Background: MicroRNA (miRNA) have been shown to regulate gene expression in many cancers. MiR-182 has recently been found to be prognostic for patients treated with radical prostatectomy for prostate cancer. We sought to assess miR-182 as a prognostic marker and understand its role in prostate cancer progression and metastasis.Methods: We analysed miR-182 expression among 147 men treated for prostate cancer using biochemical recurrence and metastasis as the endpoints. We examined miR-182 expression in prostate cancer cells and created cell lines that overexpressed miR-182 for functional assays. Finally, we examined pathways through which miR-182 may function using prediction algorithms and confirmed by Western blotting and knock-down assays.Results: We found that miR-182 was not associated with biochemical recurrence (p=0.1111) or metastasis (p=0.9268) following radical prostatectomy. However, in mechanistic assays, we found that miR-182 expression was higher among aggressive prostate cancer cells and that ectopic miR-182 expression resulted in increased proliferation, migration and invasion in vitro. We identified FOXO1 as regulated by miR-182 in prostate cancer cells, confirmed that ectopic miR-182 expression resulted in diminished FOXO1 levels, and showed that miR-182 inhibition results in increased FOXO1 levels. Expression of FOXO1 (p=0.0014) in tumors from patients who developed biochemical recurrence compared to tumors from patients who were recurrence-free five years after their radical prostatectomy.Conclusions: Our findings suggest that miR-182 may act to increase prostate cancer proliferation, migration and invasion through suppression of FOXO1. This may be valuable in the development of further therapeutic interventions.

MicroRNAs (miRNAs) are effective regulators of gene expression that have a significant role in the pathogenesis of prostate and various other cancers. The high prevalence of aberrant miRNA expression in prostate cancer, and miRNAs' distinctive properties, give much hope that they can be used as biomarkers and next generation of molecular anticancer therapeutics. Herein, we review the literature on miRNA involvement in prostate cancer pathogenesis and the current understanding of their role as oncogenes, tumor suppressors and metastasis-regulators. We also review the latest research on miRNAs in prostate cancer preclinical studies and clinical trials, and highlight the advantages and challenges of possible miRNA-based therapies. The emerging information regarding the biology of miRNAs in prostate cancer is promising, and may lead to a role(s) for these molecules as diagnostic/prognostic markers and effective therapeutic tools for better molecularly targeted treatment of prostate cancer.

MicroRNAs (miRNAs) are single-stranded, 18-24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development 1 , apoptosis 2 , and cell cycle regulation 3 . MiRNAs are estimated to regulate the expression of 30% to 90% of human genes 4 by binding to their target messenger RNAs (mRNAs) 5 . Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes 6 . Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.Methods used to investigate miRNA expression include SYBR green I dye-based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies 7 . The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis 8 .Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences.The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this...

One of the most important challenges in prostate cancer research is to identify biomarkers that are predictive of cancer aggressiveness and future tumor recurrence following radical prostatectomy. Recent studies have shown that altered expression of microRNAs (miRs) is involved in the development of prostate cancer, and among the differentially expressed miRNAs, miR-221 may be critical in metastatic induction/regulation. While the precise role of this miRNA in facilitating malignant progression remains unclear, expression of miR-221 in prostate cancer may correlate with disease state/status and serve as a surrogate biomarker for tumor recurrence and/or aggressiveness. In the present study, we sought to investigate whether miR-221 is differentially regulated in patients with aggressive and non-aggressive tumors relative to control normal prostate cell line RWPE-1 and, more specifically, whether this miRNA can be used as biomarker for disease recurrence. To address this question, we initially chose to assess the relative expression of miR-221 in a series of aggressive (n=69) and non-aggressive (n=45) primary carcinoma tissues derived from clinically resected prostates by quantitative real-time PCR. Aggressive tumors were categorized based on clinicopathological parameters such as Gleason score of 8 or higher, high PSA levels, presence of metastasis, invasion to the seminal vesicles, and recurrence after radical prostatectomy. Relative expression of mature miR-221 in tumors was quantified in comparison to control prostate epithelial cell line, RWPE-1, which was set to be 1x. Levels of miR-221 expression were found to be variable. Among all aggressive and non-aggressive cancer patients, expression of miR-221 transcript levels was differentially expressed relative to control sample. However, when we divided this group of patients on the basis of miR-221 expression levels, 72% of the patients with aggressive tumors had miR-221 less than 5-fold the RWPE-1 levels and only 28% of the patients had miR-221 expression equal to or more than 5-fold RWPE-1 levels. On the other hand, 53% of the patients with non-aggressive tumors had miR-221 less than 5-fold the RWPE-1 levels compared to 47% of the patients with miR-221 equal to or more than 5-fold RWPE-1 levels. Our results show that miR-221 is lower in majority of aggressive prostate cancer and differential expression may have utility as a biomarker for disease recurrence. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4035.

MicroRNAs (miRNAs) are single-stranded, 18-24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development 1 , apoptosis 2 , and cell cycle regulation 3 . MiRNAs are estimated to regulate the expression of 30% to 90% of human genes 4 by binding to their target messenger RNAs (mRNAs) 5 . Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes 6 . Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.Methods used to investigate miRNA expression include SYBR green I dye-based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies 7 . The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis 8 .Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences.The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol...