Aberrant Alternative Splicing Events Research Articles

Nuclear pore pathology underlying multisystem proteinopathy type 3-related inclusion body myopathy.

Multisystem proteinopathy type 3 (MSP3) is an inherited, pleiotropic degenerative disorder caused by a mutation in heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), which can affect the muscle, bone, and/or nervous system. This study aimed to determine detailed histopathological features and transcriptomic profile of HNRNPA1-mutated skeletal muscles to reveal the core pathomechanism of hereditary inclusion body myopathy (hIBM), a predominant phenotype of MSP3. Histopathological analyses and RNA sequencing of HNRNPA1-mutated skeletal muscles harboring a c.940G > A (p.D314N) mutation (NM_031157) were performed, and the results were compared with those of HNRNPA1-unlinked hIBM and control muscle tissues. RNA sequencing revealed aberrant alternative splicing events that predominantly occurred in myofibril components and mitochondrial respiratory complex. Enrichment analyses identified the nuclear pore complex (NPC) and nucleocytoplasmic transport as suppressed pathways. These two pathways were linked by the hub genes NUP50, NUP98, NUP153, NUP205, and RanBP2. In immunohistochemistry, these nucleoporin proteins (NUPs) were mislocalized to the cytoplasm and aggregated mostly with TAR DNA-binding protein 43 kDa and, to a lesser extent, with hnRNPA1. Based on ultrastructural observation, irregularly shaped myonuclei with deep invaginations were frequently observed in atrophic fibers, consistent with the disorganization of NPCs. Additionally, regarding the expression profiles of overall NUPs, reduced expression of NUP98, NUP153, and RanBP2 was shared with HNRNPA1-unlinked hIBMs. The shared subset of altered NUPs in amyotrophic lateral sclerosis (ALS), as demonstrated in prior research, HNRNPA1-mutated, and HNRNPA1-unlinked hIBM muscle tissues may provide evidence regarding the underlying common nuclear pore pathology of hIBM, ALS, and MSP.

Comparative transcriptome profiling reveals RNA splicing alterations and biological function in patients exposed to occupational noise.

Genetic factors play an important role in susceptibility to noise-induced hearing loss (NIHL). Alternative splicing (AS) is an essential mechanism affecting gene expression associated with disease pathogenesis at the post-transcriptional level, but has rarely been studied in NIHL. To explore the role of AS in the development of NIHL, we performed a comprehensive analysis of RNA splicing alterations by comparing the RNA-seq data from blood samples from NIHL patients and subjects with normal hearing who were exposed to the same noise environment. A total of 356 differentially expressed genes, including 23 transcription factors, were identified between the two groups. Of particular note was the identification of 56 aberrant alternative splicing events generated by 41 differentially expressed genes between the two groups, with exon skipping events accounting for 54% of all the differentially alternative splicing (DAS) events. The results of functional enrichment analysis showed that these intersecting DAS genes and differentially expressed genes were significantly enriched in autophagy and mitochondria-related pathways. Together, our findings provide insights into the role of AS events in susceptibility and pathogenesis of NIHL.

Roles of alternative splicing in infectious diseases: from hosts, pathogens to their interactions.

Alternative splicing (AS) is an evolutionarily conserved mechanism that removes introns and ligates exons to generate mature messenger RNAs (mRNAs), extremely improving the richness of transcriptome and proteome. Both mammal hosts and pathogens require AS to maintain their life activities, and inherent physiological heterogeneity between mammals and pathogens makes them adopt different ways to perform AS. Mammals and fungi conduct a two-step transesterification reaction by spliceosomes to splice each individual mRNA (named cis -splicing). Parasites also use spliceosomes to splice, but this splicing can occur among different mRNAs (named trans -splicing). Bacteria and viruses directly hijack the host's splicing machinery to accomplish this process. Infection-related changes are reflected in the spliceosome behaviors and the characteristics of various splicing regulators (abundance, modification, distribution, movement speed, and conformation), which further radiate to alterations in the global splicing profiles. Genes with splicing changes are enriched in immune-, growth-, or metabolism-related pathways, highlighting approaches through which hosts crosstalk with pathogens. Based on these infection-specific regulators or AS events, several targeted agents have been developed to fight against pathogens. Here, we summarized recent findings in the field of infection-related splicing, including splicing mechanisms of pathogens and hosts, splicing regulation and aberrant AS events, as well as emerging targeted drugs. We aimed to systemically decode host-pathogen interactions from a perspective of splicing. We further discussed the current strategies of drug development, detection methods, analysis algorithms, and database construction, facilitating the annotation of infection-related splicing and the integration of AS with disease phenotype.

