Comprehensive Characterization Of Alternative Splicing In Renal Cell Carcinoma And Construction Of Database For Clinically Relevant Alternative Splicing In Human Cancers

Background: Alternative splicing is an important way of transcriptional or post-transcriptional regulation of gene expression.Alternative splicing abnormalities have been implicated in tumorigenesis and progression in many cancers,including renal cell carcinoma.However,the specific and systematical clinical implication and regulatory mechanism of alternative splicing in tumors such as renal cell carcinoma need to be further studied.With the popularization of next-generation sequencing,The Cancer Genome Atlas(TCGA)in the United States has implemented and opened up multi-omics sequencing data of a large number of samples in many cancers.With the development of software for alternative splicing including Splice Seq and Spl Adder and application to the TCGA transcriptomic data,research by combining alternative splicing and multi-omics data has become a possible way for understanding tumor pathogenesis and heterogeneity.Objective: In this study,we will make full use of these alternative splicing data,which contains a large number of samples,using bioinformatics analysis methods,(1)to study the clinical significance and regulatory mechanism of alternative splicing in renal cell carcinoma,and validate by molecular biology(2)to compare the differences in the analysis results of the same data by different alternative splicing software,and construct the database of alternative splicing related to clinical and pathological characteristics.Methods: Using t-distributed stochastic neighbor embedding(t-SNE)method and alternative splicing data to perform cluster analysis and molecular subtype classification of renal cell carcinoma and to systematically study the characteristics of abnormal alternative splicing and their relationship with clinicopathological features,immune heterogeneity,genetic variation,splicing factor regulation,and molecular pathway enrichment.Molecular biology experiments were used to verify the roles of cis-sequence elements and trans-acting factors in the regulation of alternative splicing in renal cell carcinoma and the regulation of tumor cell function.Compare the similarities and differences of alternative splicing data analyzed by different software,analyze their relationship with clinicopathological features,and build an open access public database Onco Splicing.Results: Clustering analysis by alternative splicing can classify RCC into molecular subtypes with distinct clinical prognostic features.The accumulation of aberrant splicing events such as intron retention and cassette exon in the subtype with reduced splicing efficiency in RCC is associated with poor clinicopathological features,suppressive immune microenvironment,and shorter overall survival in patients.Aberrant alternative splicing in tumors can affect gene expression and function by regulating isoforms switch,altering the activity of tumor-associated molecular pathways.Splice sequence features such as splice site strength,length and GC content of alternative exon are important factors affecting the balance of alternative splicing in RCC subtypes.Genomic variation in cis-sequence elements was not significantly associated with the accumulation of aberrant splicing events in advanced RCC subtype.Altered expression of splicing factors such as TIA1 and G3BP2 is one of the causes of abnormal alternative splicing in RCC,and interfering with the expression of G3BP2 can promote the proliferation and migration of cancer cells.Alternative splicing is significantly associated with a large number of molecular pathways,and their changes are jointly affected by the immune microenvironment,and the accumulation of aberrant splicing events and consequent splicing stress can also inhibit molecular pathways.Different software produce different data,especially in the detection of genome unannotated splicing events.There are also a lot of differences for splicing events between different clinicopathological features in cancers.These splicing evnets were integrated into the open database Onco Splicing(www.oncosplicing.com).Conclusion: Alternative splicing can be used as an effective indicator for molecular subtype classfication of renal cell carcinoma,a potential biomarker for predicting clinical prognosis and immunotherapy response,and an important target for targeted therapy.Expression changes of splicing factors like TIA1 or G3BP2 are responsible for regulating aberrant alternative splicing in RCC,and the inhibitory immune microenvironment promotes the accumulation of aberrant splicing events,which can induce m RNA degradation or protein dysfunction of splicing factors or tumor-related genes.