Kev txheeb xyuas qhov systematic thiab kev sib piv ntawm Cov Qhia Qhia Txog Exosomal MiRNAs hauv npua kis nrog NADC30-zoo li PRRSV Hom

Cov ntsiab lus yooj yim:Exosomes ua lub luag haujlwm tshwj xeeb hauv kev kis tus kab mob, kev nthuav qhia antigen, thiab kev tawm tsam / txhawb lub cev tiv thaiv kab mob. Porcine reproductive and respiratory syndrome virus (PRRSV) yog ib qho ntawm cov kab mob phem tshaj plaws hauv kev lag luam npua. Ntawm no, peb tau siv PRRSV NADC30-xws li CHsx1401 strain los ua kom kis tau tus kabmob 42-hnub nyoog npua, cais cov ntshav exosomes, thiab txheeb xyuas 33 qhov sib txawv ntawm qhov qhia tawm (DE) exosomal miRNAs ntawm pawg kab mob thiab tswj, thiab 18 DE miRNAs cuam tshuam nrog tus kab mob PRRSV thiab kev tiv thaiv kab mob tau kuaj pom tias muaj peev xwm ua haujlwm molecules koom nrog kev tswj hwm tus kab mob PRRSV los ntawm exosomes.

Abstract:Exosomes yog cov hlwv hauv cov hlab ntsha zais cia thiab tso tawm los ntawm cov hlwb uas ua tus neeg nruab nrab ntawm kev sib txuas lus intercellular thiab ua lub luag haujlwm tshwj xeeb hauv kev kis tus kabmob, kev nthuav qhia antigen, thiab kev tawm tsam / txhawb lub cev tiv thaiv kab mob. Porcine reproductive and respiratory syndrome virus (PRRSV) yog ib qho ntawm cov kab mob phem tshaj plaws nyob rau hauv npua kev lag luam thiab tuaj yeem ua rau muaj kev loj hlob ntawm cov noob, kab mob ua pa hauv npua, txo kev loj hlob, thiab lwm yam kab mob ua rau npua tuag. Hauv txoj kev tshawb no, peb tau siv PRRSV NADC30-zoo li CHsx1401 strain los ua kom kis tau tus kabmob 42-hnub-hnub npua thiab cais cov ntshav exosomes. Raws li high-throughput sequencing thev naus laus zis, 305 miRNAs tau txheeb xyuas nyob rau hauv cov ntshav exosomes ua ntej thiab tom qab kis kab mob, ntawm cov 33 miRNAs tau qhia txawv txawv ntawm cov pab pawg (13 kuj upregulated thiab 20 kuj downregulated). Kev txuag kev tsom xam ib ntus ntawm CHsx1401 genome tau txheeb xyuas 8 thaj chaw khaws cia, ntawm tag nrho ntawm 16 qhov sib txawv qhia (DE) miRNAs tau kwv yees los khi rau thaj chaw khaws cia ze tshaj 30 UTR ntawm CHsx1401 genome, suav nrog 5 DE miRNAs muaj peev xwm los khi rau CHsx1401 30 UTR (ssc-miR-34c, ssc-miR-375, ssc-miR-378, ssc-miR-486, ssc-miR-6529). Kev soj ntsuam ntxiv tau nthuav tawm tias lub hom phiaj ntawm cov caj ces ntawm qhov sib txawv ntawm miRNAs tau koom nrog hauv kev ua haujlwm ntawm exosomal ntsig txog thiab kev tiv thaiv kab mob hauv lub cev, thiab 18 DE miRNAs (ssc-miR-4331-3p, ssc-miR{16}} , ssc-miR-320, ssc-miR-10b, ssc-miR-124a, ssc-miR-128, thiab lwm yam) cuam tshuam nrog PRRSV kab mob thiab tiv thaiv kab mob Raws li qhov muaj peev xwm ua tau zoo molecules koom nrog kev tswj hwm ntawm PRRSV tus kab mob kis los ntawm exosomes.

cistanche cog-nce kev tiv thaiv kab mob

Ntsiab lus:PRRSV; serum exosome; miRNAs

1. Taw qhia

Porcine reproductive and respiratory syndrome virus (PRRSV) yog ib leeg-stranded positive-strand RNA tus kab mob nrog ib lub hnab ntawv uas muaj nyob rau hauv kev txiav txim Nidovirales, tsev neeg Arteriviridae, genus Betaarterivirus [1,2]. Nws yog spherical los yog ellipsoidal nrog ib txoj kab uas hla ntawm 50-65 nm nyob rau hauv lub freezing electron microscope [3,4]. PRRSV genome yog txog 15 kb ntev nrog 50 cap thiab 30 polyA-tail thiab muaj tsawg kawg yog 10 qhib kev nyeem ntawv (ORFs) flanked los ntawm cov cheeb tsam tsis txhais lus (UTRs) ntawm ob lub 50 thiab 30 termini [5,6] thiab yog qhwv los ntawm nucleocapsid protein, nrog lipid ob txheej txheej los tsim cov kab mob. Exosomes belongs rau vesicles nrog monolayer membrane cov qauv thiab muaj tib lub qauv topological li cov hlwb [7]. Cov duab yog "khob-puab" los yog "disc-shaped" nyob rau hauv ib tug electron microscope [8,9]. Exosomes tuaj yeem nyob hauv lub cev mus ntev, thiab cov khoom hauv exosomes tuaj yeem nqus tau los ntawm cov hlwb uas nyob ib sab lossis cov receptor nyob deb thiab tom qab ntawd tswj cov receptor hlwb kom koom nrog kev sib pauv ntawm cov khoom siv caj ces ntawm cov hlwb [10,11]. Lawv feem ntau yog tsim los ntawm cov ntaub so ntswg thiab nqa cov ntsiab lus, suav nrog cell deg receptors, membrane proteins, soluble proteins, lipids, RNA (mRNA, miRNA, lncRNA, thiab viral RNA, thiab lwm yam), genomic DNA, mitochondrial DNA [12-14. ]. MicroRNAs (miRNAs) yog ib chav kawm ntawm 18–25 nucleotides (nt) evolutionarily conserved endogenous non-coding ib leeg-stranded me RNAs, uas inhibit cov txheej txheem txhais lus los ntawm inducing degradation ntawm lub hom phiaj mRNA los yog los ntawm kev khi nrog 30 UTR ntawm lub hom phiaj mRNA, ua. rau post-transcriptional gene silencing, ces tswj cov gene qhia ntawm qib post-transcriptional [15–17]. Nws kwv yees tias miRNAs tswj ntau dua 60% ntawm cov noob caj noob ces tom qab hloov pauv [18,19]. MiRNAs ua lub luag haujlwm tseem ceeb hauv kev sib txuas lus ntawm tes thiab tseem tuaj yeem siv los ua lub peev xwm ua haujlwm rau cov kab mob thiab kab mob, kis, thiab tiv thaiv [20]. Ntau qhov kev tshawb fawb tau pom tias miRNAs tuaj yeem muaj nyob hauv cov kua hauv lub cev, xws li qaub ncaug, zis, mis, thiab ntshav, thiab ua haujlwm los ntawm lub cev cov kua dej hauv lub cev [21,22]. Exosomal miRNAs raug suav hais tias yog endogenous regulators ntawm cov noob qhia thiab cov metabolism thiab tuaj yeem qhia txog ntau yam kab mob [23,24].

