The Suppression of the Epithelial to Mesenchymal Transition in Prostate Cancer through the Targeting of MYO6 Using MiR-145-5p.

Aberrant expression of miR-145-5p has been observed in prostate cancer where is has been suggested to play a tumor suppressor role. In other cancers, miR-145-5p acts as an inhibitor of epithelial-to-mesenchymal transition (EMT), a key molecular process for tumor progression. However, the interaction between miR-145-5p and EMT remains to be elucidated in prostate cancer. In this paper the link between miR-145-5p and EMT in prostate cancer was investigated using a combination of in silico and in vitro analyses. miR-145-5p expression was significantly lower in prostate cancer cell lines compared to normal prostate cells. Bioinformatic analysis of The Cancer Genome Atlas prostate adenocarcinoma (TCGA PRAD) data showed significant downregulation of miR-145-5p in prostate cancer, correlating with disease progression. Functional enrichment analysis significantly associated miR-145-5p and its target genes with EMT. MYO6, an EMT-associated gene, was identified and validated as a novel target of miR-145-5p in prostate cancer cells. In vitro manipulation of miR-145-5p levels significantly altered cell proliferation, clonogenicity, migration and expression of EMT-associated markers. Additional TCGA PRAD analysis suggested miR-145-5p tumor expression may be useful predictor of disease recurrence. In summary, this is the first study to report that miR-145-5p may inhibit EMT by targeting MYO6 in prostate cancer cells. The findings suggest miR-145-5p could be a useful diagnostic and prognostic biomarker for prostate cancer.

Targeting of AKT1 by miR-143-3p Suppresses Epithelial-to-Mesenchymal Transition in Prostate Cancer.

An altered expression of miR-143-3p has been previously reported in prostate cancer where it is purported to play a tumor suppressor role. Evidence from other cancers suggests miR-143-3p acts as an inhibitor of epithelial-to-mesenchymal transition (EMT), a key biological process required for metastasis. However, in prostate cancer the interaction between miR-143-3p and EMT-associated mechanisms remains unclear. Therefore, this paper investigated the link between miR-143-3p and EMT in prostate cancer using in vitro and in silico analyses. PCR detected that miR-143-3p expression was significantly decreased in prostate cancer cell lines compared to normal prostate cells. Bioinformatic analysis of The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA PRAD) data showed a significant downregulation of miR-143-3p in prostate cancer, correlating with pathological markers of advanced disease. Functional enrichment analysis confirmed the significant association of miR-143-3p and its target genes with EMT. The EMT-linked gene AKT1 was subsequently shown to be a novel target of miR-143-3p in prostate cancer cells. The in vitro manipulation of miR-143-3p levels significantly altered the cell proliferation, clonogenicity, migration and expression of EMT-associated markers. Further TCGA PRAD analysis suggested miR-143-3p tumor expression may be a useful predictor of disease recurrence. In summary, this is the first study to report that miR-143-3p overexpression in prostate cancer may inhibit EMT by targeting AKT1. The findings suggest miR-143-3p could be a useful diagnostic and prognostic biomarker for prostate cancer.

Non-Laboratory Project-Based Learning for Final Year Bioscience Students: Lessons From COVID-19.

