UC San Diego

Long non-coding RNAs (lncRNAs) have been implicated as regulators of cell growth and survival in a variety of cancer types, but the specific biological functions and interaction partners of many lncRNAs are still not well understood. In this dissertation, I used multiple biochemical and quantitative techniques to study how a novel cancer-associated lncRNA, termed Growth Regulator Antisense 1 (GRAS1), mechanistically regulates cell viability in cancer cells. In Chapter 2, I discussed the development of a system to investigate 5 new cancer-associated lncRNAs from screening data from multiple published studies, including linc01977, linc00883, PDCD4-AS1, linc00909, and GRAS1. More specifically, we established a working platform of overexpression and knockdown for these lncRNAs, performed cell survival screening on these lncRNAs using five different cancer cell lines, examined transcriptomic alterations in altered expression of these lncRNAs, and validated biotinylated probes to capture these lncRNAs. Based on the data in that chapter, we selected GRAS1 to be our lncRNA of interest to focus on. In Chapter 3, I reported that GRAS1 directly and specifically interacts with STAT5 in K562 cells using RNA antisense purification with mass spectrometry (RAP-MS). In addition, we found that knockdown of GRAS1 reduces phosphorylation of STAT5 as well as the activity of the STAT5 signaling pathway in K562 cells. In Chapter 4, I characterized the function of GRAS1 in non-small cell lung cancer cells. GRAS1 is critical for A549 cell survival, and depletion of GRAS1 RNA transcript induces cell death. We found that knockdown of GRAS1 resulted in a significant increase in protein levels of p53 and the DNA damage marker γH2AX. GRAS1 knockdown leads to differential expression of many mRNA transcript levels, including upregulation of DNA damage response factors involved in double strand break repair. Further, we identified the NF-κB activating protein (NKAP) as a direct interaction partner of GRAS1 using RAP-MS. Decreased GRAS1 levels in lung cancer cells caused a significant decline in NKAP level and induction of cell death through ferroptosis. The addition of the ferroptosis inhibitors ferrostatin and α-tocopherol could partially rescue the effect of GRAS1 knockdown on lung cancer cell survival. We found NKAP overexpression could completely reverse the ferroptosis as well as rescue the cell viability promoted by GRAS1 downregulation in lung cancer cells. Besides, NKAP overexpression could significantly reverse the effect of GRAS1 knockdown on lung cancer DNA damage. Chapter 4, in full, is currently being submitted for publication. Finally in Chapter 5, I listed the methods and materials we used in this study.