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

Ob xyoo dhau los, nws tau pom tias miRNAs muaj lub luag haujlwm tseem ceeb hauv kev tswj hwm lub cev tiv thaiv kab mob, kev tiv thaiv kab mob hauv lub cev, thiab tau txais kev tiv thaiv kab mob. Qee lwm cov miRNAs tau tshaj tawm tias ua rau PRRSV tsis zoo los ntawm txoj hauv kev hauv qab no, ncaj qha rau PRRSV genome lossis PRRSV receptor, lossis ua lub luag haujlwm los ntawm kev tswj hwm tus tswv tsev lub cev tiv thaiv kab mob. MiR-26 tsev neeg tuaj yeem ua rau tus kab mob rov ua dua tshiab, thiab miR-26ib tuaj yeem cuam tshuam qhov kev rov ua dua ntawm hom 1 thiab hom 2 PRRSV hom kab mob porcine alveolar macrophages (PAMs) los ntawm kev tswj hwm hom I interferon (IFN) txoj kev, uas ua tau zoo dua miR-26b [25,26]. miR-30c thiab miR-125b raug txheeb xyuas los hloov kho tus tswv tsev hauv lub cev tiv thaiv kab mob los ntawm kev tsom mus rau hom I IFN txoj kev thiab NF-κB txoj hauv kev, raws li [27–29]. MiR-23, miR-378, thiab miR{14}} yog cov tshuaj tiv thaiv kab mob tiv thaiv kab mob uas tsom rau PRRSV thiab muaj cov phiaj xwm tshwj xeeb hauv hom 2 PRRSV hom [30]. Tib lub sijhawm, tus tswv tsev miR-506 tau raug txheeb xyuas kom txwv tsis pub PRRSV rov ua dua los ntawm kev tsom ncaj qha rau PRRSV receptor CD151 hauv MARC-145 hlwb [31]. miR-181 kuj tuaj yeem cuam tshuam tsis pub PRRSV rov ua dua los ntawm kev tswj hwm PRRSV receptor CD163 hauv cov ntshav monocytes thiab PAMs [32]. Tsis tas li ntawd, miRNAs tuaj yeem txhawb nqa PRRSV replication los ntawm kev cuam tshuam nrog cov cell physiology. MiR-24-3p thiab miR-22 ncaj qha lub hom phiaj 30 UTR ntawm HO-1 thaum lub sij hawm PRRSV tus kab mob kom dim ntawm inhibition ntawm heme oxygenase-1 (HO-1), a heat shock protein (tseem hu ua HSP32) ntawm PRRSV [33,34]. Npua paub tias muaj kev cuam tshuam ntau dua rau PRRSV thiab tsis tshua muaj peev xwm tiv thaiv lawv tus kheej tawm tsam qhov nkag ntawm cov kab mob no mus rau hauv lub cev [35]. Hauv kev tshawb fawb tam sim no, kev tiv thaiv kab mob hauv lub cev thiab tau txais kev tiv thaiv kab mob ntawm npua kis tus kab mob no tau kawm ntawm qib molecular siv cov kab mob uas muaj nyob hauv thaj chaw. Cov khoom siv tshuaj tiv thaiv kab mob hauv lub cev, kis tau tus mob electron microscopy (TEM), nanoparticle tracking analysis (NTA), thiab Western blot (WB) tau siv los cais thiab txheeb xyuas cov ntshav exosomes ua ntej thiab tom qab kis kab mob nrog PRRSV, tom qab ntawd los ntawm RNA sequencing tsom xam me me, kev txheeb xyuas, thiab kev soj ntsuam ntawm cov txiaj ntsig sib txawv siv cov txheej txheem bioinformatics kom tau txais ntau PRRSV-sociated serum exosome miRNAs, tom qab los ntawm kev txheeb xyuas cov ntaub ntawv tau txais kev siv ntau npaum li cas PCR (qRT-PCR).

