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. 2021 Jan 30;22(3):1407.
doi: 10.3390/ijms22031407.

lncRNA CASC19 Contributes to Radioresistance of Nasopharyngeal Carcinoma by Promoting Autophagy via AMPK-mTOR Pathway

Hongxia Liu  1 , Wang Zheng  1 , Qianping Chen  1 , Yuchuan Zhou  1 , Yan Pan  1 , Jianghong Zhang  1 , Yang Bai  1 , Chunlin Shao  1
Affiliations

lncRNA CASC19 Contributes to Radioresistance of Nasopharyngeal Carcinoma by Promoting Autophagy via AMPK-mTOR Pathway

Hongxia Liu et al. Int J Mol Sci. .

Abstract

Nasopharyngeal carcinoma (NPC) is one of the most frequent head and neck malignant tumors and is majorly treated by radiotherapy. However, radiation resistance remains a serious obstacle to the successful treatment of NPC. The aim of this study was to discover the underlying mechanism of radioresistance and to elucidate novel genes that may play important roles in the regulation of NPC radiosensitivity. By using RNA-seq analysis of NPC cell line CNE2 and its radioresistant cell line CNE2R, lncRNA CASC19 was screened out as a candidate radioresistance marker. Both in vitro and in vivo data demonstrated that a high expression level of CASC19 was positively correlated with the radioresistance of NPC, and the radiosensitivity of NPC cells was considerably enhanced by knockdown of CASC19. The incidence of autophagy was enhanced in CNE2R in comparison with CNE2 and another NPC cell line HONE1, and silencing autophagy with LC3 siRNA (siLC3) sensitized NPC cells to irradiation. Furthermore, CASC19 siRNA (siCASC19) suppressed cellular autophagy by inhibiting the AMPK/mTOR pathway and promoted apoptosis through the PARP1 pathway. Our results revealed for the first time that lncRNA CASC19 contributed to the radioresistance of NPC by regulating autophagy. In significance, CASC19 might be a potential molecular biomarker and a new therapeutic target in NPC.

