High throughput sequencing following cross-linked immune-precipitation (HITS-CLIP) of Argonaute protein reveals novel miRNA regulatory pathways of Estrogen Receptor in breast cancer.

Micro RNAs (miRNAs) are a class of small non-coding RNAs with well-known regulatory roles in normal physiological processes and cancer. Conventionally their study has been based on defining single miRNA-mRNA target interactions using a combination of miRNA expression arrays and bioinformatic predictions of binding to the 3' untranslated regions of genes, followed by miRNA over-expression in the relevant cell type. These approaches present circumstantial evidence for binding of a particular miRNA to its target but do not allow the study of global interactions nor provide direct evidence of binding. In order to study the genome wide impact of miRNA regulation in breast cancer we chose to use a recently described biochemical technique called Cross-Linked Immune-Precipitation of Argonaute (AGO) protein followed by high throughput sequencing (HITS-CLIP). Cross-linking of RNA to adjacent protein moieties by ultraviolet (UV) light allows for stringent isolation of the miRNA-mRNA-AGO complexes by immune precipitation (IP); miRNA-mRNA interaction within the RNA-induced silencing complex (RISC) is known to occur within the folds of AGO. The isolated miRNA-mRNA-AGO complexes are then analyzed with next generation sequencing (NGS) to determine the miRNA-mRNA interactome. We performed HITS-CLIP on three well characterized breast cancer cell lines that represent ER+, Her2+ and triple negative (TN) disease (MCF7, BT474 and MDA231). To determine the role of miRNAs in coordinating the response to the estrogen receptor axis, MCF7 and BT474 cells were analyzed with or without short term (24 hour) estrogen treatment. Analysis of sequencing data revealed several novel miRNA-mRNA interactions that target the ER pathway. For example, miR-9 directly regulates ER expression and miR-193a is involved in regulating expression of the ER co-activator NCOA3. We confirmed the biological relevance of these results using functional in vitro studies where manipulation of both miR-193a and miR-9 altered the responsiveness of breast cancer cells to tamoxifen. These results were further validated by quantitation of both transcript (RT-PCR) and protein (Western Blot) levels after transfection of miRNA precursors. Functional binding between these miRNAs and putative binding targets in the 3' Untranslated Regions (3' UTRs) were also validated by luciferase-based reporter assays. Finally, we performed global analysis of miRNAs and their targets (as predicted by the HITS-CLIP datasets across all cell lines); this predicted regulation of core cellular processes such as cell proliferation, DNA repair and metabolism as being targeted by highly abundant miRNAs such as miR-27a and miR-21. In addition, our datasets confirmed previous reports of miRNA regulation of ER pathway such as miR-221 regulation of ER itself and miR-34a regulation of Myc. In summary, genome-wide biochemical approaches like HITS-CLIP allow for defining novel and clinically relevant miRNA-based regulatory pathways of endocrine responsiveness and resistance in breast cancer. In addition, breast cancer subtype specific biological pathways targeted by individual miRNAs can be predicted by this approach. [ABSTRACT FROM AUTHOR]