Long noncoding RNA NEAT1 inhibits the acetylation of PTEN through the miR-524-5p /HDAC1 axis to promote the proliferation and invasion of laryngeal cancer cells

Abstract

Long noncoding RNA nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1) is abnormally expressed in numerous tumors and functions as an oncogene, but the role of NEAT1 in laryngocarcinoma is largely unknown. Our study validated that NEAT1 expression was markedly upregulated in laryngocarcinoma tissues and cells. Downregulation of NEAT1 dramatically suppressed cell proliferation and invasion through inhibiting miR-524-5p expression. Additionally, NEAT1 overexpression promoted cell growth and metastasis, while overexpression of miR-524-5p could reverse the effect. NEAT1 increased the expression of histone deacetylase 1 gene (HDAC1) via sponging miR-524-5p. Mechanistically, overexpression of HDAC1 recovered the cancer-inhibiting effects of miR-524-5p mimic or NEAT1 silence by deacetylation of tensin homolog deleted on chromosome ten (PTEN) and inhibiting AKT signal pathway. Moreover, in vivo experiments indicated that silence of NEAT1 signally suppressed tumor growth. Taken together, knockdown of NEAT1 suppressed laryngocarcinoma cell growth and metastasis by miR-524-5p/HDAC1/PTEN/AKT signal pathway, which provided a potential therapeutic target for laryngocarcinoma.

Keywords: histone deacetylationase 1; laryngocarcinoma; long noncoding RNA nuclear paraspeckle assembly transcript 1; miR-524-5p; phosphatase and tensin homolog /protein kinase B signaling pathway.

Figure 1

NEAT1 was upregulated in laryngocarcinoma. (A) The expression of lncRNA NEAT1 in laryngocarcinoma tissues and normal adjacent tissues was detected using qPCR. (n=20). (B) QPCR was used to detect the expression of NEAT1 in laryngocarcinoma patients with different stage tumors. (C) The level of NEAT1 was validated by in situ hybridization histochemistry in tissue biopsies. (D) The levels of NEAT1 in normal nasopharyngeal epithelial cell line (NP69) and laryngocarcinoma cell lines (TU212, SNU899, M2E and SNU46). ** P < 0.01 versus normal adjacent tissues, or laryngocarcinoma patients with stage I/II, or NP69.

Figure 2

Downregulation of NEAT1 suppressed laryngocarcinoma cell proliferation and invasion. (A) The transfection efficiency was detected after transfection with si-NEAT1 for 24 h in TU212 and M2E cells. (B, C) Cell proliferation of TU212 and M2E cells were detected using CCK-8 assay after transfection with si-NEAT1 for 0, 24, 48 and 72 h. (D) Apoptosis rates of TU212 and M2E cells after transfection with si-NEAT1 were detected using Flow cytometry. (E) Transwell assay was used to evaluate cell invasion ability after transfection with si-NEAT1 for 48 h. (F) Western blot analysis was used to detect the protein expression levels of PTEN, Ac-PTEN, AKT and p-AKT after transfection with si-NEAT1. ** P < 0.01 versus Scramble.

Figure 3

NEAT1 bound to miR-524-5p and inhibited its expression. QPCR was performed to analyze the levels of miR-524-5p in human laryngocarcinoma tissues and normal adjacent tissues (A) and laryngocarcinoma patients with different stage tumors (B). (C) Spearman’s correlation analysis was used to evaluate the expression relationship between NEAT1 and miR-524-5p. (D) The predicted binding sites of NEAT1 and miR-524-5p. (E) The luciferase reporter gene assay was conducted to confirm the target relationship between NEAT1 and miR-524-5p. (F) RIP assay was conducted to examine miR-524-5p endogenously associated with NEAT1. (G) The level of miR-524-5p in normal nasopharyngeal epithelial cell line and laryngocarcinoma cell lines. (H) The expression of miR-524-5p in TU212 and M2E cells after transfection with si-NEAT1. ** P < 0.01 versus NC-mimic, Anti-lgG, normal adjacent tissues, laryngocarcinoma patients with stage I/II, NP69 or Scramble.