Background: Provision of "dry-lab" final year honours projects, based outside the laboratory, have been proposed as a viable alternative to traditional "wet-lab" projects in bioscience subjects, but their value has not been widely evaluated to date. In 2020-21, the COVID-19 pandemic meant all students in the School of Biomedical Sciences at Ulster University (UU) undertook dry-lab projects, due to campus lockdown. Therefore, this provided an ideal opportunity to evaluate the provision of dry-lab projects in a large student cohort. Methods: A pilot group of final year students (n = 4) studying Biomedical Science at UU were interviewed to evaluate their experience of conducting a dry-lab project. This evaluation and the themes that emerged were subsequently used to inform the co-creation of a survey to appraise student experience of dry-lab research project learning across the final year student cohort in School of Biomedical Sciences (n = 140). Quantitative and qualitative data was collected and analysed for trends and themes. Results: The results of this project identified four main themes related to dry-lab projects; expectations, skills & employability, quality of experience and choice. Student expectations about dry-lab projects were not dramatically changed, although initial negative opinions of some individuals were over-turned. Most students recognised that they had developed many useful employability skills through dry-lab projects, although lack of practical laboratory experience was still perceived as a drawback. Student experience was influenced by personal circumstances but students reporting poor project experience had significantly lower levels of communication with supervisor (p < 0.05). Most students agreed that choice of dry- and wet-lab projects would be valuable for future cohorts. Conclusion: This report concludes that dry-lab project provision can be a suitable and equitable alternative for wet-lab projects. Dry-lab projects can be valuable for learning new skills and may be an attractive option for some students and supervisors who prefer to work outside the laboratory setting. A choice of both dry-lab and wet-lab projects is highly recommended as it provides more choice for students to tailor their final year experience to their individual circumstances, strengths and future career aspirations.

MiR-21 Is Induced by Hypoxia and Down-Regulates RHOB in Prostate Cancer.

Tumour hypoxia is a well-established contributor to prostate cancer progression and is also known to alter the expression of several microRNAs. The over-expression of microRNA-21 (miR-21) has been consistently linked with many cancers, but its role in the hypoxic prostate tumour environment has not been well studied. In this paper, the link between hypoxia and miR-21 in prostate cancer is investigated. A bioinformatic analysis of The Cancer Genome Atlas (TCGA) prostate biopsy datasets shows the up-regulation of miR-21 is significantly associated with prostate cancer and clinical markers of disease progression. This up-regulation of miR-21 expression was shown to be caused by hypoxia in the LNCaP prostate cancer cell line in vitro and in an in vivo prostate tumour xenograft model. A functional enrichment analysis also revealed a significant association of miR-21 and its target genes with processes related to cellular hypoxia. The over-expression of miR-21 increased the migration and colony-forming ability of RWPE-1 normal prostate cells. In vitro and in silico analyses demonstrated that miR-21 down-regulates the tumour suppressor gene Ras Homolog Family Member B (RHOB) in prostate cancer. Further a TCGA analysis illustrated that miR-21 can distinguish between different patient outcomes following therapy. This study presents evidence that hypoxia is a key contributor to the over-expression of miR-21 in prostate tumours, which can subsequently promote prostate cancer progression by suppressing RHOB expression. We propose that miR-21 has good potential as a clinically useful diagnostic and prognostic biomarker of hypoxia and prostate cancer.

MiR-182 Is Upregulated in Prostate Cancer and Contributes to Tumor Progression by Targeting MITF.

Altered expression of microRNA-182-5p (miR-182) has been consistently linked with many cancers, but its specific role in prostate cancer remains unclear. In particular, its contribution to epithelial-to-mesenchymal transition (EMT) in this setting has not been well studied. Therefore, this paper profiles the expression of miR-182 in prostate cancer and investigates how it may contribute to progression of this disease. In vitro experiments on prostate cancer cell lines and in silico analyses of The Cancer Genome Atlas (TCGA) prostate adenocarcinoma (PRAD) datasets were performed. PCR revealed miR-182 expression was significantly increased in prostate cancer cell lines compared to normal prostate cells. Bioinformatic analysis of TCGA PRAD data similarly showed upregulation of miR-182 was significantly associated with prostate cancer and clinical markers of disease progression. Functional enrichment analysis confirmed a significant association of miR-182 and its target genes with EMT. The EMT-linked gene MITF (melanocyte inducing transcription factor) was subsequently shown to be a novel target of miR-182 in prostate cancer cells. Further TCGA analysis suggested miR-182 expression can be an indicator of patient outcomes and disease progression following therapy. In summary, this is the first study to report that miR-182 over-expression in prostate cancer may contribute to EMT by targeting MITF expression. We propose miR-182 as a potentially useful diagnostic and prognostic biomarker for prostate cancer and other malignancies.

A Novel Combination of Serum Markers in a Multivariate Model to Help Triage Patients Into "Low-" and "High-Risk" Categories for Prostate Cancer.

