microRNA-99b acts as a tumor suppressor in non-small cell lung cancer by directly targeting fibroblast growth factor receptor 3

Abstract

microRNAs (miRNAs) play a significant role in cancer development and progression by regulating the expression of proto-oncogenes or tumor suppressor genes. Our previous study using microarrays demonstrated that miR-99b was downregulated in patients with lung cancer. To assess whether or not miR-99b has a functional role in lung cancer, we determined the expression of miR-99b and fibroblast growth factor receptor 3 (FGFR3), which is a predicted target of miR-99b in public algorithms in human lung cancer tissues. miR-99b was downregulated and FGFR3 was upregulated in lung cancer patients. We demonstrated that the overexpression of miR-99b induced a reduction in FGFR3 expression and confirmed the target specificity between miR-99b and the FGFR3 3'-untranslated region by luciferase reporter assay. In addition, the growth rate in miR-99b precursor-treated cells was lower compared to the negative controls. Taken together, these results suggest that miR-99b may be a tumor suppressor through the downregulation of FGFR3. miR-99b may be a potent tumor suppressor and may be a potential therapeutic tool for patients with lung cancer.

Figure 1.

miR-99b and FGFR3 expressions in the normal samples differed significantly from those in the lung cancer tissue samples. Real-time RT-PCR analysis of mature miR-99b and FGFR3 expression in lung cancer and normal lung tissues prepared from the same patients. ^*p= 0.0039, ^**p= 0. 015 versus control. FGFR3, fibroblast growth factor receptor 3; RT-PCR, reverse transcription polymerase chain reaction.

Figure 2.

FGFR3 was regulated by miR-99b in lung cancer cells. (A) The signal from a luciferase reporter was significantly decreased at one miR-99b target site at the 3′UTR of FGFR3. ^*p<0.0001, versus control. (B) The effect of miR-99b on FGFR3 mRNA expression in lung cancer cells. ^*p<0.05 versus control. (C) The effect of miR-99b on HDAC1 protein expression in lung cancer cells. FGFR3, fibroblast growth factor receptor 3; RT-PCR, reverse transcription polymerase chain reaction; UTR, untranslated region.

Figure 3.

miR-99b function as inhibitor of cell proliferation in lung cancer. Inhibitory effects of miR-99b on cell proliferation in H1299, HCC95 and HCC1438 cells. OD, optical density.

Figure 4.

Anchorage-independent growth assay in H1299 cells. Results show the mean ± standard deviation (SD) of three independent experiments performed in triplicate. ^*p<0.05 versus control.