Long Noncoding RNA LINC00467 Promotes Glioma Progression through Inhibiting P53 Expression via Binding to DNMT1

Abstract

Purpose: This study aimed to investigate whether long noncoding RNA (lncRNA) LINC00467 could regulate proliferative and invasive abilities of glioma cells via p53 and DNA methyltransferase 1 (DNMT1), so as to participate in the occurrence and progression of glioma. Methods: LINC00467 expression in glioma was analyzed by GEPIA database and LINC00467 expression in glioma cell lines was detected by qRT-PCR. The regulatory effects of LINC00467 and p53 on proliferative, invasive capacities and cell cycle were conducted by CCK-8 and EdU assays, transwell assay and flow cytometry, respectively. The binding conditions between LINC00467, DNMT1 and p53 were determined by RNA immunoprecipitation (RIP) and Chromatin immunoprecipitation (ChIP) assays. Western blot was conducted to determine whether LINC00467 could regulate p53 in glioma cells. Finally, rescue experiments were carried out to evaluate whether LINC00467 regulates proliferative and invasive abilities of glioma cells through p53. Results: The expression of LINC00467 was significantly up-regulated in tumor samples than that in normal samples, which was not correlated with patient survival time. Besides, expression of LINC00467 was higher in glioma cells than that of negative control cells. Upregulation of LINC00467 promoted proliferative and invasive abilities, and accelerated cell cycle in G0/G1 phase of U87 and LN229 cells. The results of RIP and ChIP assays demonstrated that LINC00467 could bind to DNMT1 and inhibit p53 expression. Overexpression of p53 partially reversed the enhancement of LINC00467 on proliferative and invasive abilities of glioma cells. Conclusion: These results indicated that high expression of LINC00467 could promote proliferative and invasive abilities of glioma cells through targeting inhibition of p53 expression by binding to DNMT1.

Keywords: DNMT1; Glioma; Invasion; LINC00467; P53; Proliferation.

Figure 1

LINC00467 expression is remarkedly increased in glioma A. LINC00467 levels in glioma tissues (n=163) and normal tissues (n=207) analyzed in TCGA STAD database. B. The Kaplan‐Meier curve depicts the overall survival of 162 patients with glioma. Error bars indicate mean ± standard errors of the mean. *P < 0.05. TCGA, the Cancer Genome Atlas.

Figure 2

LINC00467 promoted glioma cell proliferation and invasion A. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyse the expression of LINC00467 in human normal glial cell line (HEB) and glioma cells. B. QRT-PCR analysis of LINC00467 expression in si-NC, si‐LINC00467 in U87 cells. C. QRT-PCR analysis of LINC00467 expression in pcDNA-NC, pcDNA‐LINC00467 in LN229 cells. D. The CCK8 assay was used to determine the viability of si‐LINC00467 transfected or pcDNA‐LINC00467 transfected glioma cells. E. The EdU assay was used to determine the viability of si‐LINC00467 transfected or pcDNA‐LINC00467 transfected glioma cells. F. Downregulation of LINC00467 in U87 cells inhibited cell invasion while overexpression of LINC00467 in LN229 cells promoted cell invasive capability. The data represent the mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01. CCK8, Cell-Counting Kit 8; EdU, Ethynyl deoxyuridine.

Figure 3

LINC00467 promoted cell cycle and inhibited cell apoptosis A. Flow cytometry was used to detect cell cycle. Knockdown of LINC00467 made cells arrested in G0/G1 phase while upregulation of LINC00467 promoted cell cycle. B. Flow cytometry was used to detect apoptosis rates. Knockdown of LINC00467 promoted cell apoptosis while upregulation of LINC00467 inhibited cell apoptosis. C and D. LINC00467 knockdown downregulated mRNA and protein expression of DNMT1 while overexpression of LINC00467 increased mRNA and protein expression of DNMT1 in glioma cells. The data represent the mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01. mRNA, messenger RNA; DNMT1, DNA methyltransferase 1.

Figure 4

LINC00467 inhibited p53 expression by binding to DNMT1 A and B. LINC00467 knockdown upregulated mRNA and protein expression of p53 in U87 cells, while overexpression of LINC00467 suppressed mRNA and protein expression of p53 in LN229 cells. C. Nuclear‐cytoplasmic separation assay showed that LINC00467 was mainly distributed in nuclear fractions of glioma cells. D. RIP results demonstrated that LINC00467 could be bound to DNMT1. E. ChIP results indicated that DNMT1 binds to DNAs in the p53 promoter region. F. Knockdown of LINC00467 in U87 cells downregulated the binding level of DNMT1 and p53 promoter, while overexpression of LINC00467 in LN229 cells upregulated the binding level of DNMT1 and p53 promoter. G. Transfection efficacy of si-LINC00467 and pcDNA-LINC00467 in glioma cells. H. After interfering with si-DNMT1 in U87 cells, p53 expression significantly increased. Upregulation of DNMT1 in LN229 cells, p53 expression markedly decreased. The data represent the mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. mRNA, messenger RNA; DNMT1, DNA methyltransferase 1; RIP, RNA immunoprecipitation; CHIP, Chromatin immunoprecipitation.

Figure 5

P53 reversed the anti-tumor effect of LINC00467 on glioma A, B. After transfection of si-p53 and pcDNA-p53 in glioma cells, mRNA and protein levels of p53 correspondingly changed. C and D. Cell proliferative ability was significantly decreased after downregulation of LINC00467, which was reversed by knockdown of p53. Similarly, cell proliferative ability could be enhanced after overexpression of LINC00467 and reversed by overexpression of p53. E. Cell invasive ability was significantly suppressed after knockdown of LINC00467, which was reversed by downregulation of p53. Moreover, cell invasive ability was significantly enhanced after overexpression of LINC00467, which was reversed by overexpression of p53. The data represent the mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01. mRNA, messenger RNA.