Background: Almost 50,000 men in the United Kingdom (UK) are diagnosed each year with prostate cancer (PCa). Secondary referrals for investigations rely on serum prostate-specific antigen (PSA) levels and digital rectal examination. However, both tests lack sensitivity and specificity, resulting in unnecessary referrals to secondary care for costly and invasive biopsies. Materials and Methods: Serum samples and clinical information were collected from N = 125 age-matched patients (n = 61 non-PCa and n = 64 PCa) and analyzed using Biochip Array Technology on high-sensitivity cytokine array I (IL-2, IL-4, IL-6, IL-8, IL-10, IL-1α, IL-1ß, TNFα, MCP-1, INFγ, EGF, and VEGF), cerebral array II (CRP, D-dimer, neuron-specific enolase, and sTNFR1), and tumor PSA oncology array (fPSA, tPSA, and CEA). Results: The data showed that 11/19 (68.8%) markers were significantly different between the non-PCa and the PCa patients. A combination of EGF, log10 IL-8, log10 MCP-1, and log10 tPSA significantly improved the predictive potential of tPSA alone to identify patients with PCa (DeLong, p < 0.001). This marker combination had an increased area under the receiver operator characteristic (0.860 vs. 0.700), sensitivity (78.7 vs. 68.9%), specificity (76.5 vs. 67.2%), PPV (76.2 vs. 66.7%), and NPV (79.0 vs. 69.4%) compared with tPSA. Conclusions: The novel combination of serum markers identified in this study could be employed to help triage patients into "low-" and "high-risk" categories, allowing general practitioners to improve the management of patients in primary care settings and potentially reducing the number of referrals for unnecessary, invasive, and costly treatments.

Data pertaining to aberrant intracellular calcium handling during androgen deprivation therapy in prostate cancer.

The data generated here in relates to the research article "CaV1.3 enhanced store operated calcium promotes resistance to androgen deprivation in prostate cancer". A model of prostate cancer (PCa) progression to castration resistance was employed, with untreated androgen sensitive LNCaP cell line alongside two androgen deprived (bicalutamide) sublines, either 10 days (LNCaP-ADT) or 2 years (LNCaP-ABL) treatment, in addition to androgen insensitive PC3. With this PCa model, qPCR was used to examined fold change in markers linked to androgen resistance, androgen receptor (AR) and neuron specific enolase (NSE), observing an increase under androgen deprivation. In addition, the gene expression of a range of calcium channels was measured, with only the L-type Voltage gated calcium channel, CACNA1D, demonstrating an increase during androgen deprivation. With CACNA1D knockdown the channel was found not to influence the gene expression of calcium channels, ORAI1 and STIM1. The calcium channel blocker (CCB), nifedipine, was employed to determine the impact of CaV1.3 on the observed store release and calcium entry measured via Fura-2AM ratiometric dye in our outlined PCa model. In both the presence and absence of androgen deprivation, nifedipine was found to have no impact on store release induced by thapsigargin (Tg) in 0mM Ca2+ nor store operated calcium entry (SOCE) following the addition of 2mM Ca2+. However, CACNA1D siRNA knockdown was able to reduce SOCE in PC3 cells. The effect of nifedipine on CaV1.3 in PCa biology was measured through cell proliferation assay, with no observed change in the presence of CCB. While siCACNA1D reduced PC3 cell proliferation. This data can be reused to inform new studies investigating altered calcium handling in androgen resistant prostate cancer. It provides insight into the mechanism of CaV1.3 and its functional properties in altered calcium in cancer, which can be of use to researchers investigating this channel in disease. Furthermore, it could be helpful in interpreting studies investigating CCB's as a therapeutic and in the development of future drugs targeting CaV1.3.

CaV1.3 enhanced store operated calcium promotes resistance to androgen deprivation in prostate cancer.

