Pin1-Nanog expression in human glioma is correlated with advanced tumor progression

Abstract

The stemness gene Nanog has been shown to play an important role in tumor development, including glioma. Nanog is phosphorylated at multiple Ser/Thr-Pro motifs, which promotes the interaction between Nanog and the prolyl isomerase Pin1, leading to Nanog stabilization by suppressing its ubiquitination. The present study investigated the expression and relationship of Pin1 and Nanog in human gliomas. Significantly higher mRNA and protein expression levels of Pin1 and Nanog were demonstrated in 120 glioma specimens of different pathological grades by RT-PCR, immunohistochemistry staining and western blot analysis. The relative levels of Pin1 expression, as well as Nanog expression, were significantly positively correlated with pathological grade. Moreover, a positive correlation of Pin1 and Nanog expression in human gliomas was noted. Co-localization of Pin1 and Nanog was observed in the perinuclear space in the cytoplasm of glioma cells detected by immunofluorescence staining. Significantly positive correlation between Pin1 and Nanog in gliomas indicated that Pin1 and Nanog may be related to tumorigenesis and development of glioma cells.

Figure 1

Expression of Pin1 and Nanog gene in differential pathological grade glioma tissues. (A) Expression of Pin1 mRNA by RT-PCR in different pathological grade glioma tissues (normal brain tissues as control). (B) Histogram representing relative level of Pin1 mRNA by RT-PCR (F=21.814, P<0.01, ANOVA). (C) Expression of Nanog mRNA by RT-PCR in different pathological grade glioma tissues (normal brain tissues as control). (D) Histogram representing relative level of Pin1 mRNA by RT-PCR (F=18.381, P<0.01, ANOVA). P, Pin1 (427 bp); N, Nanog (403 bp); a, β-actin (252 bp).

Figure 2

Immunohistochemical analysis of the expression patterns of Pin1 and Nanog in differential pathological grade glioma tissues. (A–D) Pin1 immunohistochemical staining of paraffin sections of gliomas (magnification, ×400). (E–H) Nanog immunohistochemical staining of paraffin sections of gliomas (magnification, ×400). (B) Expression of Pin1 in WHO grade II tissue; Pin1 expression was primarily localized in the nuclei of tumor cells (white arrow). (C) Expression of Pin1 in WHO grade III tissue; Pin1 expression was localized in both the cytoplasm and nuclei of glioma cells (white arrow). (D) Expression of Pin1 in WHO grade IV tissue; Pin1 was expressed in both the cytoplasm and nuclei of glioma cells (white arrow). (F–H) Expression of Nanog in WHO grade II, III, IV glioma tissues; Nanog showed mainly nuclear or perinuclear staining with some cytoplasmic localization (white arrow). (A and E) Normal brain tissues.

Figure 3

Expression of Pin1 and Nanog protein in differential pathological grade glioma tissues. (A) The expressions of Pin1 and Nanog protein by western blotting in different pathological grade glioma tissues (normal brain tissues as control). (B) Histogram representing the relative level of Pin1 protein as determined by western blot analysis (F=22.962, P<0.01, ANOVA). (C) Histogram representing the relative level of Nanog protein as determined by western blot analysis (F=42.691, P<0.01, ANOVA).

Figure 4

Expression of Pin1 in U87 glioma cells. (A) Expression of Pin1 mRNA as determined by RT-PCR in U87 glioma cells and WHO IV glioma tissues. (B) Histogram representing the relative level of Pin1 mRNA (P>0.05, independent Student's t-test). (C) Western blot analysis of U87 cells and WHO IV glioma tissues. (D) Histogram representing the relative level of Pin1 protein as determined by western blot analysis (P>0.05, independent Student's t-test).

Figure 5

Detection of the co-expression of Pin1 and Nanog in U87 glioma cells by immunofluorescence staining. (A) nuclei (DAPI). (B) Pin1 (FITC); Pin1 was expressed in both the cytoplasm and the nuclei of glioma cells. (C) Nanog (TRITC); Nanog showed mainly nuclear or perinuclear staining with some cytoplasmic localization. (D) Merged view; Pin1 and Nanog were co-located in the perinuclear space in the cytoplasm of glioma cells (white arrow). Magnification, ×1,000.