2. Cov ntaub ntawv thiab cov txheej txheem

2.1. Kev sim tsiaj

Rau PRRSV antigen thiab antibody ob-tsis zoo noj qab haus huv 42-hnub-laus loj npua dawb tau muab tso rau hauv npua huv pub rau kev sib cais, kev noj qab haus huv, thiab kev hloov ntawm ib puag ncig. Txhua tus npua pub dawb noj thiab haus tsis muaj kev txwv. Thaum lawv paub txog cov xwm txheej hauv qhov kev cais tawm, cov npua tau raug txhaj tshuaj nrog 2 mL 105 TCID50 / mL PRRSV NADC30-zoo li CHsx1401, uas tau hais los ntawm cov thawj coj [36,37]. Cov ntshav ntawm npua ua ntej (tswj pab pawg, n=6) thiab 7 hnub tom qab ( pab pawg kho mob, n=6) cov kab mob inoculation tau sau los ntawm anterior vena cava rau kev cais cov ntshav. Cov khib nyiab cellular hauv cov ntshav tau raug tshem tawm los ntawm centrifugation ntawm 3000 g rau 15 min. Txhua qhov kev sim tsiaj hauv peb txoj kev tshawb fawb tau pom zoo los ntawm Pawg Saib Xyuas Kev Ncaj Ncees Tsiaj ntawm Lub Tsev Haujlwm Saib Xyuas Tsiaj, Suav Academy ntawm Kev Tshawb Fawb Txog Kev Ua Liaj Ua Teb (CAAS) (Beijing, Tuam Tshoj), IAS2022-130.

2.2. Kev rho tawm thiab ua kom huv ntawm Serum Exosomes

Exosome cais thiab purification yog nqa tawm siv cov khoom siv exoEasy Maxi (QIAGEN, Hilden, Lub teb chaws Yelemees, cat. no. 76064) raws li cov chaw tsim khoom raws tu qauv.

2.3. Transmission Electron Microscopy (TEM)

Extracted exosome suspensions tau pom mus rau hauv formvar carbo-coated tooj liab mesh, thiab cov exosomes tau rinsed nrog PBS thiab raug tus qauv uranyl acetate staining rau 3 min ntawm chav tsev kub. Tom qab ziab rau ob peb feeb ntawm chav tsev kub, daim phiaj tau pom thiab yees duab ntawm 100 kV los ntawm kev xa hluav taws xob tshuab hluav taws xob (HT-7700, Hitachi-High Tech, Tokyo, Nyiv).

2.4. Nanoparticle Tracking Analysis (NTA)

Extracted exosomes tau diluted nrog 1 × PBS los ntawm kev hloov lub ntim ntawm 10 mus rau 30 µL. Tom qab kuaj cov qauv, cov concentration thiab qhov loj ntawm cov ntshav exosomes tau soj ntsuam los ntawm N30E ntws nano-analyzer ua raws li cov chaw tsim khoom cov lus qhia (NanoFCM, Xiamen, Suav).

2.5. Western Blot

Cov exosome kuaj tau muab ntxiv rau RIPA lysate tov nrog protease inhibitor (Invitrogen, Waltham, MA, USA) thiab phenylmethylsulfonyl fluoride (PMSF) kom rho tawm cov exosome protein, uas yog lysed rau dej khov rau 30 min. Tom qab ntawd, raws li cov lus qhia ntawm Bradford cov khoom siv, peb ntsuas qhov concentration ntawm cov kua qaub exosome protein. Exosome proteins underwent thermal denaturation. Tib cov protein tau muab cais tawm ntawm 12% SDS-PAGE gel thiab tom qab ntawd hloov mus rau polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). Nws tau tsau rau hauv TBST uas muaj 5% skimmed mis nyuj hmoov thiab kaw rau 1 teev ntawm chav tsev kub. Peb soaked lub membrane nyob rau hauv lub diluted thawj antibody (anti-CD9 antibody, Abcam, Boston, MA, USA, # ab92726; anti-CD81 antibody, Abcam, Boston, MA, USA, # ab109201) hmo ntuj ntawm 4 ◦C, thiab zoo tu qab tso. thawj antibody. Peb soaked daim nyias nyias nyob rau hauv diluted thib ob antibody, incubated nws nyob rau hauv chav tsev kub rau 1 h, thiab rov qab tau lub thib ob antibody. Peb tso cov yeeb yaj kiab ntxuav ntawm PBST rau ntawm cov yeeb yaj kiab tshiab, ntxiv cov ntim sib npaug sib xyaw ECL a / b chromogenic kua, thiab muab tso rau hauv chemiluminescence imager.

cistanche cov txiaj ntsig rau txiv neej-ua kom muaj zog tiv thaiv kab mob

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2.6. Exosomal Me RNA Sequencing thiab Kev Ntsuam Xyuas Cov Ntaub Ntawv

