Functional Study of the Noncoding Regulome of the Nuclear Factor Y

The nuclear factor Y (NF-Y) is a trimeric transcription factor acting as master regulator of proliferation in normal and tumor cells. Since the aberrant expression and function of noncoding RNAs (ncRNAs) are associated with tumorigenesis, the ncRNA-mediated regulation of NF-Y in cancer is worth of inspection. This project aims at functionally characterise NFYC antisense 1 (NFYC-AS1), a nuclear long ncRNA (lncRNA) transcribed head-to-head to NFYC, one subunit of NF-Y. Our expression data show that NFYC-AS1 is upmodulated in all lung cancer histotypes compared to normal tissues, with preferential association with RB1 mutation and the neuroendocrine small cell lung cancer histotype. Our functional data support an oncogenic function in promoting cancer cell proliferation by regulating the cell cycle. Additional cell-based assays will be performed to further evaluate NFYC-AS1 function in lung cancer cells. Moreover, different gain- and loss-of-function strategies will be used to modulate its expression in lung cancer cells characterized by different RB1 mutational status to dissect the mode of action and to evaluate its role in RB1-mutated cells and in the neuroendocrine commitment. In addition, this project aims at studying the microRNA regulation of NF-YA, the regulatory subunit of NF-Y, with a particular focus on the 3’UTR repertoire. Our preliminary studies show no association between NFYA splicing isoforms and a certain 3’UTR. However, prior to this is the accurate annotation of all the possible NFYA 3’UTRs and analysis of their use in different cell states and conditions. Moreover, we will identify cancer-relevant NFYA-targeting microRNAs, which will be transfected in tumor cells and consequent phenotypes will be evaluated. Overall, this project intends to functionally study the ncRNA-mediated regulation of NF-Y, focusing on the lncRNA NFYC-AS1 and the NFYA mirnome in the cancer context.