Androgen deprivation therapy (ADT) is the main treatment for advanced prostate cancer (PCa) but resistance results in progression to terminal castrate resistant PCa (CRPC), where there is an unmet therapeutic need. Aberrant intracellular calcium (Cai2+) is known to promote neoplastic transformation and treatment resistance. There is growing evidence that voltage gated calcium channel (VGCC) expression is increased in cancer, particularly CACNA1D/CaV1.3 in CRPC. The aim of this study was to investigate if increased CaV1.3 drives resistance to ADT and determine its associated impact on Cai2+ and cancer biology. Bioinformatic analysis revealed that CACNA1D gene expression is increased in ADT treated PCa patients. This was corroborated in both in vivo LNCaP xenograft mouse and in vitro PCa cell line models, which demonstrated a significant increase in CaV1.3 protein expression following ADT with bicalutamide. Expression was found to be of a shortened 170kDa CaV1.3 isoform associated with plasma and intracellular membranes, which failed to induce calcium influx following membrane depolarisation. Instead, under ADT CaV1.3 mediated a rise in basal cytosolic calcium and an increase in store operated calcium entry (SOCE). This mechanism was found to promote the proliferation and survival of ADT resistant CRPC cells. Overall, this study demonstrates for the first time in PCa that under ADT specific CaV1.3 isoforms promote an upregulation of SOCE which contributes to treatment resistance and CRPC biology. Thus, this novel oncochannel represents a target for therapeutic development to improve PCa patient outcomes.

Prognostic value of miR-21 for prostate cancer: a systematic review and meta-analysis.

Elevated levels of miR-21 expression are associated with many cancers, suggesting it may be a promising clinical biomarker. In prostate cancer (PCa), however, there is still no consensus about the usefulness of miR-21 as an indicator of disease progression. This systematic review and meta-analysis was conducted to investigate the value of miR-21 expression as a prognostic measurement in PCa patients. Medline (Ovid), EMBASE, Web of Science, Scopus and Cochrane Library databases were systematically searched for relevant publications between 2010 to 2021. Studies exploring the relationship between miR-21 expression, PCa prognosis and clinicopathological factors were selected for review. Those reporting hazard ratio (HR) and 95% confidence intervals (CIs) were subject to meta-analyses. Fixed-effect models were employed to calculated pooled HRs and 95% CIs. Risk of bias in each study was assessed using QUIPS tool. Certainty of evidence in each meta-analysis was assessed using GRADE guidelines. A total of 64 studies were included in the systematic review. Of these, 11 were eligible for inclusion in meta-analysis. Meta-analyses revealed that high miR-21 expression was associated with poor prognosis: HR = 1.58 (95% CI = 1.19-2.09) for biochemical recurrence, MODERATE certainty; HR = 1.46 (95% CI = 1.06-2.01) for death, VERY LOW certainty; and HR = 1.26 (95% CI = 0.70-2.27) for disease progression, VERY LOW certainty. Qualitative summary revealed elevated miR-21 expression was significantly positively associated with PCa stage, Gleason score and risk groups. This systematic review and meta-analysis suggests that elevated levels of miR-21 are associated with poor prognosis in PCa patients. miR-21 expression may therefore be a useful prognostic biomarker in this disease.

Biomarkers That Differentiate Benign Prostatic Hyperplasia from Prostate Cancer: A Literature Review.

Prediction of prostate cancer in primary care is typically based upon serum total prostate-specific antigen (tPSA) and digital rectal examination results. However, these tests lack sensitivity and specificity, leading to over-diagnosis of disease and unnecessary, invasive biopsies. Therefore, there is a clinical need for diagnostic tests that can differentiate between benign conditions and early-stage malignant disease in the prostate. In this review, we evaluate research papers published from 2009 to 2019 reporting biomarkers that identified or differentiated benign prostatic hyperplasia (BPH) from prostate cancer. Our review identifies hundreds of potential biomarkers in urine, serum, tissue, and semen proposed as useful targets for differentiating between prostate cancer and BPH patients. However, it is still not apparent which of these candidate biomarkers are most useful, and many will not progress beyond the discovery stage unless they are properly validated for clinical practice. We conclude that this validation will come through the use of multivariate panels which can assess the value of biomarker candidates in combination with clinical parameters as part of a risk prediction calculator. Implementation of such a model will help clinicians stratify patients with prostate cancer symptoms in primary care, with tangible benefits for both the patient and the health service.

miR-210 is induced by hypoxia and regulates neural cell adhesion molecule in prostate cells.

