MicroRNA-205-directed transcriptional activation of tumor suppressor genes in prostate cancer

Abstract

Background: MicroRNAs (miRNAs) are small noncoding RNAs that regulate the expression of approximately 60% of all human genes. They play important roles in numerous cellular processes, including development, proliferation, and apoptosis. Currently, it is believed that miRNAs elicit their effect by silencing the expression of target genes. In this study, the authors demonstrated that miRNA-205 (miR-205) induced the expression the interleukin (IL) tumor suppressor genes IL24 and IL32 by targeting specific sites in their promoters.

Methods: The methods used in this study included transfection of small RNAs; quantitative real-time polymerase chain reaction; in situ hybridization; fluorescence-labeled in situ hybridization; cell cycle, apoptosis, cell viability, migratory, clonability, and invasion assays; immunoblotting; and luciferase reporter, nuclear run-on, and chromatin immunoprecipitation assays.

Results: The results revealed that miR-205 was silenced in prostate cancer. Its re-expression induced apoptosis and cell cycle arrest. It also impaired cell growth, migration, clonability, and invasiveness of prostate cancer cells. Micro-RNA-205 induced the expression of tumor suppressor genes IL24 and IL32 at both the messenger RNA and protein levels. The induction of in vitro transcription and enrichment of markers for transcriptionally active promoters in the IL24 and IL32 genes was observed in response to miR-205.

Conclusions: In this study, a new function for miR-205 was identified that specifically activated tumor suppressor genes by targeting specific sites in their promoters. These results corroborate a newly identified function that miRNAs have in regulating gene expression at the transcriptional level. The specific activation of tumor suppressor genes (eg, IL24, IL32) or other dysregulated genes by miRNA may contribute to a novel therapeutic approach for the treatment of prostate cancer.

Figure 1

miR-205 is silenced in human prostate cancer cell lines. (A) Relative miR-205 expression levels in cell lines, assessed by qRT-PCR. (B) In-situ hybridization (ISH) analysis of miR-205 expression in prostatic cell lines. The fluorescent signals are mostly cytoplasmic in location (green) and are present only in non-malignant RWPE-1 cells indicating the expression of miR-205 compared to cancer cell lines. The nuclei were stained with DAPI (blue).

Figure 2

miR-205 is silenced in human prostate tumors. (A) Relative miR-205 RNA expression levels in BPH (n=24) and tumor (n=23) tissues, assessed by qRT-PCR indicating significant expression of miR-205 in BPH samples as compared to tumor samples. (—) represents average value. (B) miR-205 in-situ hybridiztion (ISH) on prostate tissue microarrays. Representative pictures of miR-205 expression in normal, primary and metastatic prostate tissues (Middle), U6 staining confirming the preservation of intact small RNAs in the same cases (Left); H & E stained sections allowed the identification of tumors (Right).

Figure 3

Re-expression of miR-205 induces apoptosis, cell cycle arrest, impairs cell viability, migration, clonability and invasive properties in PC3 cells. PC3 cells were transfected with a control miRNA or miR-205. Untransfected and mock transfected controls were included. (A) Representative histograms and graphical summary of cell-cycle profile changes in response to miR-205. Representative quadrants and graphical summary of apoptosis induced by miR-205. (B) Colony formation assay of PC3 cells transfected with miR-205 or control molecules. Graph is representative of 3 independent experiments. Over expression of miR-205 promotes an arrested phenotype in PC3 cells. The percent viability of metastatic PC3 cell lines decreased in response to miR-205 in a time dependent manner. (C) miR-205 significantly decreased invasive behavior of PC3 cells as compared to cont-miR transfected PC3 cells. Graph represents summary of three independent experiments. (D) PC3 cells transfected with miR-205 exhibited less migratory behavior than cont-miR transfected cells.

Figure 4

miR-205 induces expression of tumor suppressor genes IL24 and IL32. (A) Sequence of the miR-205 target sites located at -107 (IL24) and -610 (IL32) relative to the transcription start sites. Bases in bold face correspond to those bases in miR-205 that are complementary to the target sites, including G:U/T wobble base-pairing. (B) Relative IL24 and IL32 mRNA expression levels in PC3 cells transfected with 50nM concentrations of the miR-205 or cont-miR for 72 h, assessed by qRT-PCR. (C) Induction of IL24 and IL32 proteins in response to miR-205 assessed by Western blot.

Figure 5

Sequence specificity for miR-205. (A) Mutations to 8 or 11 base pairs in miR-205 resulted in miR-205-5MM and miR-205-3MM, respectively. The mutated bases are shown in bold letters. (B) Relative IL24 and IL32 mRNA expression levels in PC3 cells transfected with 50nM concentrations of each indicated miRNA duplex for 72 h, assessed by qRT-PCR. (C) IL24 is a direct target of miR-205. IL24 promoter vector containing the binding site for miR-205 was purchased from SwitchGear Genomics and cotransfected with miR-205 or cont-miR and luciferase activity was measured after 48 hours. Luciferase activity was normalized to total protein and relative expression was determined for cont-miR and miR-205. RLU (Relative Luciferase units). (D) Activation of in-vitro transcription by miR-205. Nuclear run-on experiments for nascent IL24 and IL32 mRNA transcription in PC3 cells transfected with miR- 205 or cont-miR duplexes, as measured by qRT-PCR and normalized to nascent GAPDH mRNA transcription levels.

Figure 6

Enrichment of markers for transcriptionally active promoters by miR-205. (A–B) ChIP assay in PC3 cells transfected with miR-205 or cont-miR. Quantification of immunoprecipitated IL24 and IL32 promoter regions were determined by quantitiative real-time PCR and normalized to input DNA. Signals were also confirmed by conventional PCR and PCR product for each sample was resolved on 2% agarose gel and visualized by staining with ethidium bromide. (C) Induction of downstream target genes by miR-205. Since miR-205 induced expression of IL24 and IL32 expression and caused cell cycle arrest and apoptosis in PC3 cells, we examined the expression of several downstream proteins involved in these pathways. Pro-apoptotic and cell cycle checkpoint proteins were induced in response to miR-205 compared to cont-miR.