MicroRNA-106a suppresses prostate cancer proliferation, migration and invasion by targeting tumor-derived IL-8

Abstract

Background: Tumor-derived interleukin-8 (IL-8) promotes tumorigenesis and progression of prostate cancer (PCa). MicroRNAs (miRNAs) are noncoding regulatory RNAs and their dysregulation is known to be implicated in carcinogenesis. However, the post-transcriptional mechanism of IL-8 via miRNAs is not fully understood. This study was intended to investigate whether miR-106a could affect the progression of PCa via targeting IL-8 or not.

Methods: Using bioinformatics analysis, we postulated that IL-8 might be post-transcriptionally regulated by miR-106a. This was validated by dual reporter gene assays that miR-106a could bind to the predicted site of IL-8 mRNA. To determine the biological effects of miR-106a on PCa cells (PC-3 and DU145), MTT, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), migration and invasion assays were performed.

Results: We found that miR-106a was barely expressed in PCa cells, whereas IL-8 was aberrantly upregulated. Elevated miR-106a could reduce IL-8 expression by directly binding the 3'-UTR of IL-8. Overexpression of miR-106a in PCa cells triggered cell apoptosis and suppressed cell proliferation, migration, and invasion.

Conclusions: This research showed that miR-106a could function as a tumor-suppressor by decreasing IL-8 levels in PCa.

Keywords: Prostate cancer (PCa); interleukin-8 (IL-8); miR-106a; progression.

Figure 1

The expression levels of IL-8 and miR-106a in PC-3, DU145, LNCaP, and RWPE-1. RT-PCR (A) and Q-PCR (C) showed the differential expression of IL-8 mRNA in four cell lines. Western blot analysis (B,D) showed the differential expression of IL-8 protein expression levels in four cell lines. RT-PCR (E) and Q-PCR (F) demonstrated the differential expression of miR-106a in four cell lines. (C,D) Compared to RWPE-1; (F) compared to PC-3. Double asterisks indicate P<0.001.

Figure 2

The effects of artificial miR-106a over-expression on IL-8 in PC-3 and DU145 cells. Homogenous green fluorescence protein expression (A,B), RT-PCR (C) and Q-PCR (E) demonstrated the differential expression of miR-106a expression following infected with Ad-miR-106a or Ad-control in PC-3 and DU145 cells. Western blot analysis (D) and immunocytochemistry (F) showed the changes in IL-8 protein levels following transfected with Ad-miR-106a or Ad-control in PC-3 and DU145 cells. The bar within the picture, 50 µm. Double asterisks indicate P<0.001.

Figure 3

Identifying the seed sequences of miR-106a in the IL-8 3'-UTR by dual reporter gene assays. The potential seed sequences of miR-106a were predicted in the 593-600, not on the IL-8 3'-UTR (A). After artificial over-expression of miR-106a (pre-transfected with luciferase reporter vectors) in PC-3 cells, the reporter gene activity (represented by relative luciferase activity) was significantly decreased when PGL3-IL-8 was present in the construct, whereas it was obviously restored when PGL3-Mut or PGL3-Del present. Expression of Ad-miR-106a alone (without seed sequences present) or PGL3-IL-8 (without infection of Ad-miR-106a) had no effect on reporter gene activity (B). A single asterisk indicates P<0.05.

Figure 4

The effects of miR-106a over-expression on cell proliferation and apoptosis. MTT assay showed that proliferation was significantly inhibited in PC-3 (compared to Ad-con) and DU145 (compared to Ad-con) cells after miR-106a overexpression (A), blank: normal PC-3 or DU-145 cells. TUNEL assay demonstrated that increased apoptosis following up-regulated miR-106a in PC-3 and DU145 cells (B). Cells were stained by Streptavidin-HRP and Diaminobenzidine (DAB). The bar within the picture, 50 µm. Double asterisks indicate P<0.001.

Figure 5

The effects of miR-106a overexpression on migration and invasion in the transwell culture system. The migration function of PC-3 (A) and DU145 (B) (compared to Ad-con) cells was significantly inhibited after miR-106a overexpression. The invasion capability of PC-3 (C) and DU145 (D) (compared to Ad-con) cells was significantly restrained following miR-106a overexpression. Cells were stained by methylrosanilnium chloride solution. The bar within the picture. A single asterisk indicates P<0.05. Double asterisks indicate P<0.001.