Effects Of Hsa_circ₀003863 On The Viral Replication Of Early HIV Infected Patients

Objective:Human Immunodeficiency Virus type 1?HIV-1?is a retrovirus that capable of causing Human Acquired Immunodeficiency Syndrome?AIDS?.Since the first case of infection was discovered in 1981,AIDS has spread widely around the world,seriously harming human health.Early HIV infection refers to HIV virus from the local lymph nodes to the whole body within about 6 months after infection.The HIV viral load first climbs to a very high level,then falls to stable tendency?virus setpoint?.It is considered to be a key step influencing the speed of HIV infected people progressing to the AIDS stage.Early HIV infection is characterized by viral replication,strong antiviral response,immune damage and virus diversity.The abnormal viral load during early HIV infection has led to a greater risk of HIV infection in this period.Studies of early HIV infections will help us to better understand the early antiviral responses of the host.Circular RNAs?circRNAs?is a new class of non-coding RNAs that differ from traditional linear RNAs,circRNAs form covalently bound closed loops.The expression of circRNAs is high and stable,and has certain specificity in cells,tissues and diseases.With the development of high-throughput technology such as microarray and RNA-seq,circRNAs have been found to play an important role in a variety of diseases and physiological and pathological processes.In particular,it plays a regulatory role in some viral infections.Xiang Li et al.found that mature circRNAs can bind to NF90/NF110 to form circRNP,play an important immune function in antiviral process,and have this binding NF90/NF110 regardless of endogenous circular RNA or artificially constructed circRNAs.Studies on hepatitis B-related hepatocellular carcinoma have found that circRNA-100338 can serve as a potential biomarker and therapeutic target for its diagnosis,and can play a role in regulating cancer metastasis with miR-141-3p.So far,the role of circRNAs in HIV is still unclear,but the above studies provide a strong basis for circRNAs to play an important role in HIV infection.The role of circRNAs in HIV is still unknown and needs to be explored.CircRNAs can play a regulatory role through a variety of mechanisms,including working as miRNA sponges,interacting with RNA-binding proteins,working as transcriptional and translational regulators,and competing with linear splicing to affect mRNA expression levels.Among them,circRNAs as the miRNA sponge is the earliest discovered and the most important function to date.The majority of circRNAs molecules contain dozens of miRNA target sites and thus can bind to multiple miRNAs and inhibit their function through acting as“miRNA sponges”,according to the competing endogenous RNA?ceRNA?hypothesis.The circRNA-miRNA-mRNA ceRNA regulatory network has received increasing attention and has been constructed in diseases such as Alzheimer's disease,lung cancer,and breast cancer.Currently,there is a lack of analysis of circRNAs in HIV infection.The HIV virus includes structural genes?gag,env,pol?and accessory genes?nef,tat,rev,vif,vpu and vpr?.The HIV replication cycle includes viral attachment and genomic entry,and viral RNA reverse transcribed into cDNA,cDNA into the nucleus,integration,gene transcription and translation,protein assembly and virion release,and the virus itself can only encode 15 proteins,far from meeting the needs of their own replication,the HIV virus replication process needs to include the participation of multiple host proteins.At the same time,some host restriction factors such as APOBEC3G,TRIM5?,MX2,BST2 and SAMHD1 and some miRNAs?miR-198 miR-17/92,etc.?and lncRNA?NEAT1 and NRON?also play a role in regulating viral replication.Although a number of miRNAs and lncRNAs have been reported that can play important role in HIV,the role of circRNAs in HIV,especially for HIV replication,needs to be explored.The research of the mechanism of interaction between HIV-1 and host cells is the key to revealing the pathogenesis of virus,identifying new targets for antiviral drugs,and developing highly effective antiviral drugs and vaccines.The discovery of circRNAs that can affect HIV replication will provide new hope for the early cure of HIV.Methods:1.Patient selectionA total of 3 HAART-naive early HIV-infected patients and 3 healthy controls were enrolled in this study for RNA-seq and miRNA-seq experiments.A total of 30HAART-naive early HIV-infected patients and 14 healthy controls were used to verificate differentially expressed mRNAs,circRNAs