Hypoxia in prostate tumours has been associated with disease progression and metastasis. MicroRNAs are short noncoding RNA molecules that are important in several cell processes, but their role in hypoxic signalling is still poorly understood. miR-210 has been linked with hypoxic mechanisms, but this relationship has been poorly characterised in prostate cancer. In this report, the link between hypoxia and miR-210 in prostate cancer cells is investigated. Polymerase chain reaction analysis demonstrates that miR-210 is induced by hypoxia in prostate cancer cells using in vitro cell models and an in vivo prostate tumour xenograft model. Analysis of The Cancer Genome Atlas prostate biopsy datasets shows that miR-210 is significantly correlated with Gleason grade and other clinical markers of prostate cancer progression. Neural cell adhesion molecule (NCAM) is identified as a target of miR-210, providing a biological mechanism whereby hypoxia-induced miR-210 expression can contribute to prostate cancer. This study provides evidence that miR-210 is an important regulator of cell response to hypoxic stress and proposes that its regulation of NCAM may play an important role in the pathogenesis of prostate cancer.

Appetite-regulating hormones-leptin, adiponectin and ghrelin-and the development of prostate cancer: a systematic review and exploratory meta-analysis.

BACKGROUND: Obesity has been proposed as a risk factor for prostate cancer (PCa). In obesity, serum levels of the appetite-regulating hormones-leptin, adiponectin, and ghrelin-become deregulated. OBJECTIVE: To explore whether serum levels of appetite-regulating hormones associate with the incidence of PCa, the incidence of advanced disease, or PCa-specific mortality. METHODS: PRISMA guidelines were followed. A systematic search for relevant articles published until March 2019 was performed using the databases PubMed, EMBASE, and Web of Science. Observational studies with data on serum levels of leptin, adiponectin, or ghrelin and PCa outcome were included. Meta-analysis was used to combine risk estimates. Meta-relative risks (mRRs) were calculated using random effects models. When available, raw data was pooled. Publication bias was assessed by funnel plot and Begg's test. RESULTS: Thirty-five studies were eligible for inclusion. The qualitative analysis indicated that leptin was not consistently associated with any PCa outcome, although several cohorts reported decreased adiponectin levels in men who later developed advanced PCa. Based on the meta-analysis, there was no significant effect of leptin on PCa incidence (mRR = 0.93 (95% CI 0.75-1.16), p = 0.52) or advanced PCa (mRR = 0.90 (95% CI 0.74-1.10), p = 0.30). There were insufficient studies to estimate the mRR of PCa incidence for men with the highest levels of adiponectin. The combined risk of advanced PCa for men with the highest levels of adiponectin was reduced but did not reach significance (mRR = 0.81 (95% CI 0.61-1.08), p = 0.15). CONCLUSIONS: The current evidence does not suggest an association between leptin and PCa outcome. However, there may be an inverse association between adiponectin and the incidence of advanced PCa that should be investigated by further studies. Serum ghrelin has not been largely investigated.

Serum levels of miR-199a-5p correlates with blood pressure in premature cardiovascular disease patients homozygous for the MTHFR 677C > T polymorphism.

This investigation profiled circulating serum concentrations of microRNAs (miRNAs) in premature cardiovascular disease (CVD) patients screened for the 677C > T polymorphism in methylenetetrahydrofolate reductase (MTHFR), a risk factor for hypertension. Serum samples from 75 premature CVD patients of known MTHFR genotype were analysed for CVD-related miRNA expression, to identify those that were associated with blood pressure. Samples were collected at baseline and following intervention with riboflavin as part of a randomized controlled trial. In patients with the MTHFR 677TT genotype, expression of miR-199a-5p in serum was inversely correlated with hypertension at baseline, and with change in blood pressure in TT genotype patients who responded to riboflavin intervention. These correlations were not observed in MTHFR 677CC genotype patients. In vitro experiments and in silico data analysis provided evidence that miR-199a-5p targets SMAD4. This is the first study to link miR-199a-5p expression with hypertension in a genetically at-risk cohort of premature CVD patients.

