Abstract
BackgroundRadioresistance, 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.MethodsFISH 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.ResultsWe 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.ConclusionsBased 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.
Highlights
Radioresistance, a poorly understood phenomenon, results in the failure of radiotherapy and subsequent local recurrence, threatening a large proportion of patients with esophageal squamous cell carcinoma (ESCC)
Substantial progress has been achieved in identifying novel biomarkers and therapeutic targets for improving radiation sensitivity, the molecular mechanism underlying radioresistance remains ambiguous and complex; it is proposed to involve cell cycle checkpoints that prevent cancer cells from sustaining radiation-induced DNA damage, activation of the DNA damage response, the self-renewal of cancer stem cells, the epithelialmesenchymal transition (EMT), etc. [7, 8] In this manuscript, we aimed to identify key genes that participate in sensitizing ESCC cells to radiation therapy and to lay the foundation for drug development
FISH results from 77 patients with ESCC who had been treated with radiotherapy indicated that NORAD was mainly located in the cytoplasm (Supplementary Fig. 1A)
Summary
Radioresistance, a poorly understood phenomenon, results in the failure of radiotherapy and subsequent local recurrence, threatening a large proportion of patients with ESCC. Comprehensive therapy, including radiation and chemotherapy, is introduced to either improve the effects of surgery or control the growth of cancer lesions in patients with nonresectable tumours. Radiation is an important part of combined therapy, representing an efficient method to control local recurrence and optimize surgical strategies. Preoperative chemotherapy combined with radiotherapy is required for cervical esophageal cancer [5]. Both definitive and preoperative radiation are important therapeutic strategies that prolong the overall survival of patients with ESCC [6]. Substantial progress has been achieved in identifying novel biomarkers and therapeutic targets for improving radiation sensitivity, the molecular mechanism underlying radioresistance remains ambiguous and complex; it is proposed to involve cell cycle checkpoints that prevent cancer cells from sustaining radiation-induced DNA damage, activation of the DNA damage response, the self-renewal of cancer stem cells, the epithelialmesenchymal transition (EMT), etc. [7, 8] In this manuscript, we aimed to identify key genes that participate in sensitizing ESCC cells to radiation therapy and to lay the foundation for drug development
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