Alternative splicing of PSMD13 mediated by genetic variants is significantly associated with endometrial cancer risk.

Accumulating evidence has shown that aberrant alternative splicing events are closely associated with the onset and development of cancer. However, whether genetic variants-associated alternative splicing is linked to risk of endometrial cancer remains largely uncertain. We identified single nucleotide polymorphisms (SNPs) locates in the splicing number trait locus (sQTL) of endometrial cancer using the CancerSplicing QTL database. In parallel with bioinformatics analysis, we conducted a case-control study comprising 2,000 cases and 2,013 controls to assess the association between identified SNP which possesses mRNA splicing function and endometrial cancer susceptibility. Furthermore, we used the Kaplan-Meier Plotter, The Human Protein Atlas, SPNR, and Spliceman2 databases for sQTL and differential gene expression analyses to identify the genetic variant which most potentially influence the risk of endometrial cancer through alternative splicing to reveal the potential mechanism by which candidate SNPs regulate the risk of endometrial cancer. The results indicated that SNP rs7128029 A<G was significantly associated with an increased risk of endometrial cancer (odds ratio=1.384; 95% confidence interval=1.038-1.964). Moreover, the carcinogenic effect of SNP rs7128029 A<G was consistently revealed by propensity matching analysis, an additive model, and a dominant model. Importantly, sQTL analysis showed that SNP rs7128029 could affect the transcriptional modification of PSMD13 via regulating the exon skipping of PSMD13. When the rs7128029 allele was mutated from A to G, the expression of exon 2 of PSMD13 was markedly lower (p<0.001). Furthermore, compared with participants who had higher PSMD13 expression, those who had lower PSMD13 expression had shorter survival times. These findings suggest that SNP rs7128029-mediated alternative splicing events in PSMD13 are associated with endometrial cancer risk and may be a potential early screening biomarker for endometrial cancer-susceptible populations.

Knowledge mapping of alternative splicing of cancer from 2012 to 2021: A bibliometric analysis.

As a processing method of RNA precursors, alternative splicing (AS) is critical to normal cellular activities. Aberrant AS events are associated with cancer development and can be promising targets to treat cancer. However, no detailed and unbiased study describes the current state of AS of cancer research. We aim to measure and recognize the current state and trends of AS cancer research in this study. The Web of Science Core Collection was used to acquire the articles. Utilizing three bibliometric tools (CiteSpace, VOSviewer, R-bibliometrix), we were able to measure and recognize the influence and collaboration data of individual articles, journals, and co-citations. Analysis of co-occurrence and burst information helped us identify the trending research areas related to AS of cancer. From 2012 to 2021, the total number of papers on AS of cancer published in 766 academic journals was 3,507, authored by 20,406 researchers in 405 institutions from 80 countries/regions. Research involving AS of cancer genes was primarily conducted in the United States and China; simultaneously, the Chinese Academy of Sciences, Fudan University, and National Cancer Institute were the institutions with strong research capabilities. Scorilas Andreas is the scholar with the most publications, while the most co-citations were generated by Wang, Eric T. Plos One published the most papers on AS of cancer, while J Biol Chem was the most co-cited academic journal in this field. The results of keyword co-occurrence analysis can be divided into three types: molecular (P53, CD44, androgen receptor, srsf3, esrp1), pathological process (apoptosis, EMT, metastasis, angiogenesis, proliferation), and disease (breast cancer, colorectal cancer, prostate cancer, hepatocellular carcinoma, gastric cancer). Research on AS of cancer has been increasing in intensity over the past decade. Current AS of cancer studies focused on the hallmarks of AS in cancer and AS signatures including diagnostic and therapeutic targets. Among them, the current trends are splicing factors regulating epithelial-mesenchymal transition and other hallmarks, aberrant splicing events in tumors, and further mechanisms. These might give researchers interested in this field a forward-looking perspective and inform further research.

