Changes And Roles Of RNA Alternative Splicing In Viral Infectious Diseases

Background:Alternative splicing(AS),also known as variable splicing,is a process of generating multiple m RNA splicing isomers by selecting different splicing site combinations to express different proteins.The disorder of human AS is closely related to the development of various complex diseases.Viral infection is the inducement of many diseases.They perform self-replication as well as escape from the host antiviral response by manipulating the host’s AS.However,the changes of host AS caused by novel coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)are not well understood.In addition,hepatitis B virus(HBV)infection is one of the main causes of hepatocellular carcinoma(HCC).It is worth noting that there is a high splicing difference between the tumor tissues of patients with hepatitis B virus associated hepatocellular carcinoma(HBV-HCC)and normal liver tissues,and the role of these changes in the pathogenesis of HCC is still unclear.Therefore,systematic analysis of the impact of viral infection on host AS events is expected to provide potential biomarkers for screening and monitoring COVID-19 and the occurrence and development of HCC.Objective:To systematically analyze the AS and transcript isomer changes in patients with COVID-19 or HBV-HCC,to explore the association of AS changes with host clinical features,thereby providing a biological target for screening and treatment of viral infectious diseases.Methods : Transcriptome sequencing and liquid chromatography tandem mass spectrometry were used to perform GO and KEGG functional enrichment analysis of differentially expressed proteins,genes and dysregulated transcripts.CIBERSORTx software was used to calculate the proportion of tumor infiltrating immune cells.The RMATS software was used to analyze the differential AS events,and the differential transcript isoform changes were verified by semi quantitative RT-PCR experiments.Swiss Dock simulates the binding of drug target genes of different subtypes to small molecule compounds.STRING database was used to construct a protein-protein interaction network of differentially expressed genes to explore the hub genes in HBV-HCC,and UALCAN database and HPA database were applied to verify the m RNA and protein level expression differences of hub genes.Finally,the association between alternative splicing and immune infiltration was analyzed through immunofluorescence staining.Results : SARS-Co V-2 infection resulted in widespread transcript deregulation.We observed 1,383 DTUs(differential transcript use)and 3,937 DTEs(differential transcript expression)between 9 COVID-19 samples and 10 control samples.GO functional enrichment analysis found that these DTU and DTE genes were significantly enriched in pathways such as coagulation,neutrophil activation and cytokine production.It is worth noting that in COVID-19 patients,CD74 and LRRFIP1 have significant exon skipping events,which will lead to shortened protein domains,which may be associated with reduced antiviral immunity.Validation of exon skipping events in six genes,including CD74 and LRRFIP1,was performed by semiquantitative RT-PCR and the results were largely consistent with the transcriptome sequencing results.In addition,We found that transcriptional dysregulation is also closely related to the clinical severity of COVID-19,and regulating AS abnormalities may lead to opposite therapeutic effects.In conclusion,our data emphasize that a better understanding of the full picture of AS may aid in the diagnosis and treatment of COVID-19.3,247 differentially expressed genes were identified in 5 normal samples and 5HBV-HCC samples.PPI networks of differentially expressed up-regulated and down regulated gene sets were constructed using STRING database,and 11 up-regulated and 12 down regulated hub genes were found.It was found that the abnormal expression of these hub genes was closely related to the survival time of patients.Based on the large sample HCC data in UALCAN database and HPA database,the expression differences at the m RNA and protein levels were verified.Through CMap analysis,we identified several small molecule compounds that may reverse the expression of hub genes.In addition,a total of 3,731 DTEs were identified in the case and control groups,of which 34 DTEs were significantly enriched in the spliceosome pathway.6,741 gene splicing changes were found between hepatocellular carcinoma and control samples,and 9,765 differential splicing events were detected,of which exon jumping events accounted for 38%.Importantly,we identified seven spliceosomal genes(SNRPA1,LSM2,PUF60,RBM25,HNRNPC,SF3B3 and TXNL4A)with both DTE and exon skipping events,indicating dysfunctional cellular splicing in HCC patients.In addition,we also found a close positive correlation between the level of splicing imbalance and immune infiltration,as well as the expression of PD-1 and PD-L1 in hepatocellular carcinoma.Conclusion : In this study,we found that SARS-Co V-2 infection rendered host AS dysfunction through its effect on the host spliceosome,which in turn caused extensive transcript expression abnormalities.In addition,this study identified hub genes related to HBV-HCC,significantly dysfunctional metabolism and signal pathways,and potential targeted drugs.It found that a large number of genes in HCC patients’ liver tumor tissues,including spliceosome genes,had differential AS events,and it was clear that the level of splicing disorder was associated with the immune invasion process of hepatocellular carcinoma.In conclusion,our research provides a theoretical basis for the study of alternative splicing abnormalities in viral infectious diseases.