miR-618 Inhibits Prostate Cancer Migration and Invasion by Targeting FOXP2

Abstract

miRNAs play critical role in the development and progression of prostate cancer. Here we studied the role of miR-618 in prostate cancer migration and invasion. miR-618 was downregulated in metastatic androgen-independent prostate cancer (AIPC), patients with low miR-618 had poor outcome. Overexpression of miR-618 inhibited migration and invasion and induced mesenchymal to epithelial transition (MET). Conversely, knockdown of miR-618 promoted migration and invasion and induced epithelial to mesenchymal transition (EMT). FOXP2 was the direct target of miR-618, and promoted TGF-β expression, inhibition of TGF-β reversed the effect of miR-618 knockdown. We further analyzed the correlation between miR-618 expression and FOXP2 in human prostate cancer tissues, and found there was a negative correlation between miR-618 expression and FOXP2 levels. In conclusion, we found miR-618 inhibited prostate cancer migration and invasion by targeting FOXP2 and inhibiting TGF-β.

Keywords: FOXP2; TGF-β.; miR-618; migration; prostate cancer.

Figure 1

miR-618 is downregulated in aggressive malignancy prostate cancer tissues and cells. (A). Real-time RT-PCR analyzed miR-618 expression in untreated primary prostate cancer tissues and castration-resistant prostate cancer tissues. (B). Real-time PCR determined miR-618 expression in paired ADPC tissues and AIPC tissues. (C). Kaplan-Meier overall survival curves for the patients with high versus low miR-618 expression. (D). Real-time RT-PCR analyzed miR-618 expression in primary-cultured prostate epithelial cells (indicated as N1 and N2) and prostate cancer cells. Error bars represent mean ± SD for at least three biological replicates. * P<0.05.

Figure 2

Overexpression of miR-681 inhibits migration and invasion of prostate cancer and induces MET. (A). Wound healing assay analyzed the effect of miR-618 overexpression on migration of prostate cancers. Wound closures were photographed at 0, 12 and 24 hours after wounding. (B). Transwell invasion assay analyzed the effect of miR-618 overexpression on invasion. (left) Representative micrographs of transwell invasion assay of the indicated cells, (right) quantification of indicated invading cells. (C). 3D spheroid invasion assay determined the effect of miR-618 overexpression on metastasis. (D). Western blot analyzed the expression of epithelial markers and mesenchymal markers when miR-618 was overexpression, GAPDH was used as the loading control. Error bars represent mean ± SD for at least three biological replicates. *P<0.05.

Figure 3

Knockdown of miR-618 promotes migration and invasion of prostate cancer and induces EMT. (A). Wound healing assay analyzed the effect of miR-618 knockdown on migration of prostate cancers. Wound closures were photographed at 0, 12 and 24 hours after wounding. (B). Transwell invasion assay analyzed the effect of miR-618 knockdown on invasion. (left) Representative micrographs of transwell invasion assay of the indicated cells, (right) quantification of indicated invading cells. (C). 3D spheroid invasion assay determined the effect of miR-618 knockdown on metastasis. (D). Western blot analyzed the expression of epithelial markers and mesenchymal markers when miR-618 was knock-downed, GAPDH was used as the loading control. Error bars represent mean ± SD for at least three biological replicates. *P<0.05.

Figure 4

FOXP2 is the direct target of miR-618. (A). Predicted miR-618 target sequences (blue) in the 3'UTR of FOXP2 (FOXP2-wt-3'UTR) and positions of three mutated nucleotides (cyan) in the 3'UTR of FOXP2 (FOXP2-mut-3'UTR). (B). Western blot analyzed FOXP2 expression in indicated cells transfecting miR-618 mimic or miR-618 inhibitor, β-Actin was used as the loading control. (C). Western blot analyzed GFP expression in indicated cells transfecting miR-618 mimic or miR-618 inhibitor, α-Tubulin was used as the loading control. (D). Luciferase reporter assay determined luciferase activity in indicated cells cotransfected miR-618 mimic and pGL3-FOXP2-wt-3'UTR or pGL3-FOXP2-mut-3'UTR. Error bars represent mean ± SD for at least three biological replicates. *P<0.05.

Figure 5

FOXP2 promotes cell invasion by increasing TGF-β expression. (A). Western blot analyzed TGF-β expression once miR-618 overexpression in PC3 cells. GAPDH was used as the loading control. (B). Western blot analyzed FOXP2 and TGF-β expression when miR-618 was knock-downed in PC3 and DU145 cells. GAPDH was used as the loading control. (C). when miR-618 and TGF-β were inhibited simultaneously, transwell invasion assay was used to assess cell invasion.

Figure 6

The relationship between miR-618 expression and FOXP2 expression. (A). Real-time RT-PCR assessed miR-618 expression in 12 prostate cancer tissues, western blot analyzed FOXP2 expression in the same tissues. GAPDH was used as the loading control. (B). The correlation analysis between miR-618 expression and FOXP2 expression in 12 prostate cancer tissues.