The Analysis Of High Throughput Sequencing Result Of Exosomal MicroRNA From Cytomegalovirus Infected Cells And The Correlation Between Serum Detection And Clinical Indexes Of HCMV Infected Infants

Objective:Human cytomegalovirus（HCMV）is a kind of wider spread double-stranded DNA herpes virus,not only can cause sever damage of the patients who are immunocompromised,such as HIV/AIDS,organ transplantation,or chemotherapy of malignant tumor,but also significantly leads to the congenital and perinatal infections.Children especially infants who are under six months are extremely susceptible to HCMV infection,showing various clinical manifestation of multiple organ damage,such as hepatic injury,,central nervous system involvement,pulmonary infection and so on.The immune escape mechanism and pathogenic mechanism induced by HCMV infection are very complicated,which are small regulatory non-coding RNAs that can play key role in posttranscriptional regulation of gene expression.Cell or virus encoded miRNAs can be selectively packaged into different kinds of carriers which can defend them against degradation,then released in small vesicles or exported to recipient cells.Extracellular miRNAs have been demonstrated to stably exist in most biological fluids,such as urine,saliva,blood,breast milk,amniotic fluid,cerebrospinal fluid and so on.Among the carriers,extracellular vesicles（EVs）exert a crucial influence on circulating miRNAs.So far three main types of EVs have been currently recognized on the basis of their size:apoptotic bodies（1-5um）,microvesicles（100–1000 nm）,and exosomes（40-100 nm）.An increasing evidences have indicated that EVs,especially exosomes,play an important role on conveying information to cell-to-cell communication and thus affecting various physiological and pathological process.Exosomes are released from many types of cells,containing not only proteins,lipids,but also high amounts of mRNAs,miRNAs and other non-coding RNAs.The contents of exosome are transferred by either directly fusing of exosome with the plasma membrane or endocytosis by the recipient cells.Till now,it has been reported that HCMV encodes 26 mature miRNAs from 15 precursors（www.mirbase.org,）.HCMV encoded miRNAs can regulate not only its own gene expression but also the host’s during productive infection in order to create a microenvironment applicable for viral infection and replication.Now that,given the increasing functions and importance of exosomes during viral infections,it is necessary for us to understand the profile of miRNAs in the exosomes and delivery kinetics of miRNAs via exosomes during HCMV productive infection.In this study,contents of exosomes released from HCMV infected HELF cells were analyzed,and delivery dynamics of two abundant virus miRNAs in the exosomes was detected.Methods:1.Isolation of exosomes from the cell culture supernatants and sera:exoEasy Maxi Kit（QIAGEN）was used to isolate and purify exosomes from the supernatants of HCMV infected HELF cells and sera from children.The exosomes were identified by transmission electron microscopy and by western blot.To evaluate whether the purified exosomes containing virions or not,exosomes from infected cells were incubated with HELF cells.2.MiRNA extraction:Cellular total RNA was isolated using miRNeasy Mini Kit（QIAGEN）,while total RNA from exosomes was isolated using exoRNeasy Maxi Kit（QIAGEN）according to the manufacture’s protocols.The concentration and integrity of each RNA sample was determined by Nanodrop-1000 spectrophotometer.3.Small RNA library construction and deep-sequencing data analysis:total RNA samples were independently extracted from exosomes released from mock-infected and HCMV infected cells at 72hpi according to the manufacture’s instructions.RNA samples were ligated with 3’and 5’adapters,reversely transcribed to cDNA.After amplification,the amplicons were purified and detected by Agilent 2100 Bioanalyzer to create library constructs,sequenced using an Illummina HiSeq 2500 platform.In order to select out the differentially transcribed host miRNAs in exosomes between mock-infected and HCMV infected HELFs at 72hpi,the following three principles were set out:1)absolute number of reads>10;2)the differentially transcribed miRNAs in multiples(log₂^HAN1/HELF)>2,or<0.5;3)P values<0.05.4.TaqMan assays:The relative quantities of mature hcmv-miR-US25-1-5p（MIMAT0001581,ABI）,hcmv-miR-UL112-3p（MIMAT0001577,ABI）and U6 were measured by TaqMan using Universal PCR Master Mix Kit（ABI）and specific TaqMan probes（ABI）according to the manufacturer’s instructions.U6 level was used to normalize the relative amounts of hcmv-miR-US25-1-5p and hcmv-mi R-UL112-3p by2^-△△^Ct method.The measurements were done in triplicate.5.Serum samples:after routine laboratory detection,1 ml to 4 ml of serum samples were collected from 72 children younger than 6 monthswith HCMV acute or