Downregulation of lncRNA ANRIL inhibits proliferation, induces apoptosis, and enhances radiosensitivity in nasopharyngeal carcinoma cells through regulating miR-125a

Abstract

Increasing evidence demonstrated that long non-coding RNA ANRIL serves as a fatal oncogene in many cancers, including nasopharyngeal carcinoma (NPC). However, little is known whether ANRIL regulated NPC cell radioresistance. Quantitative real-time PCR (qRT-PCR) was performed to examine the expression of lncRNA ANRIL and miR-125a in NPC tissues and cell lines. MTT assay was conducted to measure the cell viability of CNE2 and HONE1 cells. The apoptotic rate of CNE2 and HONE1 cells was determined by flow cytometry analysis. Colony survival was determined by clonogenic assay. Luciferase reporter assay was performed to verity the direct target of miR-125a. LncRNA ANRIL was evidently elevated in NPC tissues and cell lines. ANRIL inhibition suppressed proliferation, induced apoptosis, and enhanced radiosensitivity in NPC. Moreover, ANRIL could negatively modulate miR-125a expression. Furethermore, ANRIL upregulation reserved the inhibited proliferation, induced apoptosis, and enhanced radiosensitivity triggered by miR-125a overexpression. The expression of lncRNA ANRIL was upregulated in NPC tissues and cells. Moreover, knockdown of ANRIL repressed proliferation, promoted apoptosis, and improved radiosensitivity in NPC via functioning as a miR-125a sponge.

Keywords: ANRIL; NPC; apoptosis; miR-125a; proliferation; radiosensitivity.

Figure 1.

Negative Correlation between ANRIL and miR-125a in NPC tumor tissues. (A and B) qRT-PCR analysis of ANRIL and miR-125a expression in NPC patient tumor tissue samples (n = 35) and normal nasopharyngeal tissue samples (n = 35). (C) The ANRIL level and miR-125a expression had a negative correlation in NPC tumor tissues. *P < 0.05 vs. NC.

Figure 2.

ANRIL expression is upregulated and miR-125a expression is downregulated in NPC cell lines. (A) ANRIL expression was examined by qRT-PCR analysis in 5–8F, CNE1, CNE2 and HONE1 cells or HNEpC. (B) qRT-PCR analysis was conducted to determine the expression of miR-125a in 5–8F, CNE1, CNE2 and HONE1 cells or HNEpC. *P < 0.05 vs. HNEpC.

Figure 3.

ANRIL knockdown inhibits proliferation of NPC cell lines CNE2 and HONE1. CNE2 and HONE1 cells were transfected with si-control or si-ANRIL. (A and B) The level of ANRIL was detected by western blot in CNE2 and HONE1 cells. (C and D) MTT assay was performed to detect the cell viability at 24, 48 and 72 h after transfection in CNE2 and HONE1 cells. *P < 0.05 vs. si-control.

Figure 4.

ANRIL downregulation induces apoptosis, and enhances radiosensitivity in NPC cell lines CNE2 and HONE1. CNE2 and HONE1 cells were transfected with si-control or si-ANRIL. (A and B) Cell apoptosis was determined by flow cytometry analysis at 48 h after transfection in CNE2 and HONE1 cells. (C) The colony survival was determined by clonogenic assay in CNE2 and HONE1 cells treated with IR (0, 2, 4, 6 and 8 Gy). *P < 0.05 vs. si-control.

Figure 5.

ANRIL represses the miR-125a expression in CNE2 and HONE1 cells. (A) Putative miR-125a binding sequence of ANRIL was shown. (B) The relative luciferase activity was detected in CNE2 and HONE1 cells co-transfected with miR-125a mimic or miR-control and wt or mut ANRIL. (C and D) qRT-PCR analysis of miR-125a expression in si-ANRIL or pcDNA-ANRIL transfecting CNE2 and HONE1 cells. *P < 0.05 vs. controls.

Figure 6.

Knockdown of ANRIL inhibits proliferation, induces apoptosis, and enhances radiosensitivity in NPC cells through negatively regulating miR-125a expression. CNE2 and HONE1 cells were were transfected with miR-125a or co-transfected with pcDNA-ANRIL and miR-125a. (A) MTT assay was performed to determine the cell viability of CNE2 and HONE1 cells. (B) Flow cytometry analysis was performed to measure the cell apoptosis of CNE2 and HONE1 cells at 48 h after transfection. (C) Clonogenic assay was conducted to determine the colony survival in CNE2 and HONE1 cells treated with IR (0, 2, 4, 6 and 8 Gy). *P < 0.05 vs. controls.