Xov xwm

Ua tsaug rau koj tuaj xyuas Nature.com.Qhov browser version koj siv tau txwv CSS kev txhawb nqa.Rau qhov kev paub zoo tshaj plaws, peb xav kom koj siv qhov browser tshiab (lossis lov tes taw Compatibility Hom hauv Internet Explorer).Nyob rau lub sijhawm no, txhawm rau ua kom muaj kev txhawb nqa txuas ntxiv, peb yuav ua rau lub xaib tsis muaj qauv thiab JavaScript.
Cov qog nqaij hlav cancer tau hloov kho ntau yam txheej txheem los kov yeej kev nyuaj siab ntawm tes thiab txuas ntxiv mus.Protein kinase R (PKR) thiab nws cov protein activator (PACT) yog thawj cov lus teb uas saib xyuas ntau yam kev ntxhov siab uas ua rau inhibition ntawm cell proliferation thiab apoptosis.Txawm li cas los xij, txoj cai ntawm PACT-PKR txoj hauv kev hauv cov qog nqaij hlav cancer tseem tsis paub ntau.Ntawm no, peb pom tias ntev tsis-coding RNA (lncRNA) aspartyl tRNA synthetase antisense RNA 1 (DARS-AS1) ncaj qha cuam tshuam rau inhibition ntawm PACT-PKR txoj hauv kev thiab txhawb nqa cov qog nqaij hlav cancer.Siv cov kev tshuaj ntsuam xyuas loj ntawm CRISPRi 971 mob qog noj ntshav nrog lncRNA, peb pom tias DARS-AS1 tau cuam tshuam nrog kev txhim kho mob qog noj ntshav hauv cell proliferation.Yog li ntawd, DARS-AS1 knockout inhibits cell proliferation thiab txhawb kev mob qog noj ntshav cell apoptosis nyob rau hauv ntau yam kab mob qog noj ntshav hauv vitro thiab txo cov qog loj hlob hauv vivo.Mechanically, DARS-AS1 khi ncaj qha rau PACT activation domain thiab tiv thaiv PACT-PKR kev sib cuam tshuam, yog li txo PKR activation, eIF2α phosphorylation, thiab inhibiting apoptotic cell tuag.Clinically, DARS-AS1 tau nthuav dav dav hauv ntau yam qog nqaij hlav, thiab kev nthuav tawm ntau dhau ntawm lncRNA yog qhov qhia tau tias muaj qhov tsis zoo.Txoj kev tshawb no qhia txog kev kho mob qog noj ntshav tshwj xeeb ntawm PACT-PKR txoj hauv kev los ntawm DARS-AS1 lncRNA thiab muab lwm lub hom phiaj rau kev mob qog noj ntshav thiab kev kho mob.
Kev muaj peev xwm hloov tau rau kev ntxhov siab yog ib qho tseem ceeb ntawm cov kab mob qog noj ntshav thiab kev loj hlob.Kev loj hlob sai thiab metabolic cov cim qhia ntawm mob qog noj ntshav nyob rau hauv qhov hnyav microenvironments - kev noj zaub mov tsis txaus, hypoxia, thiab pH qis - uas tuaj yeem ua rau txoj kev tuag ntawm tes.Dysregulation ntawm kev ntxhov siab-rhiab cov noob xws li p535, kub poob siab proteins 6, 7, KRAS8, 9, thiab HIF-110, 11, 12, 13 yog nquag pom hauv mob qog noj ntshav, yog li thaiv apoptosis thiab txhawb kev ciaj sia.
Protein kinase R (PKR) yog ib qho tseem ceeb ntawm kev ntxhov siab sensor thiab subunit kinase ntawm eukaryotic pib yam tseem ceeb 2α (eIF2α), ib tug neeg txhais lus regulator uas txuas cellular stress rau cell tuag.PKR yog thawj zaug tau txheeb xyuas tias yog ib qho tshuaj tiv thaiv kab mob los ntawm kev tshawb pom ntawm txawv teb chaws ob-stranded RNA (dsRNA).Thaum ua kom muaj zog, PKR phosphorylates eIF2α los tiv thaiv kab mob thiab cellular protein synthesis14,15,16.PACT (PKR activator protein) tau raug txheeb xyuas tias yog thawj PKR activator protein thaum tsis muaj dsRNA17,18,19,20,21,22,23.Los ntawm kev sib cuam tshuam ncaj qha nrog PKR, PACT hloov ntau yam kev ntxhov siab (kev tshaib kev nqhis, peroxide lossis arsenite kho) rau PKR thiab cov kev taw qhia hauv qab.Ntxiv rau eIF2α phosphorylation, PACT-mediated PKR activation ua rau muaj ntau yam xwm txheej cuam tshuam nrog kev ntxhov siab, suav nrog kev hloov pauv redox ntawm txoj hauv kev PI3K/Akt24, txhim kho DNA kev puas tsuaj los ntawm p5325,26 thiab NF-κB27,28 Regulates transcription, 29. Muab lawv lub luag haujlwm tseem ceeb hauv cov lus teb kev ntxhov siab, kev loj hlob, apoptosis thiab lwm cov txheej txheem ntawm tes tseem ceeb, PKR thiab PACT tau cog lus rau kev kho mob rau ntau yam kab mob, tshwj xeeb tshaj yog mob cancer30,31,32,33.Txawm li cas los xij, txawm hais tias qhov no pleiotropic kev ua haujlwm thiab kev lom neeg tseem ceeb, kev tswj hwm ntawm PACT / PKR kev ua haujlwm hauv cov qog nqaij hlav cancer tseem tsis pom.
