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. 2010 Sep;101(9):1997-2004.
doi: 10.1111/j.1349-7006.2010.01616.x.

MicroRNA-10b regulates tumorigenesis in neurofibromatosis type 1

Guolin Chai  1 , Ning Liu, Junrong Ma, Hua Li, Janet L Oblinger, Agasanur K Prahalad, Meng Gong, Long-Sheng Chang, Margaret Wallace, David Muir, Abhijit Guha, Roger J Phipps, Janet M Hock, Xijie Yu
Affiliations

MicroRNA-10b regulates tumorigenesis in neurofibromatosis type 1

Guolin Chai et al. Cancer Sci. 2010 Sep.

Abstract

MicroRNAs (miRNAs) are frequently deregulated in human tumors, and play important roles in tumor development and progression. The pathological roles of miRNAs in neurofibromatosis type 1 (NF1) tumorigenesis are largely unknown. We demonstrated that miR-10b was up-regulated in primary Schwann cells isolated from NF1 neurofibromas and in cell lines and tumor tissues from malignant peripheral nerve sheath tumors (MPNSTs). Intriguingly, a significantly high level of miR-10b correlated with low neurofibromin expression was found in a neuroectodermal cell line: Ewing's sarcoma SK-ES-1 cells. Antisense inhibiting miR-10b in NF1 MPNST cells reduced cell proliferation, migration and invasion. Furthermore, we showed that NF1 mRNA was the target for miR-10b. Overexpression of miR-10b in 293T cells suppressed neurofibromin expression and activated RAS signaling. Antisense inhibition of miR-10b restored neurofibromin expression in SK-ES-1 cells, and decreased RAS signaling independent of neurofibromin in NF1 MPNST cells. These results suggest that miR-10b may play an important role in NF1 tumorigenesis through targeting neurofibromin and RAS signaling.