The rice blast fungus SR protein 1 regulates alternative splicing with unique mechanisms.

Serine/arginine-rich (SR) proteins are well known as splicing factors in humans, model animals and plants. However, they are largely unknown in regulating pre-mRNA splicing of filamentous fungi. Here we report that the SR protein MoSrp1 enhances and suppresses alternative splicing in a model fungal plant pathogen Magnaporthe oryzae. Deletion of MoSRP1 caused multiple defects, including reduced virulence and thousands of aberrant alternative splicing events in mycelia, most of which were suppressed or enhanced intron splicing. A GUAG consensus bound by MoSrp1 was identified in more than 94% of the intron or/and proximate exons having the aberrant splicing. The dual functions of regulating alternative splicing of MoSrp1 were exemplified in enhancing and suppressing the consensus-mediated efficient splicing of the introns in MoATF1 and MoMTP1, respectively, which both were important for mycelial growth, conidiation, and virulence. Interestingly, MoSrp1 had a conserved sumoylation site that was essential to nuclear localization and enhancing GUAG binding. Further, we showed that MoSrp1 interacted with a splicing factor and two components of the exon-joining complex via its N-terminal RNA recognition domain, which was required to regulate mycelial growth, development and virulence. In contrast, the C-terminus was important only for virulence and stress responses but not for mycelial growth and development. In addition, only orthologues from Pezizomycotina species could completely rescue defects of the deletion mutants. This study reveals that the fungal conserved SR protein Srp1 regulates alternative splicing in a unique manner.

Multi-omics analysis reveals RNA splicing alterations and their biological and clinical implications in lung adenocarcinoma

Alternative RNA splicing is one of the most important mechanisms of posttranscriptional gene regulation, which contributes to protein diversity in eukaryotes. It is well known that RNA splicing dysregulation is a critical mechanism in tumor pathogenesis and the rationale for the promising splice-switching therapeutics for cancer treatment. Although we have a comprehensive understanding of DNA mutations, abnormal gene expression profiles, epigenomics, and proteomics in lung adenocarcinoma (LUAD), little is known about its aberrant alternative splicing profiles. Here, based on the multi-omics data generated from over 1000 samples, we systematically studied the RNA splicing alterations in LUAD and revealed their biological and clinical implications. We identified 3688 aberrant alternative splicing events (AASEs) in LUAD, most of which were alternative promoter and exon skip. The specific regulatory roles of RNA binding proteins, somatic mutations, and DNA methylations on AASEs were comprehensively interrogated. We dissected the functional implications of AASEs and concluded that AASEs mainly affected biological processes related to tumor proliferation and metastasis. We also found that one subtype of LUAD with a particular AASEs pattern was immunogenic and had a better prognosis and response rate to immunotherapy. These findings revealed novel events related to tumorigenesis and tumor immune microenvironment and laid the foundation for the development of splice-switching therapies for LUAD.

Severe choline deficiency induces alternative splicing aberrance in optimized duck primary hepatocyte cultures.

