Screening And Verification Of MicroRNA In Umbilical Vein Endothelial Cells Infected By Influenza Virus And Intervention Of Mullein

MicroRNA?miRNA?is a kind of non-encoding small RNA molecule of about 22 nucleotides in length.The RISC complex,which is composed of miRNA,Ago protein family and other molecules,inhibits translation of target gene or give rise to mRNA degradation through binding to the target mRNA 3 UTR region.Recent studies have indicated that miRNA plays an important role in the replication,infection and pathogenesis of influenza virus,and it is involved in the pathogenicity of influenza virus and the pathogenesis of viral pneumonia.MiRNA is also involved in the regulation of the vascular endothelial cell functions.Studies have shown that miRNA plays an important role in virus induced-hyperpermeability of vascular endothelial cell.However,it has no detailed studies concerning the miRNA expression of vascular endothelial cells infected with influenza virus.Previous studies have demonstrated that,IV infection increased microvascular leakage in PMVECs,with the primary mechanism involving activation of the Rho/ROCK,p38MAPK,and PKC pathways,ultimately leading to ERM phosphorylation.However,the molecular mechanism of miRNA involved in the regulation of the process is not yet clear.Traditional Chinese medicine holds the belief that the flu,which is the exterior syndrome,is mainly caused by the dysfunction of lung-wei induced by the exogenous pathogenic factors or the leakage of the body's vital qi.As one of the important materia medica invigorating spleen-stomach and replenishing qi,Astragalus can replenish and restore lung qi,commonly used to treat the flu induced by qi deficiency.Research shows that Astragalus and its main ingredients,Astragalus Polysaccharide and total flavonoids of Astragalus,can protect endothelial cells,regulate immune function,and have anti-inflammatory and antiviral effects.As one of the main components of total flavonoids of Astragalus,calycosin has extensive biological activities.It can inhibit cytoskeleton remodeling in human umbilical vein endothelial cells?HUVEC?induced by LPS.It can also reduce the permeability of endothelial cells caused by catatonosis.However,the effect of calycosin on the increased permeability of HUVEC cells infected with influenza A virus has not been fully explored.ObjectiveHUVEC is commonly used to study the function of endothelial cells.In this study,we intended to explore the possible effect on the hyperpermeability and the F-actin reorganization of HUVEC induced by ?.We detected the miRNAs in HUVEC infected with IV using the miRNA microarray and RT-PCR experiment to identify the main miRNA regulating this process.Meanwhile,we predict the target genes of these functional miRNAs.And we would investigate the effect of calycosin on the permeability,F-actin and miRNAs in influenza virus infected-HUVEC,to explore whether calycosin could alleviate hyperpermeability of endothelial cell induced by IV and its possible mechanism,which could provide theoretical basis for targeted therapy of miRNA and clinical treatment of drugs for viral pneumonia.Methods1.The study of the increased permeability of HUVEC infected with IVThe infection rate of HUVEC infected with IV was detected by indirect immunofluorescence,to determine the virus strains and degrees of IV solutions.The permeability of HUVEC cells infected with PR8 or CA07 at 0 h,2 h,4 h,6 h,12 h,24 h and 36 h was detected by transmembrane resistance method.The distribution of F-actin in HUVEC infected by PR8 or CA07 at 12 h and 24 h were examined using confocal microscopy.The apoptosis of HUVEC infected with PR8 or CA07 at 24 h was detected by TUNEL method.2.The identification and validation of miRNAs in HUVEC infected with IVThe total RNA was extracted by Trizol lysis solution at 12 h and 24 h after HUVEC infected with PR8 and CA07.MiRNA chip was used to detect miRNAs expressed differently in HUVEC between the normal group and the virus group.To predict the target genes using bioinformatics website or software.To choose 8 miRNAs prepared for PCR according to the results of the microarray and target prediction to investigate whether its result was consistent with the result of miRNA array.3.The effect of calycosin on the hyperpermeability and main miRNAs of HUVEC cellsinduced by influenza virusTo test the toxicity of calycosin on HUVEC and MDCK cells and its antiviral effect.HUVEC infected with PR8?MOI,30?was incubated with 20?g/ml calycosin,then the value of transembrane resistance of HUVEC cells at different time points was monitored by Transmembrane resistance test,the