DDX3X RNA-seq - Differential Gene Expression Analysis

DDX3XRNA helicase affects breast cancer cell cycle progression by regulating expression of KLF4 Ester Cannizzaro1, Andrew John Bannister1, Namshik Han, Andrej Alendar and Tony Kouzarides*. Gurdon Institute and Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK 1Equal contribution*Corresponding Author: [e-mail] AbstractDDX3X is a multifunctional RNA helicase with documented roles in different cancer types. Here, we demonstrate that DDX3X plays an oncogenic role in breast cancer cells by modulating the cell cycle. Depletion of DDX3X in MCF7 cells slows cell proliferation by inducing a G1 phase arrest. Notably, DDX3X inhibits expression of KLF4, a transcription factor and cell cycle repressor. Moreover, DDX3X directly interacts with KLF4mRNA and regulates its splicing. We show that DDX3X-mediated repression of KLF4 promotes expression of S-phase inducing genes in MCF7 breast cancer cells. These findings provide evidence for a novel function of DDX3X in regulating expression and downstream functions of KLF4, a master negative regulator of the cell cycle.RNAseq analysis upon DDX3X knockdown in MCF7 breast cancer cells.Total RNA was extracted (as described above) from MCF7 cells transfected with either a scrambled siRNA or DDX3X targeting siRNA (#6 or #8) and harvested 72 h after transfection. Three independent biological replicates were produced for each condition. Ribosomal RNA was depleted using a Ribo-Zero rRNA removal kit (Human/Mouse/Rat) from Illumina(R), following the manufacturer’s instructions. RNA-seq libraries were produced using NEXTfex RNA-Seq kit from Bio Scientifc, following the manufacturer’s instructions. Before multiplexing, excess primer was removed with AMPure XP beads (Beckman Coulter). Before and after multiplexing, libraries were tested for both size and quantity of DNA using a Qubit dsDNA HS assay kit and a high sensitivity D1000 ScreenTape system following the manufacturer’s instructions. For differential gene expression analysis in DDX3X knockdown MCF7 cells, trimmed reads were mapped in paired end mode to the h38 human genome using tophat with the following parameters (--no-coverage- search --max-multihits 300 --report-secondary-alignments --read- mismatches 2 --library-type fr-frststrand). Multihits (reads mapping to multi loci) were fltered, along with reads mapping with quality score less than 20. Reads were counted across gene models taken from the Ensembl v86 gtf gene model list using the summarizeOverlaps function from the GenomicAlignments package in R. The strand of each read was inverted prior to counting to account for the fact that libraries represent the frst strand of synthesised cDNA. Read counts were converted into normalised fragments per kilobase mapped (FPKM) values for quality control plots. Differential expression analysis was conducted on the raw count data using the DESeq2 package in R. P values were corrected for multiple testing using the Benjamini and Hochberg FDR correction. Signifcantly changing genes were identifed based on a fold change greater than 2-fold (up or down) and an adjusted p value less than 0.05. In addition, signifcant genes were fltered to remove genes where both the control and mutant samples had an average FPKM score less than 1.