RNA M6 A Nyeem YTHDF2 Tswj NK Cell Antitumor Thiab Antiviral Immunity Part 3

YTHDF2 yog xav tau rau IL-15-mediated NK cell survival, proliferation, thiab effector functions

IL-15 yog ib qho ntawm, yog tias tsis yog qhov tseem ceeb tshaj plaws cytokine rau pleiotropicfunctions ntawm NK hlwb. Peb thiab lwm tus tau pom yav dhau los tiasIL-15 ua lub luag haujlwm tseem ceeb hauv kev tswj hwm NK cell homeostasis, ciaj sia taus, thiab cov haujlwm ua haujlwm (Becknell thiab Caligiuri, 2005; Carsonet al., 1997; Carson et al., 1994; Wang et al., 2019b; Yus et al., 2013).

Pleiotropy ntawm cov hlwb hais txog lawv lub peev xwm los ua haujlwm sib txawv hauv qhov chaw sib txawv. Cell pleiotropy yog ze ze rau kev tiv thaiv kab mob vim tias ntau lub hlwb hauv lub cev tiv thaiv kab mob tuaj yeem ua tau ntau yam sib txawv.

Hauv lub cev tiv thaiv kab mob, T hlwb yog ib hom kab mob tseem ceeb heev. Lawv paub thiab tua cov kab mob, thaum tseem tswj hwm kev ua haujlwm ntawm lwm lub cev tiv thaiv kab mob. Lub pleiotropy ntawm T hlwb tseem ceeb heev vim tias thaum lub cev tiv thaiv kab mob ntsib cov kab mob tshiab, T hlwb yuav tsum tau ua lub luag haujlwm sib txawv hauv lub sijhawm luv luv kom ua tau zoo. Piv txwv li, thaum lub cev tiv thaiv kab mob ntsib cov kab mob, T hlwb yuav tsum tau ua lub luag haujlwm ntawm kev tua cov kab mob, thiab thaum lub cev tiv thaiv kab mob ntsib tus kab mob, T hlwb yuav tsum ua lub luag haujlwm ntawm kev tswj hwm lwm cov kab mob.

Tsis tas li ntawd, nyob rau hauv lub cev tiv thaiv kab mob, ib chav kawm ntawm cov hlwb hu ua macrophages kuj muaj cov teebmeem pleiotropic. Lawv tuaj yeem phagocytose thiab rhuav tshem cov kab mob txawv teb chaws, thiab tseem tuaj yeem qhib lwm lub cev tiv thaiv kab mob los tua cov kab mob. Lub pleiotropy ntawm macrophages yog qhov tseem ceeb vim tias lawv yuav tsum tau ua lub luag haujlwm sib txawv hauv qhov chaw sib txawv. Piv txwv li, hauv kev kis kab mob, macrophages yuav tsum tau tso qee yam tshuaj los tua tus kab mob, thaum nyob rau hauv cov kab mob, macrophages yuav tsum tau engulf thiab zom cov kab mob kom tshem tawm cov kab mob.

Hauv cov ntsiab lus, muaj kev sib raug zoo ntawm cellular pleiotropy thiab kev tiv thaiv kab mob. Pleiotropic hlwb tuaj yeem ua lub luag haujlwm sib txawv hauv ib puag ncig kis los ntawm cov kab mob sib txawv, yog li txhim kho lub cev tiv thaiv kab mob muaj peev xwm teb thiab txhim kho lub cev tiv thaiv kab mob rau ntau yam kab mob. Yog li ntawd, peb yuav tsum tau xyuam xim rau pleiotropy ntawm cov hlwb thiab tseem ntxiv dag zog rau kev cog qoob loo ntawm kev tiv thaiv kab mob kom muaj kev noj qab haus huv ntawm tib neeg. Nws tuaj yeem pom tau tias peb yuav tsum txhim kho kev nco, thiab Cistanche deserticola tuaj yeem txhim kho kev nco, vim Cistanche deserticola tseem tuaj yeem tswj hwm qhov sib npaug ntawm cov neurotransmitters, xws li nce qib ntawm acetylcholine thiab kev loj hlob. Cov khoom no tseem ceeb heev rau kev nco thiab kev kawm. Tsis tas li ntawd, Cistanche deserticola tseem tuaj yeem txhim kho cov ntshav khiav thiab txhawb nqa cov pa oxygen, uas tuaj yeem ua kom lub hlwb tau txais cov as-ham txaus thiab lub zog, yog li txhim kho lub hlwb tseem ceeb thiab kev ua siab ntev.

