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Toxoplasma gondii yog ib qho kab mob intracellular protozoan parasite uas hloov cov microenvironment ntawm tus tswv tsev muaj tus kab mob thiab paub tias muaj feem cuam tshuam nrog qhov tshwm sim ntawm cov qog hlwb loj hlob.Hauv txoj kev tshawb no, peb xav tias exosomal miRNA-21 los ntawm Toxoplasma tus kab mob txhawb kev loj hlob ntawm cov qog hlwb.Exosomes los ntawm Toxoplasma-infected BV2 microglia tau pom zoo thiab internalization ntawm U87 glioma hlwb tau lees paub.Exosomal microRNA qhia profiles tau txheeb xyuas siv arrays ntawm microRNA thiab microRNA-21A-5p cuam tshuam nrog Toxoplasma gondii thiab qog sorting.Peb kuj tau tshawb xyuas cov qib mRNA ntawm cov qog uas cuam tshuam cov noob hauv U87 glioma hlwb los ntawm kev hloov miR-21 qib hauv exosomes thiab cov nyhuv ntawm exosomes rau tib neeg U87 glioma cell proliferation.Hauv exosomes ntawm U87 glioma hlwb kis nrog Toxoplasma gondii, kev qhia ntawm microRNA-21 yog nce thiab kev ua ntawm cov tshuaj tiv thaiv kab mob (FoxO1, PTEN, thiab PDCD4) txo.BV2-derived exosomes kis nrog Toxoplasma induce proliferation ntawm U87 glioma hlwb.Exosomes txhawb kev loj hlob ntawm U87 hlwb hauv tus qauv nas qog.Peb hais tias kev nce exosomal miR-21 hauv Toxoplasma-mob BV2 microglia tuaj yeem ua lub luag haujlwm tseem ceeb ua tus txhawb nqa kev loj hlob ntawm tes hauv U87 glioma hlwb los ntawm kev txo qis cov tshuaj tiv thaiv kab mob.
Nws kwv yees tias ntau dua 18.1 lab tus neeg mob qog noj ntshav tau kuaj pom thoob ntiaj teb hauv xyoo 2018, nrog txog 297,000 cov qog nqaij hlav hauv nruab nrab hauv lub paj hlwb kuaj txhua xyoo (1.6% ntawm tag nrho cov qog) 1.Cov kev tshawb fawb yav dhau los tau pom tias muaj feem cuam tshuam rau kev tsim cov qog nqaij hlav hauv tib neeg lub hlwb suav nrog ntau yam khoom siv tshuaj, tsev neeg keeb kwm, thiab ionizing hluav taws xob los ntawm lub taub hau kho mob thiab cov cuab yeej kuaj mob.Txawm li cas los xij, qhov tseeb ua rau cov mob phem no tsis paub.Kwv yees li 20% ntawm tag nrho cov qog nqaij hlav thoob ntiaj teb yog tshwm sim los ntawm cov kab mob sib kis, suav nrog cov kab mob, kab mob thiab kab mob parasites3,4.Cov kab mob kis tau cuam tshuam rau tus tswv tsev ntawm cov txheej txheem caj ces, xws li kho DNA thiab lub voj voog ntawm tes, thiab tuaj yeem ua rau mob ntev thiab puas tsuaj rau lub cev tiv thaiv kab mob 5.
Cov kab mob sib kis nrog rau tib neeg mob qog noj ntshav yog cov kab mob kis ntau tshaj plaws, suav nrog tib neeg papillomaviruses thiab kab mob siab B thiab C.Parasites kuj tuaj yeem ua lub luag haujlwm tseem ceeb hauv kev loj hlob ntawm tib neeg mob qog noj ntshav.Ntau tus kab mob parasite, xws li Schistosoma, Opishorchis viverrini, O. felineus, Clonorchis sinensis thiab Hymenolepis nana, tau cuam tshuam rau ntau hom mob qog noj ntshav hauv tib neeg 6,7,8.
Toxoplasma gondii yog ib qho intracellular protozoan uas tswj cov microenvironment ntawm cov kab mob host cell.Cov kab mob no tau kwv yees kis kwv yees li 30% ntawm lub ntiaj teb cov pej xeem, ua rau tag nrho cov pej xeem muaj kev pheej hmoo 9,10.Toxoplasma gondii tuaj yeem kis tau rau hauv lub cev tseem ceeb, suav nrog lub hauv nruab nrab lub paj hlwb (CNS), thiab ua rau muaj mob hnyav xws li mob meningitis thiab encephalitis, tshwj xeeb tshaj yog rau cov neeg mob tiv thaiv kab mob 9.Txawm li cas los xij, Toxoplasma gondii kuj tseem tuaj yeem hloov pauv qhov chaw ntawm tus neeg mob los ntawm kev hloov pauv kev loj hlob ntawm tes thiab kev tiv thaiv kab mob hauv cov tib neeg uas muaj kev tiv thaiv kab mob, ua rau kev saib xyuas ntawm tus kab mob asymptomatic 9,11.Interestingly, muab kev sib raug zoo ntawm T. gondii prevalence thiab mob qog nqaij hlav hauv hlwb, qee cov ntaub ntawv qhia tias nyob rau hauv vivo host ib puag ncig kev hloov pauv vim tus kab mob T. gondii ntev zoo li cov qog microenvironment.