Choline deficiency, one main trigger for nonalcoholic fatty liver disease (NAFLD), is closely related to lipid metabolism disorder. Previous study in a choline-deficient model has largely focused on gene expression rather than gene structure, especially sparse are studies regarding to alternative splicing (AS). In modern life science research, primary hepatocytes culture technology facilitates such studies, which can accurately imitate liver activity in vitro and show unique superiority. Whereas limitations to traditional hepatocytes culture technology exist in terms of efficiency and operability. This study pursued an optimization culture method for duck primary hepatocytes to explore AS in choline-deficient model. We performed an optimization culture method for duck primary hepatocytes with multi-step digestion procedure from Pekin duck embryos. Subsequently a NAFLD model was constructed with choline-free medium. RNA-seq and further analysis by rMATS were performed to identify AS events alterations in choline-deficency duck primary hepatocytes. The results showed E13 (embryonic day 13) to E15 is suitable to obtain hepatocytes, and the viability reached over 95% by trypan blue exclusion assay. Primary hepatocyte retained their biological function as well identified by Periodic Acid-Schiff staining method and Glucose-6-phosphate dehydrogenase activity assay, respectively. Meanwhile, genes of alb and afp and specific protein of albumin were detected to verify cultured hepatocytes. Immunofluorescence was used to evaluate purity of hepatocytes, presenting up to 90%. On this base, choline-deficient model was constructed and displayed significantly increase of intracellular triglyceride and cholesterol as reported previously. Intriguingly, our data suggested that AS events in choline-deficient model were implicated in pivotal biological processes as an aberrant transcriptional regulator, of which 16 genes were involved in lipid metabolism and highly enriched in glycerophospholipid metabolism. An effective and rapid protocol for obtaining duck primary hepatocytes was established, by which our findings manifested choline deficiency could induce the accumulation of lipid and result in aberrant AS events in hepatocytes, providing a novel insight into various AS in the metabolism role of choline.

Modulation of RNA Splicing by Oligonucleotides: Mechanisms of Action and Therapeutic Implications.

Dysregulation of RNA splicing causes many diseases and disorders. Several therapeutic approaches have been developed to correct aberrant alternative splicing events for the treatment of cancers and hereditary diseases, including gene therapy and redirecting splicing, using small molecules or splice switching oligonucleotides (SSO). Significant advances in the chemistry and pharmacology of nucleic acid have led to the development of clinically approved SSO drugs for the treatment of spinal muscular dystrophy and Duchenne muscular dystrophy (DMD). In this review, we discuss the mechanisms of SSO action with emphasis on "less common" approaches to modulate alternative splicing, including bipartite and bifunctional SSO, oligonucleotide decoys for splice factors and SSO-mediated mRNA degradation via AS-NMD and NGD pathways. We briefly discuss the current progress and future perspectives of SSO therapy for rare and ultrarare diseases.

The Catastrophic HPV/HIV Dual Viral Oncogenomics in Concert with Dysregulated Alternative Splicing in Cervical Cancer.

Cervical cancer is a public health problem and has devastating effects in low-to-middle-income countries (LTMICs) such as the sub-Saharan African (SSA) countries. Infection by the human papillomavirus (HPV) is the main cause of cervical cancer. HIV positive women have higher HPV prevalence and cervical cancer incidence than their HIV negative counterparts do. Concurrent HPV/HIV infection is catastrophic, particularly to African women due to the high prevalence of HIV infections. Although various studies show a relationship between HPV, HIV and cervical cancer, there is still a gap in the knowledge concerning the precise nature of this tripartite association. Firstly, most studies show the relationship between HPV and cervical cancer at genomic and epigenetic levels, while the transcriptomic landscape of this relationship remains to be elucidated. Even though many studies have shown HPV/HIV dual viral pathogenesis, the dual molecular oncoviral effects on the development of cervical cancer remains largely uncertain. Furthermore, the effect of highly active antiretroviral therapy (HAART) on the cellular splicing machinery is unclear. Emerging evidence indicates the vital role played by host splicing events in both HPV and HIV infection in the development and progression to cervical cancer. Therefore, decoding the transcriptome landscape of this tripartite relationship holds promising therapeutic potential. This review will focus on the link between cellular splicing machinery, HPV, HIV infection and the aberrant alternative splicing events that take place in HIV/HPV-associated cervical cancer. Finally, we will investigate how these aberrant splicing events can be targeted for the development of new therapeutic strategies against HPV/HIV-associated cervical cancer.

Alternative Splicing: A New Cause and Potential Therapeutic Target in Autoimmune Disease.

