Let-7c Inhibits the Proliferation, Invasion, and Migration of Glioma Cells via Targeting E2F5

Abstract

As a member of the miRNA family, let-7c has been identified as a tumor suppressor in many cancers. However, the molecular biological function of let-7c in glioma has not been elucidated. The aim of this study was to explore let-7c expression levels and evaluate its function in glioma cells. We first measured the expression of let-7c in four glioma cell lines and a normal cell line by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and the results showed that let-7c was downregulated in glioma cells. By applying gain-of-function and loss-of-function assays, the experiments suggested that dysregulation of let-7c could obviously affect cell proliferation, metastasis, and invasion. Based on online bioinformatics analysis and Dual-Luciferase Reporter assays, we found that E2F5 was a target gene of let-7c and contributed to the function of let-7c in glioma cells. Our investigations indicated that loss of let-7c contributed to the progression of glioma cells.

Figure 1

let-7c is downregulated in glioma cells and associated with tumor proliferation. (A) let-7c expression levels between glioma cells (U87, U251, SHG44, and A172) and the normal astrocyte cell line were measured by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). (B) let-7c mimic and inhibitor were separately transfected into U87 and SHG44 cells. (C) MTT and (D) colony formation assays were performed to measure the proliferation of glioma cells transfected with let-7c mimic and inhibitor. Error bars represented the mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01 versus control group.

Figure 2

Downregulation of let-7c enhances the migration ability of glioma cells. (A) Wound healing, (B) metastasis (migration and invasion), and (C) attachment/detachment assays were utilized to detect the migratory capability of glioma cells. (D) Flow cytometry was used to check the effect of let-7c on the cell cycle. Error bars represented the mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01 versus control (NC) group.

Figure 3

E2F5 is identified as target of let-7c. (A) The level of E2F5 in four glioma cell lines and a normal astrocyte cell line was detected. (B) Bioinformatics analysis helped us obtain the binding sites between let-7c and E2F5. (C). Luciferase reporter assays further confirmed the regulating relationship between let-7c and E2F5 after wild-type (WT) or mutant (Mut) E2F5 transfection. (D) The expression of E2F5 in response to the levels of let-7c. Error bars represent the mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01 versus control group (astrocytes or NC).

Figure 4

The function of E2F5 on the proliferation ability of glioma cells. Glioma cells were transfected with short interfering RNA for E2F5 (si-E2F5), pcDNA-E2F5, or appropriate controls (si-RNA or NC). (A) The transfection efficiency was obtained after 48 h; Western blot assay was performed to determine the protein level of E2F5. (B) MTT and (C) colony formation assays were performed to detect the cell proliferation of transfected glioma cells. Error bars represented the mean ± SD of at least three independent experiments. **p < 0.01 versus control group (si-RNA or NC).

Figure 5

The function of dysregulated E2F5 on the migration and invasion of glioma cells. Glioma cells were transfected with si-E2F5, pcDNA-E2F5, or appropriate controls (si-RNA or NC). (A) Wound healing, (B) metastasis (migration and invasion), and (C) attachment/detachment assays were utilized to detect the migratory capability of glioma cells. Error bars represented the mean ± SD of at least three independent experiments. **p < 0.01 versus control group (si-RNA or NC).

Figure 6

The effect of let-7c on glioma cells is in an E2F5-dependent manner. Glioma cells were cotransfected with pcDNA-E2F5 and pcDNA-let-7c or appropriate controls (NC). (A) MTT and (B) colony formation assays were applied to detect the proliferation ability of cotransfected glioma cells. (C) Wound healing and (D) metastasis assays were utilized to detect the migratory capability of cotransfected glioma cells. (E) Transwell assay was employed to detect the invasive ability of cotransfected glioma cells (F) Attachment/detachment assay was used to detect the migratory capability of cotransfected glioma cells. Error bars represented the mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01 versus control group (NC).