MicroRNA-181b Inhibits Cellular Proliferation and Invasion of Glioma Cells via Targeting Sal-Like Protein 4

Abstract

MicroRNAs (miRs), a class of noncoding RNAs that are 18-25 nucleotides in length, are able to suppress gene expression by targeting complementary regions of mRNAs and inhibiting protein translation. Recently, miR-181b was found to play a suppressive role in glioma, but the regulatory mechanism of miR-181b in the malignant phenotypes of glioma cells remains largely unclear. In this study, we found that miR-181b was significantly downregulated in glioma tissues when compared with normal brain tissues, and decreased miR-181b levels were significantly associated with high-grade pathology and a poor prognosis for patients with glioma. Moreover, miR-181b was downregulated in glioma cell lines (U87, SHG44, U373, and U251) compared to normal astrocytes. Overexpression of miR-181b significantly decreased the proliferation, migration, and invasion of glioma U251 cells. Sal-like protein 4 (SALL4) was identified as a novel target gene of miR-181b in U251 cells. The expression of SALL4 was significantly upregulated in glioma tissues and cell lines, and an inverse correlation was observed between the miR-181b and SALL4 expression levels in glioma. Further investigation showed that the protein expression of SALL4 was negatively regulated by miR-181b in U251 cells. Knockdown of SALL4 significantly inhibited the proliferation, migration, and invasion of U251 cells, while overexpression of SALL4 effectively reversed the suppressive effects of miR-181b on these malignant phenotypes of U251 cells. In conclusion, our study demonstrates that miR-181b has a suppressive effect on the malignant phenotypes of glioma cells, at least partly, by directly targeting SALL4. Therefore, the miR-181b/SALL4 axis may become a potential therapeutic target for glioma.

Figure 1

Downregulation of miR-181b is associated with malignant progression and poor prognosis in glioma. (A) RT-PCR was used to determine the miR-181b levels in glioma tissues (n = 74) and normal brain tissues (n = 18). **p < 0.01 versus Normal. (B) RT-PCR was used to determine the miR-181b levels in high- or low-grade glioma tissues. **p < 0.01 versus grades I–II. (C) Glioma patients with low expression of miR-181b had shorter overall survival when compared with those with high expression of miR-181b.

Figure 2

miR-181b overexpression inhibits the proliferation, migration, and invasion of glioma cells. (A) RT-PCR was used to determine the miR-181b expression in glioma cell lines (U87, SHG44, U373, and U251) compared with normal human astrocytes. **p < 0.01 versus astrocytes. U251 cells were transfected with miR-181b mimic or scramble miR (miR-NC). (B) RT-PCR was used to determine the miR-181b expression. (C) MTT, (D) wound healing, and (E) Transwell assays were conducted to examine cell proliferation, migration, and invasion. **p < 0.01 versus miR-NC (B–E).

Figure 3

SALL4 is a target gene of miR-181b in glioma cells. (A) The TargetScan software indicated that SALL4 was a putative target of miR-181b, and their targeting relationship was evolutionally conversed. (B) The luciferase reporter plasmids containing the wild type (WT) or mutant type (MT) of SALL4 3′-UTR were constructed. (C) The luciferase activity was significantly decreased in U251 cells cotransfected with the WT-SALL4-3′-UTR luciferase reporter plasmid and miR-181b mimic, when compared to the control group, which was eliminated by transfection with MT-SALL4-3′-UTR luciferase reporter plasmid. **p < 0.01 versus control.

Figure 4

SALL4 is upregulated in glioma, with an inverse correlation to miR-181b levels. (A) RT-PCR was used to determine the mRNA levels of SALL4 in glioma tissues and normal brain tissues. **p < 0.01 versus Normal. (B) The SALL4 mRNA levels were inversely correlated to the miR-181b levels in glioma tissues. (C) RT-PCR and (D) Western blot were used to examine the mRNA and protein levels of SALL4 in glioma cell lines (U87, SHG44, U373, and U251) compared with normal human astrocytes. **p < 0.01 versus astrocytes.

Figure 5

SALL4 is negatively regulated by miR-181b in glioma cells. (A) Western blot was used to examine the protein levels of SALL4 in U251 cells transfected with miR-181b mimic or scramble miR (miR-NC). **p < 0.01 versus miR-NC. U251 cells were transfected with the miR-181b inhibitor or negative control (NC) inhibitor. (B) RT-PCR was used to determine the miR-181b levels. (C) Western blot was used to examine the protein levels of SALL4. **p < 0.01 versus NC inhibitor (B–C).

Figure 6

SALL4 acts as a downstream effector in miR-181b-mediated malignant phenotypes of U251 cells. miR-181b-overexpressing U251 cells were transfected with SALL4 expression plasmid or blank vector. (A) RT-PCR and (B) Western blot were used to examine the mRNA and protein levels of SALL4. (C) MTT, (D) wound healing, and (E) Transwell assays were conducted to examine cell proliferation, migration, and invasion. **p < 0.01 versus miR-181b + blank.