Knockdown of FRAT1 expression by RNA interference inhibits human glioblastoma cell growth, migration and invasion

Abstract

Background: FRAT1 positively regulates the Wnt/β-catenin signaling pathway by inhibiting GSK-3-mediated phosphorylation of β-catenin. It was originally characterized as a protein frequently rearranged in advanced T cell lymphoma, but has recently also been identified as a proto-oncogene involved in tumorigenesis. Our previous studies showed that FRAT1 was dramatically overexpressed in gliomas and its expression level was significantly increased along with clinicopathological grades.

Methods: In the current study, we used RT-PCR and Western blotting to assess the mRNA and protein levels of FRAT1 in three glioma cell lines. In addition, to evaluate its functional role in gliomas, we examined the effects of FRAT1 knockdown on proliferation, migration and invasion in vitro and tumor growth in vivo using glioblastoma U251 cells and RNAi.

Results: FRAT1 was highly expressed in all three glioma cell lines. RNAi-mediated down-regulation of endogenous FRAT1 in human glioblastoma U251 cells resulted in suppression of cell proliferation, arrest of cell cycle, inhibition of cell migration and invasion in vitro. Moreover, FRAT1 depletion significantly impaired tumor xenograft growth in nude mice.

Conclusions: Our results highlight the potential role of FRAT1 in tumorigenesis and progression of glioblastoma. These findings provide a biological basis for FRAT1 as a potential molecular marker for improved pathological grading and as a novel candidate therapeutic target for glioblastoma management.

Figure 1. FRAT1 mRNA and protein levels in normal cultured primary astrocytes and SHG44, U87, U251 glioma cells as assessed by RT-PCR and Western blot analysis.

For RT-PCR, specific FRAT1 primers yielded a 325-bp FRAT1 cDNA fragment, and internal control primers for GAPDH yielded a 402-bp GAPDH cDNA fragment. Western blot analysis (WB) of FRAT1 detected distinct bands with apparent molecular mass of 29 kDa. β-actin was assessed as a loading control. N: human normal astrocytes; 1: SHG44; 2: U87; 3: U251.

Figure 2. RNA interference reduced the expression of FRAT1 in U251 cells.

Down-regulation of FRAT1 mRNA and protein expression in U251-S cells as compared to the parental U251, U251-NC, and U251-neo control cell lines was confirmed by RT-PCR and Western blot (WB). GAPDH was amplified as an internal control for the RT-PCR, and β-actin levels were examined as a loading control for the Western blot. 1: parental U251 cells; 2: U251-NC; 3: U251-neo; 4: U251-S.

Figure 3. RNAi-mediated knockdown of FRAT1 inhibits growth of U251 cells in vitro.

Cell viability was measured using a MTT assay. Cell growth curves were determined by absorbance at 490 nm. *, P<0.01 for U251-S relative to each of the three control lines.

Figure 4. RNAi-mediated knockdown of FRAT1 affects the cell cycle distribution of U251 cells in vitro.

Left panel: (A) parental U251, (B) U251-NC, (C) U251-neo and (D) U251-S were stained with propidium iodide. The DNA content and cell cycle were examined and analyzed by flow cytometry. Right panel: A histogram is provided showing the percentages of cells in each cell cycle phase as determined by gating of the flow cytometry.

Figure 5. FRAT1 knockdown suppresses plate colony formation and soft agar colony formation.

(A, B) Equal numbers of parental U251, U251-neo, U251-NC and U251-S cells were seeded onto 60 mm dishes. After 14 days, the cells were fixed and stained with Giemsa (A). The average number of colonies formed in three independent experiments was quantified (B). (C, D) Equal numbers of U251, U251-neo, U251-NC and U251-S cells were plated in 0.3% soft agar and cultured for 14 days. Colony formation was photographed under the microscope (C) and scored (D).

Figure 6. FRAT1 knockdown modulates migration and invasion of U251 cells.

(A) FRAT1 knockdown inhibits cell migration. Monolayers of U251, U251-neo, U251-NC and U251-S cells were mechanically wounded with a pipette tip. Repair of the lesion by cell migration was photographed 24 h later. (B) The cell migration was quantified as shown. (C) FRAT1 RNAi diminished cell invasion of U251 cells. Each of the indicated U251 cell types was assayed for cell invasion using transwell tissue culture dishes. (D) The average cell counts of invading cells from 6 high power fields are shown.

Figure 7. FRAT1 depletion decreases tumorigenicity in nude mice.

(A) BALB/c-nu mice were injected subcutaneously with 1×10⁷ of U251, U251-neo, or U251-NC control cells; or U251-S FRAT1 knockdown cells. Representative tumor formation was photographed 40 days after injection. (B) Tumor sizes were determined by measuring the tumor volume every five days from 5 to 40 days after injection. (C) Average tumor weights of mice 40 days after injection are shown. Values represent means±SD obtained from three independent experiments. (D) Immunohistochemical analysis of FRAT1 expression in tumors in nude mice 40 days following injection.