miR-133b inhibits glioma cell proliferation and invasion by targeting Sirt1

Abstract

MicroRNAs (miRs) are a class of small non-coding RNAs that function as mediators of gene expression. Dysregulations of miRs have been implicated in the development and progression of glioma. In the present study, we investigated the role of miR-133b in mediating the proliferation and invasion of glioma cells, and the potential mechanism. Real-time RT-PCR results showed that miR-133b expression was significantly decreased in glioma tissues compared with normal brain tissues. Luciferase reporter assay further identified silent information regulator 1 (Sirt1) as a novel direct target of miR-133b in glioma U87 cells. Overexpression of miR-133b suppressed Sirt1 expression and reduced the proliferation and invasion of U87 cells, which could be partly rescued by forced expression of Sirt1. In addition, the Sirt1 mRNA level was significantly higher in glioma tissues than in normal brain tissues, and was inversely correlated with miR-133b level in glioma tissues. In summary, our study sheds light on the regulatory mechanism of miR-133b in glioma growth and metastasis via direct mediation of Sirt1 expression, and suggests that Sirt1 may serve as a potential therapeutic target for glioma.

Keywords: glioma; invasion; microRNA-133b; proliferation; silent information regulator 1.

Figure 1. Real-time RT-PCR was conducted to examine the relative miR-133b levels in 21 glioma specimens and 8 normal brain tissue specimens

**P < 0.01 vs. Normal.

Figure 2. miR-133b specifically targets Sirt1 gene

(A) Targetscan software predicts that Sirt1 is a direct target gene of miR-133b. (B) The wild-type (WT) or mutant (MUT) binding sequences of miR-133b on Sirt1 3′-UTR are shown. (C) The WT or MUT Sirt1 3′-UTR was constructed and inserted into the psiCHECK ^TM2 luciferase reporter vector. (D) The luciferase activity was significantly downregulated in glioma U87 cells co-transfected with miR-133b mimic and WT Sirt1 3′UTR vector, but was unchanged in other groups. Control: U87 cells transfected with WT or MUT Sirt1 3′UTR vector. **P < 0.01 vs. Control. (E) Real-time RT-PCR was used to examine the miR-133b levels in U87 cells transfected with miR-133b mimic or inhibitor, and (F, G) western blot was performed to examine the Sirt1 protein levels in each group. Control: non-transfected U87 cells. **P < 0.01 vs. Control.

Figure 3. miR-133b overexpression could decrease the cell proliferation and invasion of glioma cells

(A) MTT assay and (B) transwell assay were conducted to determine the proliferation and invasion of U87 cells transfected with miR-133b mimic or inhibitor, respectively. Control: non-transfected U87 cells. **P < 0.01 vs. Control. bar = 100 μm.

Figure 4. Forced Sirt1 expression partly abolished the effect of miR-133b on cell proliferation and invasion

(A) Western blot was performed to examine the Sirt1 protein levels in U87 cells transfected with miR-133b mimic, or co-transfected with miR-133b mimic and Sirt1-overexpressing plasmid (miR-133b + Sirt1). (B) MTT assay and (C) transwell assay were conducted to determine the cell proliferation and invasion in each group. **P < 0.01 vs. miR-133b. bar = 100 μm.

Figure 5. Sirt1 mRNA was negatively correlated with miR-133b level in tumor tissue

(A) Real-time RT-PCR was conducted to examine the relative mRNA levels of Sirt1 in 21 glioma specimens and 8 normal brain tissue specimens. **P < 0.01 vs. Normal. (B) The reverse correlation between the miR-133b levels and the Sirt1 mRNA levels in 21 glioma specimens.