Exosomes hu ua intercellular communicators uas xa cov ntsiab lus lom, nrog rau cov proteins thiab nucleic acids, los ntawm cov hlwb nyob sib ze16,17.Exosomes tuaj yeem cuam tshuam cov kab mob ntsig txog kab mob xws li tshuaj tiv thaiv apoptosis, angiogenesis, thiab metastasis hauv qog microenvironment.Tshwj xeeb, miRNAs (miRNAs), me me tsis-coding RNAs txog 22 nucleotides ntev, yog ib qho tseem ceeb tom qab transcriptional noob regulators uas tswj ntau tshaj 30% ntawm tib neeg mRNA los ntawm miRNA-induced silencing complex (miRISC).Toxoplasma gondii tuaj yeem cuam tshuam cov txheej txheem lom neeg los ntawm kev tswj cov kab mob miRNA hauv cov kab mob.Tus tswv tsev miRNAs muaj cov cim tseem ceeb rau kev tswj hwm cov txheej txheem lom neeg kom ua tiav cov kab mob muaj sia nyob.Yog li, kawm txog kev hloov pauv hauv tus tswv tsev miRNA profile thaum kis kab mob nrog T. gondii tuaj yeem pab peb nkag siab txog kev sib cuam tshuam ntawm tus tswv tsev thiab T. gondii kom meej meej.Tseeb, Thirugnanam et al.15 tau qhia tias T. gondii txhawb lub hlwb carcinogenesis los ntawm kev hloov nws cov lus qhia ntawm cov tswv tsev tshwj xeeb miRNAs cuam tshuam nrog cov qog loj hlob thiab pom tias T. gondii tuaj yeem ua rau gliomas hauv cov tsiaj sim.
Txoj kev tshawb no tsom mus rau kev hloov pauv ntawm exosomal miR-21 hauv tus tswv tsev microglia kis Toxoplasma BV2.Peb tau pom lub luag haujlwm ua tau ntawm kev hloov pauv exosomal miR-21 hauv kev loj hlob ntawm U87 glioma hlwb vim kev tuav cia hauv lub nucleus ntawm FoxO1 / p27, uas yog lub hom phiaj ntawm overexpressed miR-21.
Exosomes muab tau los ntawm BV2 tau txais los ntawm kev siv cov centrifugation sib txawv thiab raug siv los ntawm ntau txoj hauv kev los tiv thaiv kev sib kis nrog cov khoom siv ntawm tes lossis lwm yam vesicles.SDS-polyacrylamide gel electrophoresis (SDS-PAGE) tau pom cov qauv sib txawv ntawm cov proteins uas tau muab rho tawm los ntawm BV2 hlwb thiab exosomes (Daim duab 1A), thiab cov qauv raug soj ntsuam rau lub xub ntiag ntawm Alix, uas tau soj ntsuam los ntawm Western blotting ntawm exosomal protein markers nyob rau hauv .Alix labeling tau pom nyob rau hauv exosome proteins tab sis tsis nyob rau hauv BV2 cell lysate proteins (Fig. 1B).Tsis tas li ntawd, purified RNA los ntawm exosomes muab los ntawm BV2 tau soj ntsuam siv bioanalyzer.18S thiab 28S ribosomal subunits tsis tshua pom nyob rau hauv exosomal RNA migration qauv, qhia tau hais tias purity txhim khu kev qha (Daim duab 1C).Thaum kawg, kis tau tus mob electron microscopy pom tias cov exosomes pom muaj li ntawm 60-150 nm loj thiab muaj lub khob zoo li tus qauv ntawm exosome morphology (Fig. 1D).
Cov yam ntxwv ntawm exosomes muab los ntawm BV2 hlwb.(A) Kev nyab xeeb cov ntaub ntawv nplooj ntawv.Proteins raug cais tawm ntawm BV2 hlwb lossis exosomes tau los ntawm BV2.Protein qauv txawv ntawm cov cell thiab exosomes.(B) Western blot tsom xam ntawm exosomal marker (Alix).(C) Kev ntsuam xyuas ntawm purified RNA los ntawm BV2 hlwb thiab BV2 muab tau los ntawm exosomes siv bioanalyzer.Yog li, 18S thiab 28S ribosomal subunits hauv BV2 hlwb tsis tshua pom muaj nyob hauv exosomal RNA.(D) Kev xa tawm electron microscopy pom tias exosomes cais los ntawm BV2 hlwb tau ua tsis zoo nrog 2% uranyl acetate.Exosomes yog kwv yees li 60-150 nm hauv qhov loj thiab lub khob-zoo li tus (Song thiab Jung, cov ntaub ntawv tsis tau tshaj tawm).
Cellular internalization ntawm BV2-derived exosomes rau hauv U87 tib neeg glioma hlwb raug soj ntsuam siv confocal microscopy.PKH26 sau npe exosomes yog nyob rau hauv cytoplasm ntawm U87 hlwb.Nuclei tau stained nrog DAPI (Fig. 2A), qhia tau hais tias BV2-derived exosomes tuaj yeem ua haujlwm sab hauv los ntawm cov tswv tsev cell thiab cuam tshuam rau ib puag ncig ntawm cov hlwb tau txais.
Internalization ntawm BV2-derived exosomes rau hauv U87 glioma hlwb thiab BV2-derived exosomes kis Toxoplasma RH induced proliferation ntawm U87 glioma hlwb.(A) Exosomes engulfed los ntawm U87 hlwb ntsuas los ntawm confocal microscopy.U87 glioma hlwb raug incubated nrog exosomes sau nrog PKH26 (liab) los yog tsis muaj kev tswj rau 24 teev.Lub nuclei tau stained nrog DAPI (xiav) thiab tom qab ntawd pom nyob rau hauv ib lub tshuab tsom iav confocal (scale bar: 10 μm, x 3000).(B) U87 glioma cell proliferation yog txiav txim los ntawm kev ntsuam xyuas cell proliferation.U87 glioma hlwb tau kho nrog exosomes rau lub sijhawm qhia. * P <0.05 tau txais los ntawm Tub Ntxhais Kawm qhov kev xeem. * P <0.05 tau txais los ntawm Tub Ntxhais Kawm qhov kev xeem. *P < 0,05 получено по t-критерию Стьюдента. *P <0.05 los ntawm Tub Ntxhais Kawm T-test. *P <0.05 通过学生t 检验获得. * P <0.05 * P <0,05, полученный с помощью t-критерия Стьюдента. * P <0.05 tau txais los ntawm Tub Ntxhais Kawm T-test.
