MicroRNA-133b inhibits cell migration and invasion by targeting matrix metalloproteinase 14 in glioblastoma

Abstract

Increasing evidence has suggested that microRNA-133b (miR-133b) is important in regulating the genesis of different types of cancer. However, the effects and the underlying mechanisms of miR-133b in the development of glioblastoma (GBM) remain largely unknown. The aim of the present study was to investigate the role of miR-133b in GBM and to determine the molecular mechanisms underlying its action. Reverse transcription-quantitative polymerase chain reaction was used to measure the expression levels of miR-133b in 21 human GBM samples and 9 normal brain tissue samples. A wound healing assay, and Transwell migration and invasion assays were used to evaluate the effects of miR-133b on cell migration and invasion. Western blotting and a luciferase reporter assay were used to identify the target genes of miR-133b. It was found that miR-133b suppressed GBM cell migration and invasion, and matrix metalloproteinase 14 (MMP14) was identified as a direct target gene. In conclusion, miR-133b may suppress GBM migration and invasion through directly targeting MMP14, highlighting its potential as a novel agent for the treatment of GBM invasion.

Keywords: glioblastoma; invasion; matrix metalloproteinase 14; microRNA-133b; migration.

Figure 1.

Expression of miR-133b in tissue samples. The relative expression of miR-133b in 21 GBM tissues and 9 normal brain tissues was measured by RT-qPCR, and U6 small nuclear RNA was used as an internal control (means ± standard deviation; n=3; *P<0.05). miR, microRNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; GBM, glioblastoma.

Figure 2.

Relative expression of miR-133b in GBM cell lines at 24 h post-transfection, as measured by RT-qPCR. Following miR-133b mimic transfection, miR-133b expression was markedly increased in the (A) U87 and (B) U251 cells (mean ± standard deviation; n=3; *P<0.05). miR, microRNA; NC, negative control; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; GBM, glioblastoma.

Figure 3.

miR-133b inhibits the migration and invasion of human GBM cell lines. (A and B) Wound healing assays using U87 and U251 cells transfected with miR-133b mimics or NC. (Aa and Ba) Representative images and (Ab and Bb) quantification of 3 randomly selected fields (mean ± standard deviation; n=3; *P<0.05). (C and D) Transwell migration and invasion assays using U87 and U251 cells transfected with miR-133b mimics or NC. (Ca and Da) Representative images and (Cb and Db) quantification of 3 randomly selected fields (mean ± standard deviation; n=3; *P<0.05). miR, microRNA; NC, negative control; GBM, glioblastoma.

Figure 4.

miR-133b directly targets MMP14. (A) Bioinformatics predicted interactions of miR-133b and their binding sites at the 3′UTR of MMP14 (TargetScan 6.0). (B) Luciferase activity assay demonstrated a direct targeting of the 3′UTR of MMP14 by miR-133b. Cells (U87 and U251) were co-transfected with luciferase vectors, either the wild-type MMP14 3′UTR reporter plasmid or mutated MMP14 3′UTR reporter plasmid, together with miR-133b mimics or NC. After 48 h, the luciferase activities were measured (mean ± standard deviation; n=3; *P<0.05). (C) Western blotting of MMP14 in U87 and U251 cells transfected with miR-133b mimics or NC. (a) Representative images of western blotting and (b) quantification of the bands (mean ± standard deviation; n=3; *P<0.05). miR, microRNA; NC, negative control; GBM, glioblastoma; MMP14, matrix metalloproteinase 14; UTR, untranslated region.