A whole genome map of alternative splicing events in precancerous lung dysplasia and adenocarcinomas of c-Raf mice

Alternative splicing analysis of laser micro-dissected lung dysplasia RNA samples Lung dysplasia is a precancerous condition with high risk of malignant transformation. Little is known about alternative spliced (AS) genes in dysplasia. We therefore investigated laser micro-dissected microscopic foci of non-contaminant dysplasia and/or lung adenocarcinoma of c-Raf transgenic mice and searched genome wide for AS genes using exon arrays. Notably, bioinformatics defined 34 and 36 AS genes in the comparison dysplasia versus transgenic unaltered and non-transgenic lung tissue while the comparison adenocarcinoma versus transgenic unaltered and non-transgenic lung tissue revealed 54 and 56 genes, respectively. So far only 6 of these were reported for lung cancer. Importantly, dysplasia related AS genes were also regulated in lung cancer to suggest a role in disease onset. Next to exon skipping/inclusion alternative splicing at the 3’ and 5’ was common and included genes of the splicing regulatory pathway. Disease dependent changes of variant transcripts were confirmed by RT-PCR while Western blotting identified alternative splicing to modulate protein levels of AS genes. For the AS genes Add3, Cast, Osbpl6, Nedd4l, Numb, Picalm and Slk transcript and protein level agreed well, whereas for Arhgef11, Clstn1, Dlg1, Dock9, Mbnl2, Mfge8, Npnt, Pdlim5, Ppp2r5c, Tjp1 notable differences in the abundance of variant transcripts were observed by RT-PCR and gel electrophoresis. Moreover, expression of individual variants differed between dysplasia and carcinoma to suggest their disease dependent regulation. Western blotting of IQGAP1, MYO6, PTPRM, RABGAP1L and RAD50 confirmed significant regulation of isoforms in lung cancer as compared to non-transgenic, transgenic unaltered and dysplastic lung tissue. For 46 AS genes expression of the non-variant protein was reported in human lung cancer (www.proteinatlas.org) and for 13 of these, cancer related AS events are known. Overall, new insight into lung dysplasia was obtained with 43 new cancer related AS genes to aid diagnosis and molecular intervention strategies. We analyzed and compared dysplastic (n=4) and adenocarcinoma (n=4) lesions and with transgenic but unaltered (n=4) and non-transgenic (n=5) lung tissue obtained from SPC/c-Raf transgenic mice with aid of laser micro dissection pressure catapulted (LMPC) technique. Dysplasia is a pre-neoplastic condition and developed at 5 month of age in the proposed mouse model. LMPC provides precise microscopic lesion collection without contamination. The RNA samples were analyzed using the Affymetrix Mouse Exon 1.0 ST platform. Alternative splicing was analyzed using Biotique XRAY tool. No technical replicates were performed.