Next Generation Sequencing Facilitates Quantitative Analysis of Control siRNA, RAB11A siRNA1 and RAB11A siRNA2 suppressed EBC-1 cell's Transcriptomes

This study aims to find cellular pathway changes in RAB11A suppressed in lung squamous cell carcinoma cell line EBC-1 compared to control and to evaluate downregulating genes by RAB11A suppression by siRNA. Total RNA was extracted from EBC-1 transfected with control siRNA and RAB11A specific siRNAs using the RNeasy mini kit (Qiagen, Hilden, Germany). CAGE library preparation, sequencing, mapping, and gene expression and motif discovery analysis were performed by DNAFORM (Yokohama, Japan). In brief, RNA quality was assessed by Bioanalyzer (Agilent) to ensure that RIN (RNA integrity number) is over 7.0 and A260/280 and 260/230 ratios are over 1.7. First-strand cDNAs were transcribed to the 5'end of capped RNAs, attached to CAGE “bar code” tags, and the sequenced CAGE tags were mapped to human hg19 genomes using BWA software (v0.5.9) after discarding ribosomal or non-A/C/G/T base-containing RNAs. For tag clustering, CAGE-tag 5' coordinates were input for CAGEr clustering using Paraclu algorithm with default parameters. Using CAGE analysis, we could validate the downregulation of FGF/FGFR signal-related genes in RAB11A-suppressed EBC-1 cells compared to the control cells. Finally, this study showed that RAB11 was related to tumor aggressiveness and growth in vitro and in vivo via activation of FGFR signals. Overall design: Comparison of gene expression of lung squamous cell carcinoma cell line EBC-1 with Control siRNA, RAB11A siRNA1 and RAB11A siRNA2