and miRNAs,7 HAART-naive chronic HIV-infected patients and 4 healthy controls were used for CD4+T cell sorting.The study was reviewed and approved by the the First Affiliated Hospital of China Medical University.All participants provided written informed consent prior to research participation.2.RNA and library quality controlPBMC were extracted by Ficoll density gradient centrifugation and stored in liquid nitrogen using fetal bovine serum and 10%DMSO until RNA was extracted.Total RNA was extracted using TRIzol,and DNA was removed using TurboDNase,and the RNA concentration and purity of each sample were measured using a NanoDrop ND-2000instrument.The OD260/OD280 value was used as an indicator of RNA purity.RNA integrity and gDNA contamination were detected by denaturing agarose gel electrophoresis.Library quality control and quantification were performed using a BioAnalyzer 2100 instrument.3.RNA sequencingrRNAs in total RNA were removed using RiboMinus?.The RNA was then pretreated with the TruSeq Stranded Total RNA Library Prep Kit to construct a sequencing library.Sequencing on an Illumina HiSeq sequencer.4.miRNA sequencingTotal RNA preparation miRNA sequencing library,including the following steps:1)3'linker ligation;2)5'linker ligation;3)cDNA synthesis;4)PCR amplification;5)recovery of¹50 bp PCR amplified fragment?Corresponding to²2 nt miRNAs?;and sequenced on an Illumina HiSeq sequencer.5.GO and KEGG analysis of differentially expressed mRNAFor the GO and KEGG analysis of differentially expressed mRNAs,the DAVID 6.8database was used,and P value of less than 0.05 was considered to be significantly different.6.Construction of ceRNA regulatory networkTargetScan was used to predict the miRNA binding seed sequence sites.The circRNAs and mRNAs that shared the same miRNA binding site represented circRNA-miRNA-mRNA interactions.The ceRNA network was constructed use Cytoscape software?v3.5.1?.7.CD4+T lymphocyte absolute count5 ml of peripheral blood was collected by negative pressure blood collection tube?EDTA anticoagulation?,20?l CD3/CD4/CD8 mixed dye was added to the flow tube,50?l whole blood was added,and stained at room temperature for 15 minutes in the dark.Add hemolysin 450?l,protected from light for 15 minutes at room temperature,detected by FACS Calibur flow cytometry,calculate the absolute number of CD3+/CD4+,CD3+/CD8+T lymphocytes.8.Measurement of HIV Viral Load1 ml of EDTA anticoagulated plasma was taken and the viral load was determined by qRT-PCR using a COBAS^? AmpliPrep/cobas^? TaqMan^? System automated load meter?Roche?.All operations were performed according to the instructions,with a detection range of 20-10⁷ copies/ml,and results below 20 were shown to be<20 copies/ml.9.Reverse transcription and quantitative real-time PCR?qRT-PCR?Total RNAs was extracted with TRIzol reagent?Life Technologies?,miRNAs was extracted with miRNeasy Micro Kit?QIAGEN?,DNA was removed with DNaseI?QIAGEN?,and total RNAs was reverse transcribed using Primpscript?RT reagent kit?TAKARA?kit,miRNAs reverse transcription using the Mir-XTM miRNA First Strand Synthesis Kit?TAKARA?.qRT-PCR quantification using the LightCycler480fluorescence quantitative PCR instrument?Roche?and SYBR?Premix Ex TaqTM ??Tli RNaseH Plus??TAKARA?.RPLP0,GAPDH and U6 were used as endogenous control for mRNAs,circRNAs and miRNAs respectively.The expression levels of mRNAs,circRNAs and miRNAs expression were calculated based on the change in cycling threshold with the method of 2^-??Ct.10.Primary culture cell sortingPeripheral blood mononuclear cells?PBMC?were extracted by density gradient centrifugation.PBMC of whole blood was first extracted and sorted by CD3?Percp?,CD4?APC-cy7?by BD FACS Aria ? flow cytometry.CD4+T cells?CD3+,CD4+?were sorted from PBMC,and cell purity analysis was performed using a flow cytometer LSR ?.11.Cell culture and transfection of siRNA293T cell was cultured using DMEM medium containing 10%fetal calf serum,1%ionomycin,and JURKAT cells and primary cells were cultured in RPMI 1640 medium containing 10%fetal calf serum and 1%ionomycin.All cells were cultured in a 37?,5% CO ₂ cell culture incubator.The siRNA si-hsa_circ₀003863 that synthesized by Hanheng Biotechnology Co.Ltd.was used,and the siRNA reagent was Lipofectamine RNAiMAX?Thermo Fisher?.The knockdown efficiency of si-hsa_circ₀003863 was detected by qRT-PCR after 48h.12.Package HIV pseudotyped virusPlasmid extraction:NL4-3?ENV-luc and VSV-G plasmids were extracted according to the protocol of the plasmid extraction.The day before transfection,293T cells were plated on 100 mm culture