The regulation of microRNA (miRNA) biogenesis and the role of miRNA binding proteins in breast cancer.
Lead Research Organisation:
Imperial College London
Department Name: Surgery and Cancer
Abstract
MicroRNAs are a newly discovered class of molecules that act as genetic regulators within cells. They control processes involved in cell division and cell death, and have important roles in preventing cancer. We have found that estrogen (a major regulator in breast cancer,) causes production of specific microRNAs. These microRNAs down-regulate the activity of the gene that codes for the estrogen receptor-alpha - which performs the functions of estrogen inside cells by stimulating the production of particular proteins called estrogen response elements. This means that there is never too much estrogenic activity and it is controlled, in a unique feedback loop that we have identified. In breast cancer however, the microRNAs are not properly produced, so there is no down-regulation of estrogenic activity, and the breast cancer cells continue to divide. If we can turn this process off, or give patients the microRNAs they don‘t make, we can treat breast cancer and reverse this process. Firstly, we must identify the different types of microRNAs in breast cancer and understand which factors control microRNA production. This would present exciting new possibilities for diagnosis and patient-specific treatments, and may help us to detect hard-to-diagnose cancer and determine how aggressively a cancer might behave.
Technical Summary
Aims:
The aims of the proposed project are to establish how miRNAs are regulated by the estrogen receptor- (ER ) in breast cancer and to identify the estrogen (E2)-inducible miRNA binding proteins involved.
Objectives:
1. To investigate the clinical relevance of the mir-17-92 and mir-106a-363 family of miRNAs in breast cancer.
2. To determine the mechanism(s) by which transcription of the mir-17-92 and mir-106a-363 clusters is regulated.
3. To analyse the mechanism(s) of miRNA biogenesis in breast cancer and establish the identity of the E2-inducible factor(s) involved.
Design:
Objective 1- We will use quantitative real-time PCR to measure the expression of the primary miRNA transcripts, pre-miRNAs and mature molecules belonging to the mir-17-92 and mir-106a-363 families of miRNAs, in well characterised sets of breast cancer specimens. We will study their clinical relevance by investigating whether these ER -regulated miRNAs correlate to outcomes, including survival, comparing ER -positive and ER -negative tumours.
Objective 2- We will identify ER /ER -modulated transcription factor binding sites upstream/ downstream of the mir-17-92 and 106a-363 transcriptional start sites using the UCSC genome browser and ChIP analyses prior to/following E2 stimulation. We will use chromosome capture assays to assess whether promoter and enhancer sequences are components of the same chromatin regions of identified interaction sites, and to identify the genomic localisation of the promoters/enhancers for the miRNA transcripts. The biological relevance of any regulatory binding will be investigated using siRNA knockdown of transcription factors and luciferase reporter assays.
Objective 3- We will utilise model systems to establish the identity of the estrogen-inducible factor(s) causing inhibition of miRNA processing and determine the contribution of post-transcriptional miRNA regulation in response to ER , to oncogenesis.
Methodology
The proposed project uses a range of cell and molecular biology techniques, bioinformatics and tissue handling techniques, which are well established in the host laboratory.
Scientific and Medical Opportunities
Based on previous data, we hypothesise that post-transcriptional regulation is a general mechanism of miRNA biogenesis with relevance to breast cancer, and that an estrogen-inducible mechanism is responsible for the inhibition of miRNA processing. We aim to demonstrate the involvement of miRNA binding factors in this process, and implicate them as potential new therapeutic targets. This also has general relevance to oncology, as miRNAs are major regulators of the development and growth of many cancers, although the associated mechanisms remain poorly understood. Finally, this research will increase our knowledge of the potential role of miRNAs as biomarkers in breast cancer.
The aims of the proposed project are to establish how miRNAs are regulated by the estrogen receptor- (ER ) in breast cancer and to identify the estrogen (E2)-inducible miRNA binding proteins involved.
Objectives:
1. To investigate the clinical relevance of the mir-17-92 and mir-106a-363 family of miRNAs in breast cancer.
2. To determine the mechanism(s) by which transcription of the mir-17-92 and mir-106a-363 clusters is regulated.
3. To analyse the mechanism(s) of miRNA biogenesis in breast cancer and establish the identity of the E2-inducible factor(s) involved.
Design:
Objective 1- We will use quantitative real-time PCR to measure the expression of the primary miRNA transcripts, pre-miRNAs and mature molecules belonging to the mir-17-92 and mir-106a-363 families of miRNAs, in well characterised sets of breast cancer specimens. We will study their clinical relevance by investigating whether these ER -regulated miRNAs correlate to outcomes, including survival, comparing ER -positive and ER -negative tumours.
Objective 2- We will identify ER /ER -modulated transcription factor binding sites upstream/ downstream of the mir-17-92 and 106a-363 transcriptional start sites using the UCSC genome browser and ChIP analyses prior to/following E2 stimulation. We will use chromosome capture assays to assess whether promoter and enhancer sequences are components of the same chromatin regions of identified interaction sites, and to identify the genomic localisation of the promoters/enhancers for the miRNA transcripts. The biological relevance of any regulatory binding will be investigated using siRNA knockdown of transcription factors and luciferase reporter assays.
Objective 3- We will utilise model systems to establish the identity of the estrogen-inducible factor(s) causing inhibition of miRNA processing and determine the contribution of post-transcriptional miRNA regulation in response to ER , to oncogenesis.
Methodology
The proposed project uses a range of cell and molecular biology techniques, bioinformatics and tissue handling techniques, which are well established in the host laboratory.
Scientific and Medical Opportunities
Based on previous data, we hypothesise that post-transcriptional regulation is a general mechanism of miRNA biogenesis with relevance to breast cancer, and that an estrogen-inducible mechanism is responsible for the inhibition of miRNA processing. We aim to demonstrate the involvement of miRNA binding factors in this process, and implicate them as potential new therapeutic targets. This also has general relevance to oncology, as miRNAs are major regulators of the development and growth of many cancers, although the associated mechanisms remain poorly understood. Finally, this research will increase our knowledge of the potential role of miRNAs as biomarkers in breast cancer.
