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.

Characterization of plasma exosomal microRNAs in responding to radiotherapy of human esophageal squamous cell carcinoma

“…a total of 8 and 9 mirnas with upand downregulated expression in pre-and post-radiotherapy plasma samples were identified, respectively. Certain identified mirnas in the present study have also been shown as novel circulating factors in responding to radiotherapy in other types of cancer (52,53,(60)(61)(62). Moreover, a higher level of plasma miR-339-5p, which was also identified in the present study, has been shown to be associated with radiotherapy sensitivity and favorable survival by targeting Cdc25A gene in eScc (53).…”

“…Certain identified mirnas in the present study have also been shown as novel circulating factors in responding to radiotherapy in other types of cancer (52,53,(60)(61)(62). Moreover, a higher level of plasma miR-339-5p, which was also identified in the present study, has been shown to be associated with radiotherapy sensitivity and favorable survival by targeting Cdc25A gene in eScc (53). Therefore, radiotherapy causes significant alterations in expression levels of exosomal mirnas in eScc plasma.…”

The Mechanism of Long Non-coding RNA in Cancer Radioresistance/Radiosensitivity: A Systematic Review

“…Supplementary Table S2 listed the lncRNAs involved in cancer radioresistance/radiosensitivity and their concise mechanisms. The sources of cancer included bladder cancer ( Tan et al, 2015 ; Jiang et al, 2017 ), breast cancer ( Liu et al, 2019a ; Zhang et al, 2019a ; Wang et al, 2019 ; Qian et al, 2020 ; Zhang et al, 2020a ), cardiac cancer ( Jia et al, 2019 ), gastric cancer ( Jiang et al, 2020 ; Lu et al, 2020 ; Xiao et al, 2020 ), cervical cancer ( Jing et al, 2015 ; Li et al, 2018a ; Han et al, 2018 ; Gao et al, 2019 ; Zhao et al, 2019 ; Wang et al, 2020a ; Ge et al, 2020 ), colorectal cancer ( Liu et al, 2020a ; Liu et al, 2020b ; Li et al, 2021 ; Liang et al, 2021 ), esophageal cancer ( Li et al, 2017 ; Chen et al, 2018a ; Zhang et al, 2019b ; Wang et al, 2020b ; Cheng et al, 2020 ; Liu et al, 2021a ; Sun et al, 2021 ; Yang et al, 2021 ), glioblastoma ( Li et al, 2018b ; Ahmadov et al, 2021 ; Li et al, 2022 ), glioma ( Yang et al, 2016 ; Zheng et al, 2016 ; Wang et al, 2020c ; Wang et al, 2020d ; Tang et al, 2020 ; Zheng et al, 2020 ; Gao et al, 2021 ; Tian et al, 2021 ), head and neck squamous cell cancer ( Li et al, 2020a ), laryngeal cancer ( Cui et al, 2019 ; Tang et al, 2019 ; Tang and Shan, 2019 ), nasopharyngeal cancer ( Jin et al, 2016 ; Wang et al, 2017 ; Ma et al, 2018 ; Yi et al, 2019 ; Han et al, 2020a ; Han et al, 2020b ; Wang et al, 2020e ; ...…”

“…ATM (ATM serine/threonine kinase) is an essential component of the response to DNA damage and the maintenance genome stability, which is the main repair protein involved in double-strand break (DSB) homologous recombination repair (HRR) induced by ionizing radiation ( Qin et al, 2019 ). Besides, lncRNA NORAD was reported to upregulate esophageal squamous cell cancer radioresistance via miR-199-a1/EEPD1 ( Sun et al, 2021 ). EEPD1 (endonuclease/exonuclease/phosphatase family domain containing 1) participates in DNA repair and maintains genome stability by promoting end excision and homologous recombination repair ( Wu et al, 2015 ).…”

Function and Molecular Mechanism of the DNA Damage Response in Immunity and Cancer Immunotherapy

“…The RecQ–like BLM helicase partners with EXO5 and EEPD1 nucleases for stalled DNA replication restart and maintenance of genome integrity ( 62 , 63 ). BLM deficiency in Bloom syndrome (BS) causes increased expression of inflammatory genes through the cGAS–STING–IRF3 pathway, suggesting it prevents unchecked inflammatory gene responses ( 64 ).…”