MiR-26 down-regulates TNF-α/NF-κB signalling and IL-6 expression by silencing HMGA1 and MALT1

3772–3787 Nucleic Acids Research, 2016, Vol. 44, No. 8 doi: 10.1093/nar/gkw205 Published online 28 March 2016 MiR-26 down-regulates TNF-␣/NF-␬B signalling and IL-6 expression by silencing HMGA1 and MALT1 Chyi-Ying A. Chen1 , Jeffrey T. Chang2,3 , Yi-Fang Ho1 and Ann-Bin Shyu1,* 1 Department of Biochemistry and Molecular Biology, McGovern Medical School, Houston, TX 77030, USA, Department of Integrative Biology and Pharmacology, McGovern Medical School, Houston, TX 77030, USA and 3 School of Biomedical Informatics, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA 2 Received February 18, 2016; Revised March 17, 2016; Accepted March 18, 2016 ABSTRACT MiR-26 has emerged as a key tumour suppressor in various cancers. Accumulating evidence supports that miR-26 regulates inflammation and tumourigenicity largely through down-regulating IL-6 production, but the underlying mechanism remains obscure. Here, combining a transcriptome-wide approach with manipulation of cellular miR-26 levels, we showed that instead of directly targeting IL-6 mRNA for gene silencing, miR-26 diminishes IL-6 transcription activated by TNF-␣ through silencing NF-␬B signalling related factors HMGA1 and MALT1. We demonstrated that miR-26 extensively dampens the induction of many inflammation-related cytokine, chemokine and tissue-remodelling genes that are activated via NF-␬B signalling pathway. Knocking down both HMGA1 and MALT1 by RNAi had a silencing effect on NF-␬B-responsive genes similar to that caused by miR-26. Moreover, we discovered that poor patient prognosis in human lung adenocarcinoma is associated with low miR-26 and high HMGA1 or MALT1 levels and not with levels of any of them individually. These new findings not only unravel a novel mechanism by which miR-26 dampens IL-6 production transcriptionally but also demonstrate a direct role of miR-26 in down-regulating NF-␬B signalling pathway, thereby revealing a more critical and broader role of miR-26 in inflammation and cancer than previously realized. INTRODUCTION MiR-26 exhibits tumour suppressor activity (reviewed in (1)) and has emerged as a key regulator in carcinogenesis and tumour progression. Ectopic expression of miR-26 inhibits proliferation, induces apoptosis and/or decreases tumourigenicity in multiple cancers, whereas down-regulation of miR-26 was observed across multiple tumour types (2– 5). An inverse relationship between levels of miR-26 and Interleukin-6 (IL-6) was observed in some tumour cells (6,7). It has been thought that miR-26 regulates inflammation and tumourigenicity largely through down-regulating IL-6. IL-6 is a multifunctional cytokine with important roles in many chronic inflammatory diseases (8–10). IL-6 also has pro-tumourigenic activities, including promoting tumour cell proliferation and survival, stimulating angiogenesis and inducing immune tolerance (11–15). Further, IL-6 is upregulated in many human tumours (16). All of these have focused attention on suppression of IL-6 expression as a potential anti-tumour strategy. The mechanisms for miR-26 actions in regulating IL-6 production, inflammation and tumour proliferation remain obscure. Previously, a potential miR-26 recognition site was predicted in the 3 UTR of IL-6 mRNA (17,18). Binding of miR-26 to this site was proposed to elicit rapid degradation of IL-6 mRNA and thus silence IL-6 expression in human alveolar basal epithelial A549 cells activated by TNF-␣ (18). However, the region containing this site has been reported to have little effect on IL-6 mRNA levels in monkey and mouse cell models (19). Moreover, when the predicted miR26 site in the 3 UTR of IL-6 mRNA was mutated, it had no effect on the translation of IL-6 in HeLa cells (20). These observations argue against a direct action of miR-26 on silencing the IL-6 message. Given that inflammation is a major factor contributing to malignancy and the roles of miR26 and IL-6 in this process, it is important to understand the mechanism by which miR-26 regulates IL-6 production in the context of cellular inflammatory response. In this study, we employed a variety of approaches to elucidate the mechanism underlying miR-26-mediated regulation of IL-6 production. Our results demonstrated that miR-26 does not directly target IL-6 transcript for rapid decay or translational repression in either human bronchial epithelial BEAS-2B or adenocarcinomic alveolar basal epithelial A549 cells. Rather, miR-26 down-regulates production of IL-6 via actions on NF-␬B signalling. Our data * To whom correspondence should be addressed. Tel: +713 500 6068; Fax: +713 500 0652; Email:  C The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Nucleic Acids Research, 2016, Vol. 44, No. 8 3773 further revealed that miR-26 represses IL-6 transcription through silencing the expression of MALT1 and HMGA1, two proteins with critical functions in mediating NF-␬B signalling and tumourigenicity (21–24), in BEAS-2B cells. Moreover, we discovered an inverse relationship between levels of miR-26 and of HMGA1 or MALT1 transcripts in lung adenocarcinoma (LUAD), which is linked to LUAD patient survival. Our results not only identify a novel mechanism by which miR-26 dampens IL-6 production transcriptionally through down-regulating NF-␬B signalling pathway but also point to a direct and broader role for miR26 in inflammation and malignancy. MATERIALS AND METHODS Plasmids Renilla luciferase (RL) reporter gene driven by human GAPDH promoter (pLightSwitch-Prom-GAPDH) was purchased from SwitchGear Genomics. Firefly luciferase (FL) reporter gene driven by a minimal promoter containing an NF-␬B response element (pGL4.32[luc2P/NF␬B-RE/Hygro]) was purchased from Promega. To construct pIL-6-FL, a 2.2-kb fragment carrying the human IL-6 promoter that contains the transcription elements described previously (25–27) was PCR-amplified using genomic DNA purified from BEAS-2B cells and inserted into pGL4.13[luc2/SV40]. The plasmids expressing FL (pGL4.13[luc2/SV40]) and RL (pGL4.74[hRluc/TK] or pGL4.73[hRluc/SV40]) were purchased from Promega. To create pRL-IL-6 3 UTR, pRL-IL-6 5 UTR or pRL-IL-6 ORF, the corresponding regions from a human IL-6 cDNA were inserted into the RL 3 UTR in psiCHECK2 (Promega). To construct pRL-3×26(IL-6) and pRL-3×26(GW182), DNA fragments containing three copies of the putative miR-26 recognition site (see Supplementary Figure S3A and B for the sequences) were synthesized (Integrated DNA Technologies) and inserted into the RL 3 UTR in psiCHECK2. The plasmid pRL-IL6 3 UTR(26) was created by a PCR-based mutagenesis to specifica (...truncated)