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. 2018 Mar;15(3):2921-2928.
doi: 10.3892/ol.2017.7670. Epub 2017 Dec 20.

NAC1 promotes the migration of prostate cancer cells and participates in osteoclastogenesis by negatively regulating IFNβ

Fang Chen  1 , Yinghao Yin  1 , Zhifeng Yan  1 , Ke Cao  2 , Kuangbiao Zhong  1
Affiliations

NAC1 promotes the migration of prostate cancer cells and participates in osteoclastogenesis by negatively regulating IFNβ

Fang Chen et al. Oncol Lett. 2018 Mar.

Abstract

Nucleus accumbens-associated protein 1 (NAC1), a transcriptional co-regulator, is overexpressed in advanced prostate cancer. However, the NAC1-regulated transcriptome has not been completely explored. In the present study, the functional silencing of NAC1 blocked the migration of prostate cancer cells and suppress osteoclastogenesis. The present study also determined that NAC1 was overexpressed in the highly aggressive prostate cancer cell lines PC-3, DU-145 and LNCaP. NAC1 small interfering RNA treatment of DU-145 cells decreased cell migration, but interestingly had no significant effects on cell proliferation. Furthermore, microarray analysis showed that a group of genes may be associated with the development of prostate cancer after NAC1 knockdown, including interferon-β (IFNβ), which is reported to be involved in osteoclastogenesis, an important factor affecting bone metastasis. The mechanisms of NAC1 function were further explored by co-culture studies using PC-3 and RAW264.7 osteoclast precursor cells, which demonstrated that silencing NAC1 downregulated the genes associated with the activation of osteoclasts. Furthermore, it was revealed that NAC1 had the ability to affect the release of IFNβ into the extracellular environment. Together, these findings indicated that NAC1 promoted cell migration, and that NAC1 may have a key role in osteoclastogenesis.

Keywords: interferon β; migration; nucleus accumbens-associated protein 1; osteoclastogenesis; prostate cancer.

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Figures

Figure 1.
Figure 1.
NAC1 expression is up-regulated in prostate cancer. (A) Western blot analysis of eight paired prostate cancer and matched adjacent normal tissues. (B) Negative immunohistochemical staining of NAC1 in non-cancerous prostate tissues. The marked section in the 200× image was selected for further observation at 400x. (C) Positive staining of NAC1 graded as (+) in prostate cancer tissue. (D) Positive staining of NAC1 graded as (+++) in prostate cancer tissue.
Figure 2.
Figure 2.
Silencing NAC1 expression decreased the migratory ability of prostate cancer cells. (A) Western blots showing expression of NAC1 in the highly aggressive prostate cancer cell lines PC-3, DU-145 and LNCaP. (B) Western blots showing a significant reduction of NAC1 protein in DU-145 cells transfected with NAC1 siRNAs (siNAC1-1 and siNAC1-2) compared with those transfected with negative control siRNA (NC). (C) MTT assay showing that siNAC1-1 and siNAC1-2 had no obvious effect on the cell number of DU-145 cells (P>0.05). (D) Detection of cell proliferation by plate colony formation assay in DU-145 cells transfected with NAC1 or NC siRNAs. Representative photographs show the DU-145 cell colonies in 6-well plates on the left. The cell colonies were scored visually and counted using a light microscope, as shown in the graph on the right; the siNAC1-1 and siNAC1-2 transfection groups exhibited no distinct difference in the number of colonies compared with the NC siRNA group (NS P>0.05). (E) Wound healing assay showed that NAC1 silencing affected the migration of prostate cancer DU-145 cells, as shown in the photographs on the left. The comparison of cell migration distance between siNAC1-1 or siNAC1-2 and NC siRNA transfection is shown on the right (**P<0.01). (F) Transwell migration analysis showed that NAC1 silencing affected the migration of prostate cancer cells, as shown in the photograph on the left (magnification, 200x). The quantitative analysis of the migratory ability of prostate cancer cells transfected with NAC1 or NC siRNAs is shown on the right (***P<0.001).
Figure 3.
Figure 3.
Profiling of NAC1-regulated genes in prostate cancer cells. (A) Hierarchical clustering of mRNA by the Z-score method. The results are displayed as a heat map, in which red indicates relatively high expression and green denotes relatively low expression. (B) Pathways corresponding to the up-regulated transcripts. (C) Pathways corresponding to down-regulated transcripts. (D) Western blots and RT-qPCR shown a significant reduction of NAC1 protein and mRNA in DU-145 cells transfected with NAC1 siRNAs compared with those transfected with NC (***P<0.001) (E). RT-qPCR was performed to validate the NAC1-regulated genes identified by microarray analysis in DU-145 cells transfected with siNAC1-1 or siNAC1-2 and NC. All candidate genes were validated (*P<0.05,**P<0.01,***P<0.001) by both siRNAs.
Figure 4.
Figure 4.
NAC1 promotes prostate cancer bone metastasis in a manner dependent on the induction of IFNβ. (A) NAC1 knockdown efficiency was verified in PC-3 cells by RT-qPCR and western blotting. (B) Genes associated with activation of osteoclasts were identified in RAW 264.7 cells by RT-qPCR and western blotting (**P<0.01, ***P<0.001). (C) The mRNA and protein levels of IFNβ were verified in PC-3 cells by RT-qPCR and western blotting (**P<0.01). (D) ELISA was performed to validate the protein level of IFNβ in the co-culture medium The differences between the siNAC1-1 or siNAC1-2 transfection groups and the NC group in the Figure were statistically significant (**P<0.01, ***P<0.001).
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