Nyem paub txoj hauv kev los txhim kho koj lub cim xeeb

Hauv txoj kev tshawb fawb tam sim no, peb pom tias IL-15 upregulatedmRNA thiab cov protein ntau ntawm YTHDF2 hauv NK hlwb (Fig. 6 A; thiab Daim duab 1, D-F). Yog li ntawd, peb xav tias YTHDF2 yuav tsum tau rau IL-15-mediated survival thiab effector functions ntawm NK hlwb.

Peb thawj zaug sim txheeb xyuas qhov muaj peev xwm hloov pauv cov ntsiab lus hauv qab ntawm IL-15 qhia tias ncaj qha tswj YTHDF2expression. Peb tau txheeb xyuas cov Encyclopedia of DNA Elementsusing University of California, Santa Cruz, Genome BrowserDatabase (https://genome.ucsc.edu), uas muab kev kwv yees ntawm cov chaw sib khi thoob plaws tag nrho cov genome, ua ke nrog JASPAR (http://jaspar.genereg .net).

Peb pom tias STAT5, uas yog qhov tseem ceeb hauv qab ntawm IL-15 hauv NK hlwb, muaj plaub qhov chaw nyob hauv 2 kb upstream ntawm qhov chaw pib transcription (TSS) ntawm Ythdf2, qhia tias IL-15 tuaj yeem ua tau zoo. tswj Ythdf2 transcription hauv nas NK hlwb los ntawm STAT5.

Los ntawm kev siv STAT5 inhibitor STAT5-IN-1 (Müller li al., 2008), peb pom tias inhibition ntawm STAT5 ua rau txo qis ntawm Ythdf2 ntawm obmRNA thiab protein ntau hauv murine NK hlwb (Fig. S4, A. thiab B). Txhawm rau kom paub meej ntxiv tias YTHDF2 yog qhov qis qis uas tswj hwm los ntawm STAT5, peb siv Stat5fl/fl Ncr1-iCre nas (tom qab no hu ua Stat5ΔNK nas), qhov twg STAT5 tshwj xeeb yog tshem tawm cov nas NK hlwb (Wiedemann thiab ib., 2020).

Peb kho splenicNK hlwb los ntawm Stat5WT thiab Stat5ΔNK nas nrog IL-15. Peb pom tias YTHDF2 txo qis hauv NK hlwb los ntawm Stat5ΔNKmice piv nrog NK hlwb los ntawm Stat5WT nas ntawm ob leeg RNA thiab cov protein ntau (Fig. 6, B thiab C; thiab Fig. S4, C thiab D).

Luciferase reporter assay tau pom tias ob qho tib si STAT5a thiab STAT5b qhib Ythdf2 gene transcription ncaj qha (Fig. 6, D, thiab E). Chromatin immunoprecipitation (ChIP)–qPCR cov txiaj ntsig tau qhia tias STAT5 muaj qhov tseem ceeb ntawm plaub qhov chaw tshaj li kev tswj hwm IgG ib txwm, qhia ncaj qha rau hauv cov nas NKcells (Fig. 6 F). Ua ke, peb cov txiaj ntsig tau pom tias YTHDF2 kev nthuav qhia yog tswj hwm los ntawm STAT5 nqes qis ntawm IL-15 signaling hauv NK hlwb.

Tom qab ntawd peb xav paub seb Ythdf2- tsis muaj NK hlwb puas muaj qhov tsis zoo hauv lawv cov lus teb rau IL-15. Peb cais cov splenic NK hlwb los ntawm Ythdf2ΔNK nas thiab Ythdf2WT nas thiab cultured themin vitro nyob rau hauv lub xub ntiag ntawm IL-15. Peb pom tias NK cell loj hlob tau txo qis hauv Ythdf2ΔNK nas piv nrog Ythdf2WT nas (Fig. 6 G). CellTrace Violet (CTV) labeling assayshowed that proliferation of NK cells is impaired by Ythdf2deficiency (Fig. 6 H).

