Studies Of LncRNA On Regulating Vascular Endothelial Barrier Permeability During Dengue Virus Infection

Objective Dengue virus(DENV)is enveloped positive single strand RNA virus,which belongs to the Flaviviridae and Flavivirus genus.It is mainly transmitted by insect vectors such as Aedes aegypti and Aedes albopictus.DENV infection can causes DF(Dengue fever),which can further develop to serious complications,such as dengue hemorrhagic fever / dengue shock syndrome(DHF/DSS).The mortality rate of DHF/DSS can be as high as 50%,whose pathogenesis has not yet been fully elucidated so far.Only about 2% of human genes are able to encode proteins,while the rest are transcriptional regulatory elements and non-coding RNAs.Long non-coding RNA(lnc RNA)is a type of non-coding RNA that is greater than 200 nucleotides in length,accounting for 80% of non-coding RNAs.Studies have revealed that differential expression of lnc RNAs can be induced by viral infection in host cells and regulate the occurrence and development of viral diseases.This study aimed to analyze the expression of lnc RNAs in human umbilical vein endothelial cells(HUVEC)with DENV-1 infection,as well as screening a specific lnc RNA which related to vascular permeability.In addition to exploring its regulatory function and molecular mechanism leading to changes in vascular permeability,it can provide theoretical basis and new ideas for revealing the pathogenesis of DHF/DSS and further developing molecular targeted therapy.Research contents and methods In this study,we performed a total analysis of lnc RNAs that expressed differentially after HUVEC infection with DENV-1,and screened a specific lnc RNA,named ERGAL,which was related to the permeability of the vascular endothelial barrier.The regulatory function of ERGAL was detected by biological information analysis,knockdown transfection technology,quantitative PCR,western blotting,indirect immunofluorescence,phalloidin staining,and flow cytometry.Furthermore,biological information analysis,RNA Fluorescence in situ hybridization and dual luciferase reporter genes were carried out to verify the interaction between ERGAL and mi R-183-5p for regulating vascular endothelial barrier permeability.Results and Conclusions It was found that a total of diffentially expressed 2,623 lnc RNAs in HUVECs after 24 hours of DENV-1 infection.Through performing GO,KEGG and co-expression network analysis,the differentially expressed lnc RNAs and the predicted target genes showed to be mainly involved in many biological processes,including antigen processing and presentation,apoptosis,tpye?and ? interferon signaling pathway and cell adhesion.Furthermore,a specific lnc RNA induced by DENV-1 was screened,which was named ERGAL(ERG-associated lnc RNA)due to its strong correlation with the expression of the transcription factor ERG and related to vascular permeability.The expression of ERGAL increased after DENV infection.However,the expression of the transcription factor ERG and the key intercellular proteins like VE-cadherin and claudin5 were inhibited after ERGAL knockdown;as well as ERGAL silencing could interfere with cytoskeletal remodeling,promote apoptosis and improve monolayer cell permeability.RNA fluorescence in situ hybridization showed that ERGAL was located in the cytoplasm,and its sequence contains multiple mi RNAs binding sites.Among predicted binding mi R-183-5p,the expression of mi R-183-5p also increased after DENV infection,and then it was detected that it could regulate the expression of cell junctional proteins and cytoskeletal remodeling.The relathonship between ERGAL and mi R-183-5p was verified by dual luciferase.In summary,a large amount of deregulated lnc RNA can be induced by DENV-1 infection in HUVEC,and the differentially expressed lnc RNAs were predicted to participate in the physiological process resulting in functional changes or death of vascular endothelial cells,which is closely related to the occurrence and development of DHF/DSS.The ERGAL was screened and found to bind with mi R-183-5p to regulate key intercellular junction proteins and cytoskeletal remodeling,which can promote the integrity and stability of the vascular endothelial barrier during DENV infection.