dishes,and the cells were transferred to 70%.Change the medium one hour before transfection,replaced with 1640 medium,and a total of 10?g of plasmid?NL4-3?ENV-luc:VSV-G=2:1?was mixed with jetPRIME transfection reagent,and added on the cells,the supernatant was collected 48 h later.13.HIV pseudotyped virus infected 293 and JURKAT cells that knockdown hsa_circ₀003863The prepared HIV pseudotyped virus was infected with siRNA to knock down JURKAT cells and 293T cells at 48h for hsa_circ₀003863.After 48 hours,the supernatant of JUTKAT cells were collected for P24 detection,and 293T cells were collected for DNA extraction,Alu-gag two-step PCR and HIV-1 2-LTR detection.14.Alu-gag two-step PCR and HIV-1 2-LTR detectionIntegral HIV DNA was detected using Alu-gag two-step PCR and HIV-1 2-LTR was detected by qRT-PCR to detect unintegrated HIV DNA.15.P24 detectionThe measurements of supernatant HIV P24 Antigen were performed by ELISA kit.The supernatant P24 Antigen concentration was calculated using the standard curve and multiplied by the dilution factor.16.Statistical AnalysisGraphPad Prism software was used for statistical analysis.The non-parametric Mann–Whitney U test was used to compare between group distributions.The concentration of HIV P24 Antigen in cell supernatant after siRNA knockdown and the comparison between HIV-integrated and unintegrated HIV DNA were analyzed by t test.Spearman correlation coefficient was used to analyze the correlation between variables.P<0.05 was considered statistically significant.Results:1.Differentially expressed mRNAs and circRNAs in PBMC of HAART-naive early HIV-infected patientsBased on RNA-Seq experiments in PBMC of 3 HAART-naive early HIV-infected patients and 3 healthy controls,20,315 mRNAs transcripts were identified,and 2049differentially expressed mRNAs were obtained,including 673 up-regulated mRNAs and1376 down-regulated mRNAs.At the same time,15,145 circRNAs transcripts were identified,and 1365 differentially expressed circRNAs were obtained,including 912up-regulated circRNAs and 453 down-regulated circRNAs.2.Differentially expressed miRNAs in PBMC of HAART-naive early HIV-infected patientsThe differential expression profiles of miRNAs in early HIV-infection were carried with the RNA from the same PBMC samples that were used in the mRNAs and circRNAs transcriptome study.A total of 1304 miRNAs were detected,and there were 30miRNAs differentially expressed in early HIV infection,including 12 up-regulated miRNAs and 18 down-regulated miRNAs.3.Quantitative real-time PCR validationValidation of differential expression mRNAs,circRNAs and miRNAs.Twelve differential expressed RNA were randomly selected and validated with qRT-PCR,including 4 circRNAs,4 mRNAs,and 4 miRNAs.The results showed that after expanding the number of specimens,the expression level of these differentially expressed mRNAs and miRNAs were was consistent with the results of RNA-seq and miRNA-seq.However,among the four differentially expressed circRNAs,three expression levels were consistent with the expression of RNA-seq,and there was a significant difference?P<0.05?.The change trend of another was consistent with the expression of RNA-seq,but there was no significant difference?P>0.05?.This result demonstrates the high reliability of differential expression profile of mRNAs and circRNAs obtained by RNA-seq and the differential expression profile of miRNAs obtained by miRNA-seq.4.Features of differentially expressed circRNAThe differentially expressed circRNAs are distributed on 22 autosomes and X and Y chromosomes.CircRNAs are classified into five categories according to their types,including sense overlapping circRNAs,intronic circRNAs,intergenic circRNAs,exonic circRNAs and antisense circRNAs,the constituent ratios was 8.42%?115/1365?,3.08%?42/1365?,0.37%?5/1365?,88.06%?1202/1365?and 0.07%?1/1365?respectively.The length of 89.30%?1219/1365?of the circRNAs was less than 2000 nt and the median length was above 500 nt.In each circRNAs class and length group,the number of up-regulated circRNAs was greater than that of the down-regulated circRNAs in every circRNAs category and length group.5.Construction of ceRNA regulatory network in early HIV infectionBased on the ceRNA hypothesis,we constructed a ceRNA regulatory network containing differentially expressed mRNAs,circRNAs and miRNAs.The ceRNA network contains 516 differentially expressed circRNAs,903 differentially expressed mRNAs and 21 differentially expressed miRNAs.The network is divided into two parts,one containing 366 up-regulated circRNAs,14 down-regulated