 Cell-cycle distribution analysis tau nthuav tawm ib qho tseem ceeb ntawm cov NK hlwb los ntawm Ythdf2ΔNK cov nas nyob rau theem G0/G1 tab sis qhov txo qis ntawm NKcells los ntawm Ythdf2ΔNK nas nyob rau theem S (Fig. 6 I), qhia tias Ythdf2 deficiency tshwm sim. hauv G0/G1 theem ntes hauv NK hlwb.

Tsis tas li ntawd, muaj ob-rau peb zaug nce hauv apoptotic (annexin V + Sytox-Blue +/−) NK hlwb los ntawm Ythdf2ΔNK nas piv nrog cov nas los ntawm Ythdf2WT nas (Fig. 6 J), qhia txog kev muaj sia nyob ntawm NK hlwb tom qab poob. Ythdf 2. Cov txiaj ntsig no qhia tias YTHDF2 tswj kev ua haujlwm ntawm NKcells rau IL-15 hauv vitro. Txij li thaum IL-15 tau txhais tsis zoo thiab zais hauv vivo ntawm lub xeev khov kho (Corbel li al., 1996; Fehniger li al.,2001), txhawm rau tshawb xyuas seb Ythdf2- tsis muaj NK hlwb puas muaj qhov tsis zoo hauv lawv cov lus teb. rau IL-15 hauv vivo, peb tau kho cov nas nrog IL-15.

Pom tau tias Ythdf2-deficient piv rau WT NK hlwb pom tau tias txo cov cell proliferation thiab nce cell apoptosis thaum nas tau kho nrog IL-15 (Fig. 6 K; thiab Fig. S4, E–J).Cov ntaub ntawv no qhia tias YTHDF2 tswj qhov kev teb ntawm NK hlwb rau IL-15, tshwj xeeb tshaj yog nyob rau hauv qee qhov xwm txheej nrog qib siab ntawm IL-15. Peb kuj pom tias mRNA thiab protein ntau ntawm IFN- , granzyme B, thiab perforin tau txo qis hauv Ythdf2ΔNK NK hlwb piv nrog Ythdf2WT NK hlwb teb rau IL-15 (Fig. 6, L-N), qhia tias YTHDF2 kuj pab txhawb rau IL-15-mediated NK cell effector ua haujlwm hauv vitro.

Sib sau ua ke, peb cov ntaub ntawv qhia tau hais tias YTHDF2 yuav tsum tau rau IL-15-mediated NK cell ciaj sia taus, kev loj hlob, thiab kev ua haujlwm zoo. Peb pom tias NK hlwb los ntawm Ythdf2ΔNKmice thiab Ythdf2WT nas muaj cov qib zoo sib xws ntawm IL-15 receptorsCD122 (IL-15R) thiab CD132 (IL-15R c; Fig. S4 K). IL-15 signalingis kho kom haum xeeb los ntawm tsawg kawg peb txoj kev taw qhia downstream hauv NK hlwb: Ras–Raf–MEK–ERK, PI3K–AKT–mTOR, thiab JAK1/3–STAT3/5 (Mishra et al., 2014).

Txhawm rau tshawb xyuas qhov kev taw qhia txoj kev twg yog tswj hwm los ntawm YTHDF2 hauv NK hlwb, peb tau tshuaj xyuas cov qib phosphorylation ntawm ERK, AKT, S6, STAT3, thiab STAT5 inNK hlwb los ntawm Ythdf2ΔNK nas lossis Ythdf2WT nas tom qab stimulationwith IL-15.

Cov txiaj ntsig tau pom tias qhov tsis txaus Ythdf2 tsis cuam tshuam rau ERK, AKT, S6, thiab STAT3 phosphorylation raws li IL-15stimulation (Fig. S4, L thiab M) tab sis cuam tshuam STAT5activation, raws li pov thawj los ntawm kev txo phosphorylation theem ntawmSTAT5 hauv Ythdf{{ 7}} tsis muaj NK hlwb ntau dua li hauv NK hlwb los ntawm Ythdf2WT nas (Fig. 6 O), qhia tias YTHDF2 yuav tsum tau ua kom zoo tshaj IL-15/STAT5 signaling nyob rau hauv activated NK hlwb.

Vim peb tau pom tias phosphorylated STAT5 nqes hauv qab ntawm IL-15 khi rau tus txhawb nqa ntawm Ythdf2 (Fig. 6, D–F), thiab ntawm no tau pom tias YTHDF2 yuav tsum tau ua kom zoo tshaj STAT5phosphorylation, peb cov ntaub ntawv qhia txog STAT5–YTHDF2 cov lus pom zoo rov qab rau hauv qab. ntawm IL-15 uas tuaj yeem tswj NK cellurvival, proliferation, thiab effector functions.