Alternative splicing (AS) is a complex coordinated transcriptional regulatory mechanism. It affects nearly 95% of all protein-coding genes and occurs in nearly all human organs. Aberrant alternative splicing can lead to various neurological diseases and cancers and is responsible for aging, infection, inflammation, immune and metabolic disorders, and so on. Though aberrant alternative splicing events and their regulatory mechanisms are widely recognized, the association between autoimmune disease and alternative splicing has not been extensively examined. Autoimmune diseases are characterized by the loss of tolerance of the immune system towards self-antigens and organ-specific or systemic inflammation and subsequent tissue damage. In the present review, we summarized the most recent reports on splicing events that occur in the immunopathogenesis of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) and attempted to clarify the role that splicing events play in regulating autoimmune disease progression. We also identified the changes that occur in splicing factor expression. The foregoing information might improve our understanding of autoimmune diseases and help develop new diagnostic and therapeutic tools for them.

TF-RBP-AS Triplet Analysis Reveals the Mechanisms of Aberrant Alternative Splicing Events in Kidney Cancer: Implications for Their Possible Clinical Use as Prognostic and Therapeutic Biomarkers.

Aberrant alternative splicing (AS) is increasingly linked to cancer; however, how AS contributes to cancer development still remains largely unknown. AS events (ASEs) are largely regulated by RNA-binding proteins (RBPs) whose ability can be modulated by a variety of genetic and epigenetic mechanisms. In this study, we used a computational framework to investigate the roles of transcription factors (TFs) on regulating RBP-AS interactions. A total of 6519 TF–RBP–AS triplets were identified, including 290 TFs, 175 RBPs, and 16 ASEs from TCGA–KIRC RNA sequencing data. TF function categories were defined according to correlation changes between RBP expression and their targeted ASEs. The results suggested that most TFs affected multiple targets, and six different classes of TF-mediated transcriptional dysregulations were identified. Then, regulatory networks were constructed for TF–RBP–AS triplets. Further pathway-enrichment analysis showed that these TFs and RBPs involved in triplets were enriched in a variety of pathways that were associated with cancer development and progression. Survival analysis showed that some triplets were highly associated with survival rates. These findings demonstrated that the integration of TFs into alternative splicing regulatory networks can help us in understanding the roles of alternative splicing in cancer.

Integrative analysis reveals the prognostic value and functions of splicing factors implicated in hepatocellular carcinoma

Splicing factors (SFs) play critical roles in the pathogenesis of various cancers through regulating tumor-associated alternative splicing (AS) events. However, the clinical value and biological functions of SFs in hepatocellular carcinoma (HCC) remain obscure. In this study, we identified 40 dysregulated SFs in HCC and established a prognostic model composed of four SFs (DNAJC6, ZC3H13, IGF2BP3, DDX19B). The predictive efficiency and independence of the prognostic model were confirmed to be satisfactory. Gene Set Enrichment Analysis (GSEA) illustrated the risk score calculated by our prognostic model was significantly associated with multiple cancer-related pathways and metabolic processes. Furthermore, we constructed the SFs-AS events regulatory network and extracted 108 protein-coding genes from the network for following functional explorations. Protein–protein interaction (PPI) network delineated the potential interactions among these 108 protein-coding genes. GO and KEGG pathway analyses investigated ontology gene sets and canonical pathways enriched by these 108 protein-coding genes. Overlapping the results of GSEA and KEGG, seven pathways were identified to be potential pathways regulated by our prognostic model through triggering aberrant AS events in HCC. In conclusion, the present study established an effective prognostic model based on SFs for HCC patients. Functional explorations of SFs and SFs-associated AS events provided directions to explore biological functions and mechanisms of SFs in HCC tumorigenesis.

Identification of survival-related alternative splicing signatures in acute myeloid leukemia.

