miRNA-204-5p acts as tumor suppressor to influence the invasion and migration of astrocytoma by targeting ezrin and is downregulated by DNA methylation

Abstract

microRNAs (miRNAs), through their regulation of the expression and activity of numerous proteins, are involved in almost all cellular processes. As a consequence, dysregulation of miRNA expression is closely associated with the development and progression of cancers. Recently, DNA methylation has been shown to play a key role in miRNA expression dysregulation in tumors. miRNA-204-5p commonly acts in the suppression of oncogenes in tumors. In this study, the levels of miRNA-204-5p were found to be down-regulated in the astrocytoma samples. miRNA-204-5p expression was also down-regulated in two astrocytoma cell lines (U87MG and LN382). Examination of online databases showed that the miRNA-204-5p promoter regions exist in CpG islands, which might be subjected to differential methylation. Subsequently, we showed that the miRNA-204-5p promoter region was hypermethylated in the astrocytoma tissue samples and cell lines. Then we found that ezrin expression was down-regulated with an increase in miRNA-204-5p expression in LN382 and U87MG cells after 5-aza-2'-deoxycytidine (5'AZA) treatment compared with control DMSO treatment. In addition, LN382 and U87MG cells treated with 5'AZA exhibited significantly inhibited cell invasion and migration . In a recovery experiment, cell invasion and migration returned to normal levels as miRNA-204-5p and ezrin levels were restored. Overall, our study suggests that miRNA-204-5p acts as a tumor suppressor to influence astrocytoma invasion and migration by targeting ezrin and that miRNA-204-5p expression is downregulated by DNA methylation. This study provides a new potential strategy for astrocytoma treatment.

Keywords: DNA methylation; astrocytoma; ezrin; invasion and migration; miRNA-204-5p.

Figure 1.

Relative expression of miRNA-205-5p in tissue samples and cell lines. (a) miRNA-204-5p levels in 30 freshly dissected astrocytoma samples and 10 normal brain tissue samples, as determined by qRT-PCR. The expression of miRNA-204-5p was obviously downregulated in the astrocytoma tissue samples compared with the normal brain tissue samples. ***P < 0.001. (b) miRNA-204-5p levels in normal human astrocytes (NHAs) and 2 astrocytoma cell lines, as determined by qRT-PCR. The expression of miRNA-204-5p was obviously downregulated in the 2 astrocytoma cell lines compared with the NHAs. **P < 0.01. Data are expressed as the mean ± SD of three independent experiments

Figure 2.

miRNA-204-5p acts as a tumor suppressor gene and inhibits the invasion and migration of astrocytoma cells. (a, b) The transfection efficiency of miRNA-204-5p was determined. (C, D, E, F) Invasion and migration could be inhibited by upregulating miRNA-204-5p expression in LN382 and U87MG cells. Invasion and migration could be promoted by knocking down miRNA-204-5p expression in LN382 and U87MG cells

Figure 3.

DNA methylation is present in astrocytoma tissue samples and cell lines, and the degree of methylation can be reduced by using 5-aza-2ʹ-deoxycytidine (5ʹAZA). (a, b) We used NCBI to detect miRNA-204-5p in the middle of TRPM3 and used the online databases EMBL-EBI and MethPrimer – to suggest that the miRNA-204-5p promoter regions exist in CpG islands, which is located in the area 1703–1944, and the promoter length is 242 bp. (c) The methylation statuses of 5 astrocytoma and 3 normal brain tissue samples were determined by bisulfite genomic sequencing PCR (BSP). Black dots represent methylated CpGs, and white dots represent unmethylated CpGs. The columns represent CpG dinucleotides 1–12, and the rows represent clones 1–8. (d) The miRNA-204-5p methylation statuses of 5 astrocytoma and 3 normal brain tissue samples were determined by MSP. M: PCR products specifically amplified by MSP for methylated DNA. U: PCR products specifically amplified by MSP for unmethylated DNA

Figure 4.

DNA methylation can regulate the expression of miRNA-204-5p in astrocytoma cell lines and affect the invasion and migration of these cell lines. (a) The methylation statuses of U87MG and LN382 cells were determined by BSP before and after 5ʹAZA treatment. Black dots represent methylated CpGs, and white dots represent unmethylated CpGs. The columns represent CpG dinucleotides 1–12, and the rows represent clones 1–8. (b, c) The expression of miRNA-204-5p was upregulated in LN382 and U87MG cells after 5ʹAZA treatment compared with control DMSO treatment. **P < 0.01. Data are expressed as the mean ± SD of three independent experiments. (D, E, F, G) LN382 and U87MG cells treated with 5ʹAZA had significantly inhibited cell invasion and migration compared to control DMSO-treated cells

Figure 5.

Ezrin expression is downregulated by miRNA-204-5p and regulated by DNA methylation. (a) The ezrin levels in 30 freshly dissected astrocytoma samples and 10 normal brain tissue samples were determined by qRT-PCR. The expression of ezrin was obviously upregulated in the astrocytoma tissue samples compared with the normal brain tissue samples. **P < 0.01. (b) The expression of ezrin was obviously downregulated in NHAs compared with 2 astrocytoma cell lines. *P < 0.05. Data are expressed as the mean ± SD of three independent experiments. (C, D, E, F) The expression of ezrin in a recovery experiment, in which we treated U87MG and LN382 cells with a miRNA-204-5p inhibitor +5ʹAZA, was measured. The relative expression of ezrin and miRNA-204-5p returned to near normal. **P < 0.01. Data are expressed as the mean ± SD of three independent experiments. (G, H, I, J) Cell invasion and migration were evaluated in the recovery experiment. When we treated U87MG and LN382 cells with the miRNA-204-5p inhibitor +5ʹAZA, cell invasion and migration returned to near normal levels