Genome engineering as an approach to identify crucial miRNA targets in cancer

All the cells composing the body contain the genetic information necessary for the life of each individual. Genetic information is encoded by the deoxyribonucleic acid (DNA), which represents the genomes and exerts its function via making proteins. A crucial intermediate in this process is ribonucleic acid (RNA) which is specifically called messenger RNA (mRNA). Recently, it has been shown that besides mRNA, RNA that does not produce proteins exists and its production and function in the cells are equally important to the ones employed by mRNA. Because the latter class of RNA is not created by the cells to produce proteins, but instead to have a regulative role, it is called noncoding RNA (ncRNA). Similarly to mRNA, the production and function of ncRNA ensure an accurate cellular function and consequently a healthy life. NcRNA role is to precisely regulate mRNAs when they produce proteins. This means that the production of proteins by mRNA can increase or slow down depending of particular circumstances, and this effect is mediated by the interaction between mRNA and ncRNAs. NcRNAs can be divided into different classes, depending on their length. MicroRNAs belong to the short ncRNAs class and several studies performed on microRNAs, in the last decade, have demonstrated that these short RNA molecules are important regulator of cancer development. Moreover, specific drugs can now be developed to specifically target microRNAs in the body to fight cancer or other diseases. MicroRNA function by interacting with small, specific region of mRNA to slow down protein production. In the last few years, other important discoveries, in science, are remarkably impacting our understanding of the way that the cells function as well as about what goes wrong when the cells pathologically change and form cancers. One of these new techniques is called CRISPR-CAS9 and can be used, in vitro and in vivo, to precisely edit the DNA of the cells accordingly with our needs. Because DNA generate RNA the new modification created at the level of DNA, by using CRISPR-CAS9 in the cells, will be consequently transferred to the RNA. Herein, we want to use CRISPR-CAS9 to globally disrupt the mRNA portions predicted to interact with microRNAs. Because disruption of these mRNA regions will impede functional microRNA-mRNA interaction, the mRNA will consequently produce a higher amount of protein that can be deleterious and induce cancer. In principle, mRNA that produce proteins important for cancer, can also be modulated by microRNAs. By performing specific laboratory tests and by studying changes in the growth of tumour cells seeded on specific dishes and treated with CRISPR-CAS9 we can finally identify important microRNA-mRNA interactions that can be exploited for future therapeutic intervention. By using both experimental techniques and computational approaches, this work would permit us to understand the important mRNA-microRNA interactions that regulate cancer development and this new knowledge could then be used to develop new drugs to fight cancer.

MicroRNAs (miRNAs) have a crucial role in cancer development and progression. During a particular cellular process, they act by base-pairing with the 3'UTR of hundred of mRNA targets to repress their protein translation. Notably, it has been elegantly demonstrated that in Caenorhabditis elegans, the let-7 miRNA does not function to reduce gene expression noise broadly but to directs vulva development through regulation of a single target. Whether this mode of miRNA regulation also occurs in other systems, such as human, remains largely unclear. Prediction of miRNA targets, experimental validation and rescue experiments have suggested that this could be the case, but this has never been addressed systematically. Considering that a single miRNA can regulate thousands of transcripts, using this method to precisely address this question, for any single putative target, would be very time consuming. Herein, we aim to develop CRISPR-CAS9 pooled screening technologies that specifically and rapidly address this important biological question. The discovery of key targets of important miRNAs in cancer or other diseases can reveal new therapeutic targets/biomarkers. This proposed work follows from our recent study pending revision, in Nature Communication, where we discovered that two miRNAs, miR-100 and miR-125b, are induced by TGFb in advanced PDAC and are important effectors of the tumourigenic TGFb response. We developed a novel method to identify all the targets of these miRNAs, that we called RIP-USE, which indicated that these two miRNAs co-regulate hundreds of transcripts after TGFb induction, but could not identify the key targets of such miRNAs. Therefore, we now want to develop a series of CRISPR/CAS9 screening methods that would permit to identify the crucial miRNA targets. We will focus on the oncogenic miR-100 and miR125b as well the tumour suppressor miR-205 and miR-203 to develop methods that can be used to discover key miRNA targets for any miRNA of choice.

Our proposed research will principally impact on four different aspects listed below:
1. Basic science. We will positively impact on basic science. MiRNA activity represents one of the most important layer of gene regulation. With our new CRISPR-CAS9 technologies that we want to develop here, we will fill gaps surrounding the basic biology of miRNA mechanism of action. This research would permit us and the research community to further understand how miRNAs regulate cellular processes and reveal which are the crucial miRNA-mRNA interactions that occur in pancreatic cancer for selected, important miRNAs. Additionally, because we aim to introduce tumour-sphere formation assay as selective pressure during CRISPR-CAS9 pooled screenings, we would provide to the research community a novel method that couple CRISPR-CAS9 pooled screening with an in vitro assay that checks for stemness and tumourigenic-related processes. Essentially this assay could also be used by others for discovery of stemness related genes or stemness controlled regions by using CRISPR-CAS9 loss (CRISPRi) or gain of function (CRISPRa) pooled screening assays that target coding genes or cys-regulative regions instead of miRNA-mRNA interactions.
2. Pancreatic cancer. The proposed study will have a strong positive impact on pancreatic cancer biology which is still not enough characterised to permit the development of an effective cure. Pancreatic cancer is a deadly disease mainly because it does not cause symptoms at the early stages. Current therapeutic approaches ensure five-year survival rate for only 5% of cases and this catastrophic statistic has remained stable from several years. We and others believe that this is due to a lack of the understanding of the molecular mechanisms that drive this disease which seems to act different than other cancer. MiRNAs play an important role in pancreatic cancer. We have impacted significantly on this filed also by revealing two oncogenic miRNAs that mediate the tumourigenic TGFb response. We also discovered the transcripts regulated by those miRNAs providing a source of candidate TGFb effectors to the pancreatic cancer research community that could represent important therapeutic targets/biomarkers. It is now central to evaluate which of these miRNA targets are crucial for the action of the two miRNAs during pancreatic cancer progression. Because TGFb is "undraggable" and because it is well known that TGFb induces pancreatic cancer aggressiveness and metastasis, we believe that the identification of crucial TGFb effectors could significantly help with the developing of an effective cure, especially for patients with advanced disease.
3. The patients and carers. It is crucial that patients and carers are continuously informed on the results coming from cancer research and how this research can impact on the cure of this deadly disease.
4. Researchers. We will impact on researcher's knowledge of miRNAs mode of action and pancreatic cancer biology. Taking from this study other researchers can add their own new ideas to further develop projects that in turn will increase knowledge and so on, exactly as we did to mature the proposed project. This is essential to improve our understanding of pancreatic cancer and molecular biology in general.