Aberrant RNA alternative splicing (AS) variants play critical roles in tumorigenesis and prognosis in human cancers. Here, we conducted a comprehensive profiling of aberrant AS events in acute myeloid leukemia (AML). RNA AS profile, including seven AS types, and the percent spliced in (PSI) value for each patient were generated by SpliceSeq using RNA-seq data from TCGA. Univariate followed by multivariate Cox regression analysis were used to identify survival-related AS events and develop the AS signatures. A nomogram was developed, and its predictive efficacy was assessed. About 27,892 AS events and 3,178 events were associated with overall survival (OS) after strict filtering. Parent genes of survival-associated AS events were mainly enriched in leukemia-associated processes including chromatin modification, autophagy, and T-cell receptor signaling pathway. The 10 AS signature based on seven types of AS events showed better efficacy in predicting OS of patients than those built on a single AS event type. The area under curve (AUC) value of the 10 AS signature for 3-year OS was 0.91. Gene set enrichment analysis (GSEA) confirmed that these survival-related AS events contribute to AML progression. Moreover, the nomogram showed good predictive performance for patient's prognosis. Finally, the correlation network of AS variants with splicing factor genes found potential important regulatory genes in AML. The present study presented a systematic analysis of survival-related AS events and developed AS signatures for predicting the patient’s survival. Further studies are needed to validate the signatures in independent AML cohorts and might provide a promising perspective for developing therapeutic targets.

The Clinical and Biological Implications of Aberrant Alternative Splicing in Patients with De Novo, Non-M3 Acute Myeloid Leukemia

BackgroundAberrant alternative splicing (AS) were reported important in the pathogenesis and prognosis of acute myeloid leukemia (AML). Previous studies reported inferior outcomes in AML patients carrying mutations in genes encoding RNA-splicing proteins, which accounted for 18% of AML patients. However, whether aberrant AS is correlated with these mutations or carried clinical and biological significances in AML patients remain unknown. In this study, we aimed to answer these questions by a set of array data from a large cohort.Materials and MethodsA total of 171 newly diagnosed non-M3 AML patients, who received standard induction chemotherapy and had complete clinical and genetic information were enrolled. There were 20 healthy marrow donors for comparison. We extracted RNA from mononuclear cells (MNC) in the bone marrow (BM), followed by hybridization on the microarrays of Affymetrix Human Transcriptome Array 2.0, which contained >6 million distinct probes covering 44710 coding, 22829 non-coding transcripts, and exon-exon junctions. This comprehensive coverage facilitated investigation of the global splicing patterns. The splicing index (SI) of a probe in a gene was derived from the ratio between AML patients and normal controls in terms of the probe intensity divided by the gene expression level, i.e., SI = [(intensity of probe A in gene A) / (gene A expression level)] of AML patient / [(intensity of probe A in gene A) / (gene A expression level)] of normal control. When the value is less than 1, SI is defined as the negative reciprocal. SI had to be less than -2 or more than 2 to be denoted as an aberrant AS event. A gene with at least one aberrant AS event would be called an aberrant AS gene. SI values were log2 transformed and analyzed for the association with overall survival (OS) by the univariable Cox proportional hazards model. Functional annotation for significant aberrant AS events was performed by the Database for Annotation, Visualization and Integrated Discovery (DAVID) web tool with default settings. The aberrant AS events were then merged into a Least Absolute Shrinkage and Selection Operator (LASSO) regression, which identified the best subset of aberrant AS events for the prediction of OS. We adopted a leave-one-out cross validation scheme to identify the subset with the best performance. A beta value was assigned to each of the selected aberrant AS events by LASSO. Based on the results, we constructed a scoring system by a weighted sum of the selected features, where the weights were LASSO beta values. The scoring system was applied to predict OS and relapse-free survival (RFS).ResultsAt first, univariable Cox analysis identified 95 OS-associated aberrant AS events (P<0.01; n=171) in 69 coding and 6 non-coding genes. A DAVID analysis was performed to explore the functional relevance of these events. Of note, 41 of the involved coding genes were known involved in RNA splicing pathways (P=0.049). Furthermore, the aberrant AS genes were associated with AMPK signaling pathway (P=0.007), PI3K-Akt signaling pathway (P=0.025), and HIF-1 signaling pathway (P=0.042). We used a LASSO regression model to identify the subset of aberrant AS events that best predicted OS by a leave-one-out cross validation test. As a result, a simple scoring system of 9 aberrant AS events was developed. The system was composed of a weighted sum of aberrant AS events on APP, CD93, CT45A3, CUX2, PIK3R3, RORA, SCRN1, and ZSCAN31, and a non-coding transcript. A high score was predictive of inferior OS (Figure 1A; P<0.001; median survival, 18.0 and 108.1 months) and RFS (Figure 1B; P=0.005; median survival, 10.2 vs. 88.8 months; n=137). Patients with detectable mutations in spliceosome (n=16) did not have a higher aberrant AS score (P=0.589). Age, hemogram, cytogenetics, and FAB classification were not correlated with aberrant AS score, either. However, a higher score was predictive of lower complete remission rate (P=0.01), a higher relapse rate (P=0.006), and higher incidence of FLT-3 -ITD. In addition, multivariate analyses revealed a higher aberrant AS score as an unfavorable prognostic factor for OS and RFS (both P< 0.001) independent of age, WBC counts, cytogenetics, genetic mutations of NPM1 and CEBPA and FLT-3 -ITD (Table 1).We present the clinical significance and prognostic impact of an aberrant AS scoring system in de novo, non-M3 AML patients. Large prospective cohorts are needed to confirm our observations. [Display omitted] DisclosuresTang:Celgene International Sàrl: Research Funding. Yao:Celgene International Sàrl: Research Funding. Li:Celgene International Sàrl: Research Funding. Huang:Celgene International Sàrl: Research Funding. Ko:Celgene International Sàrl: Research Funding. Hsu:Celgene International Sàrl: Research Funding. Lin:Celgene International Sàrl: Research Funding. Wu:Celgene International Sàrl: Research Funding. Tsai:Celgene International Sàrl: Research Funding. Tien:Celgene International Sàrl: Research Funding.

