LncRNA ASTILCS as an important regulator of liver carcinoma cell survival [shRNA cassette seq]

Extensive genomic studies have significantly improved our understanding of hepatocellular carcinoma (HCC) biology and have led to the discovery of multiple protein-coding genes driving hepatocarcinogenesis. In addition, these studies have identified thousands of new non-coding transcripts deregulated in HCC, suggesting these transcripts are important for disease progression. Long non-coding RNA is a large class of non-coding transcripts which participate in the regulation of virtually all cellular functions. However, a majority of lncRNAs remain dramatically understudied. Here, we applied a pooled shRNA-based screen to identify lncRNAs essential for HCC cell survival. We validated our screening results using RNAi, CRISPRi, and antisense oligonucleotides. We found a lncRNA, termed ASTILCS, is critical for HCC cell growth and is overexpressed in tumors from HCC patients. We demonstrated that HCC cell death upon ASTILCS knockdown is associated with apoptosis induction and downregulation of a neighboring gene, Protein Tyrosine Kinase 2 (PTK2), a crucial protein for HCC cell survival. Taken together, our study describes a novel regulator of HCC progression providing valuable insight in HCC biology. Overall design: shRNA cassette seq of Huh7 liver cell line RNA Input library - The input pooled shRNA plasmid library before virus production consisting of 7873 shRNA vectors allowing to do knockdown of the 1618 lncRNAs based on RNAi. Selected - The shRNA plasmid library packaged in lentivirus and applied on Huh7 cells. Two days after exposure, infected cells were selected for four days on puromycin and then maintained for four weeks under selection followed by DNA isolation.