Tom qab paub meej tias lub internalization ntawm BV2-derived exosomes rau hauv U87 glioma hlwb, peb tau ua cell proliferation assays los soj ntsuam lub luag hauj lwm ntawm BV2-derived Toxoplasma-derived exosomes nyob rau hauv kev loj hlob ntawm tib neeg glioma hlwb.Kev kho mob ntawm U87 hlwb nrog exosomes los ntawm T. gondii-mob BV2 hlwb tau pom tias T. gondii-mob BV2-derived exosomes ua rau muaj kev loj hlob ntawm U87 hlwb ntau dua piv rau kev tswj hwm (Fig. 2B).
Tsis tas li ntawd, kev loj hlob ntawm U118 hlwb tau zoo ib yam li U87, raws li Toxoplasma stimulated exosomes ua rau cov qib siab tshaj plaws ntawm kev loj hlob (cov ntaub ntawv tsis qhia).Raws li cov ntaub ntawv no, peb tuaj yeem qhia tau tias BV2-derived Toxoplasma-exosomes ua lub luag haujlwm tseem ceeb hauv glioma cell proliferation.
Txhawm rau tshawb xyuas cov txiaj ntsig ntawm Toxoplasma-infected BV2-derived exosomes ntawm qog hlav, peb tau txhaj U87 glioma hlwb rau hauv cov nas liab qab rau tus qauv xenograft thiab txhaj tshuaj BV2-derived exosomes lossis RH-infected BV2-derived exosomes.Tom qab cov qog tau tshwm sim tom qab 1 lub lis piam, txhua pab pawg sim ntawm 5 nas tau muab faib raws li qog loj los txiav txim siab qhov pib pib, thiab cov qog loj tau ntsuas rau 22 hnub.
Hauv cov nas nrog tus qauv U87 xenograft, cov qog loj thiab qhov hnyav tau pom nyob rau hauv BV2-derived RH-infected exosome pawg hnub 22 (Daim duab 3A,B).Ntawm qhov tod tes, tsis muaj qhov sib txawv ntawm cov qog loj ntawm BV2-derived exosome pawg thiab pawg tswj xyuas tom qab kev kho exosome.Tsis tas li ntawd, nas txhaj nrog glioma hlwb thiab exosomes pom cov qog loj tshaj plaws nyob rau hauv pawg ntawm RH-infected BV2-derived exosomes (Fig. 3C).Cov txiaj ntsig no qhia tau tias BV2-derived Toxoplasma-infected exosomes ua rau glioma loj hlob hauv tus qauv nas qog.
Oncogenesis (AC) ntawm BV2-derived exosomes hauv U87 xenograft nas qauv.Cov qog loj (A) thiab qhov hnyav (B) tau nce ntau hauv BALB / c cov nas liab qab kho nrog RH-infected exosomes muab los ntawm BV2.BALB/c liab qab nas (C) tau txhaj subcutaneously nrog 1 x 107 U87 hlwb raug tshem tawm hauv Matrigel sib tov.Rau hnub tom qab txhaj tshuaj, 100 μg ntawm BV2-derived exosomes tau kho hauv cov nas.Cov qog loj thiab qhov hnyav tau ntsuas ntawm cov hnub qhia thiab tom qab kev txi, feem. * P <0.05. * P <0.05. * 0,05. * P <0.05. * P <0.05. * P <0.05. * 0,05. * P <0.05.
Cov ntaub ntawv qhia tau hais tias 37 miRNAs (16 overexpressed thiab 21 downexpressed) cuam tshuam nrog kev tiv thaiv kab mob lossis qog kev loj hlob tau hloov pauv hauv microglia tom qab kis tus kab mob Toxoplasma RH (Fig. 4A).Cov txheeb ze qhia theem ntawm miR-21 ntawm cov hloov pauv miRNAs tau lees paub los ntawm lub sijhawm tiag tiag RT-PCR hauv exosomes tau los ntawm BV2, exosomes kho nrog BV2 thiab U87 hlwb.Kev nthuav qhia ntawm miR-21 pom tau tias muaj kev nce ntxiv hauv exosomes los ntawm BV2 hlwb kis Toxoplasma gondii (RH strain) (Fig. 4B).Cov txheeb ze qhia theem ntawm miR-21 hauv BV2 thiab U87 hlwb tau nce tom qab hloov pauv exosomes (Fig. 4B).Cov txheeb ze ntawm miR-21 qhia nyob rau hauv lub hlwb cov ntaub so ntswg ntawm cov neeg mob qog thiab nas kis tus kab mob Toxoplasma gondii (ME49 strain) yog siab tshaj nyob rau hauv kev tswj, raws li (Fig. 4C).Cov txiaj ntsig no cuam tshuam nrog qhov sib txawv ntawm qhov qhia theem ntawm kev kwv yees thiab paub tseeb microRNAs hauv vitro thiab hauv vivo.
