Inactivation of Par3/Hippo pathway promotes prostatic symmetric cell division and tumorigenesis

Purpose: mRNA sequencing has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare mRNA level of WT and Par3KO derived prostate tissues Methods: prostate mRNA profiles of 8-week-old wild-type (WT) and Par3 knockout (Par3-/-) mice were generated by sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA). Results: Using an optimized data analysis method, we mapped about 20 million sequence reads per sample to the mouse genome and identified 12,014 transcripts in the prostate of WT and Par3-/- mice with BWA workflow and 24,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 132 known housekeeping genes, and 120 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than three orders of magnitude and a goodness of fit (R2) of 0.9120. Approximately 10% of the transcripts showed differential expression between the WT and Par3-/- retina, with a fold change =2.0 and p value <0.05. Altered expression of 32 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Conclusions: Our results reveal that Par3 KO promotes prostatic epithelium proliferation. Overall design: prostate mRNA profiles of 8-week old wild type (WT) and Par3-/- mice were generated by deep sequencing, in triplicate, using Illumina GAIIx.