The landscape and biological relevance of aberrant alternative splicing events in esophageal squamous cell carcinoma.

Aberrant alternative splicing events (AASEs) are key biological processes for tumorigenesis and the rationale for designing splice-switching oligonucleotides (SSOs). However, the landscape of AASEs in esophageal squamous cell carcinoma (ESCC) remains unclear, which undermines the development of SSOs for ESCC. Here, we profiled AASEs based on 125 pairs of RNA-seq libraries. We identified 14,710 AASEs in ESCC, most of which (92.67%) affected coding genes. The first exon of transcripts was frequently changed in ESCC. We constructed a regulatory network where 74 RNA-binding proteins regulated 2142 AASEs. This network was enriched in apoptotic pathways and various adhesion/junction-related processes. Somatic mutations in ESCC regulating ASEs were mainly through trans-regulatory mode and were enriched in intron regions. Isoform switches of apoptotic genes and binding genes both tended to induce "noncoding transcripts" and "domain loss," disrupting the apoptotic and Hippo signaling pathways. All ESCC samples were grouped into three clusters with different AASEs patterns and the second cluster was identified as "cold tumor," with a low abundance of immune cells, activated immune pathways, and immunomodulators. Our work comprehensively profiled the landscape of AASEs in ESCC, revealed novel AASEs related to tumorigenesis and immune microenvironment, and suggested promising directions for designing SSOs for ESCC.

Identification of prognostic alternative splicing signatures in uveal melanoma.

Alternative splicing (AS) events were reportedly associated with the development of multiple cancers. The study was designed to provide a comprehensive analysis of AS events and explore their potential prognostic value in uveal melanoma (UM). The prognostic AS events, identified based on the data of 80 UM patients obtained from The Cancer Genome Atlas, were further screened and analyzed for construction of prognostic signatures by using LASSO regression and multivariate Cox model. Kaplan-Meier survival analysis was used to evaluate the prognostic value. The AS events-related functional pathways were explored by gene set enrichment analysis (GSEA). The difference between two subgroups in terms of treatment options was investigated. The regulatory network between prognostic AS events and splicing factors (SFs) was then constructed. A total of 1014 AS events were identified as prognostic AS events. Five prognostic AS events were involved in the construction of prognostic signatures, including AKAP2/87175/AP, RGMA/32575/ES, DNASE1L1/90581/ES, BIN1/55198/ES and ERCC2/50430/AT. UM patients were then divided into two subgroups. Prognostic AS signatures had an excellent performance in predicting the survival of UM patients, with an area under curve (AUC) of 0.962. GSEA results suggested several splicing-associated mechanisms, including cellular metabolic process and apoptosis. Low-risk subgroup could be more sensitive to drugs. A higher expression of immune checkpoint genes was observed in high-risk group than in low-risk group. SFs-AS regulatory network also revealed significant association between AS events and SFs. Aberrant AS events in UM patients might serve as prognostic predictors.