Tag nrho RNA los ntawm exosomes tau muab rho tawm nrog Trizol raws li cov chaw tsim khoom cov lus qhia. Tom qab ntawd peb tau kuaj pom qhov RNA concentration thiab optical density (OD) tus nqi thiab kuaj pom qhov degradation thiab purity ntawm RNA nrog 1% agarose gel electrophoresis. Lub caij no, Agilent Bioanalyzer 2100 tau siv los txheeb xyuas qhov ncaj ncees ntawm RNA. Peb siv tag nrho RNA ntawm exosomes tom qab kev soj ntsuam zoo. Raws li cov chaw tsim khoom cov lus qhia, peb siv NEB NEXT multiplex me me RNA tsev qiv ntawv npaj rau Illumina® (Illumina, San Diego, CA, USA). Cov khoom siv tau npaj ib lub tsev qiv ntawv RNA cDNA me me thiab ua kom nws ua 50 nt ib-kawg nyeem los ntawm Illumina Novaseq 6000 platform. Tag nrho cov txheej txheem rau kev npaj tsev qiv ntawv me me RNA tau ua tiav los ntawm Novogene (Beijing, Suav). Cov ntaub ntawv tom qab kev tswj xyuas zoo tau ua raws li cov porcine siv genome (Sus scrofa 11.1) siv bowtie. Paub miRNAs tau txheeb xyuas los ntawm miRbase (v22.0) database [38] (https://www.mirbase.org, nkag rau 14 Lub Ib Hlis 2022), miRdeep2 (v0.0.5) [39], thiab miRevo (v1.1. ) [40] thiab tau siv los kwv yees miRNAs tshiab. Nyob rau tib lub sijhawm, kev tshuaj xyuas qhov sib txawv ntawm miRNAs tau ua los ntawm DESeq (v1.24.0) [41], xav tau|hloov pauv| > 1.6 thiab p <0.05. Kev sib tw tau ua tiav siv MEGA (V11) [42] tom qab los ntawm ib qho kev sib tw hauv paus siv PHAST (v1.6.9) [43] thiab kev soj ntsuam ntawm thaj chaw khaws cia tshaj plaws ntawm 10 tus kab mob, suav nrog WUH3 (GenBank accession no. HM853973), VR2332 ( GenBank accession no. U87392), JXA1 (GenBank accession no. EF112445), CH-1a (GenBank accession no. AY032626), NADC30 (GenBank accession no. HN654459), HUN4 (GenBank accession no. 22-1812 (GenBank accession no. MN648450), SC/DJY (GenBank accession no. MT075480), thiab Lelystad (GenBank accession no. M96262.2). RNAhybrid (V2.0) [44] tau siv los kwv yees qhov kev sib txuas ntawm cov cim miRNA ib ntus rau 3 0 UTR ntawm CHsx1401 tus kab mob genome. Miranda (v3.3a) thiab RNAhybrid tau siv los tsom rau kev kwv yees cov noob. Pawg profile [45] R pob tau siv rau GO (Gene Ontology) kev ua kom muaj txiaj ntsig zoo ntawm lub hom phiaj cov noob thiab KEGG (Kyoto Encyclopedia of Genes thiab Genomes) txoj kev txhim kho kev txheeb xyuas.

2.7. Kev lees paub ntawm miRNA Kev nthuav qhia los ntawm RT-qPCR

Tag Nrho RNA raug cais tawm los ntawm cov ntshav exosomes siv Trizol (Invitrogen, Shanghai, Tuam Tshoj) raws li cov chaw tsim khoom raws tu qauv. Cov RNA cais tau raug txheeb xyuas los ntawm RT-qPCR ntawm cov qauv (n=6 ib pawg). cDNA tau tsim tawm raws li cov lus qhia ntawm miRNA 1st strand cDNA synthesis (los ntawm qia-loop) cov khoom siv (Vazyme, Nanjing, Tuam Tshoj), thiab cov fluorescence kom muaj nuj nqis tau ua los ntawm ABI 7500 raws li cov lus qhia ntawm miRNA universal SYBR qPCR master mix (Vazyme, Nanjing, Suav). Cov thermal voj voog tsis siv tau raws li hauv qab no: thawj theem: 95 ◦C rau 30 s; Theem 2: 95 ◦C rau 5 s, 60 ◦C rau 34 s, thiab 40 cycles; Kauj Ruam 3: 95 ◦C rau 15 s, 60 ◦C rau 1 min, thiab 95 ◦C rau 15 s. Primer sequences ntawm miRNAs, U6 gene, tau siv los ua ib qho kev siv [46] thiab teev nyob rau hauv Cov Lus Ntxiv S1. Txhua qhov kev txheeb xyuas qRT-PCR tau ua tiav siv peb qhov kev hloov pauv roj ntsha thiab nrog peb qhov rov ua dua rau txhua tus qauv. Cov txheeb ze ntau ntawm cov ntawv sau tseg tau suav los ntawm 2-Ct txoj kev, thiab SPSS (v22.0) thiab GraphPad Prism (v8.0) tau siv rau kev txheeb xyuas cov ntaub ntawv thiab kev kos duab, raws li. p <0.05 txhais tau tias qhov sib txawv yog qhov tseem ceeb.

3. Cov txiaj ntsig

3.1. Tus nqi txheeb ze ntawm Antigen thiab Antibody tom qab tus kab mob Inoculation

Cov txiaj ntsig ntawm PRRSV cov tshuaj tiv thaiv kab mob thiab tshuaj tiv thaiv kab mob ua ntej (hnub 0) thiab tom qab (hnub 7) kev sib tw tau qhia hauv Table 1. Kev kuaj xyuas serological ntawm PRRSV antigen thiab cov tshuaj tiv thaiv kab mob ua ntej qhov kev sib tw tsis zoo, thiab cov antigen yog Qhov zoo tom qab qhov kev sib tw, qhia tau hais tias cov npua tau ua tiav nrog CHsx1401.

3.2. Kev rho tawm thiab kev txheeb xyuas cov ntshav Exosomes

Cov vesicles cais los ntawm cov ntshav tau tshawb pom los ntawm TEM. Feem ntau cov vesicles tuaj yeem pom cov concave saucer- lossis disc-shaped exosomes nyob nruab nrab. Lub membrane ntug ntawm exosomes pom, thiab cov morphology kuj ua tiav (Daim duab 1A, B). Kev soj ntsuam nanoparticles pom tias 95.73% ntawm cov exosomes muaj txoj kab uas hla ntawm 30-150 nm, feem ntau nyob ib ncig ntawm 72.25 nm, nrog qhov nruab nrab txoj kab uas hla ntawm 76.22 nm, uas yog raws li qhov loj ntawm exosomes (Daim duab 1C). Qhov loj me no zoo ib yam li qhov kuaj pom los ntawm TEM thiab tau lees paub ntxiv tias tus kheej ntawm cov vesicles li exosomes. Western blot tsom xam pom tias cov vesicles cais los ntawm cov ntshav kuaj tau zoo rau CD9 thiab CD81 proteins (Daim duab 1D). Cov yam ntxwv saum toj no ua raws li cov qauv kev txheeb xyuas exosome tsim los ntawm International Society for Extrace Vesicles (ISEV) hauv MISEV2018 [47].

Table 1. Antigen thiab antibody ntawm (hnub 0) thiab (hnub 7) nrog cov kab mob sib tw.

