The role of MAPK signaling pathway in the Her-2-positive meningiomas

Abstract

Meningiomas are common types of adult nerve system tumors. Although most cases are considered benign, due to its high rate of recurrence and easy malignant progression to anaplastic meningioma they present a puzzle for the current treatment. The HER-2 oncogene has important value for meningioma cells development and progression. So far, little is known about the effect on the exact underlying signal pathway and molecular mechanisms of HER-2-positive meningioma cells. The goal of the present study was to determine the effects of HER-2 gene and possible involvement of MAPK signal pathway in human malignant meningioma. We applied q-PCR analysis, immunofluorescence (IF) staining, western blot analysis, animal model, MAPK inhibition, MTT assay and cell invasion analysis for the investigation. The results demonstrated that the downregulation of the expression of HER-2 significantly inhibited cell motility and proliferation of human meningioma cells in vivo. Accordingly, in the HER-2-overexpression meningioma cells with the inhibition of ERK1/2, ERK5, JNK, in the cells with the ERK1/2, ERK5 inhibition, protein expression was markedly suppressed as well as the cell proliferation resistance. No difference was observed in the HER-2-overexpression meningioma cells with the inhibition of JNK. These findings suggest that HER-2 gene can affect the proliferation ability of human meningioma cells in vivo and MAPK signal pathway may contribute to the carcinogenesis and development of human meningiomas combinating with HER-2.

Figure 1

Effect of the HER-2 gene on the expression of HER-2 in IOMM-Lee cells. (A) The levels of HER-2 in different cell lines. Compared with NC-sh, the levels of HER-2 in the IOMM-Lee cell lines transfected with HER2-sh1, HER2-sh2, HER2-sh3 were decreased, obviously HER2-sh2 (^**P<0.01). (B) The mRNA levels of HER-2 in the IOMM-Lee malignant meningioma cells. Compared with the NC-overexpression, the expression of HER-2 in the IOMM-Lee cells transfected with HER-2-over was significantly increased (^**P<0.01). (C and D) The protein expression of HER-2 in IOMM-Lee cells transfected with HER-2-sh and HER-2-over were analyzed by western blotting. (C) The protein expression levels of HER-2 in the IOMM-Lee cell lines transfected with HER-2-sh2 obviously decreased (^**P<0.01). The data are expressed as the mean standard deviation from three independent experiments. (D) The protein expression levels of HER-2 in the IOMM-Lee cell lines transfected with HER-2-over obviously increased (^**P<0.01). The data are expressed as the mean standard deviation from three independent experiments. Blank, blank control; NC, negative; sh, short hairpin, over, overexpression.

Figure 2

The IOMM-Lee cells transfected with the HER-2-overexpression lentiviral reflected by immunofluorescence staining. (A and B) Immunofluorescence staining of HER-2 in the blank group showed HER-2 expression in the IOMM-Lee cells. (C and D) Immunofluorescence staining of HER-2 in the HER-2-over group presented strong effects on the IOMM-Lee cells transfected with HER-2-overexpression lentiviral. Blank, blank control; over, overexpression.

Figure 3

HER-2 affects protein expression levels of ERK5, JNK, ERK1/2, P38 and Ras in IOMM-Lee cells. (A) Protein levels of ERK5, p-ERK5, JNK, p-JNK, ERK1/2, p-ERK1/2, P38, p-P38, Ras in the IOMM-Lee cells were determined using western blot analysis 72 h post-transfection. β-actin was used as an internal loading control. (B) Protein levels of ERK5, p-ERK5, JNK, p-JNK, ERK1/2, p-ERK1/2, Ras in the HER-2-sh group were significantly decreased. However, in the HER-2-over group, the protein levels of ERK5, p-ERK5, JNK, p-JNK, ERK1/2, p-ERK1/2, Ras were increased, compared with NC control and following normalization against β-actin (P<0.05). No difference was observed between the protein expression of P38, p-P38 in the HER-2-sh and HER-2-over cells with that in the NC control. The data are expressed as the mean standard deviation from three independent experiments. NC, negative control; sh, short hairpin; over, overexpression; ERK5, extracellular signal-regulated kinase 5; JNK, Jun N-terminal kinase; ERK1/2, extracellular regulated protein kinases 1/2; p-, phosphorylated.

Figure 4

Tumorigenesis of HER-2-overexpression in IOMM-Lee cells in vivo. (A) growth situation of tumor-burdened nude mice. (B) Time - a tumor-burdened nude mouse tumor growth curve (^*P<0.05, ^**P<0.01). (C) Tumor number and volume of each group. Blank, blank control; NC, negative; sh, short hairpin, over, overexpression.

Figure 5

MTT assay present the inhibition of the cell proliferation. (A) MTT assay at 72 h, in HER-2-over with concentration of 40 µg/l PD98059 (ERK1/2 inhibition) cell resistance was 1.54-fold higher than that of HER-2-over without inhibition (P<0.01), whereas the resistance of the HER-2-over with concentration of 40 µg/l of PD98059 (ERK1/2 inhibition) cells significantly decreased 45%, compared with the blank group with the same concentration of PD98059 (P<0.01). (B) At 72 h, HER-2-over with concentration of 10 µg/l XMD8-92 (ERK5 inhibition) cell resistance was decreased 73.8% compared with the HER-2-over without inhibition (P<0.01), whereas the resistance of the HER-2-over with concentration of 10 µg/l XMD8-92 (ERK5 inhibition) cells significantly decreased 24%, compared with the blank group with the same concentration PD98059 (P<0.01). (C) At different time-point (24, 48, 72 h), MTT assay revealed that, no difference was observed in the groups with the different concentration of SP600125 (10, 20, 40 µg/l) (P>0.05). Blank, blank control; over, overexpression.

Figure 6

Inhibition of the IOMM-Lee cells transfected with the HER-over lentiviral vector cell invasion. (A) Representative images of Transwell invasion assays, demonstrating that invasion of HER-2-over cells with the concentration of 40 µg/l PD98059 was less than the HER-2-over group by 58.5% (^**P<0.05). (B) Transwell invasion assays with the concentration of 10 µg/l of XMD8-92 (ERK5 inhibition), the invasion of HER-2-over cell decreased 46.1% (^*P<0.05). (C) No difference was observed in the invasion ability of SP600125 group contrast to the HER-2-over group (P>0.05). Blank, blank over, overexpression.

Figure 7

Inhibitions of protein expression of ERK1/2 and ERK5 through ERK1/2 (MAPK) pathways in HER-2-positive IOMM-Lee meningioma cells. Protein level of ERK1/2 in the HER-2-over IOMM-Lee cells were determined using western blot analysis at 72 h post-transfection. β-actin was used as an internal loading control. (A) Western blot assay demonstrated that PD98059 (ERK1/2 inhibition) inhibited the protein expression of ERK1/2 at the best inhibition concentration of 40 µg/l (^*P<0.05). (B) Western blot assay demonstrated XMD8-92 (ERK5 inhibition) inhibited the protein expression of ERK5 the component of ERK1/2 signaling at the best inhibition concentration of 10 µg/l (^*P<0.01). (C) No effect we can observed on the JNK at different inhibition concentration of SP600125. The data are expressed as the mean standard deviation from three independent experiments. -, Blank group; 0, the inhibition of 0 µg/l; 5, the inhibition of 5 µg/l; 10, the inhibition of 10 µg/l; 20, the inhibition of 20 µg/l; 40, the inhibition of 40 µg/l.