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Research Article
Xiaozhi Zhang
Xi'an JiaoTong University
Corresponding Author
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Background Radioresistance, a poorly understood phenomenon, results in the failure of radiotherapy and consequent local recurrence, threatening a large proportion of ESCC patients. To date, lncRNAs have been found to be involved in diverse biological processes, including radioresistance.
Methods ELISA was used to evaluated the H3 modifications in radio-resistant ESCC cells. FISH and qRT-PCR were adopted to examine the expression and localization of lncRNA-NORAD, pri-miR-199a and miR-199a. Electron microscopy and Nanoparticle tracking analysis (NTA) was conducted to observe and identify exosomes. High-throughput RNA sequencing and TMT mass spectrometry were performed to identify the functional lncRNAs and proteins involved in ESCC radioresistance. A series of in vitro and in vivo experiments were performed to investigate the biological effect of NORAD. CHIP, qPCR-RIP, co-IP and dual-luciferase reporter assays were used to explore the interaction of related RNAs and proteins.
Results We show here that a DNA damage activated non-coding RNA-NORAD, which is critical for ESCC radio-resistance. NORAD was highly expressed in radio-resistant ESCC cells and tissues. Irradiation treatment promotes NORAD expression via enhancing H3K4me2 enrichment on its region. NORAD knockdown cells exhibit significantly hypersensitivity to irradiation in vivo and in vitro. NORAD is required for initiating repair and restart of stalled forks, G2 cycle arrest and homologous recombination repair upon irradiation treatment. Mechanistically, NORAD inhibits miR-199a expression by competitively binding PUM1 from pri-miR-199a, inhibiting the process of pri-miR-199a. Mature miR-199a in NORAD-knockdown cells can be packaged into exosomes; miR-199a restores the radiosensitivity of radioresistant cells by targeting EEPD1, then inhibiting ATR/Chk1 signaling pathway. Simultaneously, NORAD knockdown blocks the ubiquitination of PD-L1, leads to the better response for radiation and anti-PD-1 treatment in mouse model.
Conclusion This study raises the possibility that LncRNA-NORAD could be a potential treatment target for improving the efficiency of immunotherapy in combination with radiation in ESCC.
Keywords
Esophageal squamous cell carcinoma, Radioresistance, NORAD, pri-miR-199a, EEPD1
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This is a list of supplementary files associated with this preprint. Click to download.
- supplementary1.tif
Supplementary Fig.1 A. The enrichment of H3K4me2 on NORAD region in MCF-7, HCT-116, SK-N-SH and KMS-11 cancer cells based on UCSC. B. The expression correlation of NORAD with multiple H3K4 methyltransferases, including Ash1, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, Set1A, Set1B, SETD7, SMYD2, SMYD3, WDR5.s
- supplementary2.tif
Supplementary Fig.2 A. SDS-PAGE measurement of total proteins extracted from KYSE-150-sh-nc and KYSE-150-sh-NORAD cells. B. Heatmap for differential expressed proteins between KYSE-150-sh-nc and KYSE-150-sh-NORAD cells. C. KEGG analysis of the downregulated proteins in NORAD knockdown cells. D. Gene Ontology analysis of the downregulated proteins in NORAD knockdown cells. E. The protein-protein interaction for the potential interacted proteins of EEPD1 are mapped by STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database (version 11.0). F. Gene Ontology Analysis of proteins potentially interacted with EEPD1 and 15 terms in Biological Process, 6 terms in Molecular Function and 1 GO-term in Cellular Component are significantly enriched.
- supplementary3.tif
Supplementary Fig.3 A. The infiltration fraction of immune cells (B cells, CD4+T cells, CD8+T cells and Macrophages) based on CIBERSORT between NORAD high expression and NORAD low expression ESCC patients’ group. B. Representative IHC for CD8+ T cells (I) and CD4+ T cells in NORAD knockdown and control group of allograft tumors. The scale bar represents 50 μm.