Kev hloov pauv hauv kev qhia ntawm exosomal miP-21a-5p hauv microglia kis Toxoplasma gondii (RH).(A) Qhia txog kev hloov pauv tseem ceeb hauv siRNA cuam tshuam nrog kev tiv thaiv kab mob los yog qog nqaij hlav tom qab kis kab mob T. gondii RH.(B) Tus txheeb ze miR-21 qhia theem tau kuaj pom los ntawm lub sijhawm RT-PCR hauv BV2-derived exosomes, BV2-kho exosomes, thiab U87 hlwb.(C) Cov txheeb ze miR-21 qhia qib tau pom nyob rau hauv lub hlwb cov ntaub so ntswg ntawm cov neeg mob qog (N=3) thiab nas kis tus kab mob Toxoplasma gondii (ME49 strain) (N=3). * P <0.05 tau txais los ntawm Tub Ntxhais Kawm qhov kev xeem. * P <0.05 tau txais los ntawm Tub Ntxhais Kawm qhov kev xeem. *P < 0,05 было получено с помощью t-критерия Стьюдента. * P <0.05 tau txais los ntawm Tub Ntxhais Kawm T-test. *P <0.05 通过学生t 检验获得. * P <0.05 * P <0,05, полученный с помощью t-критерия Стьюдента. * P <0.05 tau txais los ntawm Tub Ntxhais Kawm T-test.
Exosomes los ntawm RH-mob BV2 hlwb ua rau kev loj hlob ntawm gliomas hauv vivo thiab hauv vitro (Fig. 2, 3).Txhawm rau txheeb xyuas cov mRNAs cuam tshuam, peb tau tshuaj xyuas mRNA qib ntawm cov tshuaj tiv thaiv cov noob caj noob ces, lub thawv forkhead O1 (FoxO1), PTEN, thiab cov txheej txheem cell tuag 4 (PDCD4) hauv U87 hlwb kis nrog exosomes tau los ntawm BV2 lossis RH BV2.Kev tshuaj xyuas bioinformatics tau pom tias ntau cov noob qog muaj feem cuam tshuam, suav nrog FoxO1, PTEN, thiab PDCD4 noob, muaj miR-2121,22 qhov chaw sib khi.mRNA qib ntawm antitumor hom noob tau txo qis hauv RH-mob BV2-derived exosomes piv rau BV2-derived exosomes (Fig. 5A).FoxO1 pom tau tias txo cov protein ntau hauv RH-mob BV2-derived exosomes piv rau BV2-derived exosomes (Daim duab 5B).Raws li cov txiaj ntsig no, peb tuaj yeem lees paub tias exosomes tau los ntawm RH-infected BV2 downregulate anti-oncogenic genes, tswj lawv lub luag haujlwm hauv qog loj hlob.
Toxoplasma RH-infected BV2-derived exosomes induce suppression of antitumor genes in U87 glioma cells by Toxoplasma RH-infected BV2-derived exosomes.(A) Real-time PCR ntawm FoxO1, PTEN thiab PDCD4 qhia hauv exosomes muab los ntawm T. gondii RH-infected BV2 piv rau PBS exosomes.β-actin mRNA tau siv los ua kev tswj hwm.(B) FoxO1 qhia tau txiav txim siab los ntawm Western blotting thiab densitometry cov ntaub ntawv raug soj ntsuam los ntawm kev siv ImageJ program. * P <0.05 tau txais los ntawm Tub Ntxhais Kawm qhov kev xeem. * P <0.05 tau txais los ntawm Tub Ntxhais Kawm qhov kev xeem. *P < 0,05 было получено с помощью t-критерия Стьюдента. * P <0.05 tau txais los ntawm Tub Ntxhais Kawm T-test. *P <0.05 通过学生t 检验获得. * P <0.05 * P <0,05, полученный с помощью t-критерия Стьюдента. * P <0.05 tau txais los ntawm Tub Ntxhais Kawm T-test.
Txhawm rau nkag siab txog cov txiaj ntsig ntawm miP-21 hauv exosomes ntawm cov qog-koom nrog cov noob kev tswj hwm, U87 hlwb tau hloov nrog tus inhibitor ntawm miP-21 siv Lipofectamine 2000 thiab cov hlwb tau sau 24 teev tom qab hloov pauv.FoxO1 thiab p27 qhia qib hauv cov hlwb hloov nrog miR-21 inhibitors tau muab piv rau cov hlwb kho nrog BV2-derived exosomes siv qRT-PCR (Fig. 6A,B).Kev hloov pauv ntawm miR-21 inhibitor rau hauv U87 hlwb ua rau txo qis FoxO1 thiab p27 qhia (Daim duab 6).
RH-infected exosomal BV2-derived miP-21 hloov FoxO1/p27 qhia hauv U87 glioma hlwb.U87 hlwb tau kis nrog miP-21 inhibitor siv Lipofectamine 2000 thiab cov hlwb tau sau 24 teev tom qab hloov pauv.FoxO1 thiab p27 qhia qib hauv cov hlwb hloov nrog miR-21 inhibitors tau muab piv rau qib hauv cov hlwb kho nrog BV2-derived exosomes siv qRT-PCR (A, B).
