Identification of LATS transcriptional targets in HeLa cells using whole human genome oligonucleotide micorarray

Human LATS1 and LATS2 (LATS1/2) are tumor suppressors that have been shown to be mutated or downregulated in several human cancers including leukemia, lung, prostate and breast cancers. However, the precise mechanisms and the proteins modulated by LATS1/2 that are responsible for these events remain largely unknown. To elucidate potential signaling pathways, the current study investigated the expression profile in HeLa cells with reduced expression of LATS1/2. Using RNA-mediated intereference, both LATS1 and LATS2 were substantially knocked-down, and accordingly, this lead to an increase in multiple phenotypes associated with tumor progression, including enhanced cell proliferation, resistance to drug-induced cell death, and increase cell migration. Using whole human genome oligo (60-mer) arrays (Agilent), gene modulated by loss of LATS1/2 were identified and functionally grouped into categories including cell proliferation, cell death, cell adhesion and motility, as well as cell communication. Selected genes, including known tumor suppressor genes and oncogenes such as CDKN1A, WISP2, SLIT2, TP53INP1, BIRC4BP, SPRY2, SPRY4, SPRED1, FAT4,and CYR61 were confirmed by qRT-PCR to be significantly differentially expressed. Importantly, the collection of genes identified suggests that LATS1/2 functions through diverse mechanisms and multiple signaling pathways including the Hippo signaling pathway, as well as the p53, Ras-ERK, or WNT networks, in inhibit tumor progression. HeLa cells were treated with a negative control siRNA with scrambled sequence (siRNA-Control) or a combination of siRNA targeting LATS1 and siRNA targeting LATS2 (siRNA-LATS1/2). Total RNA was extracted using TRIzol and RNA was further purified with RNease Mini Kit (Qiagen). Whole Human Genome Oligo (60-mer) array gene expression analysis was performed with siRNA-LATS1/2 and siRNA-Control HeLa cells a total of 4 times. Two biological replicates were performed, in which LATS1/2 were knocked-down in separate experiments and two technilogical replicates were performed in which the Cy3 and Cy5 dyes were flipped. Data were normalized by the linear lowess method. Genes that were 1.5-fold differentially expressed on 3 of the 4 arrays were scored as significant. Furthermore, genes must meet a p-value of ≤ 0.05 based on a student t-test to be included in further analysis. Molecular functions of genes were classified according to Gene Ontology.