Abstract B33: Regulation and function of Nrf2-associated long noncoding RNA
Authors: Johnson Gavin , Beaver Laura , Williams David E. , Ho Emily , Dashwood Roderick H. 
Published: October 2015

We hypothesized that the long non coding RNA (lncRNA), Loc344887, is induced by Nrf2 activation and acts as a transcriptional co-activator of the Nrf2 target gene NQO1. Nrf2 is a transcription factor that is important in oxidative stress responses. Upon oxidative stress, Nrf2 is released from its binding partner Keap1. Nrf2 translocates to the nucleus, partners with small Maf proteins, binds to antioxidant response elements (ARE) and induces detoxifying and metabolizing genes such as NQO1, HMOX1, and GSTs. We propose that Loc344887 is acting as a co-activator of Nrf2, either directly in the Nrf2-Maf complex, or indirectly by some other downstream mechanism, in order to regulate NQO1 expression.

Loc344887 has significantly reduced expression in human colon tumors when compared to normal colon tissue (TCGA database). Human HCT116 colon cancer cells and CCD841 non-cancer colonic epithelial cells replicate this expression difference between tumor and non-transformed. We used these cell lines to study the regulation and function of Loc344887.

HCT116 and CCD841 cells were treated with 15 μM sulforaphane (SFN), a dietary isothiocyanate known to have chemopreventative properties due to Nrf2 activation. Loc344887 increased 40-fold in HCT116 cells, but was not induced in CCD841 cells. Loc344887 was also induced by oltipraz and tert-butylhydroquinone, known Nrf2 activators. Keap1 knockdown upregulated Loc344887, and inducibility of Loc344887 by SFN was lost when Nrf2 was knocked down using RNAi. The gene promoter of Loc344887 contains putative AREs, and chromatin immunoprecipitation assays identified putative Nrf2 and MafK binding regions.

Because Loc344887 is regulated by Nrf2, we hypothesized that Loc344887 may be involved in NRF2 gene activation. Knockdown of Loc344887 caused a loss of NQO1 induction by both SFN and Keap1 knockdown; however, this effect was not seen for HMOX1. Time-course assays showed that Loc344887 is induced as early as 1 h post-SFN treatment and peaks at ~8 h, similar to HMOX1. NQO1, however, was induced much slower, peaking after 24 h. This suggests that other factor(s), such as Loc344887, needs to be present for full induction to occur.

This study shows that Loc344887 is an Nrf2 target gene and is required for complete NQO1 induction, but not for HMOX1 activation. Thus, there is a differential response for Nrf2 target genes that depends, in part, on lincRNAs as co-regulators. Further studies are required to access the mechanism of NQO1 regulation. We hypothesize that once Loc344887 is induced, it acts as a scaffold for MafK, Nrf2 and/or other transcription factors on the NQO1 promoter. Loc344887 localizes primarily to the nucleus, and preliminary RNA immunoprecipitation experiments show that Loc344887 binds directly to MafK. Further studies will also test whether Loc344887 is required for regulation of other Nrf2 target genes, or if other lncRNAs are involved in Nrf2 target gene induction.

Studies supported in part by Chancellor's Research Initiative funding from Texas A&M University, and by NIH grants CA090890, CA122959, P30 ES00210, P30 ES023512.

Citation Format: Gavin Johnson, Laura Beaver, David E. Williams, Emily Ho, Roderick H. Dashwood. Regulation and function of Nrf2-associated long noncoding RNA. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B33.