Txhawm rau kom dim ntawm tus tswv lub cev tiv thaiv kab mob, Toxoplasma parasite hloov mus rau hauv cov ntaub so ntswg.Lawv parasitize ntau cov ntaub so ntswg, nrog rau lub hlwb, lub plawv, thiab cov leeg pob txha, thoob plaws hauv lub neej ntawm tus tswv tsev thiab hloov kho lub cev tiv thaiv kab mob.Tsis tas li ntawd, lawv tuaj yeem tswj hwm lub voj voog ntawm tes thiab apoptosis ntawm cov tswv tsev cell, txhawb lawv txoj kev loj hlob 14,24.Toxoplasma gondii feem ntau kis tus tswv tsev dendritic hlwb, neutrophils, thiab monocyte / macrophage kab, nrog rau lub hlwb microglia.Toxoplasma gondii induces qhov sib txawv ntawm macrophages ntawm M2 phenotype, cuam tshuam rau qhov txhab kho tom qab kab mob, thiab tseem cuam tshuam nrog hypervascularization thiab granulomatous fibrosis.Qhov kev coj tus cwj pwm pathogenesis ntawm tus kab mob Toxoplasma tuaj yeem cuam tshuam nrog cov cim cuam tshuam nrog kev loj hlob ntawm qog.Qhov chaw tsis txaus ntseeg uas tswj hwm los ntawm Toxoplasma tuaj yeem zoo li tus kab mob sib kis.Yog li, nws tuaj yeem xav tias tus kab mob Toxoplasma yuav tsum ua rau kev loj hlob ntawm cov qog hlwb.Qhov tseeb, tus nqi siab ntawm Toxoplasma kab mob tau tshaj tawm hauv cov ntshav ntawm cov neeg mob uas muaj ntau yam qog hlwb.Tsis tas li ntawd, Toxoplasma gondii tej zaum yuav yog lwm yam carcinogenic effector thiab ua synergistically los pab lwm cov kab mob carcinogens tsim cov qog hlwb.Hauv qhov no, nws tsim nyog sau cia tias P. falciparum thiab Epstein-Barr tus kab mob sib koom ua ke rau kev tsim ntawm Burkitt's lymphoma.
Lub luag haujlwm ntawm exosomes ua tus tswj hwm hauv thaj chaw ntawm kev tshawb fawb mob qog noj ntshav tau raug tshawb xyuas ntau.Txawm li cas los xij, lub luag haujlwm ntawm exosomes ntawm cov kab mob parasites thiab cov neeg muaj kab mob tseem nkag siab tsis zoo.Txog tam sim no, ntau tus neeg tswj hwm, suav nrog cov protein uas zais cia, tau piav qhia txog cov txheej txheem lom neeg uas cov kab mob protozoan tiv thaiv tus tswv tsev tawm tsam thiab kis kab mob mus ntev.Tsis ntev los no, tau muaj lub tswv yim loj hlob uas protozoan-koom nrog microvesicles thiab lawv cov microRNAs cuam tshuam nrog cov tswv tsev los tsim ib qho chaw zoo rau lawv txoj sia nyob.Yog li, kev tshawb fawb ntxiv yog xav tau los tshawb pom kev sib raug zoo ntawm kev hloov pauv exosomal miRNAs thiab glioma cell proliferation.Kev hloov pauv ntawm MicroRNA (cluster genes miR-30c-1, miR-125b-2, miR-23b-27b-24-1 thiab miR-17-92) khi rau STAT3 txhawb nqa hauv cov neeg mob toxoplasma-mob macrophages, yog tswj thiab induces los tiv thaiv -apoptosis teb rau Toxoplasma gondii kab mob 29.Tus kab mob Toxoplasma nce qhov kev qhia ntawm miR-17-5p thiab miR-106b-5p, uas cuam tshuam nrog ntau yam kab mob hyperproliferative 30.Cov ntaub ntawv no qhia tias tus tswv tsev miRNAs tswj los ntawm Toxoplasma kab mob yog cov tseem ceeb molecules rau parasite ciaj sia taus thiab pathogenesis nyob rau hauv tus tswv tsev lom neeg tus cwj pwm.
Hloov miRNAs tuaj yeem cuam tshuam ntau yam kev coj cwj pwm thaum pib thiab kev loj hlob ntawm cov hlwb tsis zoo, suav nrog gliomas: tus kheej txaus ntawm cov cim kev loj hlob, insensitivity rau kev loj hlob-inhibiting signals, apoptosis evasion, unlimited replicate muaj peev xwm, angiogenesis, ntxeem tau thiab metastasis, thiab o.Hauv glioma, hloov pauv miRNAs tau pom nyob rau hauv ntau qhov kev qhia profileing kev tshawb fawb.
Hauv txoj kev tshawb fawb tam sim no, peb tau lees paub qhov siab ntawm miRNA-21 qhia hauv toxoplasma-tus kab mob cov kab mob.miR-21 tau raug txheeb xyuas tias yog ib qho ntawm feem ntau tshaj tawm microRNAs hauv cov qog nqaij hlav, suav nrog gliomas, 33 thiab nws cov lus qhia cuam tshuam nrog qib ntawm glioma.Cov ntaub ntawv pov thawj qhia tau hais tias miR-21 yog ib qho tshiab oncogene uas ua raws li kev tiv thaiv apoptotic hauv kev loj hlob glioma thiab muaj ntau dhau ntawm cov ntaub so ntswg thiab ntshav ntawm tib neeg lub hlwb malignancies.Interestingly, miR-21 inactivation nyob rau hauv glioma hlwb thiab cov ntaub so ntswg ua rau inhibition ntawm cell proliferation vim caspase-dependent apoptosis.Bioinformatic tsom xam ntawm miR-21 kwv yees lub hom phiaj tau qhia ntau yam qog nqaij hlav hauv cov noob caj noob ces cuam tshuam nrog txoj hauv kev apoptosis, suav nrog programmed cell tuag 4 (PDCD4), tropomyosin (TPM1), PTEN, thiab forkhead box O1 (FoxO1), nrog rau miR-2121 binding site.22.38 Nws.