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Received 06 Jun, 2021
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On 27 May, 2021
Reviews received
Received 18 May, 2021
Reviewer #1 agreed
On 17 May, 2021
Reviewers invited
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This preprint is under consideration at Journal of Experimental & Clinical Cancer Research. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research Article
Xiaozhi Zhang
Xi'an JiaoTong University
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 14 May, 2021
No community comments so far
Editorial decision: Major revision
On 11 Jun, 2021
Review #2 received
Received 10 Jun, 2021
Review #1 received
Received 06 Jun, 2021
Reviewer #2 agreed
On 27 May, 2021
Reviews received
Received 18 May, 2021
Reviewer #1 agreed
On 17 May, 2021
Reviewers invited
Invitations sent on 12 May, 2021
Editor assigned
On 11 May, 2021
First submitted
On 11 May, 2021
Submission checks complete
On 10 May, 2021
Editor invited
On 10 May, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background Radioresistance, a poorly understood phenomenon, results in the failure of radiotherapy and consequent local recurrence, threatening a large proportion of ESCC patients. To date, lncRNAs have been found to be involved in diverse biological processes, including radioresistance.
Methods ELISA was used to evaluated the H3 modifications in radio-resistant ESCC cells. FISH and qRT-PCR were adopted to examine the expression and localization of lncRNA-NORAD, pri-miR-199a and miR-199a. Electron microscopy and Nanoparticle tracking analysis (NTA) was conducted to observe and identify exosomes. High-throughput RNA sequencing and TMT mass spectrometry were performed to identify the functional lncRNAs and proteins involved in ESCC radioresistance. A series of in vitro and in vivo experiments were performed to investigate the biological effect of NORAD. CHIP, qPCR-RIP, co-IP and dual-luciferase reporter assays were used to explore the interaction of related RNAs and proteins.
Results We show here that a DNA damage activated non-coding RNA-NORAD, which is critical for ESCC radio-resistance. NORAD was highly expressed in radio-resistant ESCC cells and tissues. Irradiation treatment promotes NORAD expression via enhancing H3K4me2 enrichment on its region. NORAD knockdown cells exhibit significantly hypersensitivity to irradiation in vivo and in vitro. NORAD is required for initiating repair and restart of stalled forks, G2 cycle arrest and homologous recombination repair upon irradiation treatment. Mechanistically, NORAD inhibits miR-199a expression by competitively binding PUM1 from pri-miR-199a, inhibiting the process of pri-miR-199a. Mature miR-199a in NORAD-knockdown cells can be packaged into exosomes; miR-199a restores the radiosensitivity of radioresistant cells by targeting EEPD1, then inhibiting ATR/Chk1 signaling pathway. Simultaneously, NORAD knockdown blocks the ubiquitination of PD-L1, leads to the better response for radiation and anti-PD-1 treatment in mouse model.
Conclusion This study raises the possibility that LncRNA-NORAD could be a potential treatment target for improving the efficiency of immunotherapy in combination with radiation in ESCC.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
This is a list of supplementary files associated with this preprint. Click to download.
- supplementary1.tif
Supplementary Fig.1 A. The enrichment of H3K4me2 on NORAD region in MCF-7, HCT-116, SK-N-SH and KMS-11 cancer cells based on UCSC. B. The expression correlation of NORAD with multiple H3K4 methyltransferases, including Ash1, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, Set1A, Set1B, SETD7, SMYD2, SMYD3, WDR5.s
- supplementary2.tif
Supplementary Fig.2 A. SDS-PAGE measurement of total proteins extracted from KYSE-150-sh-nc and KYSE-150-sh-NORAD cells. B. Heatmap for differential expressed proteins between KYSE-150-sh-nc and KYSE-150-sh-NORAD cells. C. KEGG analysis of the downregulated proteins in NORAD knockdown cells. D. Gene Ontology analysis of the downregulated proteins in NORAD knockdown cells. E. The protein-protein interaction for the potential interacted proteins of EEPD1 are mapped by STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database (version 11.0). F. Gene Ontology Analysis of proteins potentially interacted with EEPD1 and 15 terms in Biological Process, 6 terms in Molecular Function and 1 GO-term in Cellular Component are significantly enriched.
- supplementary3.tif
Supplementary Fig.3 A. The infiltration fraction of immune cells (B cells, CD4+T cells, CD8+T cells and Macrophages) based on CIBERSORT between NORAD high expression and NORAD low expression ESCC patients’ group. B. Representative IHC for CD8+ T cells (I) and CD4+ T cells in NORAD knockdown and control group of allograft tumors. The scale bar represents 50 μm.
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