Daim duab 1. Cov yam ntxwv tseem ceeb ntawm cov ntshav ntshav exosomes. (A, B) qhia cov yam ntxwv morphological ntawm vesicles los ntawm TEM. Scale bars yog 500 nm thiab 100 nm, feem. (C) NTA qhia txog txoj kab uas hla thiab concentration ntawm feem ntau vesicles. (D) Western blot pom tias muaj cov cim exosome CD81 thiab CD9 hauv cov ntshav exosomes. Nco tseg: Mix hauv WB cov txiaj ntsig yog cov qauv sib xyaw ua ke raug rho tawm los ntawm exoEasy Maxi cov khoom siv

3.3. Me me RNA Sequencing ntawm Serum Exosomes

Rau txhua tus qauv, cov ntaub ntawv huv tau mus txog 0.5 Gb, thiab Q30 qhov feem pua ​​​​tau siab dua 96.20%. Cov ntawv nyeem huv ntawm txhua tus qauv tau ua raws li tus npua siv genome. Ntawm 12 tus qauv, pawg tswj hwm tau txais 10,920,887, 10,248,696, 10,109,117, 10,655,494, 9,217,285, thiab 9,782,523 nyeem, raws li. Cov pab pawg kho mob tau txais 11,889,518, 10,593,504, 10,593,504, 12,846,080, 10,105,325, 11,729,451, thiab 9,789,542 nyeem, feem. Qhov nruab nrab, 77.96% ntawm tag nrho cov ntawv nyeem huv muaj 19–22 nucleotides (nt) ntev (Daim duab 2A). Cov ntawv nyeem tom qab kev tswj xyuas tau zoo suav nrog ntau dua 92.59% ntawm tag nrho cov ntawv nyeem. Cov ntawv nyeem huv tau ua tiav raws li cov porcine siv genome, thiab daim ntawv qhia tus nqi ntawm 12 lub tsev qiv ntawv ntawm cov genome yog ntau dua 92.30%, thiab tus nqi mapped yog 94.98% (Daim duab 2B). Nws tau qhia tias lub tsev qiv ntawv serum exosomal miRNA yog qhov zoo thiab tsim nyog rau kev tshuaj xyuas ntxiv. Cov ntsiab lus tau teev nyob rau hauv Cov Lus Qhia Ntxiv S2.

Daim duab 2. Txheej txheem cej luam ntawm cov ntaub ntawv RNA transcriptome me me. (A) Ntev ntev ntawm kev nyeem cov lej ntawm cov kua qaub exosome (nt=nucleotides); (B) tus nqi ntawm 12 qhov piv txwv mapped rau cov genome siv

3.4. Differentially Expression Analysis ntawm miRNAs

Tom qab kev soj ntsuam ntau ntawm cov kev qhia miRNA, miRNAs tau raug tshuaj xyuas los ntawm cov theem pib tau piav qhia yav dhau los hauv Tshooj 2.6. Tag nrho ntawm 305 miRNAs tau txais ua ntej thiab tom qab inoculation ntawm CHsx1401 hom (tswj, n=6; kho, n=6). Tag nrho ntawm 33 qhov sib txawv qhia (DE) miRNAs tau txheeb xyuas los ntawm ob pawg, 13 DE miRNAs tau tswj hwm, thiab 20 DE miRNAs tau raug txo qis hauv pawg kho mob (Daim duab 3 thiab Cov Lus Ntxiv S3).

3.5. Functional Enrichment Analysis ntawm miRNA Target Genes

Tag nrho ntawm 7283 lub hom phiaj cov noob tau kwv yees los ntawm 33 DE miRNAs, thiab cov haujlwm ntawm cov noob caj noob ces yog cov tseem ceeb hauv kev tswj hwm zoo ntawm MAPK cascade, cov txheej txheem lipid metabolism, kev tswj ntawm intracellular signal transduction, ERK1 thiab ERK2 cascade, thiab lwm yam. (Daim duab 4A ). Hais txog kev ua haujlwm ntawm molecular, qhov sib txawv ntawm miRNA lub hom phiaj cov noob tsuas yog tsom rau GTP-enzyme kev tswj hwm kev ua ub no, kev ua haujlwm kinase, nucleoside triphosphatase kev tswj hwm kev ua ub no, thiab lwm yam haujlwm ntsig txog kev hloov pauv thiab lub zog metabolism (Daim duab 4B). Tsis tas li ntawd, ntawm cov khoom ntawm tes, lub hom phiaj cov noob feem ntau koom nrog kev ua haujlwm lom neeg ntawm supramolecular polymers, Golgi, autophagosomes, cell nto, endosomes thaum ntxov, thiab lwm yam. (Daim duab 4C). Lub luag haujlwm ntawm cov khoom no muaj feem cuam tshuam nrog kev tsim ntawm exosomes, uas kuj piav qhia qhov tseeb ntawm qhov sib txawv. KEGG txoj kev txhim kho txoj kev tsom xam pom tau tias lub hom phiaj cov noob tau nthuav dav hauv endocytosis, MAPK signaling txoj kev, Rap1 signaling pathway, sphingolipid signaling pathway, thiab PI3K Akt signaling pathway (p < 0.05) (Daim duab 5A ). Nyob rau tib lub sijhawm, txoj hauv kev muaj txiaj ntsig tau muab cais thiab txheeb xyuas. Cov txiaj ntsig tau pom tias KEGG txoj hauv kev ntawm lub hom phiaj gene yog qhov tseem ceeb hauv kev ua cov ntaub ntawv ib puag ncig, tib neeg cov kab mob, thiab cov kab mob lom (Daim duab 5B).

Daim duab 3. Kev qhia txawv ntawm miRNAs hauv exosomes. (A) Volcano zaj duab xis ntawm miRNAs ntawm kev tswj thiab pab pawg kho mob; (B) hierarchical clustering heatmap ntawm DE miRNAs ntawm pawg tswj thiab kev kho mob.

