miR-23a promotes invasion of glioblastoma via HOXD10-regulated glial-mesenchymal transition

Abstract

Glioblastoma is the most aggressive and invasive brain tumor and has a poor prognosis; elucidating the underlying molecular mechanisms is essential to select molecular targeted therapies. Here, we investigated the effect of microRNAs on the marked invasiveness of glioblastoma. U373 glioblastoma cells were infected with 140 different microRNAs from an OncomiR library, and the effects of the invasion-related microRNAs and targeted molecules were investigated after repeated Matrigel invasion assays. Screening of the OncomiR library identified miR-23a as a key regulator of glioblastoma invasion. In six glioblastoma cell lines, a positive correlation was detected between the expression levels of miR-23a and invasiveness. A luciferase reporter assay demonstrated that homeobox D10 (HOXD10) was a miR-23a-target molecule, which was verified by high scores from both the PicTar and miRanda algorithms. Forced expression of miR-23a induced expression of invasion-related molecules, including uPAR, RhoA, and RhoC, and altered expression of glial-mesenchymal transition markers such as Snail, Slug, MMP2, MMP9, MMP14, and E-cadherin; however, these changes in expression levels were reversed by HOXD10 overexpression. Thus, miR-23a significantly promoted invasion of glioblastoma cells with polarized formation of focal adhesions, while exogenous HOXD10 overexpression reversed these phenomena. Here, we identify miR-23a-regulated HOXD10 as a pivotal regulator of invasion in glioblastoma, providing a novel mechanism for the aggressive invasiveness of this tumor and providing insight into potential therapeutic targets.

Fig. 1

MiR-23a promotes invasion of GBM cells. a The Matrigel invasion assays were performed using six human GBM cell lines: LN443, U373, LN308, U87, U251, and KMG4. *P < 0.05, **P < 0.005, and ***P < 0.0005 vs. LN443. b Schematic diagram identifying microRNAs to confer marked invasion of GBM cells. U373 cells were infected with the OncoMir Precursor Virus Library, and the Matrigel invasion assay was repeated three times to enrich the cells that acquired elevated invasion ability. Total RNA was isolated from the cells and subjected to semi quantitative RT-PCR using OncoMir Precursor Library primers, followed by sequencing. c Endogenous expression levels of miR-23a in the six GBM cell lines were examined by semi quantitative RT-PCR. GAPDH was utilized as an internal control. d In the six GBM cell lines shown in c, the correlations between the invasion ability and expression levels of miR-23a were analyzed. R² = 0.95741. e The scores for HOXD10, Sprouty2, and Marcks from the PicTar and miRanda algorithms are shown

Fig. 2

MiR-23a directly targets the HOXD10-3’UTR in GBM cells. a U373 and LN443 cells were infected with miR-23a-producing lentivirus or control lentivirus, and the expression levels of miR-23a and HOXD10 mRNA were examined by semi quantitative RT-PCR. GAPDH was utilized as an internal control. b The expression levels of HOXD10 protein were examined by immunoblotting in U373 and LN443 cells with or without forced expression of miR-23a. α-Tubulin was used as a loading control. c Diagram of the luciferase reporter vector fused to the 3’UTR of HOXD10 utilized in the luciferase assay. The sequences of miR-23a and the targeted HOXD10-3’UTR are shown. d Dual luciferase assay. HOXD10-3′UTR luciferase activity were measured in miR-23a-overexpressing U373 and LN443 cells. *P < 0.01 and **P < 0.001 vs. without miR-23a. e The expression levels of sprouty2 mRNA in miR-23a-overexpressing U373 and LN443 cells were examined by semi quantitative RT-PCR. f The phosphorylation levels of ERK were investigated by immunoblotting in the indicated cells. g The cell proliferation of U373 and LN443 cells with or without forced miR-23a was investigated and graphed as the means ± SD. * P < 0.05 vs. control cells

Fig. 3

MiR-23a regulates expression of invasion- and glial-mesenchymal transition (GMT)-related genes via HOXD10. a The expression levels of uPAR, RhoA, and RhoC mRNAs were examined in control and miR-23a-overexpressing U373 and LN443 cells by semi quantitative RT-PCR. GAPDH was utilized as an internal control. b, c In U373 and LN443 cells with or without miR-23a overexpression, the mRNA expression levels of the indicated GMT-related genes were investigated by semi quantitative RT-PCR (b) and real-time RT-PCR (c). *P < 0.05 and **P < 0.005 vs. without miR-23a. d, e The expression levels of miR-23a and the indicated molecules were examined by semi quantitative RT-PCR (d) and immunoblotting (e) in LN443 cells with or without miR-23a and HOXD10 overexpression

Fig. 4

MiR-23a produces mesenchymal changes in cell morphology and affects the polarity of focal adhesions. a The expression levels of miR-23a, HOXD10 mRNA, and HOXD10 protein were examined by semi quantitative RT-PCR (upper three panels) and immunoblotting (lower two panels) in U373 and LN443 cells. GAPDH and α-tubulin were utilized as internal controls for semi quantitative RT-PCR and immunoblotting, respectively. b Photomicrographs of U373 and LN443 cells with or without miR-23a or HOXD10-overexpression are displayed. The scale bars indicate 100 µm. c Immunofluorescence of focal adhesions. (Left panels) U373 and LN443 cells with or without forced miR-23a or HOXD10 expression were subjected to immunofluorescence analysis for paxillin (green) and actin (red). (Right panels) The paxillin counts in the indicated cells are shown. d Cells stained with anti-paxillin Ab were divided into three regions by angles of 120°, and the “A” region was set as the movement direction based on cell morphology and the structures of actin filaments. In U373 and LN443 cells with or without forced miR-23a or HOXD10 expression, the paxillin counts were determined and are shown

Fig. 5

MiR-23a promotes tumor invasion of glioblastoma via reduced HOXD10. a Wound-healing assays were performed with miR-23a-overexpressing U373 and LN443 cells and their respective control cells. Representative photomicrographs at 0 and 24 h are shown. b The distances moved are displayed as the mean ± SD. N.S. indicates not statistically significant. c Matrigel invasion assays were performed with both U373 and LN443 cells with or without forced miR-23a or HOXD10 expression. Micrographs of invading cells stained with crystal violet are displayed. d In the Matrigel invasion assays, the invaded cells under the filter were counted in three randomly selected regions, and graphed as the mean ± SD. e (Left) Micrographs of U87 cells with or without forced HOXD10 expression are displayed. (Right) In the Matrigel invasion assays, invaded cells were counted, and the data are presented as the mean ± SD. * P < 0.01 vs. control cells

Fig. 6

Mechanisms of miR-23a-regulated promotion of GBM invasion through targeting of HOXD10. a, b KMG4 cells were transfected with anti-miR-23a and scramble DNA as a control, and the expression levels of Snail, MMP2, MMP9, and MMP14 (a) and invasion ability (b) were investigated. *P < 0.05, **P < 0.005, and ***P < 0.0005 vs. the indicated samples. c miR-23a directly targets the HOXD10-3′-UTR, triggering dramatic alterations in the expression of genes associated with invasion (uPAR, MMP14, RhoA, and RhoC) and glial-mesenchymal transition (GMT) events (Snail, Slug, MMP2, MMP9, and E-cadherin), and inducing polarity of focal adhesions, ultimately resulting in cooperatively aggressive invasion of GBM