Regulation of a long noncoding RNA MALAT1 by aryl hydrocarbon receptor in pancreatic cancer cells and tissues

Abstract

Environmental toxicants such as dioxins and polycyclic aromatic carbons are risk factors for pancreatitis and pancreatic cancer. These toxicants activate aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, of which activation regulates many downstream biological events, including xenobiotic metabolism, inflammation, and cancer cell growth and transformation. Here, we identified that environmental toxicant-activated AHR increased expression of metastasis associated lung adenocarcinoma transcript 1 (MALAT1) in pancreatic cancer cells and pancreatic tissues. The MALAT1 is a long noncoding (lnc) RNA which interacts with Enhancer of Zeste 2 (EZH2), a histone methyltransferase with epigenetic silencer activity, and the MALAT1-EZH2 interaction increased its epigenetic silencing activity. In contrast, AHR antagonist, CH223191 or resveratrol, counteracted the AHR-mediated MALAT1 induction and MALAT1-enahnced EZH2 activity. Collectively, these results revealed a novel pathway of how environmental exposure leads to epigenetic alteration via activation of AHR-MALAT1-EZH2 signaling axis under pancreatic tissue- and cancer cell-context.

Keywords: Aryl hydrocarbon receptor; EZH2; Environmental toxicants; Epigenetic regulation; MALAT1; Pancreas.

Figure 1.. MALAT1-EZH2 interaction and environmental toxicant-induced MALAT1 induction.

A schematic diagram for RIP assay. EZH2-associated lncRNAs were pulled down with EZH2 antibody (A). To measure the strength of binding interactions between EZH2 and the pull-downed lncRNAs, a RT-qPCR assay was performed with each lncRNA primer set (B). TCDD-mediated lncRNA induction in Panc-1 and AsPC-1 human pancreatic cancer cells (C). BaP-mediated MALAT1 induction in Panc-1 and AsPC-1 cells (D). Values are represented as mean ± SD. *Significant difference or induction P < 0.05.

Figure 2.. AHR-mediated MALAT1 induction.

TCDD treatment induced MALAT1 while co-treatment of AHR antagonist, either CH223191 or resveratrol, inhibited the MALAT1 induction (A). Transfection of siRNAs targeting AHR (siAHR I and II) reduced AHR protein and mRNA levels but not with scrambled control siRNA (siCT) (B). AHR depletion with siAHR I and II significantly decreased the MALAT1 induction (C). A schematic diagram of human MALAT1 gene proximal promoter region. A DRE site is shown as square. Black bar indicated the location of ChIP primer in the MALAT1 gene promoter region (D, top). A ChIP assay showed that TCDD treatment facilitated recruitment of AHR and RNA polymerase II to the DRE site (D, bottom). *Significant induction or **repression P < 0.05.

Figure 3.. MALAT1 depletion or AHR-mediated MALAT1 induction on EZH2 level and activity.

Cells were transfected with siRNAs targeting MALAT1 (siMALAT1 I & II) or control siRNA (siCT). Effects of MALAT1 depletion on the EZH2 protein levels (A, top). The MALAT1 depletion was confirmed by RT-qPCR (A, bottom). Effects of MALAT1 depletion on H3K27me3 marks (B, top) and H3K27me3 enzymatic activities (B, bottom). Effects of TCDD ± CH223191 (5 μM) or resveratrol (20 μM) for 24 hours on the levels of EZH2 protein and H3K27me3 (C) and H3K27me3 enzymatic activities (D).

Figure 4.. Effects of TCDD-activated AHR on MALAT1-EZH2 signaling in vivo.

Immunostaining analysis of control-, TCDD-, and TCDD plus Resveratrol-treated pancreatic tissue sections (N = 4-5 per treatment) with EZH2 and H3K27me3 antibodies. Scale bar, 50 μm (A). TCDD-mediated induction of MALAT1 and its inhibition by resveratrol in pancreatic tissues (B, top). Immunoblotting analysis of pancreatic tissues treated with TCDD, TCDD plus Resveratrol, or control with EZH2 or H3K27me3 antibody. Beta-Actin (Actin) or histone 3 were used as controls (B, bottom). The H3K27me3 enzymatic assay was performed with the pancreatic tissue nuclear lysates treated with TCDD, TCDD plus Resveratrol, or control (C). The RT-qPCR with TCDD, TCDD plus Resveratrol, or control pancreatic tissue RNA lysates was performed to determine the miR-200b level changes (D).