AUTHOR=Simmers Mark D. , Jima Dereje D. , Tsuji Yoshiaki , Cowley Michael 

TITLE=LncRNA Tuna is activated in cadmium-induced placental insufficiency and drives the NRF2-mediated oxidative stress response

JOURNAL=Frontiers in Cell and Developmental Biology

VOLUME=11

YEAR=2023

URL=https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2023.1151108

DOI=10.3389/fcell.2023.1151108

ISSN=2296-634X

ABSTRACT=<p>Cadmium (Cd) is a toxic heavy metal found throughout the environment and one of the top ten toxicants of major public health concern identified by the World Health Organization. <italic>In utero</italic> Cd exposure causes fetal growth restriction, malformation, and spontaneous abortion; however, the mechanisms by which Cd impacts these outcomes are poorly understood. Cd accumulates in the placenta, suggesting that these negative outcomes may be a consequence of disrupted placental function and placental insufficiency. To understand the impact of Cd on gene expression within the placenta, we developed a mouse model of Cd-induced fetal growth restriction through maternal consumption of CdCl<sub>2</sub> and performed RNA-seq on control and CdCl<sub>2</sub> exposed placentae. The top differentially expressed transcript was the <italic>Tcl1 Upstream Neuron-Associated</italic> (<italic>Tuna</italic>) long non-coding RNA, which was upregulated over 25-fold in CdCl<sub>2</sub> exposed placentae. <italic>Tuna</italic> has been shown to be critical for neural stem cell differentiation. However, within the placenta, there is no evidence that <italic>Tuna</italic> is normally expressed or functional at any developmental stage. To determine the spatial expression of Cd-activated <italic>Tuna</italic> within the placenta, we used <italic>in situ</italic> hybridization as well as placental layer-specific RNA isolation and analysis. Both methods confirmed the absence of <italic>Tuna</italic> expression in control samples and determined that Cd-induced <italic>Tuna</italic> expression is specific to the junctional zone. Since many lncRNAs regulate gene expression, we hypothesized that <italic>Tuna</italic> forms part of the mechanism of Cd-induced transcriptomic changes. To test this, we over-expressed <italic>Tuna</italic> in cultured choriocarcinoma cells and compared gene expression profiles to those of control and CdCl<sub>2</sub> exposed cells. We demonstrate significant overlap between genes activated by <italic>Tuna</italic> overexpression and genes activated by CdCl<sub>2</sub> exposure, with enrichment in the NRF2-mediated oxidative stress response. Herein we analyze the NRF2 pathway and show that <italic>Tuna</italic> increases <italic>NRF2</italic>/NRF2 both at the transcript and protein levels. <italic>Tuna</italic> drives increased NRF2 target gene expression, a result that is abrogated with the use of an NRF2 inhibitor, confirming that <italic>Tuna</italic> activates oxidative stress response genes through this pathway. This work identifies the lncRNA <italic>Tuna</italic> as a potential novel player in Cd-induced placental insufficiency.</p>