Daim duab 4. GO function enrichment analysis of DE miRNAs target genes. (A) Cov txheej txheem lom neeg ntawm DE miRNAs phiaj cov noob; (B) molecular functions ntawm DE miRNAs hom noob caj noob ces; (C) cellular Cheebtsam ntawm DE miRNAs tsom cov noob

Daim duab 5. KEGG pathway enrichment tsom xam ntawm cov hom phiaj. (A) Kev nthuav dav KEGG tseem ceeb nrog cov hom phiaj ntawm DE miRNAs; (B) Kev faib tawm ntawm txoj kev KEGG muaj txiaj ntsig zoo.

3.6. Targeting Prediction of Serum Exosomal miRNA thiab PRRSV CHsx1401 Genome

Raws li phastCons qhab nia ntawm ib lub hauv paus tom qab kev sib raug zoo los ntawm PHAST, tag nrho ntawm yim feem ntau khaws cia (dub bands saum toj kawg nkaus daim ntawv qhia) tau txais ntawm cov kab mob genomes (Daim duab 6). Tag nrho ntawm 31 DE miRNAs tau pom los khi rau ntu kev txuag los ntawm kev kwv yees cov miRNAs khi rau ntu kev txuag. Ntawm lawv, hauv thaj chaw khaws cia (14,644–15,020 nt) ze tshaj rau 30 UTR (14,870–15,020) ntawm CHsx1401 genome, 16 DE miRNAs raug kwv yees los khi rau nws, suav nrog 5 miRNAs ({15}mAR} c, ssc-miR-375, ssc-miR-378, ssc-miR-486, thiab ssc-miR-6529) uas tuaj yeem khi rau 30 UTR ntawm CHsx1401. Ntawm cov miRNAs no, tsuas yog ssc-miR-223 tau tswj hwm tom qab kis kab mob, thiab lwm yam miRNAs tau raug txo qis tom qab kis kab mob. Saib Ntxiv Table S4 kom paub meej.

Daim duab 6. Conserved segments nyob rau hauv lub genome ntawm CHsx1401 hom kwv yees los ntawm PHAST

3.7. Kev tshuaj ntsuam DE miRNAs ntsig txog Exosome Function thiab PRRSV

Ntau qhov sib txawv ntawm miRNAs hais txog kev ua haujlwm ntawm exosomes thiab PRRSV tau pom los ntawm kev ua kom muaj txiaj ntsig zoo ntawm cov hom phiaj. Ntawm lawv, 11 DE miRNAs xws li ssc-miR-4331-3p, ssc-miR-744, thiab ssc-miR-320 tau koom nrog hauv exosome uptake, thiab lawv cov hom phiaj tseem ceeb yog tsom rau hauv tsev neeg Ras gene, tsev neeg annexin, thiab ADP ribosylation gene tsev neeg. Kaum yim DE miRNAs, suav nrog sscmiR-10b, ssc-miR-124a, thiab ssc-miR-128, koom nrog txoj hauv kev tiv thaiv kab mob, thiab lawv lub hom phiaj cov noob yog tsom rau hauv MAPK tsev neeg gene, PIK3 tsev neeg, thiab tsev neeg protein phosphatase. Thaum 11 DE miRNAs koom nrog kev cuam tshuam tus kab mob, cov noob caj noob ces muaj feem xyuam rau hauv MAPK gene tsev neeg thiab tsev neeg protein phosphatase. Tsis tas li ntawd, ntau qhov sib txawv ntawm miRNAs, xws li tshiab_102. Rau DE miRNAs, suav nrog ssc-miR-320, ssc-miR-423-5p, ssc-miR-4331-3p, ssc-miR-7137-3p, thiab ssc-miR{{ 25 }}, yog co-exosome muaj nuj nqi, PRRSV virus ntxeem tau, thiab txoj kev tiv thaiv kab mob, raws li qhia nyob rau hauv daim duab 7. Cov ntsiab lus muaj nyob rau hauv Supplementary Table S5.

Daim duab 7. DE miRNAs ntsig txog exosome uptake, PRRSV ntxeem tau, thiab kev tiv thaiv

3.8. QRT-PCR Assay ntawm DE miRNAs ntawm Ob Pawg

Tsib DE miRNAs raug xaiv los ua pov thawj. Raws li cov txiaj ntsig qRT-PCR, kev qhia ntawm ssc-miR-19a thiab ssc-miR-32 tau nce hauv pawg kho mob, thaum ssc-miR-124a, ssc-miR{ {8}}, thiab ssc-miR-34c tau qhia ntau dua nyob rau hauv pawg tswj hwm, raws li cov ntaub ntawv sib txuas (Daim duab 8).

Daim duab 8. Tsib DE miRNA validated los ntawm qRT-PCR

4. Kev sib tham

PRRSV tseem yog cov kab mob tawv ncauj hauv kev lag luam npua thoob ntiaj teb, ua rau muaj kev lag luam loj hauv ntiaj teb. Tam sim no, kev txhaj tshuaj tiv thaiv feem ntau yog siv los tiv thaiv thiab tswj PRRSV, ntawm cov tshuaj tiv thaiv kab mob hloov pauv nyob (MLV) yog siv dav tshaj plaws [48]. Txawm hais tias cov tshuaj tiv thaiv no tau zoo los txo cov kab mob PRRS thiab qhov tshwm sim, nws kuj tseem ua rau muaj kev hloov pauv caj ces thiab ntau haiv neeg ntawm tus kab mob thiab ua rau muaj kev sib kis ntawm cov kab mob qus thiab cov kab mob nyob hauv thaj chaw [49,50]. Nyob rau hauv xyoo tas los no, kev sib kis thiab nthuav dav ntawm tus kab mob recombinant NADC30-zoo li PRRSV strain tau ua rau muaj ntau yam kab mob porcine reproductive thiab ua pa syndrome hauv Suav teb. Qhov zoo sib xws ntawm CHsx1401 thiab NADC30 siv hauv txoj kev tshawb no tseem nyob ntawm 92.2–99.1%. Txij thaum ntawd los, nws tau dhau los ua kab mob sib kis hauv Suav teb. Exosomes, raws li tus neeg nruab nrab ntawm kev sib txuas lus ntawm tes, tau dav pom nyob rau hauv ntau yam kua hauv lub cev thiab muaj qhov tshwj xeeb zoo hauv kev kuaj kab mob thiab kev kho mob [51,52]. Raws li cov ntaub ntawv dhau los, exosomes ua lub luag haujlwm tseem ceeb hauv kev sib txuas lus hauv kev nthuav qhia antigen [53], lub cev tiv thaiv kab mob [53,54], tus kab mob rov ua dua tshiab [54], mob qog noj ntshav [55], kab mob neurodegenerative [56], angiogenesis [57], qog cell tsiv teb tsaws [58] thiab ntxeem tau [59], thiab muaj txiaj ntsig tshawb fawb siab.

