In Vitro Long Noncoding RNA Responsiveness to Ischemic Conditions in Fetal Sheep Islets

Abstract

Fetal growth restriction (FGR) predisposes offspring to long-term health risks, such as Type 2 Diabetes and obesity. They have a higher risk of developing glucose intolerance due to impaired insulin secretion. Placental insufficiency causes fetal hypoxemia and hypoglycemia in FGR fetus leading to β-cell dysfunction from reduced β-cell mass. Long noncoding RNAs (lncRNA) are regulatory molecules that modulate transcriptional and post-transcriptional processes, and high-throughput RNA sequencing data identified several differentially expressed lncRNA in FGR islets versus controls. The objective of this study was to determine ischemic responsiveness of these differentially expressed FGR lncRNAs in control islets in vitro and develop an in vitro hypoxic/ischemic cell line model that shows responsive MALAT1 expression and NF-kB activity. The islets were isolated from fetal sheep and were cultured in ischemic and optimal conditions. MIN6 and INS832 cells were cultured in hypoxic conditions (200-250 uL/mol CoCl2 or 1% O2) for 24 hours. Western blot was conducted to measure p50 and p65 subunit translocation and luciferase assay was conducted to measure NF-κB response. oFUVECs were cultured in 1% O2 for 24 hours. We found that in vitro islet ischemia significantly altered for six of the nine lncRNAs. Among these, MALAT1 and H19 concentrations were higher (P<0.05), and SI-linc20-39a, LINC28868, SI-linc9-103, and RUNX1T1 Carmen concentrations were lower (P<0.05). Hypoxic oFUVECs only showed 7-fold high expression in H19 and no changes the remaining lncRNAs. MALAT1 and NF-kB expressions did not change in response to hypoxic (1% O2) in insulinoma cells lines. NF-kB activity increased with CoCl2-treated MIN6 cells. We found no alternative splicing for MALAT1 transcript and confirmed the coding potential for five lncRNAs. Our data confirmed the MALAT1 as a lncRNA with minimal coding potential with no detection of alternative splicing. Because we could not find a working insulinoma cell that shows responsive MALAT1 and NF-kB, primary fetal sheep islets may be the ideal in vitro model to investigate the regulatory roles of MALAT1 in FGR islets.