Figure 4

NEAT1 promoted laryngocarcinoma growth and metastasis via sponging miR-524-5p. (A) TU212 and M2E cells were transfected si-NEAT1 or/and miR-524-5p inhibitor, and the transfection efficiency was detected using qPCR. (B, C) CCK-8 assay was performed to analyze cell proliferation. (D) Apoptosis rates in each group were detected using Flow cytometry. (E) The invasion ability was evaluated using Transwell invasion assay. (F) Western blot analysis was used to detect the protein levels of PTEN, Ac-PTEN, AKT and p-AKT. * P < 0.05, ** P < 0.01 versus Scramble and NC-inhibitor or Scramble and miR-524-5p inhibitor.

Figure 5

Overexpression of miR-524-5p reversed the promotive effects of NEAT1 overexpression on cell proliferation and invasion. (A) The level of miR-524-5p was analyzed after TU212 cells transfected with pcDNA-NEAT1 or/and miR-524-5p mimic. (B) Cell proliferation was detected with CCK-8 assay. (C) Transwell assay was used to evaluate cell invasion ability. (D) Apoptosis rates of TU212 cells. * P < 0.05, ** P < 0.01 versus Vector and NC-mimic or Vector and miR-524-5p mimic.

Figure 6

HDAC1 was a target gene of miR-524-5p. (A) The binding sites between miR-524-5p and HDAC1. (B) The relative luciferase activity in HEK-293T cells co-transfected with HDAC1-WT or HDAC1-Mut and with miR-524-5p mimic or NC-mimic. The mRNA levels of HDAC1 in laryngocarcinoma tissues and normal adjacent tissues (n=20) (C), laryngocarcinoma patients with different stage tumor (D) and laryngocarcinoma cell lines (E). (F) The protein expression of HDAC1 in laryngocarcinoma cell lines. (G–J) The mRNA and protein levels were detected with qPCR and Western blotting after TU212 and M2E cells transfected with si-NEAT1 or/and miR-524-5p inhibitor. * P < 0.05, ** P < 0.01 versus NC-mimic, normal adjacent tissues, laryngocarcinoma patients with stage I/II, NP69 or Scramble and NC-inhibitor.

Figure 7

Overexpression of HDAC1 reversed the inhibitory effects of miR-524-5p overexpression and NEAT1 silence on cell proliferation and invasion. (A) The protein level of HDAC1 was detected after TU212 cells transfected with pcDNA-HDAC1 or/and miR-524-5p mimic. (B) Cell proliferation was detected with CCK-8 assay. (C) Transwell assay was used to evaluate cell invasion ability. (D) Apoptosis rates of TU212 cells. (E) The protein level of HDAC1 was detected after TU212 cells transfected with pcDNA-HDAC1 or/and si-NEAT1. (F) Cell proliferation was detected with CCK-8 assay. (G) Transwell assay was used to evaluate cell invasion ability. (H) Apoptosis rates of TU212 cells. * P < 0.05, ** P < 0.01 versus Vector and NC-mimic, Scramble and Vector, NC-mimic and pcDNA-HDAC1, and Scramble with pcDNA-HDAC1.

Figure 8

NEAT1 promoted laryngocarcinoma cell proliferation and invasion by inhibiting histone acetylation. (A) TU212 and M2E cells were transfected with pcDNA-NEAT1 or/and si-HDAC1, and the mRNA level of HDAC1 was detected using qPCR. (B, C) Cell proliferation was assessed with CCK-8 assay in TU212 and M2E cells. (D) Apoptosis rates were detected using Flow cytometry. (E) The invasion ability was evaluated using Transwell invasion assay. (F, G) Western blot was used to detect the protein expression levels of HDAC1, H3, Ac-H3, H4, Ac-H4, PTEN, Ac-PTEN, AKT and p-AKT. ** P < 0.01 versus Vector and Scramble or Vector and si-HDAC1.

Figure 9

Silence of NEAT1 suppressed tumor growth in vivo. (A) Tumor growth curves in two groups. (B) Representative images of TU212 cells from nude mice. (C) The effect of NEAT1 silence on tumor weight. The expression of NEAT1 (D), miR-524-5p (E), PTEN, Ac-PTEN, AKT, p-AKT and HDAC1 (F) in tumor tissues was detected with Western blotting. * P < 0.05, ** P < 0.01 versus shRNA.