miRNAs,and 378up-regulated mRNAs;the other containing 150 down-regulated circRNAs,7up-regulated miRNAs,and 525 down-regulated mRNA.6.Bioinformatics analysis of ceRNA regulatory networks in early HIV infectionGene oncology?GO?analysis and KEGG pathway analysis were performed on 903differentially expressed mRNAs involved in ceRNA network construction.GO analysis showed that the ten most enriched biological processes were immune response,inflammatory response,response to lipopolysaccharide,interferon-gamma-mediated signaling pathway,positive regulation of NF-kappaB import into nucleus,defense response to virus,type I interferon signaling pathway,negative regulation of G1/S transition of mitotic cell cycle and T cell costimulation.Most of the above biological processes are closely related to HIV infection,especially immune response,inflammatory response and defense response to virus have been shown to play an important role in early HIV infection.7.Discovery of hsa_circ₀003863By analyzing ceRNA network,it was found that hsa_circ₀003863 has a higher fold change,and hsa_circ₀003863 potentially regulated target genes that include a number of genes that have been reported to play important roles in HIV,such as IL-15,CD38,TNFSF10,RAC1 and so on.8.Identification of circular RNA hsa_circ₀003863To ensure that we measured circRNAs,rather than its linear mRNAs,we designed a back-to-back primer containing its cyclization site for hsa_circ₀003863,the PCR product was detected by Sanger Sequencing and sequencing results confirmed that the cyclization site and the cyclization site of the amplified product were consistent with the information in the circBase database.The measured hsa_circ₀003863 results are authentic.9.Expression of hsa-circ₀003863 in early HIV infectionThe expression of hsa-circ₀00386 in early HIV infection was validted by qRT-PCR in18 early-infected patients and 9 healthy controls.The results showed that hsa-circ₀003863 was significantly expressed between 18 early HIV-infected patients and 9 healthy controls?P<0.05?,this result was consistent with the transcriptome sequencing results.10.The expression level of hsa_circ₀003863 in HIV-infected patients can be related to the level of virus setting pointIn the absence of ART treatment,the virus setting point is an important indicator affecting the progress of HIV disease.The results suggest that the expression of hsa_circ₀00386 can predict disease progression.11.The expression level of hsa_circ₀003863 in HIV-infected patients can predict the decline of CD4+T cellsThe rate of decline of CD4+T cells in HIV-1 infected patients is a key factor in predicting the progression of the disease.Using Kaplan-meier survival analysis to analyze the relationship between the expression level of hsa_circ₀003863 and the time to the absolute count of CD4+T cells decreased to 500 cells/?l,we found that the expression level of hsa_circ₀003863 in HIV-infected patients can predict CD4+T cells decline.12.The expression level of hsa-circ₀003863 in CD4+T cells was positively correlated with VL.We selected the main target cell CD4+T of HIV virus by flow cytometry,qRT-PCR and detected the expression level of hsa-circ₀003863.The experiment found that the expression level of hsa-circ₀003863 in CD4+T cells was positive correlated with VL?P<0.05?.13.hsa-circ₀003863 affects HIV replicationIn JURKAT cells,hsa-circ₀003863 was down-regulated with siRNA and infected with NL4-3 pseudovirus to collect supernant 48 hours after infection.The measurements of supernatant P24 were performed by ELISA kit.The results showed that JURKAT cells showed a significant decrease in P24 levels 48 hours after down-regulation of hsa-circ₀003863.In 293T cells,hsa-circ₀003863 was down-regulated with siRNA and infected with NL4-3 pseudovirus to collect cells 48 hours after infection,DNA was extracted,and Alu-step two-step PCR and qRT-PCR were used.Alu-gag two-step PCR results showed that 293T cells decreased the cDNA of HIV integration after 48 hours of down-regulation of hsa-circ₀003863,suggesting that hsa-circ₀003863 may affect the integration phase of viral replication.Conclusion:1.The ceRNA regulatory network of circRNA-miRNA-mRNA exists in PBMC of HIV-infected individuals,and circRNAs may be involved in the pathogenesis of multiple early HIV infections.2.The expression level of hsa_circ₀003863 in HIV-infected patients was positively correlated with the level of viral replication and can predicted the decrease in the number of CD4+T cells.3.hsa-circ₀003863 may regulate HIV replication by affecting the integration phase of HIV replication.