Kev txheeb xyuas dav dav qhia Tardbp raws li YTHDF2 lub hom phiaj hauv NK hlwb

Txhawm rau hais txog cov txheej txheem molecular uas YTHDF2 tswj hwm NK hlwb, peb thawj zaug ua RNA sequencing (seq) inIL-2- nthuav tawm Ythdf2WT thiab Ythdf2ΔNK NK hlwb. Kev tshem tawm ntawm Ythdf2 hauv NK hlwb ua rau 617 qhov sib txawv qhia cov noob, suav nrog 252 cov noob hloov pauv thiab 365 cov noob tsis muaj zog (Fig. S5 A).

Gene ontology (GO) tsom xam pom tias qhov sib txawv ntawm cov noob tau nthuav dav hauv cell-cycle, cell-division, thiab cell-division-related processes, suav nrog mitoticcytokinesis, chromosome cais, spindle, nucleosome, midbody, thiab chromosome (Fig. A).

Gene set enrichment analysis (GSEA) pom tau tias muaj txiaj ntsig zoo ntawm E2F lub hom phiaj, G2 / Mcheckpoint, thiab mitotic spindle hallmark gene sets hauv Ythdf2ΔNKNK hlwb (Fig. S5 B). Cell-cycle thiab cell-division-related genes, suav nrog Aurka, Aurkb, Cdc20, Cdc25b, Cdc25c, Cdk1, E2f2, thiab Plk1(Bertoli li al., 2013), tau txo qis hauv NK hlwb los ntawm Ythdf2ΔNK nas (Fig. ).

Spindle thiab chromosome segregationgenes xws li Anln, Aspm, Birc5, Bublb, Cenpe, Esco2, Ska1, Ska3, thiab Tpx2 (Gorbsky, 2015) kuj tseem txo qis hauv NKcells los ntawm Ythdf2ΔNK nas piv nrog NK hlwb los ntawm Ythddf2gWT.

Tsis tas li ntawd, cov noob ntawm cov ciaj sia taus, suav nrog Birc5 (Niu et al., 2010), Septin4 (Larisch li al., 2000), thiab Rffl (Yanget al., 2007), tau txo qis hauv NK hlwb los ntawm Ythdf2ΔNK nas (Fig. 7D). NK cell effector muaj nuj nqi cov noob, suav nrog Klrk1, Ncr1, CD226, thiab Gzma (Bezman li al., 2012), kuj tseem txo qis hauv NK hlwb los ntawm Ythdf2ΔNK nas (Fig. 7 D).

Cov ntaub ntawv no txhawb nqa peb lub luag haujlwm ntawm YTHDF2 hauv kev tswj hwm NK cell proliferation, ciaj sia taus, thiab effector functions.Peb mam li ua m6A-seq hauv IL-2– nthuav NK hlwb los ntawm Ythdf2WT thiab Ythdf2ΔNK nas. Thawj qhov kev tshuaj ntsuam xyuas pom tau tias peb qhov kev hloov pauv roj ntsha ntawm txhua tus genotype pawg ua ke (cov ntaub ntawv tsis qhia), qhia txog kev rov ua tau zoo ntawm m6A-seq cov qauv.

Hypergeometric optimization ntawm motif enrichment (HOMER) tsom xam pom qhov m6A kev pom zoo motif (GGAC), qhia txog kev vam meej ntawm m6A-modifiedtranscripts (Fig. 7 E). m6A cov kev hloov kho tau pom muaj nyob hauv cov ntawv teev cov protein-coding, thiab cov peaks tau ntxiv rau hauv 59 thaj chaw uas tsis tau txhais lus (UTR) thiab 39UTR, tshwj xeeb tshaj yog nyob ib ncig ntawm qhov pib thiab nres codons (Fig. 7 F thiab Fig. S5 C).

GOenrichment tsom xam ntawm cov noob nrog m6A peaks qhia tias feem ntaum6A-cim cov ntaub ntawv hauv NK hlwb tau ua kom muaj txiaj ntsig hauv txoj hauv kev koom nrog hauv lub voj voog ntawm tes thiab kev loj hlob ntawm tes (Fig. S5 D).

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