miR-30c-5p inhibits glioma proliferation and invasion via targeting Bcl2

Abstract

Background: Glioma is a highly malignant brain tumor, characterized by the poor prognosis and high recurrence rates. Previous studies have confirmed that miRNA-30c-5p is closely associated with tumor cell biological properties. The present study explored the biological role of miR-30c-5p in human glioma malignant behavior and underlying mechanisms.

Methods: Levels of miR-30c-5p were detected in glioma tissues and adjacent normal tissues. Two glioma cell lines including U87 and U251 were transfected with miR-30c-5p mimic or inhibitors. Cell proliferation was evaluated by MTT assay and colony formation assay. Cell apoptosis and invasive potential of glioma cells were assessed by flow cytometry and transwell assays, respectively. Luciferase reporter assay was performed to validate the target gene of miR-30c-5p.

Results: Levels of miR-30c-5p were dramatically decreased in glioma tissues as compared to the adjacent normal tissues. Upregulation of miR-30c-5p significantly suppressed cell growth and colony formation, and induced apoptosis in glioma cells. In contrast, inhibition of miR-30c-5p promoted the proliferation and inhibited apoptosis in tumor cells. Furthermore, miR-30c-5p strongly suppresses the invasion of glioma cells. Western blot showed that Bcl-2 was significantly decreased following treatment with miR-30c-5p mimics and increased after miR-30c-5p inhibitor treatment. Moreover, luciferase reporter assays indicated that transfection of miR-30c-5p led to a marked reduction of luciferase activity, but had no effect on Bcl-2 3'-UTR mutated fragment. Mechanically, miR-30c-5p promoted the activation of caspase 3 and caspase 9 in glioma cells. Furthermore, miR-30c-5p promoted apoptosis and inhibited colony formation and migration, and knockdown of Bcl2 further increased the number of apoptotic cells and suppressed colony formation and migration of glioma cells. By contrast, miR-30c-5p inhibitors decreased apoptosis and increased colony formation and migration, and restored Bcl2 expression further suppressed glioma cell apoptosis and enhanced colony formation and migration.

Conclusions: These results demonstrated that miR-30c-5p regulated growth, apoptosis and migration in glioma cells by targeting Bcl2, suggesting that miR-30c-5p might serve as a novel target for glioma therapy.

Keywords: Bcl2; Glioma; miRNA.

Figure 1

miR-30c-5p suppresses the cell viability of U87 and U251 glioma cells. (A) Expression of miR-30c-5p in glioma tissues and normal tissues was determined using qRT-PCR. ***, P<0.001, compared to normal. Two glioma cell lines including U87 and U251 were transfected with miR-30c-5p mimic or inhibitors (B). The cell viability of glioma cells was evaluated by MTT assay in different time points (C,D). *, P<0.05; **, P<0.01.

Figure 2

miR-30c-5p suppresses colony formation of U87 and U251 glioma cells. Glioma cell lines U87 (A,B) and U251 (A,C) were transfected with miR-30c-5p mimic or inhibitors. The colony formation of glioma cells was evaluated by colony formation assay. Colonies were stained with crystal violet. Magnification 5×. **, P<0.01.

Figure 3

miR-30c-5p induces glioma cell apoptosis of U87 and U251 glioma cells. Glioma cell lines U87 and U251 were transfected with miR-30c-5p mimic or inhibitors. Flow cytometry was used to detect the cell apoptosis of U87 (A,B) and U251 (A,C) glioma cells. **, P<0.01.

Figure 4

miR-30c-5p inhibits glioma cell invasive potential of U87 and U251 glioma cells. Glioma cell lines U87 and U251 were transfected with miR-30c-5p mimic or inhibitors. Transwell invasion assay was used to evaluate the invasive potential of U87 (A,B) and U251 (A,C) glioma cells. **, P<0.01. Magnification 200×.

Figure 5

Bcl2 is a direct target of miR-30c-5p in glioma cells. The 3'UTR of Bcl2 gene contained the binding sites for miR-30c-5p (A). U87 cells were transfected with miR-30c-5p mimic or inhibitors. (B) The protein expression of Bcl2 was detected by western blot and then quantified to the control GAPDH. (C) Luciferase reporter assay was used to validate that Bcl2 is a direct target of miR-30c-5p. (D) The protein expression of caspase 3/8/9 and cleaved caspase 3/8/9 was detected by western blot. **, P<0.01; ***, P<0.001.

Figure 6

miR-30c-5p regulates glioma cell colony formation and invasion via Bcl2. Glioma cell lines U87 and U251 were transfected with miR-30c-5p mimic or inhibitor alone, or miR-30c-5p inhibitor plus Bcl-2 overexpression and miR-30c-5p mimics plus Bcl-2 knockdown. Cell colony formation and migration were detected by colony formation assay (A,B) and Transwell invasion assay (C,D), respectively. Colonies and invaded cells were stained with crystal violet. Magnification 200×. **, P<0.01; ***, P<0.001.

Figure 7

miR-30c-5p induces glioma cell apoptosis of glioma cells via Bcl2. Glioma cell lines U87 and U251 were transfected with miR-30c-5p mimic or inhibitor alone, or miR-30c-5p inhibitor plus Bcl-2 overexpression and miR-30c-5p mimics plus Bcl-2 knockdown. Flow cytometry was used to detect the cell apoptosis of U87 (A,C) and U251 (B,D) glioma cells. **, P<0.01; ***, P<0.001.