1,721,028 research outputs found
Do miRNAs play a role in androgen-induced phenotypic expression in prostate epithelial cells?
Get PDFActivation of the androgen receptor (AR) results in a phenotypic change in androgen-responsive cells; for example, in the human prostate carcinoma cell line LNCaP, androgens mediate proliferation. MicroRNAs (miRNA) are a class of short non-coding RNA that act as post-transcriptional regulators of gene expression; their role in the androgenic phenotype is yet to be fully explored.
In the present study, I examined the hypothesis that androgen-mediated effects on gene expression and cellular proliferation are in part mediated through miRNAs. Therefore, exposure to a synthetic AR agonist (mibolerone), a physiological AR agonist (dihydrotestosterone) and a therapeutic AR antagonist (bicalutamide) was studied in LNCaP at four, eight, 24 and 120 hours. The cellular response to these compounds was assessed in terms of proliferation, prostate-specific antigen (PSA) expression and alteration of the miRNA profile.
Microarray analysis revealed that a number of miRNAs were altered in a compound-specific and temporal manner. Of note, androgen-induced proliferation and PSA secretion were associated with repression of miR-221 and induction of miR-210.
In silico methods identified putative targets of miR-221, such as the transcription factor IRX5. Immunoblotting and a luciferase reporter construct provided preliminary evidence that IRX5 is indeed a novel target of miR-221. Down-regulation of IRX5 is anti-proliferative and pro-apoptotic therefore androgen-mediated repression of miR-221 may contribute to growth.
miR-210, a hypoxia-inducible miRNA, was induced under androgenic stimuli with concomitant repression of the Myc-inhibitor Mnt. Efforts were made to elucidate the mechanism of miR-210 induction outside the context of classical HIF1 activation, but further research is required.
miR-720 had the highest expression of any individual miRNA in LNCaP cells. DNMT3A was identified as a potential target implicating miR-720 in androgen insensitivity. However, analysis of androgen-insensitive cell lines did not reveal differential expression and its role in prostate epithelial biology remains to be discovered.
In summary, the research confirms the hypothesis that androgens can mediate phenotypic change through mechanisms that include miRNA-mediated responses. The data offers insights into androgen-induced processes and identifies new avenues for future research. [For supplementary files please contact author]
Evaluating the genotoxic potential of oligonucleotide pharmaceuticals
Get PDFAccording to regulatory guidelines, routine genotoxicity tests are not appropriate for biotechnology derived pharmaceuticals, including oligonucleotide based therapeutics, as they are not expected to interact with genomic DNA. However, reports of oligonucleotides capable of binding duplex DNA in a sequence specific manner to form triple-helix (triplex) or displacement-loop (D-loop) structures that in turn cause mutation have raised concern. The European Medicines Agency (EMA) has questioned the capability of antisense oligonucleotide (ASO) therapeutics to form such structures at genomic DNA.
Additionally, concern has been expressed regarding the fate of chemically modified ASO degradation products (nucleotide analogues). It is well established that non-canonical antiretroviral nucleoside analogues, employed in antiretroviral therapy, result in gross chromosome aberrations following incorporation into genomic DNA.
This study has addressed these concerns by evaluating the genotoxic potential of a triplex forming oligonucleotide (TFO) and a D-loop forming ASO targeting genomic DNA. Furthermore, the incorporation efficiency and genotoxicity of nucleotide analogues derived from ASO degradation was investigated.
Data presented here demonstrate a TFO targeting genomic DNA was not capable of inducing mutation above the detection limit of this assay. However, a biologically active ASO molecule induced sequence specific mutation ~4.4 fold above control in a system where RAD51 protein expression was induced. Additionally, DNA polymerase was capable of incorporating various ASO derived nucleotide analogues into a primed DNA template with reduced efficiency. Treatment with phosphorothioate nucleotide analogue, one of the most common chemical modifications used in ASO design, induced mutation ~100 fold above control.
