Development of Biotechnical Tools for Cancer Research

This project has three components 1) identification and validation of microRNA targets by a genetic selection system; 2) RNA-guided gene activation and suppression by transcription factors based on modified CRISPR-Cas system and 3) identification of long non-coding RNA RoR as a p53 negative regulator.;Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) system is a bacterial adaptive immune defense against foreign nucleic acids from viruses, plasmids, and other mobile genetic elements. There are three types of CRISPR systems; type II uses single effector enzyme, Cas9, to cleave double-stranded DNA (dsDNA). Because of this unique feature, CRISPR/Cas9 system has been widely used as a genetic engineering tool from bacteria to mammals. In this system, Cas9 nuclease can bind and cleave genome directed by sequence-specific guide RNA (gRNA). While the wild type Cas9 can cleave double stranded DNA, the mutant Cas9 at D10A and H804A can only bind to but cannot cleave the DNA. Given this unique feature, we tried to repurpose Cas9 as a gene activation or suppression tool. To achieve this, we generated DMCas9 (double-mutant Cas9), and fused it with VP16AD (VP16 activation domain) or VP64AD (VP64 activation domain) to explore its utility for gene activation. To generate a Cas9-based gene silencing tool, we fused DMCas9 with Krab, a well-known protein domain with suppression function. Using a reporter system carrying a specific sequence that was recognized by a given gRNA and mCherry as a readout, we demonstrated that co-expression of VP16AD-DMCas9 and specific gRNAs leads to a at least three-fold induction of mCherry signal. We also found that DMhCas9-VP64AD combined with myoD-gRNAs induced endogenous myoD drastically. In contrast, DMhCas9-Krab along with the specific gRNA resulted in a significant suppression of mCherry activity. Furthermore, we showed that DMCas9-Krab combined with gRNAs suppressed endogenous expression of AR and ER in MCF-7 cells, causing cell growth inhibition. Together, these results demonstrate the feasibility of Cas9-based gene activation and gene suppression.;It is well known that upon stress, the level of the tumor suppressor p53 is remarkably elevated. However, despite extensive studies, the underlying mechanism involving important inter-players for stress-induced p53 regulation is still not fully understood. We present evidence that the human lincRNA-RoR (RoR) is a strong negative regulator of p53. Unlike MDM2 that causes p53 degradation through the ubiquitin-proteasome pathway, RoR suppresses p53 translation through direct interaction with the heterogeneous nuclear ribonucleoprotein I (hnRNP I). Importantly, a 28-base RoR sequence carrying hnRNP I binding motifs is essential and sufficient for p53 repression. We further show that RoR inhibits p53-mediated cell cycle arrest and apoptosis. Finally, we demonstrate a RoR-p53 autoregulatory feedback loop where p53 transcriptionally induces RoR expression. Together, these results suggest that the RoR-hnRNP l-p53 axis may constitute an additional surveillance network for the cell to better respond to various stresses.;MicroRNAs are a group of naturally-occurring small non-coding RNAs. They have been categorized as an important class of negative gene regulators by means of their major function, binding to the 3'UTRs of mRNAs and causing mRNA degradation or translational repression. Overwhelming evidence has indicated that microRNAs can play a fundamental role in regulation of diverse cellular functions and dysregulation of microRNA expression could lead to a variety of disorders including human cancer. Although significant progress has been made in the past years in discovery of microRNAs and their biogenesis, and their role in many cellular phenotypes, it is not fully understood how microRNAs exert their cellular functions. Hence, identification of microRNA targets is a critical step toward understanding of molecular mechanisms of microRNA-mediated gene expression in normal and disease processes. Here we reported a selection system for microRNA target identification and validation. We used Sleeping Beauty (SB) transposon system to generate selection vectors. This SB-based vector carries tetO-EF1a promoter driving GFP/or mCherry-T2A-Pu and tetR-Krab under CMV promoter. We examined the feasibility of these constructs in 293T and HeLa cells. Using GFP or m-Cherry along with Pu (puromycin resistance) as a reporter, we found that the construct carrying a given UTR or microRNA sponge provides a significant selection advantage over the vector alone. We also generated a pSSMT5-based cDNA library with the capacity of nearly 17,000 3'UTR and tested this library in a 293 miR-21 Knockout cell line and found that exogenous expression of miR-21 combined with pSSMT5-target library confers the cells with higher level of puromycin resistance. Together, these results suggest that this system provides proof of principle, and it can be used as a powerful tool for identification and validation of microRNA targets.