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. 2021 Jan 4;12(1):69.
doi: 10.1038/s41419-020-03302-2.

LncRNA linc00312 suppresses radiotherapy resistance by targeting DNA-PKcs and impairing DNA damage repair in nasopharyngeal carcinoma

Zhen Guo  1   2   3   4 , You-Hong Wang  1   2   3 , Heng Xu  5 , Chun-Su Yuan  6   7 , Hong-Hao Zhou  1   2   3 , Wei-Hua Huang  1   2   3 , Hui Wang  8 , Wei Zhang  9   10   11
Affiliations

LncRNA linc00312 suppresses radiotherapy resistance by targeting DNA-PKcs and impairing DNA damage repair in nasopharyngeal carcinoma

Zhen Guo et al. Cell Death Dis. .

Abstract

Radioresistance is the main obstacle in the clinical management of nasopharyngeal carcinoma (NPC). linc00312 is deregulated in a number of human cancers, including NPC. However, the detailed functions and underlying mechanisms of linc00312 in regulating radiosensitivity of NPC remains unknown. In this study, cox regression analysis was used to assess the association between linc00312 and NPC patients' survival after radiotherapy. Our results reveal that linc00312 is significantly down-regulated in NPC tissues and patients with higher expression of linc00312 are significantly associated with longer overall survival and better short-term radiotherapy efficacy. Overexpression of linc00312 could increase the sensitivity of NPC cells to ionizing radiation, as indicated by clonogenic survival assay, comet assay, and flow cytometry. Mechanistically, RNA pull down and RNA immunoprecipitation were performed to investigate the binding proteins of linc00312. linc00312 directly binds to DNA-PKcs, hinders the recruitment of DNA-PKcs to Ku80, and inhibits phosphorylation of AKT-DNA-PKcs axis, therefore inhibiting the DNA damage signal sensation and transduction in the NHEJ repair pathway. In addition, linc00312 impairs DNA repair and cell cycle control by suppressing MRN-ATM-CHK2 signal and ATR-CHK1 signal. In summary, we identified DNA-PKcs as the binding protein of linc00312 and revealed a novel mechanism of linc00312 in the DNA damage response, providing evidence for a potential therapeutic strategy in NPC.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. linc00312 is down-regulated in NPC and functions as a tumor suppressor.
a The expression of linc00312 was detected by real-time RT-PCR in 92 NPC tissue samples and 10 normal nasopharyngeal tissue samples with chronic inflammation. b Real-time RT-PCR was used to determine the expression of linc00312 in patients with NPC, with radiosensitive response (PR/CR) and radioresistance response (PD/SD). c Cox regression analysis of the association between linc00312 expression level and NPC patients’ 3-year overall survival. d The subcellular localization of linc00312 in HNE1 and HONE1 cells was detected by cytoplasm/nuclear separation assay followed by real-time RT-PCR. GAPDH was utilized as the control of cytoplasm, and U6 was utilized as the control of nuclear. e Representative images of FISH showing the subcellular localization of linc00312 in HNE1 and HONE1 cells. f Cell viability of NPC cells transfected with linc00312 overexpression vector or control vector were measured by CCK-8 assay in series of time points. g The colony-formation ability of NPC cells transfected with linc00312 overexpression vector or control vector were detected by colony formation assay. h Flow cytometry was used to detect the cell cycle of NPC cells transfected with linc00312 overexpression vector or control vector. i The apoptosis of NPC cells transfected with linc00312 overexpression vector or control vector were measured by Hoechst staining. *P < 0.05, **P < 0.01.
Fig. 2
Fig. 2. linc00312 overexpression improves radiosensitivity of NPC cells in vitro.
a The clonogenic survival assay was used to determine the radiosensitivity of NPC cells. b The irradiation dose–survival curve was fitted using the single-hit multitarget model. c Cells were exposed to a series of irradiation dose, and flow cytometry was used to detect cell apoptosis. d The histogram of cell apoptotic rate in different treatment groups. e Real-time RT-PCR was used to detect the expression of linc00312 in NPC cells after 8 Gy irradiation at different points of time. f The irradiation-induced linc00312 overexpression effect was more remarkable if the cells were transfected with linc00312 overexpression vector before irradiation. *P < 0.05, **P < 0.01.
Fig. 3
Fig. 3. linc00312 overexpression enhances the sensitivity of xenograft tumor to radiation in vivo.
a The isolated xenograft tumors from four groups with different treatments (linc00312 overexpression, control, linc00312 overexpression + 6 Gy irradiation, control + 6 Gy irradiation). b The tumor growth and volume in different treatment groups were measured every 3 days. c The weight of tumor in different treatment groups were weighted when the mice were sacrificed. d Representative images of γH2AX detected by immunohistochemical analysis and apoptotic cells detected by TUNEL assay in tumor sections from different treatment groups. e H-score was used to indicate the γH2AX-positive cells in tumor sections from different treatment groups. f The apoptotic rate detected by TUNEL assay was calculated by optical density. *P < 0.05, **P < 0.01.
Fig. 4
Fig. 4. linc00312 directly binds to DNA-PKcs and impairs the formation of DNA–PK complex in response to DSBs.
a The silver-stained PAGE gel that showed the separated proteins pulled down by linc00312. b Pathway enrichment analysis of the pulled down proteins. c The IP products from RIP assay were quantified by real-time RT-PCR. % Input reflects the enrichment efficiency of target genes in IP group compared with Input group. d Pearson’s correlation coefficients of images of internalized linc00312 and DNA-PKcs in NPC cells. e Representative confocal images of linc00312 (red) and DNA-PKcs (green) in NPC cells. f Pearson’s correlation coefficients of images of internalized DNA-PKcs (Ser2056) and Ku80 in NPC cells. g Representative confocal images of DNA-PKcs (Ser2056) (red) and Ku80 (green) at the indicated time points in linc00312 overexpressed cells or control cells that were treated with 8 Gy irradiation. The last column shows the scatterplot of red and green pixel intensities of NPC cells in different treatment groups. *P < 0.05, **P < 0.01.
Fig. 5
Fig. 5. linc00312 affects radiosensitivity through regulating DNA damage response pathways.
a The DNA repair efficiency was detected by NHEJ reporter assay. b Representative images of comet assay in NPC cells transfected with linc00312 overexpression vector or control vector at the indicated time points. c Ionizing radiation-induced DNA damage was quantified by the tail moment. d Western blotting of key proteins involved in DNA damage repair pathway that were affected by linc00312. e The clustering diagram of the six NPC cell samples: orange represents the three cell samples that received 8 Gy irradiation after transfection of linc00312 overexpression vector; red represents the three cell samples that received 8 Gy irradiation after transfection of control vector. f The differential genes (P < 0.05) identified by microarray between linc00312 + IR and control + IR groups were enriched by KEGG pathway analysis and displayed as bubble map. *P < 0.05, **P < 0.01.
Fig. 6
Fig. 6. The role of linc00312 in regulating radiosensitivity of NPC.
linc00312 directly binds to DNA-PKcs, hinders the recruitment of DNA-PKcs to Ku80, and inhibits phosphorylation of AKT–DNA–PKcs axis, therefore inhibiting the DNA damage signal sensation and transduction in the NHEJ repair pathway. In addition, linc00312 impairs DNA repair and cell cycle control by suppressing MRN–ATM–CHK2 signal and ATR–CHK1 signal.
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