Radiation induces NORAD expression to promote ESCC radiotherapy resistance via EEPD1/ATR/Chk1 signalling and by inhibiting pri-miR-199a1 processing and the exosomal transfer of miR-199a-5p

Abstract

Background: Radioresistance, a poorly understood phenomenon, results in the failure of radiotherapy and subsequent local recurrence, threatening a large proportion of patients with ESCC. To date, lncRNAs have been reported to be involved in diverse biological processes, including radioresistance. Methods: FISH and qRT–PCR were adopted to examine the expression and localization of lncRNA-NORAD, pri-miR-199a1 and miR-199a-5p. Electron microscopy and nanoparticle tracking analysis (NTA) were conducted to observe and identify exosomes. High-throughput microRNAs sequencing and TMT mass spectrometry were performed to identify the functional miRNA and proteins. A series of in vitro and in vivo experiments were performed to investigate the biological effect of NORAD. ChIP, RIP-qPCR, co-IP and dual-luciferase reporter assays were conducted to explore the interaction of related RNAs and proteins. Results: We show here that DNA damage activates the noncoding RNA NORAD, which is critical for ESCC radioresistance. NORAD was expressed at high levels in radioresistant ESCC cells. Radiation treatment promotes NORAD expression by enhancing H3K4me2 enrichment in its sequence. NORAD knockdown cells exhibit significant hypersensitivity to radiation in vivo and in vitro. NORAD is required to initiate the repair and restart of stalled forks, G2 cycle arrest and homologous recombination repair upon radiation treatment. Mechanistically, NORAD inhibits miR-199a-5p expression by competitively binding PUM1 from pri-miR-199a1, inhibiting the processing of pri-miR-199a1. Mature miR-199a-5p in NORAD knockdown cells is packaged into exosomes; miR-199a-5p restores the radiosensitivity of radioresistant cells by targeting EEPD1 and then inhibiting the ATR/Chk1 signalling pathway. Simultaneously, NORAD knockdown inhibits the ubiquitination of PD-L1, leading to a better response to radiation and anti-PD-1 treatment in a mouse model. Conclusions: Based on the findings of this study, lncRNA-NORAD represents a potential treatment target for improving the efficiency of immunotherapy in combination with radiation in ESCC.