Dysregulation of mRNA splicing can cause human disease, and more and more studies showed that targeting alternative splicing could lead to the development of novel therapeutics . To date, accumulated data have already highlighted the crucial roles of AS in many human diseases, including cancer, neurodegenerative disorders, immune and infectious diseases [33–35]. Herein, we presented a comprehensive analysis of AS events in CD using a well-established public dataset and a validation dataset from sequencing a Chinese cohort. In our study, we found SE is the most significantly enriched AS event in CD. This suggested that the CD patients’ transcriptome is much more complex and chaotic compared to the healthy controls, due to a more sophisticated mRNA isoform system. The PCA plot also demonstrated that even if only using different AS events as the principal components, we were still able to differentiate the CD group and control group, suggesting that the mRNA AS event itself, other than the expression level, could be a solid comprehensive signature for CD patients. All these results proved our hypothesis that dysregulated AS events may contribute to the etiology of CD.
Among the AS event related significantly enriched pathways, “Lysine degradation” pathway is the most enriched pathway. EZH1 and EZH2 are two genes involved in this pathway, which have been previously linked to the progression of IBD [36, 37]. They both have dysregulated SE events in CD patients. “Sphingolipids metabolism” is the most significantly enriched pathways in A3SS events. Interestingly, sphingolipids have recently recognized as mediators of inflammation and potential therapeutic targets for IBDs . Our results suggested that metabolism of sphingolipids in CD patients may differ from the healthy people due to AS, resulting in a dysregulated sphingolipid pool. Moreover, the three most dysregulated genes in inflammatory response related pathways were all previously linked to IBD. IRF1, as an interferon regulatory factor, has been correlated with CD activity index and CD endoscopic index of severity . In a cohort study, IRF1 has a 72% increase in gene expression among CD patients compared to the controls . STAT3, as a central component in immune response signal transduction, if has gain-of-function mutation, will cause multi-organ early auto-immune diseases . Notably, STAT3 has also been indicated as one of the crucial targets for treating IBDs, although activation of STAT3 is likely to occur in both innate and acquired cell types . MAOA, as a monoamine oxidase, though is not directly associated with IBDs, is highly related with inducing oxidative stress in obese people who are suffering chronic inflammation . Recently, dysregulation of MAOA has also been linked to specific microbe alteration . In conclusion, both top AS related significantly enriched pathways and significantly dysregulated genes are highly related with IBD and CD. Our results provided new insights that post-transcriptional regulation could also contribute to IBD via regulating these key pathways and genes.
Although our study provided a comprehensive AS analysis on colon tissues from CD patients, and also have identified nearly 3,000 unique AS events in CD patients, our studies still have certain limitations. In the first place, although we validated the AS events of STAT3 and IRF1 genes, the experimental validation of discovered AS events in many other genes is still lacking. Moreover, whether these AS events affect protein diversity also requires experimental validation. The validation studies should include examining the protein level of these AS regulated genes, and the detecting the downstream effect on certain signaling transduction pathways. Finally, even if we have detected several splicing factors were dysregulated in CD patients, a RIP-seq or CLIP-seq study should be performed to further detect the responsible splicing factors, RNA binding proteins for CD related AS events. Nonetheless, our study demonstrated a new potential pathophysiology of CDs which is regulated by AS. Our study also presented the first landscape of AS in CD patients. Our data also suggested that drugs that target AS related genes and splicing factors should be considered for further screening for CD patients.