Downregulation of FHL1 protein in glioma inhibits tumor growth through PI3K/AKT signaling

Figures

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  Figure 1.. Expression of FHL1 in human glioma specimen is negatively associated with pathological grades and survival time. (A) The expression of FHL1 in different pathological grades of glioma specimen (grades I–IV) was determined by immunohistochemistry staining. Its expression was indicated by brown staining in the cytoplasm (n=114). Magnifications, ×100 and ×400 (white boxes). (B) Overall survival curves of patients with glioma with high or low/negative expression of FHL1 were calculated using the Kaplan-Meier method. The difference in survival time between the two groups was examined using a log-rank test, and low expression of FHL1 was identified to be significantly associated with poor prognosis. FHL1, four-and-a-half LIM domains 1.

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  Figure 2.. FHL1 inhibits the proliferation of glioma cells in vitro. (A) The mRNA and protein levels of FHL1 in 2 glioma U87 and U251 cell lines were examined using an reverse transcription polymerase chain reaction assay and western blot analysis, respectively. The expression of β-actin was used as an internal control. (B and C) The (B) stable FHL1-overexpressing U87 cell line and (C) stable FHL1-knockdown U251 cell line were confirmed by western blot analysis with anti-FHL1 antibody. These experiments were repeated 3 times. (D) Cell proliferation was examined every 24 h in the stable FHL-overexpressed U87 cells and stable FHL1-knockdown U251 cells using an MTT assay. (E) Cell cycle progression of 2 stable cell lines was determined by fluorescence-activated cell sorting following propidium iodide staining. The error bars represent the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001. FHL1, four-and-a-half LIM domains 1; siRNA, small interfering RNA.

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  Figure 3.. FHL1 affects glioma cell growth through PI3K/Akt signaling. (A) Western blot analysis for the phosphorylation levels of Akt and total AKT expression in the two types of stable cell lines and corresponding control cell lines, with β-actin as an internal control. (B) FHL1 interacts with AKT in U87 cells. U87 cells were transfected with 0.5 µg FLAG-FHL1 expression vector or 0.5 µg FLAG empty vector. The IP analysis was performed with anti-FLAG antibody, and the WB was performed with anti-AKT antibody and anti-p110α antibody. FHL1, four-and-a-half LIM domains 1; p, phosphorylated; siRNA, small interfering RNA; IP, immunoprecipitation; WB, western blot analysis.

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  Figure 4.. FHL1 overexpression inhibits tumor growth in vivo through PI3K/AKT signaling. (A) U87 cells stably transfected with FHL expression or control vector were inoculated subcutaneously in nude mice respectively (n=6). (B) The tumor growth was monitored by measuring tumor size twice a week from the 7th day after tumor cell inoculation. (C) Tumors were dissected from mice on the last day of the experiments and images were captured. (D) Tumor tissue was lysed using lysis buffer, and the expression level of FHL1 and phosphorylated AKT (p-AKT) was analyzed by western blot analysis. Scale bars represent the mean ± standard deviation. *P<0.05 and **P<0.01. n=6. FHL1, four-and-a-half LIM domains 1; p, phosphorylated.