Identification of Wnt2 targets in dendritic cell

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to identify the key moleculars or pathways which are changed in dendritic cells undergoing the treatment of CAF-secreted Wnt2. Methods: mRNA profiles of mouse dendritic cells (mDCs) with treatment of Wnt2, CAF culture medium (CAF.CM), or CAF culture medium + Wnt2 Ab(CAF.CM+Wnt2 Ab), were generated by deep sequencing, in triplicate, using Illumina. 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. The overlap changes of mRNA profiles in Wnt2-treated mDCs and CAF.CM-treated mDCs compared with control mDCs, revealed the possible changed cellular pathways induced by CAF-secreted Wnt2. The analysis of the mRNA profiles which were changed after the blocking of Wnt2 activities in CAF.CM would be used to comfirmed the changed cellular pathways induced by CAF-secreted Wnt2. Results: Using an optimized data analysis workflow, we found that 768 transcripts and 2068 transcripts were significantly changed in Wnt2-treated DCs and CAF.CM-treated respectively compared with untreated DCs. And 393 changed transcripts overlapped in Wnt2-treated DCs and CAF.CM-treated compared with untreated DCs. Moreover, treatment of CAF.CM with Wnt2 Ab induced differential expression of 497 genes. Conclusions: Our study represents the first detailed analysis of Wnt2-induced changes of DC transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of Wnt2- or CAF.CM- induecd expression profiles. Overall design: mRNA profiles of mouse dendritic cells (mDCs) with treatment of Wnt2, CAF culture medium (CAF.CM), or CAF culture medium + Wnt2 Ab(CAF.CM+Wnt2 Ab), were generated by deep sequencing, in triplicate, using Illumina.