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Figures

Figure 1
Figure 1
Differential miRNA expression profiles in neurofibromatosis type 1 (NF1) versus non‐NF1 malignant peripheral nerve sheath tumor (MPNST) cell lines. (a–e) miRNA expression in non‐NF1 (STS26T) and NF1 (ST88‐14, T265p21) associated MPNST cells was investigated by qRT‐PCR. Expression levels of miR‐10b (a), miR‐155 (b), and miR‐335 (c) were significantly higher in NF1 MPNST cells than in non‐NF1 MPNST cells, while expression levels of let‐7a (d) and let‐7b (e) were significantly lower in NF1 MPNST cells. The result was consistent with microarray data. ST88‐14 or T265p21 versus STS26T: *P < 0.05, **P < 0.01, ***P < 0.001, n = 3. (f) Western blotting showed that STS26T expressed a significant amount neurofibromin, but a lower level of TWIST1; T265p21 and ST88‐14 expressed a very low to undetectable level of neurofibromin, but a higher level of TWIST1.
Figure 2
Figure 2
miRNA profiles in neurofibromatosis type 1 (NF1) tumor tissues. Human NF1 tumor samples were divided into an NF1 neurofibroma group (control, n = 13), a non‐NF1 malignant peripheral nerve sheath tumor (MPNST) group (n = 4), and an NF1 MPNST group (n = 8) according to clinical and pathological diagnosis. miRNA expression was studied by qRT‐PCR. Compared to the neurofibroma control group, the expression level of miR‐10b was significantly higher, while expression levels of let‐7a and let‐7b were significantly lower in NF1 MPNST tissues. Compared to non‐NF1 MPNST, the expression level of miR‐10b was significantly higher in NF1 MPNST. There were no differences in expression levels of miR‐155 and miR‐335 among the three groups. (a) Compared to NF1 neurofibromas, P < 0.05; (b) compared to non‐NF1MPNST, P < 0.05.
Figure 3
Figure 3
miRNA profiles in primary Schwann cells from neurofibromatosis type 1 (NF1) neurofibromas. Primary Schwann cells were isolated from normal human adult sciatic nerves, and NF1 dermal and plexiform neurofibromas. Neurofibromatosis type 1 (NF1) malignant peripheral nerve sheath tumor (MPNST) cell lines ST88‐14 and T265p21 were also used for comparison. miRNA expression was studied by qRT‐PCR. miR‐10b expression level was significantly higher in NF1 dermal Schwann cells than in normal Schwann cells. Neurofibromatosis type 1 (NF1) plexiform Schwann cells also showed higher miR‐10b expression, but the difference was not statistically significant (P = 0.07). The highest expression level of miR‐10b was observed in NF1 MPNST cells. Expression levels of miR‐155 and miR‐335 were significantly higher, and expression levels of let‐7b and let‐7a were significantly lower in NF1 MPNST cells. There were no significant differences in the expression levels of miR‐155, miR‐335, and let‐7a between NF1 neurofibroma cells and normal Schwann cells. SC, Schwann cells. (a) P < 0.05 versus normal SC; (b) P < 0.05 versus dermal or plexiform SC; n = 3–4.
Figure 4
Figure 4
Antisense inhibition of miR‐10b corrected abnormal growth behaviors of neurofibromatosis type 1 (NF1) malignant peripheral nerve sheath tumor (MPNST) cells. The inhibitors for miR‐10b, miR‐155, miR‐335, or the enhancers for let‐7a or negative controls were transfected into NF1 MPNST cells (ST88‐14). (a) Inhibiting miR‐10b expression significantly reduced cell proliferation at days 4–7 (n = 5 each time point; MTT assay). (b) Inhibiting miR‐10b decreased colony formation. (c) Restoring miR‐10b or miR‐335 or let‐7a significantly decreased cell invasion (n = 3). (d) Inhibiting miR‐10b significantly decreased cell migration (n = 3). Inhibitors or enhancers versus negative controls: *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
Figure 5
miR‐10b directly targeted neurofibro‐matosis type 1 (NF1) 3′UTR. (a) The 3′UTR of the NF1 gene was analyzed by TargetScan and a target sequence for miR‐10b was identified. This target sequence was highly conserved across species. (b) Neurofibromatosis type 1 (NF1) 3′UTR was cloned into the 3′UTR of the Luc reporter vector and the resulting construct was stably transfected into 293T cells. miR‐10b cotransfection significantly reduced expression of the Luc reporter containing NF1 3′UTR. miR‐10b could not reduce luciferase activity if its targeted seed sequence in the NF1 3′UTR was mutated (NF1 3′UTRm). These results indicate that miR‐10b directly targeted NF1 3′UTR. (c) The MDH1‐PGK‐GFP/microRNA‐10b (vector/miR‐10b) or MDH1‐PGK‐GFP vector (vector) was transfected into 293T cells. Overexpressing miR‐10b suppressed neurofibromin expression. *P < 0.05 versus control; n = 3. (d) miR‐10b enhancers were transiently transfected into STS26T cells. Enhancing miR‐10b significantly increased invasion. This result indicates that overexpression of miR‐10b directly regulates the cellular tumor characteristics.
Figure 6
Figure 6
Antisense inhibition of miR‐10b restored neurofibromin expression. Expressions of miR‐10b and neurofibromin in some human tumor cell lines were studied by qRT‐PCR and western blotting separately. Ewing sarcoma cells (SK‐ES1) expressed the highest miR‐10b (a), but very low neurofibromin (b). Inhibiting miR‐10b restored neurofibromin expression at 48 and 72 h (c). These results suggest that miR‐10b directly suppresses neurofibromin in SK‐ES1.
Figure 7
Figure 7
miR‐10b modulated RAS signaling. (a) The MDH1‐PGK‐GFP/miR‐10b (vector/miR‐10b) or MDH1‐PGK‐GFP vector (vector) was transfected into 293T cells. Transfected cells were stimulated with serum free medium (control) or medium supplemented with 20% FBS for 15 min, and phosphorylated ERK and total ERK were analyzed by western blotting. Overexpressing miR‐10b induced higher phosphorylated ERK expression. (b) The miR‐10b inhibitors or negative controls were transfected into NF1 MPNST cells (ST88‐14). Cells were serum‐starved for 24 h, and then incubated in serum‐free medium (control) or medium plus 20% FBS for 15 min. Phosphorylated ERK and total ERK were detected by western blotting. Compared to controls, inhibiting miR‐10b expression reduced phosphorylated ERK in response to serum stimulation.
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