FoxO1, raws li ib qho ntawm cov kev hloov pauv hloov pauv (FoxO), tau koom nrog hauv kev txhim kho ntau yam ntawm tib neeg mob qog noj ntshav thiab tuaj yeem tswj hwm qhov kev qhia ntawm cov qog qog nqaij hlav xws li p21, p27, Bim, thiab FasL40.FoxO1 tuaj yeem khi thiab qhib lub voj voog ntawm tes inhibitors xws li p27 kom txo qis kev loj hlob ntawm tes.Ntxiv mus, FoxO1 yog ib qho tseem ceeb ntawm PI3K / Akt signaling thiab tswj ntau cov txheej txheem lom neeg xws li kev loj hlob ntawm tes thiab kev sib txawv ntawm tes los ntawm kev ua kom p2742 transcription.
Hauv kev xaus, peb ntseeg tias exosomal miR-21 muab los ntawm Toxoplasma-mob microglia tuaj yeem ua lub luag haujlwm tseem ceeb ua tus tswj kev loj hlob ntawm glioma hlwb (Daim duab 7).Txawm li cas los xij, kev tshawb fawb ntxiv yog xav tau los nrhiav kev sib txuas ncaj qha ntawm exosomal miR-21, hloov Toxoplasma kab mob, thiab kev loj hlob glioma.Cov txiaj ntsig no xav tias yuav muab qhov pib rau kev kawm txog kev sib raug zoo ntawm Toxoplasma kab mob thiab qhov tshwm sim ntawm glioma.
Daim duab schematic ntawm cov txheej txheem ntawm glioma (lub hlwb) carcinogenesis yog npaj rau hauv txoj kev tshawb no.Tus sau kos hauv PowerPoint 2019 (Microsoft, Redmond, WA).
Txhua qhov kev sim cov txheej txheem hauv txoj kev tshawb fawb no, suav nrog kev siv tsiaj, tau ua raws li Seoul National University Kev Saib Xyuas Tsiaj thiab Cov Neeg Siv Khoom Tus Txheej Txheem Kev Cai Coj Ncaj Ncees thiab tau pom zoo los ntawm Pawg Saib Xyuas Kev Tshawb Fawb ntawm Seoul National University Tsev Kawm Ntawv Tshuaj (IRB tus lej SNU- 150715).-2).Tag nrho cov txheej txheem sim tau ua tiav raws li ARRIVE cov lus pom zoo.
BV2 nas microglia thiab U87 tib neeg glioma hlwb tau coj los ua kab ke hauv Dulbecco's Modified Eagle's Medium (DMEM; Welgene, Seoul, Kauslim) thiab Roswell Park Memorial Institute's Medium (RPMI; Welgene), feem, txhua tus muaj 10% fetal bovine serum, 4 mM l- glutamine, 0.2 mM penicillin thiab 0.05 mM streptomycin.Cells raug coj mus rau hauv lub incubator nrog 5% CO2 ntawm 37 ° C.Lwm glioma cell kab, U118, tau siv rau kev sib piv nrog U87 hlwb.
Txhawm rau cais cov exosomes los ntawm T. gondii-infected RH thiab ME49 hom, T. gondii tachyzoites (RH strain) tau sau los ntawm lub plab kab noj hniav ntawm 6-lub lim tiam-laus BALB/c nas txhaj 3-4 hnub ua ntej.Tachyzoites tau ntxuav peb zaug nrog PBS thiab purified los ntawm centrifugation hauv 40% Percoll (Sigma-Aldrich, St. Louis, MO, USA)43.Kom tau txais tachyzoites ntawm hom ME49, BALB / c nas tau txhaj tshuaj intraperitoneally nrog 20 cov ntaub so ntswg hlwv thiab tachyzoite hloov hauv cov hlwv tau sau los ntawm kev ntxuav lub plab kab noj hniav ntawm 6-8 hnub tom qab kis kab mob (PI).Cov nas kis tau PBS.ME49 tachyzoites tau loj hlob hauv hlwb ntxiv nrog 100 μg / ml penicillin (Gibco / BRL, Grand Island, NY, USA), 100 μg / ml streptomycin (Gibco / BRL), thiab 5% fetal bovine serum (Lonza, Walkersville, MD) .., USA) ntawm 37 ° C thiab 5% carbon dioxide.Tom qab cog hauv Vero hlwb, ME49 tachyzoites tau dhau ob zaug los ntawm 25 koob ntsuas thiab tom qab ntawd los ntawm 5 µm lim kom tshem tawm cov khib nyiab thiab cov hlwb.Tom qab ntxuav, cov tachyzoites raug tshem tawm hauv PBS44.Cov kab mob hlwv ntawm Toxoplasma gondii strain ME49 tau khaws cia los ntawm kev txhaj tshuaj intraperitoneal ntawm cov hlwv cais tawm ntawm lub hlwb ntawm tus kab mob C57BL/6 nas (Orient Bio Animal Center, Seongnam, Kauslim).Lub hlwb ntawm ME49-mob nas tau sau tom qab 3 lub hlis ntawm PI thiab minced nyob rau hauv lub tshuab ntsuas kom cais cov hlwv.Cov nas kis tau raug khaws cia nyob rau hauv cov xwm txheej tshwj xeeb tsis muaj kab mob (SPF) ntawm Seoul National University School of Medicine.
Tag nrho RNA tau muab rho tawm los ntawm BV2-derived exosomes, BV2 hlwb thiab cov ntaub so ntswg siv miRNeasy Mini Kit (Qiagen, Hilden, Lub teb chaws Yelemees) raws li cov chaw tsim khoom cov lus qhia, suav nrog lub sijhawm incubation rau cov kauj ruam elution.RNA concentration tau txiav txim siab ntawm NanoDrop 2000 spectrophotometer.Qhov zoo ntawm RNA microarrays tau soj ntsuam siv Agilent 2100 bioanalyzer (Agilent Technologies, Amstelveen, Netherlands).
