Study of the transcriptional effect of ANO1 knock-down in two head and neck squamous cell carcinoma cell lines by RNA-sequencing of samples cultured in three dimensional collagen type I

ANO1 is located in the 11q13 region that is commonly amplified in head and neck squamous cell carcinoma. However, the exact mechanism of ANO1's involvement in oncogenesis and cancer proliferation remains unclear. In this study, we discovered novel genes which have an interplay in ANO1 cancer signalling pathways and were significantly differentially expressed upon ANO1 knockdown. All cell lines were cultured in three dimensional collagen I, a matrix that closely resembles tumor microenvironment. The aim of this study was to reveal novel mediators and pathways in ANO1 cancer cell signalling, and therefore gain insight on novel molecules that can present potent therapeutic targets. We performed RNA sequencing to identify differentially expressed genes upon ANO1 knockdown in head and neck squamous cell carcinoma (HNSCC) cell lines cultivated in three dimensional collagen type I. HNSCC cell lines used in this study harbor amplification and high expression of ANO1 gene. Each UT-SCC cell line used in the RNA sequencing of this study was transduced using as control three shScramble (shScr) lentiviral transduction replicates, as well as three replicates of shANO1 constructs, including at least once the constructs shANO1 65 and shANO1 66. Transduced cell lines were selected with puromycin. Cells were grown as inert in three dimensional collagen I cultures for up to five days until microscopicaly visible colonies have formed. RNA was extracted and further prosessed for RNA sequencing analysis. Illumina NextSeq1 x 75 bp was used.