Keywords: AMPK/mTOR; autophagy; lncRNA CASC19; nasopharyngeal carcinoma; radioresistance.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Validation of the radioresistant CNE2R cells by the measurements of colony formation, apoptosis, and cell migration. (A) Colony formation assay of the survival curve of CNE2R cells and its parent cell line CNE2 irradiated with different doses of γ-rays. (B) Flow cytometry assay of apoptosis of CNE2 and CNE2R cells irradiated with 4 Gy of γ-rays. (C) Migration assay and invasion assay of CNE2 and CNE2R cells. * p < 0.05 between indicated groups.
Figure 2
Figure 2
RNA-seq analysis of the paired radiosensitive and radioresistant nasopharyngeal carcinoma (NPC) cell lines CNE2 and CNE2R. (A,B) The Circos plots of total DElncRNAs and DEmRNAs between CNE2 and CNE2R cells. (C) The heat map plot of the distinguishable DElncRNAs with the threshold of log2 (fold changes) > 1.5 (p < 0.05). (D) KEGG pathway analysis of the DElncRNAs co-expressed mRNAs with a correlation coefficient larger than 0.995 (p < 0.05). (E) The expression abundance (FPKM, FragmentsPer Kilobase per Million) of the top 20 upregulated DElncRNAs in radioresistant cell line CNE2R. (F) Kaplan–Meier analysis of the relationship between the expression of CASC19 in head and neck squamous cell carcinoma (HNSC) and overall survival (OS) of the patients (data from GEPIA).
Figure 3
Figure 3
Relationship of lncRNA CASC19 expression with radiosensitivity of NPC cells. (A) Reverse-transcription quantitative PCR (RT-qPCR) assay of CASC19 expression in HONE1, CNE2, and CNE2R cells. (B) The survival factions of HONE1, CNE2, and CNE2R cells detected by clone formation assay. (C) The efficiency of CASC19 siRNA transfection in CNE2R cells. (D) Dose responses of survival factions of CNE2R cells after transfection of siCASC19-1, siCASC19-2, and control siRNA, respectively. (E,G) The influence of si-CASC19-2 on the expression of CASC19 in CNE2 cells and the survival fraction of CNE2 cells after irradiation. (F,H) The influence of si-CASC19-2 on the expression of CASC19 in HONE1 cells and the survival fraction of HONE1 cells after irradiation. * p < 0.05 between indicated groups.
Figure 4
Figure 4
CASC19 knockdown-enhanced radiosensitivity of NPC xenograft tumors. (A) Tumor growth curves and representative images of HONE1, CNE2, and CNE2R xenografts in nude mice. The xenografts were irradiated with 8 Gy × 3 under a volume of about 100 mm3. * p < 0.05 between indicated groups of same cell line; # p < 0.05 between indicated different cell lines before and after irradiation. (B) RT-qPCR assay of the CASC19 expression in the CNE2R xenograft tumors that were intratumorally injected with cholesterol-modified CASC19 siRNA every 3 days from the time-point of the tumor having a volume of about 50 mm3 until the mice were sacrificed. (C) Tumor growth curves and representative image of CNE2R xenograft tumors under different treatments of siNC, siCASC19, siNC + ionizing radiation (IR), and siCASC19 + IR, respectively. * p < 0.05 between same cells before and after irradiation. # p < 0.05 between CNE2R siNC group and CNE2R siCASC19 group before and after irradiation.
Figure 5
Figure 5
Autophagy potentiated the radioresistance of NPC cells. (A) Fluorescence images of HONE1, CNE2, and CNE2R cells transfected with mRFP-GFP-LC3-tagged adenovirus (×40). (B) Western blot assay of P62 and LC3 proteins in HONE1, CNE2, and CNE2R cells. (C) Western blot assay of LC3 proteins in HONE1, CNE2, and CNE2R cells after 24 h of 4 Gy radiation. (D) Efficiency of siLC3 transfection in CNE2R cells. (E) Influence of siLC3 transfection in the survival of CNE2R cells after radiation. (F) Fluorescence images siLC3-interfered CNE2R cells transfected with mRFP-GFP-LC3-tagged adenovirus. * p < 0.05 between indicated groups.
Figure 6
Figure 6
CASC19 knockdown by siRNA inhibited autophagy incidence via the AMPK/mTOR pathway and increased apoptosis in CNE2R cells. (A) Fluorescence images of CNE2R cells transfected with siCASC19 and mRFP-GFP-LC3-tagged adenovirus (×40). 3-MA was applied as a negative control for autophagy inhibitor and rapamycin was used as a positive control for autophagy induction. (B) Western blot assay of P62, LC3, p-mTOR, mTOR, p-AMPK, and AMPK proteins and their relative expression levels in CNE2R cells after transfection with siCASC19-1 and siCASC19-2. (C) Micronucleus formation in CNE2, CNE2R, and relevant siCASC19-transfected cells after 4 Gy irradiation. (D) Western blot assay of γH2AX protein in CNE2, CNE2R cells, and siCASC19-transfected CNE2R cells at 0.5 h after 4 Gy irradiation. (E) Flow cytometry assay of apoptosis in 4 Gy-irradiated CNE2R cells that were transfected with siCASC19 and siNC. (F) Western blot analysis of PARP1 and cleaved-caspase3 proteins in 4 Gy-irradiated CNE2R cells that were transfected with siCASC19 and siNC, respectively. * p < 0.05 between indicated groups.
Figure 7
Figure 7
Regulation pathway of CASC19 in the radioresistance of NPC. Irradiation (IR) increased the expression of CASC19 in NPC cell lines. CASC19 contributes to autophagy via activating the AMPK/mTOR signaling pathway while inhibiting apoptosis by depressing PARP1 and cleaved-caspase3.
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