lncRNAs yog cov ntawv sau loj dua 200 nucleotides uas tsis muaj protein ntau-coding peev xwm.Txij li thaum tag nrho cov genome sequencing tej yaam num tau txheeb xyuas ntau txhiab tus lncRNAs, 35,36 tau siv zog ntau los qhia lawv cov haujlwm lom neeg.Lub cev loj hlob ntawm kev tshawb fawb tau pom tias lncRNAs koom nrog ntau cov txheej txheem lom neeg37 suav nrog kev tswj hwm ntawm X-chromosome inactivation38,39, imprinting40, transcription41,42, translation43 thiab txawm tias mob qog noj ntshav 44,45,46,47.Cov kev tshawb fawb no tau tshaj tawm tias ntau lub lncRNAs koom nrog PACT/PKR txoj hauv kev.Ib qho kev tshawb fawb no tau pom tias lncRNA ASPACT inhibited PACT transcription thiab nce nuclear tuav ntawm PACT mRNA.Lwm cov kev tshawb fawb tau pom tias lncRNA nc886 khi rau PKR thiab inhibits nws cov phosphorylation49,50.Txog tam sim no, lncRNA tswj PACT-mediated PKR activation tsis tau tshaj tawm.
Aspartyl-tRNA synthetase antisense RNA 1 (DARS-AS1) tau raug txheeb xyuas tias yog ib qho oncogenic lncRNA51,52,53,54.Los ntawm kev tswj hwm ntawm miP-194-5p53, miP-12952 thiab miP-532-3p51, DARS-AS1 tau pom tias yuav txhawb kev loj hlob ntawm cov cell cell carcinoma, thyroid carcinoma thiab non-small cell lung carcinoma, ntsig txog.Tong thiab cov npoj yaig kuj pom tias DARS-AS1 txhawb nqa kev loj hlob ntawm myeloma los ntawm kev tswj xyuas qhov ruaj khov ntawm cov protein 39 (RBM39) RNA-binding motif.Txawm li cas los xij, tsis muaj kev tshawb fawb tau tshawb fawb txog seb qhov lncRNA no puas koom nrog kev tswj hwm ntawm PACT-PKR ua kom muaj zog thiab cov lus teb kev ntxhov siab ntawm cov qog nqaij hlav cancer.
Ntawm no, peb tau ua qhov kev ntsuam xyuas loj-ntawm-kev ua haujlwm uas siv CRISPRi system thiab txiav txim siab tias DARS-AS1 lncRNA txhawb kev loj hlob ntawm ntau hom qog nqaij hlav cancer.Tsis tas li ntawd, peb tau txheeb xyuas cov txheej txheem tseem ceeb: DARS-AS1 khi ncaj qha rau PACT, inhibits PACT thiab PKR khi, tiv thaiv phosphorylation ntawm eIF2α, qis PKR substrate, thiab thaum kawg inhibits apoptotic cell tuag.Hauv kev xaus, peb txoj haujlwm nthuav tawm DARS-AS1 lncRNA ua tus tswj hwm ntawm PACT-PKR txoj hauv kev thiab lub hom phiaj muaj peev xwm kho mob qog noj ntshav thiab kuaj mob.
Cov kev tshawb fawb genomic profileing dav dav tau txheeb xyuas ntau pua tus lncRNAs cuam tshuam nrog mob qog noj ntshav.Txawm li cas los xij, lawv txoj haujlwm tseem tsis tau paub ntau56.Txhawm rau txheeb xyuas cov neeg sib tw muaj txiaj ntsig lncRNA koom nrog hauv kev mob qog noj ntshav, peb tau ua qhov kev ntsuam xyuas poob ntawm kev ua haujlwm kom txo qis kev loj hlob hauv SW620 kab mob qog nqaij hlav hauv plab uas siv CRISPRi system (Fig. 1a).Qhov tshwj xeeb tshwj xeeb ntawm SW480 thiab SW620 kab mob qog noj ntshav hauv plab yog tias lawv tau muab los ntawm cov qog nqaij hlav hauv thawj thiab theem nrab hauv ib tus neeg mob.Qhov no muab qhov kev sib piv tseem ceeb rau kev kawm txog kev hloov pauv caj ces hauv kev loj hlob ntawm kev mob qog noj ntshav siab.Yog li, peb tau txheeb xyuas cov ntawv sau tseg ntawm cov kab mob qog nqaij hlav hauv plab (SW480 thiab SW620) siv RNA sequencing thiab sau qee qhov ua tau zoo lncRNAs los ntawm cov ntaub ntawv luam tawm.Raws li cov txiaj ntsig no, peb tau tsim ib lub tsev qiv ntawv sgRNA uas muaj 7355 sgRNA oligos tsom 971 mob qog noj ntshav nrog lncRNAs thiab 500 yam tsis muaj sgRNA oligos rau kev tswj tsis zoo (Cov ntaub ntawv ntxiv 1).