Nyob rau hauv txoj kev tshawb no, high-throughput sequencing thev naus laus zis tau siv los tsim cov miRNA qhia profile ntawm serum exosomes, thiab 33 DE miRNAs tau txheeb xyuas. Raws li peb txhua tus paub, tus tswv tsev-encoded miRNA tuaj yeem khi nrog tus kab mob genome thiab tom qab ntawd tswj kev rov ua dua, kev sib txuas, thiab tso tawm tus kab mob kom txwv tsis pub kis kab mob thiab cuam tshuam rau cov txheej txheem pathological [15]. Kev tshawb fawb ntawm miRNAs tsom rau tus kab mob genome kuj tau rov qhia dua hauv cov tsiaj. gga-miR-454 thiab gga-miR-130b nyob rau hauv qaib kis kab mob bursal tuaj yeem tsom tus kab mob genome los tiv thaiv kab mob sib kis, thaum gga-miR-21 ncaj qha tsom rau cov kab mob kis kab mob VP1 los tiv thaiv kab mob protein translation [60,61]. Hauv PRRSV cov kev tshawb fawb, ssc-miR-181 tshwj xeeb khi rau thaj chaw khaws cia siab heev hauv qab ntawm tus kab mob genome ORF4 thiab cuam tshuam PRRSV replication [62]. Hauv txoj kev tshawb no, qhov sib txawv ntawm kev qhia ntawm ssc-miR-181 ntawm ob pawg tsis tau mus txog qib tseem ceeb. Hauv peb txoj kev tshawb fawb, cov genomes ntawm cuaj tus kab mob PRRSV sib txawv tau muab piv nrog cov kab mob CHsx1401, thiab yim feem ntau khaws cia tau txheeb xyuas. Nws tau kwv yees tias 31 DE miRNAs tuaj yeem khi rau 8 qhov kev txuag feem ntau ntawm CHsx1401, thiab 16 DE miRNAs tuaj yeem khi rau cov kev khaws cia ze li ntawm 30 UTR ntawm CHsx1401. Ntawm lawv, 5 DE miRNAs (ssc-miR-34c, ssc-miR-375, ssc-miR-378, ssc-miR-486, thiab ssc-miR{{ 36}}) tuaj yeem khi ib txhij rau CHsx1401 30 UTR. Tsis tas li ntawd, qhov kev qhia tawm ntawm ssc-miR-223 tau kwv yees los khi rau 30 UTR lub hom phiaj ntawm PRRSV genome. Cov txiaj ntsig tau pom tias qhov kev khaws cia ntawm tus kab mob genome tuaj yeem ua lub luag haujlwm tseem ceeb hauv nws cov kab mob, thiab cov miRNAs uas tuaj yeem khi rau cov kev khaws cia ntawm cov genomes ntawm ntau hom PRRSV yuav muaj qhov tseem ceeb hauv kev tswj cov kab mob ntawm tus kab mob. Qee qhov sib txawv ntawm miRNAs tau raug pov thawj tias muaj feem xyuam nrog PRRSV los ntawm cov kev tshawb fawb yav dhau los thiab tseem muaj kev koom tes ncaj qha rau kev tswj hwm ntawm PRRSV, suav nrog ssc-miR-10b [63], ssc-miR-378 [30] , ssc-miR-124a [64], let-7f-5p [65], ssc-miR-744 [66], thiab ssc-miR{{57 ib [67].

PRRSV tuaj yeem zam kev tiv thaiv tus tswv los ntawm kev cuam tshuam nrog lub cev tiv thaiv kab mob. Cov txheej txheem no yog tswj hwm los ntawm ntau txoj kev taw qhia, suav nrog MAPK signaling pathway, PI3K Akt signaling pathway, autophagy, chemokine, thiab TNF signaling pathway. Tam sim no, txoj kev taw qhia MAPK suav nrog peb txoj hauv kev tseem ceeb: ERK1/2, JNK, thiab p38 txoj hauv kev. Kev ua kom lub MAPK cascade tuaj yeem txhawb nqa tus tswv tsev cell apoptosis, pab tus kab mob hauv kev khiav tawm ntawm tus tswv tsev tiv thaiv kab mob, thiab txhawb nqa PRRSV replication [68]. Tsis tas li ntawd, kev ua kom muaj c-Jun N-terminal kinases (JNKs) thiab p38 tuaj yeem txhawb kev tso tawm ntawm qhov mob IL-10 [68–70] thiab txhim kho cov nyhuv inflammatory. Ntxiv nrog rau inducing apoptosis, PRRSV tseem tuaj yeem ua rau autophagy, uas tuaj yeem txhawb nqa PRRSV replication. Kev ua kom PI3K / Akt yog qhov tsim nyog rau kev nkag mus rau tus kab mob thiab txhawb nqa tus kab mob rov ua dua, thiab PRRSV-activated Akt inhibits tus tswv tsev cell apoptosis los ntawm kev tswj tsis zoo ntawm JNK txoj kev [71]. TNF Nws tuaj yeem ua lub luag haujlwm tseem ceeb hauv kev txhawb nqa thiab kev tswj hwm ntawm inflammatory teb ua ke nrog rau lwm yam inflammatory yam, tab sis TNF Kev nthuav qhia cuam tshuam los ntawm cov kev cai tsis zoo ntawm PRRSV replication [72]. Hauv txoj kev tshawb no, miRNAs (ssc-miR-10b, ssc-miR-122-5p, ssc-miR-124a, ssc-miR-128, ssc-miR{ {24}}a-5p, thiab lwm yam.) enriched nyob rau hauv cov kev no muaj feem xyuam rau PRRSV-induced apoptosis, autophagy, thiab o thiab yog ze ze rau cov kab mob tiv thaiv kab mob, tiv thaiv kab mob, thiab replication.