F-actin distribution was captured by laser scanning confocal microscope,the expression of has-miR-92a-1-5p and has-miR-29b-1-5p in HUVEC cells was detected by qRT-PCR.Results1.The study of the increased permeability of HU VEC infected with IV1.1 Four different strains of IV?A/PR/8/1934,A/Ca/07/2009,A/BJ/501/2009,A/BJ/HZ01/2011??MOI,1?were used to infect HUVEC.And their infection rates were approximately 10%.15%,8%,3%.1.2 PR8 at an MOI of 0.1,1,10 were used to infect HUVEC.The infection rates of PR8 were approximately 5%,13%,41%.1.3 PR8 at an MOI of 20,30,40 were used to infect HUVEC.The infection rates detected at 12 h were 32%,46%,51%,at 24 h were 50%,62%,68%.1.4 The permeability of HUVEC infected with IV cell was increased,and the F-actin was reorganized.1.5 Flow cytometry showed that apoptosis rate of HUVEC in control group and virus group has no significantly different.2.The identification and validation of miRNAs in HVEC infected with IV2.1 At 12 h after infected with PR8,expression of 30 miRNAs in HUVEC in virus group showed significant difference compared with the control group.Among them,expression of 13 miRNAs was decreased,while the expressions of others increased.At 24 h,expression of 198 miRNAs in HUVEC in virus group has obvious difference compared with the control group.Among them,expression of 126 miRNAs was decreased,the others'was increased.4 miRNAs at two time point occurred a significant change.2.2 At 12 h after infected with CA07,expression of 305 miRNAs in HUVEC in virus group showed significant difference compared with the control group.Among them,expression of 146 miRNAs was decreased,the others' was increased.At 24 h,expression of 262 miRNAs in HUVEC in virus group has obvious difference compared with the control group.Among them,expression of 198 miRNAs was decreased,the others' was increased.129 miRNAs at two time point occurred a significant change.2.3 At 12 h after infected with CA07 or PR8,3 of the differentially miRNAs in HUVEC infected with different virus had the same ID.At 24 h,66 of the differentially miRNAs inHUVEC infected with different virus had the same ID.2.4 KO and KEGG enrichment results show that miRNAs corresponded to multiple target genes and were involved in the regulation of varied pathways.The target genes involved in regulating the HUVEC permeability were the genes of PKC,ROCK1,CAMKII,actin,etc.The pathway involved in were PKC pathway,Rho/ROCK pathway,Ca²⁺pathway,etc.Accordingly,we select 8 miRNAs,whose target gene maybe involved in regulating permeability of endothelial cells.2.5 PCR experiment result showed that,at 12 h,there were 5 miRNAs,whose expression decreased significantly in virus group compared to those in control group,and they were has-miR-138-5p?P<0.05?,has-miR-21-5p?P<0.01?,has-miR-29a-3p?P<0.01?,has-miR-200a-3p?P<0.01?and has-miR-181a-5p?P<0.001?;at 24 h,there were 3 miRNAs,whose expressions decreased significantly in virus group compared with those in control group,and they were has-miR-181a-5p?P<0.01?,has-miR-29b-1-5p?P<0.001?and has-miR-147b?P<0.001?,while the expression of has-miR-138-5p and has-miR-99a-5p was reduced insignificantly.3.The effect of calycosin on the hyperpermeability and main miRNAs of HUVEC cells induced by influenza virus3.1 Calycosin at the concentration of 40 ?g/ml had no toxicity to HUVEC.Calycosin of 80?g/ml was non-toxic to MDCK cells.3.2 Calycosin at a concentration of 20 ?g/ml can inhibit the increase of HUVEC cell permeability induced by ?.3.3 Calycosin at a concentration of 20 ?g/ml can reverse the reorganization of F-actin in HUVEC induced by ?.3.4 Calycosin at a concentration of 10 ?g/ml,20?g/ml,40 ?g/ml demonstrated no anti-viral effect.3.5 Calycosin at a concentration of 20 ?g/ml can increase the expression of has-miR-92a-1-5p and has-miR-29b-1-5p in HUVEC cells infected with influenza virus.Conclusions1.IV could cause the redistribution of F-actin in HUVEC cells,thus resulting in the increase of endothelial permeability.The HUVEC infected with PR8 or CA07?MOI,30?was used in the experiment at 12 h or 24 h.2.8 differentially miRNAs detected by PCR had similar expression trend with those detected by microarray.3.The result showed that calycosin at a concentration of 20?g/ml,which had no anti-viral effect,can inhibit the cytoskeleton reorganization and hyperpermeability of HUVEC infected by IV,and up-regulate the expression of has-miR-29b-1-5p?P<0.05?and has-miR-92a-1-5p.It indicated that calycosin could protect endothelial function through ameliorating the increased permeability of HUVEC infected with IV via has-miR-92a-1-5p,which needed to be confirmed by functional verification.