FRAT1 Enhances the Proliferation and Tumorigenesis of CD133+Nestin+ Glioma Stem Cells In Vitro and In Vivo

Abstract

Glioma stem cells (GSCs) are considered the source for development, recurrence, and poor prognosis of glioma, so treatment targeted GSCs is of great interest. The frequently rearranged in advanced T cell lymphomas-1 (FRAT1) gene is an important member of the Wnt/β-catenin signaling transduction pathway, and aberrantly activation of Wnt signaling has been identified to contribute to the tumorigenesis, proliferation, invasion of a variety kinds of cancer stem cells. However, correlations between FRAT1 and GSCs and the specific mechanisms remain unclear. In this study, we aimed to investigate the effect of FRAT1 on GSCs proliferation, colony formation, sphere formation and tumorigenesity in vitro and in vivo and its underlying mechanism. Lentiviral transfection was used to construct GSCs with low FRAT1 expression. The expression of FRAT1 on GSCs proliferation in vitro was assessed by cell counting kit-8(CCK-8). Colony formation and sphere formation assays were conducted to assess the colony and sphere formation ability of GSCs. Then, an intracranial glioma nude mouse model was built to measure the effect of low FRAT1 expression on GSCs proliferation and tumorigenesity in vivo. Real-time PCR, Western blot, and Immunohistochemistry were processed to detect the mRNA and protein expressions of FRAT1, β-catenin in the glioma tissue of xenograft mice to study their correlations. The functional assays verifed that low FRAT1 expression inhibited CD133⁺Nestin⁺ GSCs proliferation, colony formation, sphere formation ability in vitro. In vivo GSCs xenograft mice model showed that low FRAT1 expression suppressed the proliferation and tumorigenesity of CD133⁺Nestin⁺ GSCs and reduced β-catenin mRNA and protein expression. Furthermore, the expression of FRAT1 and β-catenin were positively correlated. Altogether, results indicate that FRAT1 enhances the proliferation, colony formation, sphere formation and tumorigenesity of CD133⁺Nestin⁺ glioma stem cells in vitro and in vivo as well as the expression of β-catenin. Therefore, inhibiting proliferation of GSCs and FRAT1 may be a molecular target to GSCs in treating human glioma in the future.

Keywords: Glioma stem cells (GSCs); Proliferation; The frequently rearranged in advanced T cell lymphomas-1(FRAT1); Wnt/β-catenin pathway.

Figure 1

Immunofluorescent assay was performed and low expression of FRAT1 inhibited CD133⁺Nestin⁺ GSCs proliferation, colony and sphere formation ability in vitro. (A) Immunofluorescent assay to measure CD133 and nestin expression in GSCs (400×). Fluorescent microscopy for CD133-positive staining (green fluorescence) and nestin-positive staining (green fluorescence) in cytoplasm of GSCs; DAPI-positive staining (blue fluorescence) in nucleus of GSCs. (B) Immunofluorescent assay for FRAT1 and CD133 expression in GSCs (400×). Fluorescent microscopy for CD133-positive staining (green fluorescence) and FRAT1-positive staining (red fluorescence) in cytoplasm of GSCs. FRAT1 and CD133 proteins co-localized in GSCs. (C-D) Real-time PCR for mRNA expression of FRAT1 and β-catenin in GSCs in vitro. GAPDH was used as an internal reference. (E) Western blot of FRAT1 and β-catenin protein expression in GSCs in vitro. β-Tubulin was used as an internal reference. (F) CCK-8 assay detected the effect of low FRAT1 expression on the viability of in vitro GSCs. A450 OD of GSCs was continuous monitored from days 0 to 6. (G) Representative images of GSCs colony formation and the relative colony number of GSCs are shown. (H) Representative images of GSCs sphere formation are shown (100×) as well as the sphere number of GSCs. *p < 0.05, **p < 0.01; ***p < 0.001; ns, non-significant.

Figure 2

Low expression of FRAT1 inhibited GSCs proliferation in vivo and also reduced the expression of β-catenin. (A) Small animal MRI for low FRAT1 expression and in vivo GSCs proliferation. MRI for axial and coronal intracranial gliomas (red arrows) of all groups of xenograft mice on day 21 after inoculation. (B) RadiAnt DICOM Viewer used to measure intracranial glioma volumes of all groups of xenograft mice on days 7, 14, and 21 after inoculation to prepare a growth curve for intracranial gliomas. (C-D) Real-time PCR for mRNA expression of FRAT1 and β-catenin in glioma tissues of xenograft mice. GAPDH was used as an internal reference. (E) Western blot of FRAT1 and β-catenin protein expression in glioma tissues of xenograft mice. β-Tubulin was used as an internal reference. (F) Representative images showing the immunohistochemistry of FRAT1 and β-catenin protein expression in glioma tissues of xenograft mice (×400). FRAT1-positive protein staining localized in the cytoplasm of glioma cells. β-catenin-positive protein staining localized in the cytoplasm and (or) nucleus of glioma cells. (G) Scatter plot of correlation between FRAT1 IRS and β-catenin IRS in the glioma tissues of xenograft mice. Pearson correlation analysis showed that there was a positive correlation between FRAT1 IRS and β-catenin IRS in the glioma tissues of xenograft mice. *p < 0.05, **p < 0.01; ***p < 0.001; ns, non-significant.