To conclude, ASO and their putative degradation products appeared to be capable of off-target mutagenesis providing favourable conditions were met. However, as genotoxicity data has been presented for a single ASO and nucleotide analogue, it seems plausible to suggest this work can provide a foundation to test future ASO therapeutics and putative degradation products
MicroRNA mediated biological effects in response to bariatric surgery
Get PDFBariatric surgery offers sustained dramatic weight loss and remission of diabetes, yet the mechanisms of these health benefits are not clear. In the present study, I profiled circulating and colorectal miRNAome in response to bariatric surgery (Roux-en-Y gastric bypass). Indeed, the response of circulating and colorectal miRNA profiles to RYGB were striking and selective. Fourteen circulating microRNA and thirteen colorectal microRNA exhibited significantly alteration post RYGB. Interestingly, circulating miR-122 decreased dramatically (56 fold) post RYGB surgery. The expression of hepatic miR-122 and its metabolic targets were examined both in in vivo RYGB surgical model and in an in vitro mechanistic model. Manipulation of miRNA-122 cold induce changes of key enzymes involved in energy metabolism, glucose transport, glycolysis, TCA cycle, pentose phosphate shunt, fatty acid oxidation and gluconeogenesis, suggesting an overall increased energy expenditure status after RYGB. Furthermore, potential mechanisms involved in the control of hepatic miR-122 were investigated, with focus on metabolites (glucose, and fatty acids), hormones (glucocorticoid) and transcription factors (PPARs). Finally, by correlating the circulating miRNAome and metabolome data, we were able to generate a comprehensive landscape of the crosstalk between miRNAs and metabolic pathways. Follow-up studies will allow a detailed understanding of miRNAs responsible for regulating specific metabolic pathways, and conversely identifying metabolites capable of regulating the expression and activity of specific miRNAs.Open Acces
Mechanistic studies on the molecular toxicology of the carcinogen PhIP: the role of microRNAs
Get PDFThe effect of environmental factors in cellular processes has been an area of
interest for decades. Within this research field, emerging research niches appear
concominantly with advances in molecular biology and genetics. One such field is the
one of molecular toxicology, which investigates the molecular and cellular events that
certain chemicals trigger. Chemicals that present genotoxic properties are of particular
interest based on the potential contribution to the aetiology of cancer that the
discoveries of their effects might provide. A group of chemicals that belong to this
category are heterocyclic amines, organic compounds that are formed during the
cooking of red meat from the pyrolysis of aminoacids. Research into heterocyclic
amines has created a well established description for their ability to create DNA
adducts that lead to mutations. Recent reports from our laboratory however, indicated
that certain heterocyclic amines, and in particular 2-amino-1-methyl-6-phenylimidazo
[4,5-b] pyridine (PhIP), present with molecular effects that are non genotoxic but
nevertheless carcinogenic and are very similar to those induce by the natural hormone
estradiol in breast cancer cells. Based on these findings this thesis set out to
investigate whether these non-genotoxic effects of PhIP also extend to epigenetic
mechanisms of gene expression control, and more specifically microRNA expression
regulation. MicroRNAs are a class of small non-coding RNA molecules that posttrasncriptionally
regulate gene expression and are involved in an array of process
including carcinogenesis. Our results showed that PhIP, as well as estradiol, drive
differential regulation of microRNAs and that these effects are very similar amongst
the two compounds. This deregulation of microRNAs could be an attributing factor to
the cancer promoting characteristics of both estradiol and PhIP and they extend the
estrogenic character of this heterocyclic amine to the area of epigenetic regulation,
and microRNAs in particular.Open Acces
The role of miRNA deregulation in non-genotoxic chemical induced carcinogenesis
