Direct regulation of LAMP1 by tumor-suppressive microRNA-320a in prostate cancer - PubMed (original) (raw)

Direct regulation of LAMP1 by tumor-suppressive microRNA-320a in prostate cancer

Atsushi Okato et al. Int J Oncol. 2016 Jul.

Abstract

Advanced prostate cancer (PCa) metastasizes to bone and lymph nodes, and currently available treatments cannot prevent the progression and metastasis of the disease. Therefore, an improved understanding of the molecular mechanisms of the progression and metastasis of advanced PCa using current genomic approaches is needed. Our miRNA expression signature in castration-resistant prostate cancer (CRPC) revealed that microRNA-320a (miR‑320a) was significantly reduced in cancer tissues, suggesting that miR‑320a may be a promising anticancer miRNA. The aim of this study was to investigate the functional roles of miR‑320a in naïve PCa and CRPC cells and to identify miR‑320a-regulated genes involved in PCa metastasis. The expression levels of miR‑320a were significantly reduced in naïve PCa, CRPC specimens, and PCa cell lines. Restoration of mature miR‑320a in PCa cell lines showed that miR‑320a significantly inhibited cancer cell migration and invasion. Moreover, we found that lysosomal-associated membrane protein 1 (LAMP1) was a direct target of miR‑320a in PCa cells. Silencing of LAMP1 using siRNA significantly inhibited cell proliferation, migration, and invasion in PCa cells. Overexpression of LAMP1 was observed in PCa and CRPC clinical specimens. Moreover, downstream pathways were identified using si-LAMP1-transfected cells. The discovery of tumor-suppressive miR‑320a-mediated pathways may provide important insights into the potential mechanisms of PCa metastasis.

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Figures

Figure 1

Figure 1

Expression levels of miR-320a in clinical prostate cancer specimens and cell lines. Expression levels of miR-320a were determined by RT-PCR. RNU48 was used for normalization.

Figure 2

Figure 2

Effects of miR-320a restoration on the proliferation, migration, and invasion of PCa cells. (A) Cell proliferation was determined 72 h after transfection using XTT assays. (B) Cell migration activity was determined 48 h after transfection using Boyden chamber migration assays. (C) Cell invasion activity was determined 48 h after transfection using Matrigel invasion assays. *P<0.0001. Experiments were performed triplicate. Bars indicate SDs.

Figure 3

Figure 3

Strategy for identification of candidate target genes of miR-320a. TargetScan Release 7.0 was used to identify genes containing the miR-320a seed sequence in the 3′-UTR. Next, we analyzed the gene expression dataset using the GEO database (GEO accession no. GSE29079). Finally, we attempted to identify miR-320a target genes using _miR-320a_-transfected PC3 cells (GEO accession no. GSE77790).

Figure 4

Figure 4

Expression of LAMP1 in clinical PCa specimens using a tissue microarray. (A) Representative immunohistochemical staining for LAMP1 in a prostate cancer tissue microarray (Table III; nos. 8, 32, 61 and 70). LAMP1 was strongly expressed in PCa. (B) Comparison of immunohisto-chemical staining of LAMP1 in prostate cancer tissues using IHC scores. LAMP1 expression was significantly higher in PCa tissues than in normal prostate tissues.

Figure 5

Figure 5

Expression of LAMP1 in clinical CRPC specimens. Representative immunostaining for LAMP1 in CRPC clinical specimens. PSA and AR were used to confirm prostate cancer metastasis.

Figure 6

Figure 6

Direct regulation of LAMP1 by miR-320a. (A) LAMP1 mRNA expression was determined at 96 h after transfection of miR-320a. GAPDH was used as an internal control. (B) LAMP1 protein expression 72 h after transfection with miR-320a. GAPDH was used as a loading control. (C) Luciferase reporter assays using a vector encoding the putative miR-320a target site in the LAMP1 3′-UTR (wild-type and deletion constructs). *P=0.0038, **P=0.0002, and ***P<0.0001. Experiments were performed in triplicate. Bars indicate SDs.

Figure 7

Figure 7

Effects of silencing LAMP1 on cell proliferation, migration, and invasion in PCa cell lines. (A) LAMP1 mRNA expression was determined at 72 h after transfection with si-LAMP1. GAPDH was used as an internal control. (B) LAMP1 protein expression was evaluated by western blotting at 72 h after transfection with si-LAMP1. GAPDH was used as a loading control. (C) Cell proliferation was determined by XTT assays. (D) Cell migration activity was determined by Boyden chamber migration assays. (E) Cell invasion activity was determined by Matrigel invasion assays. *P<0.0001. Experiments were performed in triplicate. Bars indicate SDs.

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