The unidirectional hypoxia-activated prodrug OCT1002 inhibits growth and vascular development in castrate-resistant prostate tumors.

BACKGROUND: OCT1002 is a unidirectional hypoxia-activated prodrug (uHAP) OCT1002 that can target hypoxic tumor cells. Hypoxia is a common feature in prostate tumors and is known to drive disease progression and metastasis. It is, therefore, a rational therapeutic strategy to directly target hypoxic tumor cells in an attempt to improve treatment for this disease. Here we tested OCT1002 alone and in combination with standard-of-care agents in hypoxic models of castrate-resistant prostate cancer (CRPC). METHODS: The effect of OCT1002 on tumor growth and vasculature was measured using murine PC3 xenograft and dorsal skin fold (DSF) window chamber models. The effects of abiraterone, docetaxel, and cabazitaxel, both singly and in combination with OCT1002, were also compared. RESULTS: The hypoxia-targeting ability of OCT1002 effectively controls PC3 tumor growth. The effect was evident for at least 42 days after exposure to a single dose (30 mg/kg) and was comparable to, or better than, drugs currently used in the clinic. In DSF experiments OCT1002 caused vascular collapse in the PC3 tumors and inhibited the revascularization seen in controls. In this model OCT1002 also enhanced the anti-tumor effects of abiraterone, cabazitaxel, and docetaxel; an effect which was accompanied by a more prolonged reduction in tumor vasculature density. CONCLUSIONS: These studies provide the first evidence that OCT1002 can be an effective agent in treating hypoxic, castrate-resistant prostate tumors, either singly or in combination with established chemotherapeutics for prostate cancer.

Targeting Hypoxic Prostate Tumors Using the Novel Hypoxia-Activated Prodrug OCT1002 Inhibits Expression of Genes Associated with Malignant Progression.

Purpose: To understand the role of hypoxia in prostate tumor progression and to evaluate the ability of the novel unidirectional hypoxia-activated prodrug OCT1002 to enhance the antitumor effect of bicalutamide.Experimental Design: The effect of OCT1002 on prostate cancer cells (LNCaP, 22Rv1, and PC3) was measured in normoxia and hypoxia in vitroIn vivo, tumor growth and lung metastases were measured in mice treated with bicalutamide, OCT1002, or a combination. Dorsal skin fold chambers were used to image tumor vasculature in vivo Longitudinal gene expression changes in tumors were analyzed using PCR.Results: Reduction of OCT1002 to its active form (OCT1001) decreased prostate cancer cell viability. In LNCaP-luc spheroids, OCT1002 caused increased apoptosis and decreased clonogenicity. In vivo, treatment with OCT1002 alone, or with bicalutamide, showed significantly greater tumor growth control and reduced lung metastases compared with controls. Reestablishment of the tumor microvasculature following bicalutamide-induced vascular collapse is inhibited by OCT1002. Significantly, the upregulation of RUNX2 and its targets caused by bicalutamide alone was blocked by OCT1002.Conclusions: OCT1002 selectively targets hypoxic tumor cells and enhances the antitumor efficacy of bicalutamide. Furthermore, bicalutamide caused changes in gene expression, which indicated progression to a more malignant genotype; OCT1002 blocked these effects, emphasizing that more attention should be attached to understanding genetic changes that may occur during treatment. Early targeting of hypoxic cells with OCT1002 can provide a means of inhibiting prostate tumor growth and malignant progression. This is of importance for the design and refinement of existing androgen-deprivation regimens in the clinic. Clin Cancer Res; 23(7); 1797-808. ©2016 AACR.

Regulation of miR-200c and miR-141 by Methylation in Prostate Cancer.