Get PDFMicroRNAs (miRNAs) are short, non-protein coding RNAs. These small-RNAs can regulate gene expression by either inhibiting translation or promoting mRNA degradation, they are also involved in diverse physiological and pathological events. Previous data from our group, showed that phenobarbital (PB, a CAR activator), induces dysregulation of the hepatic miRNAome in rat livers. In this study, we aim to determine if non-genotoxic rodent carcinogens can deregulate miRNA expression. Thus, the liver miRNAome associated with a novel chemical (SYN) and the non-genotoxic rodent carcinogen PB was explored at different doses and time points using high-throughput sequencing of small-RNAs (miRNA seq), a powerful tool for discovery and profiling of miRNA expression, providing a deeper understanding of molecular toxicology. Male Han Wistar rats were treated with PB (50 or 1000 ppm) or SYN (50, 500, and 3000 ppm) in the diet for up to 28 days. Control animals were fed regular diet. Rats were sacrificed after 1, 3, and 28 days. Total RNA was isolated from frozen livers and library constructs were generated with the Illumina platform. The miRanalyzer algorithm and DESeq were used for bioinformatics (for known and novel miRNAs) and statistical analysis. Around 200 known miRNAs were detected per sample. Three key miRNAs related to the epithelial-to-mesenchymal transition (EMT), were detected at the 3rd day –miR-200s, miR 30b, and miR-21. Consequently downregulation of targets related to these RNAs and EMT (ZEB1/2, Snail-1, PTEN, and E-cadherin) was seen. These findings suggest activation of a homeostatic response in EMT control in response to PB/SYN treatment, presumably in order to preserve the epithelial nature of hepatocytes. Analysis of putative novel miRNAs sequences determined three candidates homologous to miR-4488 and a putative isomiRNA of miR-6215. The miRNA-seq approach enables a more detailed exploration of miRNAs and their response to hepatotoxicants, such as PB, helping elucidate underlying pathway perturbations.Open Acces
Role of IL-6 and ERα in the genotoxicity of environmental carcinogens in mammary cells
Get PDFWorldwide, breast cancer is the most commonly diagnosed cancer in women. Inherited genes (15%) have a role in the prevalence of breast cancer while diet and lifestyle play major roles (around 85%). Environmental factors such as consumption of red or processed meat, cigarette smoke and inflammation are major risk factors of this disease. Despite the known breast cancer risk factors, the underlying mechanisms are not yet well studied. Benzo(a)pyrene B(a)P and PhIP (2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine) are among the most abundant procarcinogens present in cooked and processed meat and are linked with the incidence of breast cancer. Upregulation of cytochrome P450 (CYP450) enzymes that activate these procarcinogens leading to increased DNA damage has been observed in breast cells. Interleukin-6 (IL-6) is one of the most stable inflammatory biomarkers upregulated in breast cancer and linked with the poor prognosis of breast cancer. IL-6 plays a key role in tumour progression through tumour-promoting signaling pathways and epigenetic gene regulation. I hypothesized that IL-6 can regulate CYP enzymes that can modulate the genotoxicity of procarcinogens. This hypothesis was investigated by mechanistic studies in in-vitro cell culture. Increase in the genotoxicity of pro-carcinogens via CYP regulation was observed in the presence of IL-6 in breast cells. Moreover, I investigated the role of Estrogen receptor in the genotoxicity and generation of oxidative stress by PhIP. Hormone responsive breast tumours are among the most fatal and widespread of cancer types and estrogens are well-known to increase tumourigenesis. PhIP is known to exhibit powerful estrogenic activities. The presence of estrogen receptor (ER) can increase the susceptibly of cells to oxidative stress that can play a vital role in tumour initiation and progression. I therefore hypothesized that the presence of a functional ER receptor can increase PhIP genotoxicity and ability to generate oxidative stress. This hypothesis was tested using in-vitro techniques in ER positive MCF-7 and ER negative MDA-MB-231 cells. Increased genotoxicity and oxidative stress via CYP regulation was observed in MCF-7 compared to MDA-MB-231 cells following PhIP treatments. Interestingly, I also found PhIP can augment the potential of ethanol to generate oxidative stress via CYP regulation leading to increase in DNA damage. Ethanol is one of the most important modifiable risk factors of breast cancer. Lastly, I investigated if B(a)P can independently induce the production of inflammatory cytokines, leading to increased migration and invasion in breast cancer cells. Increase in the production of inflammatory mediators, migration and invasion of breast cells following B(a)P treatments was observed in MCF-7 and MDA-MB-231 cells. Induction of IL-6 by B(a)P leading to deregulation of miRNAs, that can regulate the metastatic potential of breast cells was also observed. This shows that B(a)P can initiate tumours not only via genotoxicity but also by non-genotoxic epigenetic events. Overall, this thesis suggests novel genetic and epigenetic mechanisms by which procarcinogens can initiate and potentiate tumourigenesis.Open Acces
