Up-Regulation of microRNA-183 Promotes Cell Proliferation and Invasion in Glioma By Directly Targeting NEFL

Abstract

Glioblastoma multiforme (GBM) is the most common and lethal type of primary malignant brain tumor. In recent years, increasing reports suggest that discovery of microRNAs (miRNAs) might provide a novel therapeutical target for human cancers, including GBM. The expression and roles of microRNA-183 (miR-183) has been explored in several types of human cancers, including in GBM, and plays important roles in tumor initiation and progression. However, its biological functions in GBM remain largely unknown. In this study, we demonstrated that miR-183 was significantly up-regulated in astrocytoma tissues and glioblastoma cell lines. Introduction of miR-183 mimics into U251 cells could promoted, while its antisense oligos inhibited cell proliferation and invasion. Moreover, we identified neurofilament light polypeptide (NEFL) as a novel target gene of miR-183. The expression levels of NEFL are inversely correlated with that of miR-183 in human astrocytoma clinical specimens. In addition, NEFL-siRNA could significantly attenuate the inhibitory effects of knockdown miR-183 on the proliferation and invasion of U251 cells via mTOR signaling pathway. Overall, This study revealed that miR-183 promotes glioma cell proliferation by targeting NEFL, and also demonstrated that miR-183 could be a potential target for GBM treatment.

Keywords: Cell proliferation and invasion; Glioblastoma; NEFL; miR-183.

Fig. 1

Expression of miR-183 in human astrocytoma samples and cell lines. a Analysis of miR-183 expression in 44 human astrocytoma samples and 20 normal brain tissues. b qRT-PCR analysis of miR-183 expression in the human glioma cell lines (U251, U87, and LN229) as well as NHA (norm the normal human astrocytes cells). The data represent the mean ± SDs of three replicates. ***p < 0.001

Fig. 2

The expression of miR-183 could affect the proliferation and invasion of glioblastoma cells. a qRT-PCR analyzes the expression of miR-183 in U251 cells upon miR-183 mimic transfection. b qRT-PCR analyzes the expression of miR-183 in U251 cells upon miR-183 inhibitor transfection. c CCK8 assay showing the increased proliferation of U251 cells transfected with miR-183 mimics. d CCK8 assay showing the reduced proliferation of U251 cells transfected with miR-183 inhibitor. e Overexpression of miR-183 promotes tumor cell migration, as determined by in vitro wound healing assays. f Knockdown miR-183 inhibits tumor cell migration, as determined by in vitro wound healing assays. g Matrigel chamber invasion assay showing promoted invasion of U251 cells after being transfected with miR-183 mimics. h Matrigel chamber invasion assay showing reduced invasion of U251 cells after being transfected with miR-183 inhibitor. The data represent the mean ± SDs of three replicates. *p < 0.05; **p < 0.01;***p < 0.001

Fig. 3

miR-183 negatively regulates NEFL expression in GBM. a Schematic of the interaction sites of miR-183 in the 3′-UTRs of NEFL. b Luciferase assays of HEK293 and U251 cells co-transfected with pMIR-REPORT-WT/MT 3′-UTR NEFL and miR-183 or the negative control, as indicated. c qRT-PCR analysis showing the mRNA level of NEFL after miR-183 mimics or inhibitors were transfected into U251 cells for 24 h. miR-183 down-regulated the mRNA level of NEFL. d Western blot analysis showing the protein expression of NEFL, p-p70S6K, and p70S6K after the miR-183 mimics or inhibitor were transfected into U251 cells for 48 h. miR-183 decreased the protein expression of NEFL and p-p70S6K; GAPDH was used as a loading control. e qRT-PCR analysis showing that the mRNA level of NEFL was significantly reduced in astrocytoma tissues compared to normal brain tissues. f Spearman’s correlation analysis was used to determine the correlation between the expression levels of NEFL and miR-183 in human astrocytomas; Spearman’s correlation, r = −0.615 (n = 44). g Spearman’s correlation analysis was used to determine the correlation between the expression levels of NEFL and miR-183 in human normal brain tissue; Spearman’s correlation, r = 0.135 (n = 20). The data represent the mean ± SDs of three replicates. ***p < 0.001

Fig. 4

NEFL-siRNA reverses the inhibitory effects of knockdown miR-183 in U251 cells. a Western blot analyzes the protein expression level change of NEFL, phosphorylated p70S6K, and total p70S6K cells with the miR-183 inhibitor or NEFL-siRNA transfected in U251. b CCK8 assay showing the proliferation of U251 cells that were forced to repress the expression of NEFL using siRNA after being transfected with miR-NC or miR-183 inhibitor. The results showed that transfected miR-183 inhibitor decreased the proliferation of U251 cells. NEFL-siRNA attenuate the inhibitory effects of miR-183 inhibitor to U251 cells. c Matrigel chamber invasion assay showing reduced invasion of U251 cells that were forced to repress the expression of NEFL using siRNA after being transfected with miR-NC or miR-183 inhibitor. The results showed that transfected miR-183 inhibitor decreased the invasion of U251 cells. NEFL-siRNA attenuates the inhibitory effects of miR-183 inhibitor to U251 cells. The data represent the mean ± SDs of three replicates. Asterisk (*) indicates a significant difference compared to the miR-NC + siR-NC group, #indicates a significant difference compared to the miR-183 inhibitor + siR-NEFL group. *p < 0.05; **p < 0.01; ***p < 0.001; #p < 0.05; ##p < 0.01; ###p < 0.001