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Identification, Expression Profiling and Functional Analysis of MicroRNAs in Breast Cancer
MicroRNAs (miRs) are a recently discovered family of noncoding 18-25 nucleotide RNAs. In animal cells, miRs have been shown to regulate gene expression by binding to partially complementary sites within the 3' untranslated regions of target messenger RNAs (mRNAs). This binding causes inhibition of translation without affecting significantly the stability of the target mRNA molecules. In humans, many of the more than 200 miRs are thought to target transcription
factors and to participate in regulation of development and differentiation.
Emerging evidence links abnormal expression of miR genes with some types of human cancer, including colorectal neoplasia and chronic lymphocytic leukemia. As potential negative regulators of gene expression, miR loss or
reduced expression could contribute to oncogenesis by allowing increased production of oncogenic proteins in cases where miRs target proto-oncogenes. Conversely, overexpression of miRs could reduce production of tumor suppressor
proteins where tumor suppressor genes are targets of miR regulation.
The primary objective of this proposal is to identify miRs with regulatory roles in breast cancer. Functional analysis of differentially-expressed miRs is likely to show the importance of miR regulatory action in inhibiting or contributing to breast tumor formation. First, I will characterize the expression profile of previously reported miRs and novel miRs that I will identify using cloning and bioinformatic approaches. I will construct size-selected RNA libraries from non- and tumorigenic breast cell lines (BCLs). I will characterize by Northern and microarray analyses the expression of miRs in normal and malignant BCLs subjected to different hormonal, drug, and/or growth factor treatments. Then, I will analyze miRs functionally: an existing retroviral system will be employed to overexpress miRs and 2'-O-methyl oligonucleotides complementary to the miRs will be used to abrogate their function. The effects of miR overexpression or depletion on cell biological parameters (growth rate, sensitivity to apoptosis, etc.) and on global changes of gene expression will be determined using cellular assays and microarray technology, respectively.
Unraveling the mechanisms of miR action during breast cancer progression should provide a better understanding of the molecular basis of this disease and set the stage for exploring the diagnostically or prognostically utility of miR expression patterns in breast cancer in a clinical environment.
Genes produce messenger RNA (mRNA) molecules that contain the information to manufacture proteins, which control critical cellular processes such as cell proliferation, differentiation and death. In breast cancer and other cancers,
misregulation or mutation of critical genes results in overproduction, absence, or malfunction of proteins that maintain normal growth regulation. MicroRNAs (miRs) are a recently discovered novel class of RNAs. miRs do not code for
proteins and are very short (18-25 nucleotides long), whereas mRNA molecules are between 1000 and 30,000 nucleotides long. miRs can bind to mRNAs of target genes and block the production of proteins.
Hundreds of miRs have been discovered in the human genome. Thus, the production of hundreds to thousands of proteins is likely to be regulated by miRs. This underscores the relevance of miR discovery and the importance of their study. Recent studies of several types of cancer including colon cancer and leukemia found that the expression of some miRs was affected by the malignant status of the cells--some miRs were newly expressed and others no longer expressed. These results strongly suggest that specific miRs contribute to the etiology of some types of cancer. In this proposal, the potential roles of miRs in the regulation of breast cancer will be investigated.
The breast is composed of epithelial, fat, muscle, and connective tissues. However, the majority of breast cancers arise from epithelial cells. Epithelial cells can be isolated from normal breast and breast tumors and cultured in the laboratory. I will determine which miRs are expressed both in normal breast cell lines (BCLs) and in those derived from malignant breast cancers. A complete list of expressed miRs in normal and malignant cells will help to pinpoint those miRs that are most likely to participate in the formation or prevention of breast cancer. Then, I will study how manipulating the quantity of miR expression levels affects BCLs. For example, if a cell expresses much more or much less of a miR than normal: does it divide more often and acquire tumor-like properties? Does it stop growing and differentiate, or is it more susceptible to cell death? Does it express a different set of protein-encoding genes?
Positive results of these studies would demonstrate an important role for miRs in breast cancer and provide an excellent foundation for future projects analyzing miRs in a clinical setting to diagnose, predict outcome or even treat breast cancer.