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

Lub cell plasma membrane yog nplua nuj nyob rau hauv ntau yam lipid rafts, thiab sphingolipid- thiab cholesterol-nplua nuj sphingolipids (sphingomyelin thiab glycosphingolipids) yog cov molecules tseem ceeb ntawm lipid rafts. Kev lees paub ntawm lipids los ntawm qee cov proteins ntawm tus kab mob tuaj yeem yog qhov tsim nyog rau kev nkag mus ntawm tus kab mob [73]. Cov kab mob hauv lub hnab ntim cov kab mob glycoproteins nkag mus rau hauv lipid rafts ntawm theem ntawm tus kab mob nkag mus, cuam tshuam nrog cov receptors nyob rau hauv lipid rafts, los yog hloov los ntawm lawv lub xeev ntuj mus rau activated daim ntawv los pib los yog txhawb cov kab mob internalization / fusion, xws li HSV, SARS coronavirus, thiab piglet epidemic diarrhea virus [73,74]. Cov kev tshawb fawb yav dhau los pom tias kev tshem tawm cov roj cholesterol los ntawm qhov chaw ntawm MARC-145 hlwb txo qis PRRSV tus kab mob, ua kom pom tias inhibition ntawm tus kab mob PRRSV tshwj xeeb yog kho los ntawm kev tshem tawm cov roj cholesterol hauv cellular. Kev txo qis ntawm cell membrane cov roj cholesterol ua rau muaj kev cuam tshuam tus kab mob nkag mus, tshwj xeeb tshaj yog cov kab mob txuas, thiab tso tawm [75]. Sphingolipid metabolism tuaj yeem tswj cov txheej txheem membrane thiab adhesion, uas yog qhov tseem ceeb hauv PRRSV tus kab mob ntxeem tau. Endocytosis yog qhov tseem ceeb tshaj plaws hauv kev tshawb fawb no. Endocytosis yog ib qho tseem ceeb mechanism ntawm exosome uptake los ntawm lub hom phiaj hlwb. Cov kev tshawb fawb yav dhau los tau pom tias exosome uptake yog ib qho kev xav tau ntawm lub zog thiab cytoskeleton-dependent txheej txheem, uas qhia txog lub luag haujlwm ntawm endocytosis hauv cov txheej txheem no [76]. Nws tau raug pov thawj tias ntau txoj hauv kev tuaj yeem kho cov txheej txheem no, suav nrog phagocytosis, macropinocytosis, clathrin, thiab lwm yam. [77,78], uas ua rau muaj kev faib tawm thiab lub luag haujlwm ntawm cov tshuaj endocytosed. Kev nthuav dav ntawm qhov sib txawv ntawm exosomal miRNAs nyob rau hauv txoj kev no qhia tau hais tias exosomes ua lub luag haujlwm tseem ceeb hauv PRRSV tus kab mob, thiab kev tswj hwm cov ntsiab lus ntawm kev thauj mus los hauv exosomes tuaj yeem ua rau muaj kev hloov pauv pathophysiological hauv lub hom phiaj hlwb thiab lub cev.

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

5. Cov lus xaus

Los ntawm kev txheeb xyuas thiab kev txheeb xyuas bioinformatics ntawm serum exosomal miRNAs los ntawm PRRSV-mob npua, ntau yam ntawm PRRSV-txog txoj hauv kev thiab sib txawv qhia miRNAs tau txais hauv txoj kev tshawb no, xws li ssc-miR-4331-3p, ssc-miR{{ 5}}, sscmiR-320, ssc-miR-10b, ssc-miR-124a, ssc-miR-128, thiab lwm yam, uas ua lub luag haujlwm muaj peev xwm ua haujlwm hauv PRRSV -induced lub cev tiv thaiv kab mob, ntxeem tau, thiab exosome uptake. Tsis tas li ntawd, vim hais tias ib leeg miRNA tuaj yeem tsom ntau cov noob thiab ib lub noob kuj tseem tswj hwm los ntawm ntau lub miRNAs, ntau miRNAs ua haujlwm ntau yam hauv txoj hauv kev saum toj no. Qee cov miRNAs tau raug txheeb xyuas los tswj tus kab mob PRRSV los ntawm kev ua haujlwm ntawm cov receptors tseem ceeb lossis ncaj qha rau cov kab mob genome, xws li ssc-miR-10b, ssc-miR-378, miR-124a, cia-7f-5p, ssc-miR-744, ssc-miR-19a, thiab lwm yam. Meanwhile, txoj kev tshawb no kuj tau kwv yees ntau yam miRNAs uas tuaj yeem khi rau qhov kev txuag feem ntau ntawm 30 UTR ntawm CHX1401 tus kab mob genome, suav nrog ssc-miR-34c, ssc-miR-375, ssc-miR-378, ssc-miR{{34} }, thiab ssc-miR-6529, uas tej zaum yuav yog ib qho tseem ceeb rau kev tswj cov kab mob kis kab mob.

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