congenital infection from Shengjing Hospital of China Medical University.Diagnostic criteria for HCMV acute infection were HCMV IgM positive and urine HCMV DNA copies higher than 10³ per milliliter in routine Chemiluminescence immunoassay（Diasorin,Italy）and QRT-PCR detections（DaAn Gene,China）.Sera from 22 age matched children who lack HCMV acute infection indicators were also collected as controls.This study was specifically approved by the Ethical Committee of Shengjing hospital.6.Statistical analysis:All the experimental data were processed by SPSS statistics 22and GraphPad Prism 5 software,the difference was considered statistically significant when P<0.05.Results:1.Exosomes were extracted from HELF cells infected by HCMV Han BAC.Result of transmission electron microscopy showed that the purified exosomes have the expected size as it has been reported before.Exosomal membrane marker TSG101 in exosomes extracted from HELF cells infected with HAN BAC were detected by Western blot;no specific indicators GFP was observed in the cells.2.High-throughput sequencing of all small RNAs（smRNA-seq）libraries by the Illummina HiSeq 2500 platform generate33074443 and s27976426 clean reads for mock-infected and HCMV infected HELFs,respectively;According to the criteria given in the material and method section,a total of 58 host miRNAs were significantly abundant in exosomes from infected cells,of which 32 miRNAs were determined only in the exosome from infected samples compared to the mock-infected samples.Among them,miR-182-5p and miR-4634 stood out,showing 857.42 folds and 367.46 folds increase,respectively.While the quantities of 70 host miRNAs were lower in the exosomes from infected cells,of which 22 miRNAs were determined only in the mock-infected exosome samples.The lowest one was miR-561-5p,showing 180 folds decrease,followed by miR-598-3p and miR-589-5p with 128 folds decrease in the exosomes from HCMV infected cells compared to those in the exosomes from mock infected cells.In our comprehensive result of high-throughput sequencing,16 HCMV mature miRNAs were detected,among them,the most abundant HCMV miRNAs are miR-US25-1-5p and miR-UL112-3p.3.HCMV miR-US25-1-5p and miR-UL112-3p were measured by TaqMan in both the exosomes and cells infected with HAN BAC at 6,24,48,72 and 96 hpi.The two miRNAs began to be detected in exosomes as early as 6 hpi and increased gradually with elongation of infection time.Similarly,the transcription levels of the two miRNAs in the infected cells also grew continuously.And the abundances of the two HCMV miRNAs within the exosomes were positively correlated with those in the infected cells.Exosomal membrane marker TSG101 in exosomes extracted from HELF cells infected with HAN BAC at the corresponding time points were detected by Western blot and found that the level of of exosome not increased with elongation of infection time.4.Purified exosomes from cells infected with HAN BAC at 72hpi were incubated with uninfected HELFs.HCMV miR-US25-1-5p and miR-UL112-3p were quantitatively measured by TaqMan in RNAs extracted from mock-infected and infected cells at 2,4,8,12,16,20 and 24 hours after treatment.5.HCMV miR-US25-1-5p and miR-UL112-3p levels were measured by TaqMan in circulating exosomes from 72 patients with HCMV acute infection and 22 non infected infants as controls.Results showed that 64 samples from acute infected children were positive for miR-US25-1-5p while 60 positive samples for miR-UL112-3p,and there5 samples were both negative for HCMV miR-US25-1-5p and miR-UL112-3p.the amounts of miR-UL112-3p and miR-US25-1-5p in the circulating exosomes respectively showed a positive correlation with the copy numbers of urine HCMV DNA during HCMV acute infection.In addition,the relative quantity of miR-US25-1-5p was positively correlated with the biochemical indexes of cholestasis..Conclusion:1.HCMV infection can alter the profile of host miRNAs in the released exosome or transcription of host miRNAs in infected cells;2.16 HCMV mature miRNAs could be detected in the exosomes from HCMV infected cells.Among them,the most abundant HCMV miRNAs are miR-US25-1-5p and miR-UL112-3p.3.The abundances of the two HCMV miRNAs within the exosomes were positively correlated with those in the infected cells.It seems that exosomes did not have a significant enrichment effect on viral nucleic acid packaging.4.HCMV miRNAs could be transferred from infected cells to uninfected cells via exosomes.5.HCMV-miR-US25-1-5p and HCMV-miR-UL112-3p could be detected from exosomes isolated from the peripheral sera of HCMV acute infected infants;the relative expression of the HCMV miRNAs had a positive correlation with the copy numbers of urine HCMV DNA.6.The relative quantity of miR-US25-1-5p was positively correlated with the biochemical indexes of cholestasis,such asγ-glutamyl transpeptidase、conjugated bilirubin and bile acid.