleavage in miR-29b mimics-transfected MM cells, which was abrogated by constitutive expression of HDAC4. Finally, we provided evidence that miR-29b over-expression potentiated, whereas its stable inhibition dampened, apoptosis and autophagy triggered by vorinostat. In conclusion, our findings shed light on the oncogenic role of HDAC4, which can be targeted through miR-29b-based therapeutic approaches, and identify miR-29b as a relevant effector of vorinostat activity in MM cells.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
