LncRNA NEAT1/let-7a-5p axis regulates the cisplatin resistance in nasopharyngeal carcinoma by targeting Rsf-1 and modulating the Ras-MAPK pathway

Abstract

The long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) was reported to be upregulated and be involved in oncogenic growth and drug resistance in nasopharyngeal carcinoma (NPC). However, the exact roles of NEAT1 and its underlying mechanisms in the drug resistance of NPC remain largely unclear. In this study, the expressions of NEAT1, let-72-5p and Rsf-1 mRNA were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The effects of NEAT1 and let-72-5p on cell proliferation and cisplatin resistance of NPC cells were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and 5-ethynyl-20-deoxyuridine (EdU) assay. Western blot analysis was performed to detect the protein levels of Rsf-1, Ras, p-Raf1, Raf1, p-MEK1, MEK1, p-ERK1/2 and ERK1/2. Xenograft tumor assay was done to elucidate the role of NEAT1 involved in NPC tumor growth in vivo. We found that NEAT1 was upregulated and let-7a-5p was downregulated in NPC tissues, as well as NPC cell lines. Inhibition of NEAT1 markedly repressed the cisplatin resistance of NPC cells. NEAT1 was demonstrated to interact with let-7a-5p. Besides, a negative correlation between NEAT1 and let-7a-5p expression was observed in NPC tissues. Rsf-1 was confirmed as a target of let-7a-5p. NEAT1 remarkably reversed the inhibitory effect of let-7q-5p on the cisplatin resistance of NPC cells in vitro. Additionally, NEAT1 knockdown inhibited the Ras-MAPK pathway in NPC cells. NEAT1 knockdown suppressed tumor growth in the presence of cisplatin in vivo. Overall, these findings suggest that NEAT1/let-7a-5p axis regulates the cisplatin resistance in NPC by targeting Rsf-1 and modulating the Ras-MAPK signaling pathway.

Keywords: Ras-Mapk pathway; Rsf-1; cisplatin; let-7a-5p; long non-coding RNA; nasopharyngeal carcinoma; nuclear paraspeckle assembly transcript 1.

Figure 1.

The expression levels of NEAT1 and let-7a-5p in NPC tissues and cells. RT-qPCR analysis of expression of NEAT1 (A) and let-7a-5p (B) in 24 paired of NPC tissues and corresponding normal tissues. RT-qPCR analysis of NEAT1 (C) and let-7a-5p (D) expression in NPC cell lines (CNE-1, S18, 5–8F, HK-1) and NPE cell line NP-69. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 2.

NEAT1 knockdown in hibited cisplatin resistance of NPC cells. The expression level of NEAT1 in S18 (A) and 5–8F (B) cells was obviously downregulated by transfecting with sh-NEAT1. Cell viability was determined by MTT assay in S18 (C) and 5–8F (D) cells after transfection with sh-NEAT1 or sh-NC, following exposing to 10 μg/ml of cisplatin. The representative fluorescent images of EdU assay in S18 (E) and 5–8F (F) cells were shown. EdU assay showed that downregulation of NEAT1 inhibited cell proliferation ability in S18 (G) and 5–8F (H) cells in the presence of 10 μg/ml of cisplatin. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3.

Verification of NEAT1 as a direct target of let-7a-5p. (A) The sequence of the predicted let-7a-5p targeting site within NEAT1 3′-UTR and the mutated sites were shown. (B) After co-transfection with pMIR-report plasmid and let-71-5p, anta-let-7a-5p or matched controls, the relative luciferase activity of pMIR-report plasmid containing Wt or Mut NEAT1 were evaluated in 5–8F cells. (C) The effect of pcDNA-NEAT1 on NEAT1 expression was validated by RT-qPCR. (D) RT-qPCR analysis of let-7a-5p expression in 5–8F cells transfected with pcDNA-NEAT1, sh-NEAT1 or matched controls. (E) The correlation analysis showed that let-7a-5p was negatively associated with NEAT1 in NPC tissues. **P < 0.01, and ***P < 0.001.

Figure 4.

Rsf-1 was a direct target of let-7a-5p. (A) The predicted let-7a-5p complementary site within the Rsf-1 3′-UTR and the mutated sites were shown. (B) let-7a-5p obviously repressed the luciferase activity of the pMIR-Rsf-1-Wt but not that of pMIR-Rsf-1-Mut. Upregulation of let-7a-5p decreased, but anta-let-7a-5p increased, the Rsf-1 mRNA level (C) and protein level (D) in 5–8F cells. ***P < 0.001.

Figure 5.

NEAT1 reversed the inhibitory effect of let-7a-5p on the cisplatin resistance of NPC cells. MTT assay was used to assess cell viability in S18 (A) and 5–8F (B) cells stably transfected with let-7a-5p alone or combined with pcDNA-NEAT1, following exposure to 10 μg/ml cisplatin for 48 h. EdU assay was performed to detect the cell proliferation in S18 (C) and 5–8F (D) cells transfected with let-7a-5p or pcDNA-NEAT1, following treatment with 10 μg/ml cisplatin for 48 h. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 6.

Knockdown of NEAT1 inhibited the Ras-MAPK pathway. Western blot analysis was conducted to measure protein levels of Ras, p-Raf1, Raf1, p-MEK1, MEK1, p-ERK1/2 and ERK1/2 in S18 (A) and 5–8F (B) cells transfected with sh-NEAT1 or sh-NC.

Figure 7.

Knockdown of NEAT1 suppresses NPC tumor growth in vivo. The xenograft tumor model was established by subcutaneously injecting with 5–8F cells stably expressing sh-NEAT1 or sh-NC, following administration with cisplatin or phosphate buffered saline every three days. (A) The tumor size was monitored through measuring the length and width every week. (B) RT-qPCR analysis of NEAT1 expression in xenografted tumors. (C) Mice were sacrificed at 5 weeks after injection and tumors were excised, pictured and weighed. Scale bar: 1 mm. **P < 0.01 and ***P < 0.001.