BACKGROUND: In prostate cancer (PCa), abnormal expression of several microRNAs (miRNAs) has been previously reported. Increasing evidence shows that aberrant epigenetic regulation of miRNAs is a contributing factor to their altered expression in cancer. In this study, we investigate whether expression of miR-200c and miR-141 in PCa is related to the DNA methylation status of their promoter. METHODS: PCR analysis of miR-200c and miR-141, and CpG methylation analysis of their common promoter, was performed in PCa cell-lines and in archived prostate biopsy specimens. The biological significance of miR-200c and miR-141 expression in prostate cancer cells was assessed by a series of in vitro bioassays and the effect on proposed targets DNMT3A and TET1/TET3 was investigated. The effect on promoter methylation status in cells treated with demethylating agents was also examined. RESULTS: miR-200c and miR-141 are both highly elevated in LNCaP, 22RV1, and DU145 cells, but significantly reduced in PC3 cells. This correlates inversely with the methylation status of the miR-200c/miR-141 promoter, which is unmethylated in LNCaP, 22RV1, and DU145 cells, but hypermethylated in PC3. In PC3 cells, miR-200c and miR-141 expression is subsequently elevated by treatment with the demethylating drug decitabine (5-aza-2'deoxycytidine) and by knockdown of DNA methyltransferase 1 (DNMT1), suggesting their expression is regulated by methylation. Expression of miR-200c and miR-141 in prostate biopsy tissue was inversely correlated with methylation in promoter CpG sites closest to the miR-200c/miR-141 loci. In vitro, over-expression of miR-200c in PC3 cells inhibited growth and clonogenic potential, as well as inducing apoptosis. Expression of the genes DNMT3A and TET1/TET3 were down-regulated by miR-200c and miR-141 respectively. Finally, treatment with the soy isoflavone genistein caused demethylation of the promoter CpG sites closest to the miR-200c/miR-141 loci resulting in increased miR-200c expression. CONCLUSIONS: Our findings provide evidence that miR-200c and miR-141 are under epigenetic regulation in PCa cells. We propose that profiling their expression and methylation status may have potential as a novel biomarker or focus of therapeutic intervention in the diagnosis and prognosis of PCa. Prostate 76:1146-1159, 2016. © 2016 Wiley Periodicals, Inc.

miR-24 regulates CDKN1B/p27 expression in prostate cancer.

BACKGROUND: MicroRNAs (miRNAs) are small, non-coding RNA molecules with an important role in cancer. In prostate cancer, several miRNAs are expressed abnormally suggesting they may be useful markers for diagnosis, prognosis, and potential therapeutic intervention in this disease. However, the contribution of individual miRNAs to the development and progression of this disease remains poorly understood. This study investigated the role of miR-24, which has not been extensively studied in relation to prostate cancer. METHODS: We used PCR to investigate the expression of miR-24 in a panel of prostate cancer cell-lines and in a series of clinical prostate biopsy specimens. The biological significance of miR-24 expression in prostate cancer cells was assessed by a series of in vitro bioassays and the effect on proposed targets p27 (CDKN1B) and p16 (CDK2NA) was investigated. RESULTS: We showed that miR-24 expression was significantly lower in prostate cancer cell lines compared to a normal prostate epithelial cell line. Decreased expression of miR-24 was also more frequently observed in both needle core and prostatectomy tumor tissue relative to matched normal tissue. Low miR-24 expression correlated with high PSA serum levels and other markers of increased prostate cancer progression. Importantly, over-expression of miR-24 inhibited cell cycle, proliferation, migration, and clonogenic potential of prostate cancer cells, as well as inducing apoptosis. p27 and p16 were confirmed as targets of miR-24 in prostate cancer cells and a significant inverse correlation between miR-24 and p27 was revealed in clinical prostatectomy specimens. CONCLUSIONS: These findings provide evidence that miR-24 has a tumor suppressor role in prostate cancer and also targets p27 and p16 in prostate cancer cells. We propose that it may be a useful progression biomarker or focus of therapeutic intervention for this disease.

Nitric Oxide Up-Regulates RUNX2 in LNCaP Prostate Tumours: Implications for Tumour Growth In Vitro and In Vivo.