Metabolic profiling studies of tumour cell phenotypes
Get PDFOvarian cancer is a devastating disease affecting millions of women worldwide. Initially successful chemotherapy using platinum-based drugs is often followed by a relapse after which platinum therapy is usually ineffective and prognosis poor. The mechanism by which platinum resistance develops and its reversal to a more sensitive phenotype are of major interest in modern medical oncology, especially for ovarian cancer which has overall low survival rates. In this study, different types of in vivo-derived and in vitro cell models of platinum resistance were examined within a metabolic context using ¹H NMR spectroscopy and bioinformatics-based approaches to test the hypothesis that a metabolic signature of a platinum resistance phenotype could be defined. The in vitro-derived cell models of platinum resistance focused on the study of the Mlh1 gene, whose loss of expression has been shown to play a role in the development of resistance to platinum drugs and mitochondrial dysfunction. A comparative metabolic analysis of a transient Mlh1 expression cell model (HEK-293T) involving kidney cell lines of common genetic background and the ovarian cancer cell lines A2780 and its platinum resistant sub-line CP70 of well-established Mlh1 status was conducted. This resulted in the definition of a metabolic signature associated with Mlh1 expression, which included metabolites such as glutathione, alanine, myo-inositol and phosphocholine. It also achieved to associate the loss of MLh1 to mitochondria. The in vivo-imitating cell model focused on the normoxic & hypoxic baseline metabolic profiling of isogenic ovarian cancer cells that were clinically sensitive (PEA1 and PEO1) or resistant (PEA2 and PEO4) to platinum. The ¹H NMR-generated results showed oxygen level-related changes in glucose, lactate, pyruvate, acetate, and choline-related metabolites. Overall, this study managed to contribute towards the understanding of platinum resistance in ovarian cancer through a metabolic spectrum using a variety of analytical and methodological tools
Using liver miRNA profiles to predict chemical hepatocarcinogenesis
Get PDFIndustrial, agricultural, and pharmaceutical requirements drive the development of a plethora of new chemical entities each year, many of which - for example drugs, pesticides, and food additives - have to be assessed for potential human health hazard. The current benchmark for risk assessement is the lifetime rodent bioassay which is expensive, time-consuming, laborious, requires the sacrifice of numerous animals, and is often irrelevant to humans. Hence alternative strategies to the rodent lifetime bioassay for prediction of chemical carcinogens are being pursued, especially for the liver which is an organ frequently affected by exogenous chemicals due to its detoxifying and metabolic roles. Numerous studies in recent years support the important role of microRNAs in cancer development, including hepatocellular carcinoma. The principal hypothesis of this project was that hepatic microRNA signatures can contribute to the earlier prediction of chemical hepatocarcinogens. Examination of livers from male Fischer rats treated with six chemical hepatocarcinogens, with diverse mode of actions for 90 days revealed that all the tested hepatocarcinogens affected the liver miRNAome from that early stage. Interestingly, a small set of microRNAs were identified whose expression was frequently deregulated by the hepatocarcinogens. Bioinformatic analysis indicates that these microRNAs can regulate pathways which are important in hepatocellular carcinoma. A more detailed investigation of one of those hepatocarcinogens, phenobarbital, showed that its effects on liver microRNAs were both dose and time dependent, with a progressive induction of specific microRNA clusters.Thus this study was the first to investigate in depth the effects of chemical hepatocarcinogens on the liver miRNAome and supports the potential usefulness of hepatic microRNA signatures in risk assessment
