MiR-181d acts as a tumor suppressor in glioma by targeting K-ras and Bcl-2

Abstract

Purpose: Recently, several microRNAs (miRNAs) were reported to be involved in the modulation of glioma development. The aim of our study was to determine the effect of miR-181d on the growth of glioma and to investigate whether this growth is modulated by targeting K-ras and Bcl-2.

Methods: Real-time PCR was used to analyze the expression of miR-181d in human glioma samples and glioma cell lines. Apoptosis, cell cycle, and proliferation (MTT) assays were performed to assess the phenotypic changes in glioma cells. Immunohistochemistry was used to determine the expression of K-ras and Bcl-2 in glioma tissues, and a luciferase reporter assay was carried out to confirm whether K-ras and Bcl-2 are direct targets of miR-181d. Western blotting was used to identify the potential signaling pathways affected glioma cell growth by miR-181d. In vivo, xenograft tumors were examined for an anti-glioma effect of miR-181d.

Results: MiR-181d was down-regulated in human glioma samples and up-regulated in transfected glioma cells. Ectopic expression of miR-181d suppressed proliferation and triggered cell cycle arrest and apoptosis in glioma cell lines. K-ras and Bcl-2 were identified as direct targets of miR-181d and were up-regulated in glioma samples. The results showed evidence linking the tumor suppressor activity of miR-181d in glioma cells with the K-ras-related PI3K/AKT and MAPK/ERK pathways. Furthermore, xenograft tumors from miR-181d-treated U251 cells were suppressed in vivo.

Conclusion: MiR-181d may act as a glioma suppressor by targeting K-ras and Bcl-2.

Fig. 1

MiR-181d is down-regulated in glioma tissue samples and up-regulated in transfected cells. a The relative levels of miR-181d in glioma samples were measured using real-time quantitative RT-PCR. Normal brain tissues were counted as control. b U251 and LN229 cells at 70% confluence were transfected with miR-181d mimics, and miR-181d expression was analyzed 48 h after transfection. **P < 0.001 compared to the blank control

Fig. 2

MiR-181d induced apoptosis, cell cycle arrest and inhibits cell growth in U251 and LN229 cells. a Cells were transfected with miR-181d and analyzed by apoptosis assay. Data are presented as the mean of triplicate experiments. b Cells were transfected with miR-181d, and the distribution of cells within the stages of the cell cycle was determined. Data are presented as the mean of triplicate experiments. c, d Graphical representation of a. e, f Graphical representation of b. g U251 cells were transfected with miR-181d and assessed for changes in cell proliferation using an MTT assay. h LN229 cells were transfected with miR-181d and assessed for changes in cell proliferation using an MTT assay. Data are presented as the mean of triplicate experiments. *Significant difference (P < 0.05)

Fig. 3

K-ras and Bcl-2 are targets of miR-181d. a Two binding sites for miR-181d were found in K-ras 3′-UTR. Underlined sequences represent the homology between miR-181d seed sequence in various mammals (Hs, human; Pt, chimpanzee; Cf, dog; Gg, goat). b Two binding sites for miR-181d were found in Bcl-2 3′-UTR. Underlined sequences represent the homology between miR-181d seed sequence in various mammals (Hs, human; Pt, chimpanzee; Sa, shrew; Gg, goat). The expression levels of K-ras, Bcl-2 and GAPDH were determined by western blotting. c, e The Luciferase reporter results of K-ras in U251 and LN229 cells. d, f The Luciferase reporter results of Bcl-2 in U251 and LN229 cells. g Over-expression of miR-181d decreased K-ras and Bcl-2 expressions in U251 and LN229 cells. *Significant difference (P < 0.05)

Fig. 4

K-ras and Bcl-2 are over-expressed in glioma samples. Immunohistochemistry was used to assess the expression levels of K-ras and Bcl-2 for each grade of glioma. Magnification, ×200 Bar

Fig. 5

MiR-181d affects the K-ras pathway in U251 and LN229 cells. Western blotting analysis of c-Raf, p-Akt, Akt, p-ERK, and ERK in U251 and LN229 untransfected cells or cells transfected with miR-181d or scrambled oligo. GAPDH serves as a loading control

Fig. 6

U87 cell xenograft tumor experiment. a Growth of U251 cell xenografts. Injection of miR-181d inhibited tumor growth. b Immunohistochemical analysis of U251 xenograft tumors. K-ras and Bcl-2 expressions were suppressed simultaneously. Magnification, ×200 Bar. *Significant difference (P < 0.05). **P < 0.001