Signature of gene aberrant alternative splicing events in pancreatic adenocarcinoma prognosis.

Alternative splicing (AS), as an effective and universal mechanism of transcriptional regulation, is involved in the development and progression of cancer. Therefore, systematic analysis of alternative splicing in pancreatic adenocarcinoma (PAAD) is warranted. The corresponding clinical information of the RNA-Seq data and PAAD cohort was downloaded from the TCGA data portal. Then, a java application, SpliceSeq, was used to evaluate the RNA splicing pattern and calculate the splicing percentage index (PSI). Differentially expressed AS events (DEAS) were identified based on PSI values between PAAD cancer samples and normal samples of adjacent tissues. Kaplan-Meier and Cox regression analyses were used to assess the association between DEAS and patient clinical characteristics. Unsupervised cluster analysis used to reveal four clusters with different survival patterns. At the same time, GEO and TCGA combined with GTEx to verify the differential expression of AS gene and splicing factor. After rigorous filtering, a total of 45,313 AS events were identified, 1,546 of which were differentially expressed AS events. Nineteen DEAS were found to be associated with OS with a five-year overall survival rate of 0.946. And the subtype clusters results indicate that there are differences in the nature of individual AS that affect clinical outcomes. Results also identified 15 splicing factors associated with the prognosis of PAAD. And the splicing factors ESRP1 and RBM5 played an important role in the PAAD-associated AS events. The PAAD-associated AS events, splicing networks, and clusters identified in this study are valuable for deciphering the underlying mechanisms of AS in PAAD and may facilitate the establishment of therapeutic goals for further validation.

Prognostic significance of survival-associated alternative splicing events in gastric cancer.

Alternative splicing events are a major source of transcript and protein diversity in eukaryotes. Aberrant alternative splicing events have been increasingly reported in various cancers, including gastric cancer. To further explore the prognostic significance of alternative splicing events in gastric cancer patients, a comprehensive and systematic investigation was conducted by integrating alternative splicing event data and clinical information. Univariate Cox regression analysis identified 1383 alternative splicing events to be significantly associated with the overall survival of gastric cancer patients. Then, least absolute shrinkage and selection operator (LASSO) and multivariate Cox analyses were performed for the development of prognostic signatures. The final prognostic signature based on all seven types of alternative splicing events can act as an independent prognostic indicator after multivariate adjustment of several clinical parameters. Furthermore, the correlation and function analysis identified CELF2, BAG2, RBFOX2, PTBP2 and QKI as hub splicing factors, and the focal adhesion signaling pathway was most significantly correlated with survival-associated alternative splicing events. The results of this study may establish a foundation for further research investigating the underlying mechanism of alternative splicing events in the progression of gastric cancer.

The Mutational and Transcriptional Landscapes of Hepatocarcinogenesis in a Rat Model.

SummaryHepatocellular carcinoma (HCC) initiation is characterized by stepwise accumulation of molecular alterations, during which the early events are largely unknown. Here, we presented a comprehensive genomic and transcriptomic landscape at stages of hepatitis, cirrhosis, and HCC by using a diethylnitrosamine-induced rat HCC model. We observed the early occurrence of gene instability and aberrant cancer associated signaling pathways in liver hepatitis. We further characterized the progressive molecular changes during hepatocarcinogenesis, wherein the intense rivalry between tumor-suppressive and oncogenic strengths occurred in cirrhosis stage. Despite the significant pathological difference, mutation signatures and expression landscape are highly similar between hepatitis and cirrhosis stages. Furthermore, we identified PI3K-Akt signaling pathway as a key pathway in the process of hepatocarcinogenesis through integrative analysis, and PIK3CD is a potential biomarker indicating HCC recurrence. The dynamic immune response during hepatocarcinogenesis, such as continuous decline of monocytes, suggests an immunological intervention strategy beyond chemoprevention for liver cancer.