Differential expression of filamin B splice variants in giant cell tumor cells

Abstract

Giant cell tumor of bone (GCT) is the most commonly reported non-malignant bone tumor in Hong Kong. This kind of tumor usually affects people aged 20-40 years. Also, it is well known for recurrence locally, especially when the tumor cannot be removed completely. Filamins are actin-binding proteins which contain three family members, filamin A, B and C. They are the products of three different genes, FLNA, FLNB and FLNC, which can generate various transcript variants in different cell types. In this study, we focused on the effects of FLNBv2 and FLNBv4 toward GCT cells. The only difference between FLNBv2 and FLNBv4 is that FLNBv4 does not contain hinge 1 region. We found that the relative abundance of FLNBv4 varies among different GCT cell lines while the expression level of FLNBv4 in normal osteoblasts was only marginally detectable. In the functional aspect, overexpression of FLNBv4 led to upregulation of RANKL, OCN, OPG and RUNX2, which are closely related to GCT cell survival and differentiation. Moreover, FLNBv4 can have a negative effect on cell viability of GCT cells when compare with FLNBv2. In conclusion, splicing variants of FLNB are differentially expressed in GCT cells and may play a role in the proliferation and differentiation of tumor cells.

Figure 1.

Expression level of filamin B (FLNB) transcript variants in giant cell tumor of bone stromal cells (GCTSC) by transcriptome analysis. (A) Expression level of different FLNB transcripts in three different GCT cell lines and MSC. (B) The morphology of GCT cell lines G33 and G35 by light microscopy.

Figure 2.

Expression level of filamin B (FLNB) transcript variants in giant cell tumor of bone stromal cells (GCTSC) by real-time PCR. Relative expression of FLNB variants determined by real-time PCR using 16 cDNA samples of GCT cell lines and one normal osteoblast cell line. GAPDH was used as a reference gene to normalize the expression level. Error bars represent standard deviations of triplicate assays.

Figure 3.

Differential expression of filamin B variant4 (FLNBv4) in liver cancer and adjacent normal tissues. (A) The relative expression levels of FLNBv4 and other FLNB variants were determined in five pairs of liver cancer tissues and their adjacent normal tissues by real-time PCR. GAPDH was used for normalization. T, tumor tissue; N, adjacent normal tissue. (B) The ratio of transcript level of FLNBv4 to other FLNB variants in liver cancer tissues and their adjacent normal tissues is shown. A significant difference in the ratio between liver cancer and normal tissues was found using paired t-test (*P<0.05). Error bars represent standard deviations of five different samples.

Figure 4.

Changes in gene expression triggered by filamin B variant2 (FLNBv2) and FLNBv4. Relative expression of four genes after the transfection of G33, G51 and G62 cells by FLNBv2, FLNBv4 and empty vector as revealed by real-time PCR. Error bars represent standard deviations of triplicate assays. (A) RANKL; (B) OCN; (C) OPG; (D) RUNX2 (n=3; *P<0.05, **P<0.01 and ***P<0.001 using one way ANOVA when comparing the effects of each group).

Figure 5.

Correlation of expression levels of filamin B variant4 (FLNBv4) and RUNX2 in giant cell tumor (GCT) cells. Relative expression of FLNBv4 and RUNX2 determined by real-time PCR using 9 cDNA samples of GCT cell lines. GAPDH was used as a reference gene to normalize the expression level. A positive correlation between FLNBv4 and RUNX2 was identified (P=0.0174 and r=0.8167).

Figure 6.

Effects of transfection of filamin B variant2 (FLNBv2) and FLNBv4 on the cell viability in giant cell tumor (GCT) cell lines. Cells were incubated for 2 and 4 days and the cell viability was then examined by cell counting kit-8 (CCK-8) assay. Cell viability in each experiment is normalized by cells transfected by empty plasmid. Error bars represent standard deviations of triplicate assays. (A) G33; (B) G51; (C) G62 (*P<0.05, **P<0.01 and ***P<0.001 using one way ANOVA).