Downregulation of lncRNA X Inactive Specific Transcript (XIST) Suppresses Cell Proliferation and Enhances Radiosensitivity by Upregulating mir-29c in Nasopharyngeal Carcinoma Cells

Abstract

BACKGROUND LncRNA X inactive specific transcript (XIST) was reported to function as an oncogene in nasopharyngeal carcinoma cells (NPC) by sponging miR-34a-5p. However, the role of XIST in modulating the radiosensitivity of NPC cells and its mechanism still remain undefined. MATERIAL AND METHODS The expressions of XIST and miR-29c in NPC cells were evaluated by qRT-PCR. CNE1 and CNE2 cells were transfected with si-XIST, pcDNA-XIST, miR-29c mimics, anti-miR-29c, or respective controls by Lipofectamine 2000. The effects of XIST knockdown and miR-29c overexpression on cell proliferation, survival fraction, and γ-H2AX expression were investigated by CCK-8 assay, colony formation assay, immunofluorescence, and Western blot, respectively. Luciferase reporter assay and qRT-PCR analysis were performed to confirm whether XIST interacts with miR-29c and regulates its expression. RESULTS XIST was upregulated and miR-29c was downregulated in NPC cells. The expressions of XIST and miR-29c changed reversely in response to irradiation. Knockdown of XIST and miR-29c overexpression both resulted in a dramatic suppression of cell proliferation, a marked enhancement of radiosensitivity, and an obvious increase of γ-H2AX foci formation in NPC cells. Luciferase reporter assay and qRT-PCR analysis demonstrated that XIST interacts with miR-29c and negatively regulates its expression. Moreover, miR-29c inhibition abrogated XIST knockdown-induced cell proliferation inhibition and radiosensitivity increase in NPC cells. CONCLUSIONS XIST knockdown suppressed cell proliferation and enhanced radiosensitivity of NPC cells by upregulating miR-29c, providing a novel therapeutic target to improve radiotherapy efficiency for patients with NPC.

Figure 1

Expression alteration of XIST and miR-29c in NPC cells in response to irradiation. (A) qRT-PCR was performed to examine the expressions of XIST and miR-29c in NPC cell lines (CNE1 and CNE2) and primary normal human nasal epithelial line HNEpC. qRT-PCR was carried out to analyze the expressions of XIST (B) and miR-29c (C) in CNE1 and CNE2 cells at indicated time points after 4-Gy irradiation treatment. * P<0.05.

Figure 2

Effect of XIST knockdown on cell proliferation and radiosensitivity of NPC cells. (A) Transfection efficiency of si-XIST-1 and si-XIST-2 in CNE1 and CNE2 cells was detected by qRT-PCR. (B) CCK-8 assay was performed to determine cell proliferation at 24 h, 48 h, and 72 h in si-XIST- or si-con-transfected CNE1 and CNE2 cells. (C) Colony formation assay was used to measure colony survival rate 2 weeks after CNE1 and CNE2 cells transfected with si-XIST or si-con were exposed to the indicated single doses of irradiation (0, 2, 4, 6, or 8 Gy). (D) CNE1 and CNE2 cells transfected with si-XIST or si-con were irradiated with 4-Gy X-rays and then subjected to immunofluorescent staining for γ-H2AX expression. (E) Western blot analysis of γ-H2AX expression level in CNE1 and CNE2 cells transfected with si-XIST or si-con under 4 Gy irradiation. * P<0.05.

Figure 3

Effect of miR-29c overexpression on cell proliferation and radiosensitivity of NPC cells. (A) CCK-8 assay was carried out to evaluate cell proliferation at 24 h, 48 h, and 72 h in CNE1 and CNE2 cells transfected with miR-29c or miR-con. (B) The clonogenic survival curves were compared in CNE1 and CNE2 cells transfected with miR-29c or miR-con with the indicated single doses of irradiation (0, 2, 4, 6, or 8 Gy) treatment. (C) Immunofluorescent staining for γ-H2AX expression in CNE1 and CNE2 cells transfected with miR-29c or miR-con under 4-Gy irradiation. (D) Western blot analysis of γ-H2AX level in CNE1 and CNE2 cells transfected with miR-29c or miR-con with 4-Gy irradiation. * P<0.05.

Figure 4

Mutual effect between XIST and miR-29c. (A) The predicted wild-type or mutant binding sites of miR-29c on XIST. (B) CNE1 and CNE2 cells were cotransfected with miR-29c or miR-con and pGL3-XIST-wt or pGL3-XIST-mut, and luciferase reporter assay was performed. (C) The expression of miR-29c in CNE1 and CNE2 cells transfected with si-XIST, pcDNA-XIST, or respective controls was evaluated by qRT-PCR. * P<0.05.

Figure 5

miR-29c downregulation abated the effects of si-XIST on proliferation and radiosensitivity of NPC cells. (A) Cell viability was determined by CCK-8 assay in CNE1 and CNE2 cells transfected with si-XIST or combined si-XIST and anti-miR-29c. (B) CNE1 and CNE2 cells transfected with si-XIST or simultaneous si-XIST and anti-miR-29c were exposed to the indicated single doses of irradiation (0, 2, 4, 6, or 8 Gy), and colony formation assay was used to measure colony survival rate 2 weeks later. * P<0.05, si-XIST vs. si-con; ^# P<0.05, si-XIST+anti-miR-29c vs. si-XIST+anti-miR-con.