Next Generation Sequencing Analysis of lung and spleen tissue transcriptomes from Wild Type KrasLA2 and miR-301a-/- Kras mice

Purpose: we used next generation sequencing to analyze gene expression profiles of lung and spleen tissues wild-type KrasLA2(WT-KrasLA2) and miR-301a knockout KrasLA2 (miR301aKO-KrasLA2) mice. The goals of this study are to compare the different gene expression profiles of lung tumorigenesis between WT-KrasLA2 and miR301aKO-KrasLA2 mice. Methods: WT-KrasLA2 and miR301aKO-KrasLA2 mice were sacrificed at 9 weeks and lung and spleen tissue were obtained and total RNA was extracted. mRNA profiles were generated by deep sequencing spleen tissue in single, lung tissue from WT and miR-301a KO mice in single, lung tissue from WT-KrasLA2 and miR301aKO-KrasLA2 mice in duplicate using High-seq 2000 Illumina sequencing platform. 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) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: After quality filtering of raw sequencing data, we obtained 47,865,482 (95%) out of 50,579,239 tags of lung, 34,855,916 (96%) out of 36,426,771 tags of spleen from WT-KrasLA2 mice, and 44,632,009 (95%) out of 46,771,631 tags of lung, 44,306,242 (94%) out of 47,032,715 tags of spleen from miR301aKO-KrasLA2 mice. We then mapped these tags to the mouse genome. We identified 1,641 DEGs (1,166 upregulated and 475 downregulated) in lung between WT-KrasLA2 and miR301aKO-KrasLA2 mice with a fold change more than 2.0 (up-regulation) or less than 0.5 (down-regulation). Conclusions: We identified 1166 up-regulation and 475 down-regulation differentially expressed genes (DEGs) between WT-KrasLA2 and miR301aKO-KrasLA2 mice. Immune system response and cell cycle were major pathways involved in the protection role of miR-301a deletion in lung tumorigenesis. Runx3 activation is an early event identified in miR301aKO-KrasLA2 mice compared to WT-KrasLA2 mice. Lung and spleen mRNA profiles of WT-KrasLA2 and miR301aKO-KrasLA2 mice were generated by deep sequencing, spleen tissue in single, lung tissue from WT and miR-301a KO mice in single, lung tissue from WT-KrasLA2 and miR301aKO-KrasLA2 mice in duplicate using High-seq 2000 Illumina sequencing platform.