Aberrant expression of the transcription factor RUNX2 in prostate cancer has a number of important consequences including increased resistance to apoptosis, invasion and metastasis to bone. We previously demonstrated that hypoxia up-regulated RUNX2 in tumour cells, which in turn up-regulated the anti-apoptotic factor Bcl-2. Here, we investigate the impact of nitric oxide (NO) on RUNX2 and Bcl-2 expression in prostate cancer and further, how RUNX2 over-expression can impact tumour growth, angiogenesis and oxygenation in vivo. The effect of NO levels on RUNX2 and thus Bcl-2 expression was examined in prostate cancer cells in vitro using methods including gene and protein expression analyses, nitrite quantitation, protein-DNA interaction assays (ChIP) and viability assays (XTT). The effect of RUNX2 over-expression on tumour physiology (growth, oxygenation and angiogenesis) was also assessed in vivo using LNCaP xenografts. A low (but not high) concentration of NO (10 µM) induced expression of RUNX2 and Bcl-2, conferring resistance to docetaxel. These effects were induced via the ERK and PI3K pathways and were dependent on intact AP-1 binding sites in the RUNX2 promoter. RUNX2 over-expression in LNCaP tumours in vivo decreased the time to tumour presentation and increased tumour growth. Moreover, these tumours exhibited improved tumour angiogenesis and oxygenation. Low levels of NO increase expression of RUNX2 and Bcl-2 in LNCaP prostate tumour cells, and in vivo up-regulation of RUNX2 created tumours with a more malignant phenotype. Collectively, our data reveals the importance of NO-regulation of key factors in prostate cancer disease progression.

miR-24 and miR-205 expression is dependent on HPV onco-protein expression in keratinocytes.

A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miR-24 and miR-205. We investigated how expression of Human Papilloma Virus Type-16 (HPV16) onco-proteins E6 and E7 affected expression of miR-24 and miR-205 during proliferation and differentiation of HFKs. We show that the induction of both miR-24 and miR-205 observed during differentiation of HFKs is lost in HFKs expressing E6 and E7. We demonstrate that the effect on miR-205 is due to E7 activity, as miR-205 expression is dependent on pRb expression. Finally, we provide evidence that miR-24 effects in the cell may be due to targeting of cyclin dependent kinase inhibitor p27. In summary, these results indicate that expression of both miR-24 and miR-205 are impacted by E6 and/or E7 expression, which may be one mechanism by which HPV onco-proteins can disrupt the balance between proliferation and differentiation in keratinocytes.

Use of the comet-FISH assay to compare DNA damage and repair in p53 and hTERT genes following ionizing radiation.

The alkaline single cell gel electrophoresis (comet) assay can be combined with fluorescent in situ hybridisation (FISH) methodology in order to investigate the localisation of specific gene domains within an individual cell. The number and position of the fluorescent signal(s) provides information about the relative damage and subsequent repair that is occurring in the targeted gene domain(s). In this study, we have optimised the comet-FISH assay to detect and compare DNA damage and repair in the p53 and hTERT gene regions of bladder cancer cell-lines RT4 and RT112, normal fibroblasts and Cockayne Syndrome (CS) fibroblasts following γ-radiation. Cells were exposed to 5Gy γ-radiation and repair followed for up to 60 minutes. At each repair time-point, the number and location of p53 and hTERT hybridisation spots was recorded in addition to standard comet measurements. In bladder cancer cell-lines and normal fibroblasts, the p53 gene region was found to be rapidly repaired relative to the hTERT gene region and the overall genome, a phenomenon that appeared to be independent of hTERT transcriptional activity. However, in the CS fibroblasts, which are defective in transcription coupled repair (TCR), this rapid repair of the p53 gene region was not observed when compared to both the hTERT gene region and the overall genome, proving the assay can detect variations in DNA repair in the same gene. In conclusion, we propose that the comet-FISH assay is a sensitive and rapid method for detecting differences in DNA damage and repair between different gene regions in individual cells in response to radiation. We suggest this increases its potential for measuring radiosensitivity in cells and may therefore have value in a clinical setting.