DMEM nrog 10% exosome-poor FBS tau npaj los ntawm ultracentrifugation ntawm 100,000g rau 16 teev ntawm 4 ° C thiab lim los ntawm 0.22 µm lim (Nalgene, Rochester, NY, USA).BV2 hlwb, 5 × 105, tau coj mus kuaj hauv DMEM muaj 10% exosome-depleted FBS thiab 1% tshuaj tua kab mob ntawm 37 ° C thiab 5% CO2.Tom qab 24 teev ntawm incubation, tachyzoites ntawm strain RH los yog ME49 (MOI = 10) tau ntxiv rau lub hlwb thiab cov kab mob uas tsis yog invading raug tshem tawm nyob rau hauv ib teev thiab refilled nrog DMEM.Exosomes los ntawm BV2 hlwb raug cais los ntawm kev hloov kho qhov sib txawv centrifugation, txoj kev siv dav tshaj plaws.Tshem tawm cov exosome pellet nyob rau hauv 300 µl PBS rau RNA los yog protein tsom xam.Qhov siab ntawm cov exosomes cais tau txiav txim siab siv BCA protein kuaj cov khoom (Pierce, Rockford, IL, USA) thiab NanoDrop 2000 spectrophotometer.
Precipitates los ntawm BV2 hlwb los yog exosomes muab tau los ntawm BV2 tau lysed nyob rau hauv PRO-PREP ™ protein extraction tov (iNtRon Biotechnology, Seongnam, Kauslim) thiab cov proteins twb loaded rau Coomassie ci ntsa iab xiav stained 10% SDS polyacrylamide gels.Tsis tas li ntawd, cov protein tau pauv mus rau PVDF daim nyias nyias rau 2 teev.Western blots tau lees paub siv Alix antibody (Cell Signaling Technology, Beverly, MA, USA) ua tus cim exosomal.HRP-conjugated tshis anti-nas IgG (H + L) (Bethyl Laboratories, Montgomery, TX, USA) thiab LAS-1000 ntxiv rau luminescent duab analyzer (Fuji Photographic Film, Tokyo, Nyiv) tau siv los ua cov tshuaj tiv thaiv thib ob..Kev xa tawm electron microscopy tau ua los kawm txog qhov loj thiab morphology ntawm exosomes.Exosomes cais tawm ntawm BV2 hlwb (6.40 µg / µl) tau npaj rau ntawm cov pa roj carbon coated meshes thiab tsis zoo stained nrog 2% uranyl acetate rau 1 min.Cov qauv npaj tau pom ntawm qhov ntsuas hluav taws xob nrawm ntawm 80 kV siv JEOL 1200-EX II (Tokyo, Nyiv) nruab nrog lub koob yees duab ES1000W Erlangshen CCD (Gatan, Pleasanton, CA, USA).
BV2-derived exosomes tau stained siv PKH26 Red Fluorescent Linker Kit (Sigma-Aldrich, St. Louis, MO, USA) rau 15 feeb ntawm chav tsev kub.U87 hlwb, 2 × 105, nrog PKH26-labeled exosomes (liab) los yog tsis muaj exosomes raws li kev tswj tsis zoo, tau incubated ntawm 37 ° C rau 24 teev nyob rau hauv 5% CO2 incubator.U87 cell nuclei tau stained nrog DAPI (xiav), U87 hlwb tau kho nyob rau hauv 4% paraformaldehyde rau 15 min ntawm 4 ° C thiab tom qab ntawd soj ntsuam hauv Leica TCS SP8 STED CW confocal microscope system (Leica Microsystems, Mannheim, Lub teb chaws Yelemees).saib tau.
cDNA tau tsim los ntawm siRNA siv Mir-X siRNA thawj strand synthesis thiab SYBR qRT-PCR cov khoom siv (Takara Bio Inc., Shiga, Nyiv).Lub sij hawm tiag tiag PCR tau ua tiav siv iQ5 lub sijhawm PCR tshawb pom qhov system (Bio-Rad, Hercules, CA, USA) siv cov primers thiab cov qauv sib xyaw nrog SYBR Premix.DNA tau nthuav dav rau 40 lub voj voog ntawm denaturation ntawm 95 ° C rau 15 s thiab annealing ntawm 60 ° C rau 60 s.Cov ntaub ntawv los ntawm txhua qhov tshuaj tiv thaiv PCR tau txheeb xyuas siv cov ntaub ntawv tsom xam module ntawm iQ™5 optical system software (Bio-Rad).Cov txheeb ze hloov pauv hauv cov noob qhia ntawm cov hom phiaj xaiv noob thiab β-actin / siRNA (thiab U6) tau suav nrog siv tus qauv nkhaus txoj kev.Cov txheej txheem primer siv tau qhia hauv Table 1.
3 x 104 U87 glioma hlwb tau cog rau hauv 96-zoo daim hlau thiab sib xyaw nrog Toxoplasma-infected exosomes tau los ntawm BV2 (50 μg / mL) lossis nonpulse exosomes tau los ntawm BV2 (50 μg / mL) raws li kev tswj ntawm 12, 18 teev thiab .Tus nqi ntawm cov cell proliferation tau txiav txim siab siv Cell Counting Kit-8 (Dojindo, Kumamoto, Nyiv) (Cov duab ntxiv S1-S3) 46 .