Epigenetic control of the post-bariatric phenotype: the role of microRNAs
Get PDFThe rising obesity pandemic and the concomitant rise in its co-morbidities are leading causes of global morbidity and mortality. Bariatric surgery is a form of gastrointestinal surgery that leads to sustained weight loss, diabetes resolution, reduction in cancer risk and other improvements in health. MicroRNAs are a family of small, endogenous, non-coding RNAs that regulate gene expression at the post-transcriptional level. MicroRNAs control expression of over half the human transcriptome and are involved in processes fundamental to both normal physiology and disease, including obesity and diabetes. This study hypothesizes that microRNAs are biomarkers for health improvements following bariatric surgery. Using quantitative PCR, a microRNA baseline was established in the serum and urine of an obese human population and increases in three anti-fibrotic microRNAs were found in urine following bariatric surgery. Circulating microRNA profiles were characterised in Roux-en-Y gastric bypass patients preoperatively and at 5 timepoints postoperatively. Roux-en-Y gastric bypass significantly altered circulating microRNA profiles in a time dependent manner. Relative to preoperative levels, of the 159 circulating microRNAs assayed 2 were significantly deregulated 1 month postoperatively, 5 were deregulated at 3 months, 10 at 6 months, 28 at 9 months and 31 at 12 months. Target prediction and pathway analysis revealed that these differentiated microRNAs regulate biological pathways that are involved in obesity and that microRNAs may contribute to the development of the beneficial post-bariatric phenotype. Both circulating and urinary post-bariatric microRNA levels correlated with measured clinical biomarkers such as BMI and blood glucose. These results indicate that bariatric operations fundamentally alter microRNA expression both in urine and in circulation and suggest that microRNAs represent not only potentially novel biomarkers for improvements in health following surgery, but are possible biological effectors that contribute to the mechanisms behind bariatric surgery. MicroRNA expression profiles could potentially be used to monitor operative outcomes and understanding the role of these differentially expressed microRNAs could shed light behind the mechanism by which bariatric surgery improves health.Open Acces
Effect of the inflammatory mediator TNF-alpha on colorectal cancer epithelial cells development and metastasis, role of ietary carcinogens and miRNA
Get PDFColorectal cancer (CRC) is the third most common cancer world-wide and second leading cause of mortality. The majority of CRC cancer cases result from epigenetic and genetic alterations that promote development and metastasis of the disease. Exposure to environmental and dietary carcinogens are strongly associated with CRC. Also, inflammatory mediators are known as a major risk factor for CRC, however the underlying mechanisms are still understudied. Upregulation of pro-inflammatory mediators and dysregulation of miRNAs in the tumour microenvironment (TME) have been observed in CRC. Tumour necrosis factor alpha (TNF-α) is a pleiotropic cytokine thought to play numerous roles in tumour progression including epigenetic gene regulation, activation of tumour promoting signalling pathways, thus presence of TNF- in CRC tumour microenvironment may be key to promoting CRC progression. I hypothesise that the presence of TNF- α in the TME could regulate miRNAs and enzyme expression to induce DNA damage caused by dietary carcinogens, thereby stimulating changes that promote CRC development and metastasis. The present study investigated this hypothesis through a mechanistic approach with in vitro cell line culture. Effects of TNF-α on phenotypic changes were observed and the potential involvement of miRNAs were determined. The results showed that TNF-α enhances dietary carcinogen-induced DNA damage through activation of JNK signalling pathway. Also, TNF-α induced metastatic phenotype cell proliferation and migration through miRNA regulation. Moreover TNF-α regulated expression of CYP450 enzymes through miRNA regulation, which can promote chemical carcinogen genotoxicity. Taken together, the data indicated that CRC progression and metastasis may be related to epigenetic and inflammatory mediators active at the tumour site. Understanding these molecular mechanisms could provide better prevention and therapeutic strategies.Open Acces
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