5-lub lim tiam poj niam BALB/c liab qab nas tau yuav los ntawm Orient Bio (Seongnam-si, Kaus Lim Qab Teb) thiab khaws cia ib leeg rau hauv lub tawb tsis muaj menyuam ntawm chav sov (22 ± 2 ° C) thiab av noo (45 ± 15 ° C).%) ntawm chav sov (22 ± 2 ° C) thiab av noo (45 ± 15%).Lub voj voog 12-teev lub teeb thiab lub voj voog 12-teev tsaus ntuj tau ua raws li SPF (Seoul National University School of Medicine Animal Center).Cov nas tau muab faib ua peb pawg ntawm 5 nas thiab txhua pawg tau txhaj tshuaj subcutaneously nrog 400 ml ntawm PBS uas muaj 1 x 107 U87 glioma hlwb thiab kev loj hlob zoo txo ​​BD Matrigel™ (BD Science, Miami, FL, USA).Rau hnub tom qab txhaj tshuaj, 200 mg ntawm exosomes tau los ntawm BV2 hlwb (nrog / tsis muaj tus kab mob Toxoplasma) tau txhaj rau hauv qhov chaw qog.Nees nkaum ob hnub tom qab qog nqaij hlav, cov qog loj ntawm cov nas hauv txhua pab pawg tau ntsuas nrog lub caliper peb zaug hauv ib lub lis piam, thiab cov qog ntim tau suav los ntawm cov mis: 0.5 × (dav) × 2 × ntev.
MicroRNA qhia kev tsom xam siv miRCURYTM LNA miRNA array, tiam 7 muaj, mmu thiab rno arrays (EXIQON, Vedbaek, Denmark) npog 1119 tus cwj pwm zoo nas ntawm 3100 tib neeg, nas thiab nas miRNA ntes probes.Thaum lub sijhawm txheej txheem no, 250 mus rau 1000 ng ntawm tag nrho RNA raug tshem tawm ntawm 5′-phosphate los ntawm kev kho mob nrog calf plab hnyuv alkaline phosphatase ua raws li daim ntawv lo nrog Hy3 ntsuab fluorescent dye.Cov ntawv sau npe tom qab ntawd hybridized los ntawm kev thauj cov microarray slides siv cov khoom siv hybridization chamber (Agilent Technologies, Santa Clara, CA, USA) thiab cov khoom siv hybridization slides (Agilent Technologies).Hybridization tau ua tiav rau 16 teev ntawm 56 ° C, tom qab ntawd cov microarrays raug ntxuav raws li cov neeg tsim khoom pom zoo.Cov txheej txheem microarray slides tau raug tshuaj xyuas siv Agilent G2565CA microarray scanner system (Agilent Technologies).Cov duab luam theej tau raug xa tuaj siv Agilent Feature Extraction software version 10.7.3.1 (Agilent Technologies) thiab fluorescence siv ntawm txhua daim duab tau suav nrog siv GAL cov ntaub ntawv sib txuas ntawm Exiqon raws tu qauv.Microarray cov ntaub ntawv rau txoj kev tshawb fawb tam sim no tau muab tso rau hauv GEO database raws li tus lej GPL32397.
Kev nthuav qhia cov ntaub ntawv ntawm mature exosomal miRNAs hauv microglia ntawm RH lossis ME49 hom kab mob Toxoplasma tau txheeb xyuas siv ntau yam cuab yeej hauv network.miRNAs cuam tshuam nrog kev loj hlob ntawm cov qog tau txheeb xyuas siv miRWalk2.0 (http://mirwalk.umm.uni-heidelberg.de) thiab lim tawm nrog cov teeb liab siv zog (log2) ntau dua 8.0.Ntawm cov miRNAs, qhov sib txawv ntawm miRNAs tau pom tias muaj ntau dua 1.5-fold hloov los ntawm lim tsom xam ntawm miRNAs hloov los ntawm RH lossis ME49 hom kab mob T. gondii.
Cells tau noob nyob rau hauv 6-zoo daim hlau (3 x 105 hlwb/zoo) hauv opti-MEM (Gibco, Carlsbad, CA, USA) siv Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).Cov kab mob kis tau raug coj mus rau 6 teev thiab tom qab ntawd qhov nruab nrab tau hloov mus rau qhov nruab nrab tshiab.Cells tau sau tseg 24 teev tom qab hloov pauv.
Kev txheeb cais feem ntau yog ua los ntawm Tub Ntxhais Kawm T-test nrog Excel software (Microsoft, Washington, DC, USA).Rau kev soj ntsuam tsiaj txhu, ob txoj kev ANOVA tau ua tiav siv Prism 3.0 software (GraphPad Software, La Jolla, CA, USA). P-tus nqi <0.05 raug suav hais tias yog qhov tseem ceeb. P-tus nqi <0.05 raug suav hais tias yog qhov tseem ceeb. Значения P <0,05 считались статистически значимыми. P qhov tseem ceeb <0.05 tau suav tias yog qhov tseem ceeb. P 值 < 0.05 被认为具有统计学意义. P <0.05 Значения P <0,05 считались статистически значимыми. P qhov tseem ceeb <0.05 tau suav tias yog qhov tseem ceeb.
Txhua qhov kev sim cov txheej txheem siv hauv txoj kev tshawb no tau pom zoo los ntawm Pawg Saib Xyuas Kev Tshawb Fawb ntawm Seoul National University Tsev Kawm Ntawv Tshuaj (IRB tus lej SNU-150715-2).
The data used in this study are available upon reasonable request from the first author (BK Jung; mulddang@snu.ac.kr). And the microarray data for the current study is deposited in the GEO database under registration number GPL32397.
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