Schematic sawv cev ntawm kev tshuaj ntsuam siv CRISPRi system.b sgRNA enrichment tom qab kuaj.Kab rov tav dotted sawv cev rau log2 (fold hloov) = ± 0.58.Txoj kab ntsug dotted qhia p tus nqi = 0.05.Cov dots dub sawv cev tsis yog lub hom phiaj sgRNA (tsim los ua NC).Cov dots liab yog sgRNAs tsom DARS-AS1.Blue dots yog sgRNAs tsom rau LINC00205, yav dhau los piav txog oncogenic lncRNA.fold hloov = (normalized nyeem ntawv, hnub 17) / (normalized nyeem ntawv, hnub 0).c DARS-AS1 sgRNA knockdown inhibited cell loj hlob.Cov kab yuam kev sawv cev ± tus qauv sib txawv ntawm peb qhov kev sim.* p ≤ 0.05, ** p ≤ 0.01 ob-tailed Student's t-test.d DARS-AS1 qhia hauv cov qog (TCGA dataset).em Kev nthuav qhia ntawm DARS-AS1 nyob rau hauv ua ke ib txwm thiab qog qog los ntawm cov neeg mob nrog BLCA, KIRC, PRAD, LUSC, UCEC, LUAD, LIHC, KIRP, thiab COAD, raws li (TCGA dataset).p-tus nqi tau txais los ntawm kev siv ob-tailed Tub Ntxhais Kawm T-test.
Tom qab tsim cov plasmid thiab ntim cov kab mob lentivirus, peb tau hloov pauv dCas9-SW620 kab mob qog nqaij hlav hauv plab nrog lub tsev qiv ntawv saum toj no hauv plaub qhov kev sim kis kab mob ywj pheej.Qhov sib txawv ntawm kev kis kab mob (MOI) rau cov kab mob no yog 0.1-0.3, qhia tias txhua lub xov tooj tsuas tuaj yeem kis tau nrog ib qho sgRNA.Tom qab 18 hnub ntawm kev coj noj coj ua hauv vitro, qhov kev nthuav dav ntawm lub hom phiaj sgRNAs txo qis lossis nce ntxiv tom qab kev tshuaj ntsuam, thaum tus naj npawb ntawm cov tswj tsis tau tswj hwm oligonucleotides tseem tsis tau hloov pauv piv rau qhov kev tshuaj ntsuam ua ntej, qhia tias peb lub hom phiaj muaj kev tshuaj ntsuam xyuas tshwj xeeb. tsev qiv ntawv.Rice.1b thiab ntxiv cov lus 1). LINC00205, uas tau tshaj tawm yav dhau los los txhawb kev mob ntsws cancer thiab lub siab mob qog noj ntshav58,59,60, tau raug tshuaj xyuas (log2 (foldchange) < −0.58, p tus nqi < 0.05), lees paub qhov kev ntseeg tau ntawm qhov kev tshuaj ntsuam no (Fig. 1b). LINC00205, uas tau tshaj tawm yav dhau los los txhawb kev mob ntsws cancer thiab lub siab mob qog noj ntshav58,59,60, tau raug tshuaj xyuas (log2 (foldchange) < −0.58, p tus nqi < 0.05), lees paub qhov kev ntseeg tau ntawm qhov kev tshuaj ntsuam no (Fig. 1b). LINC00205, о котором ранее сообщалось, что он способствует прогрессированию рака легких и рака печени58, 59, 60, был исключен (log2 (кратное изменение) <-0,58, значение p <0,05), что подтверждает надежность этого скрининга (рис . 1b). LINC00205, yav dhau los tau tshaj tawm los txhawb txoj kev loj hlob ntawm mob ntsws cancer thiab mob qog noj ntshav 58,59,60, raug cais tawm (log2 (fold hloov) <-0.58, p-value <0.05), lees paub qhov muaj zog ntawm qhov kev tshuaj ntsuam no (Fig. .1b) . LINC00205 之前被报道可促进肺癌和肝癌进展58,59,60，被筛选掉的（log2（倍数变化）. LINC00205 之前被报道可促进肺癌和肝癌进展58,59,60，被筛选掉的（log2（倍数变化）. LINC00205, о котором ранее сообщалось, что он способствует прогрессированию рака легких и печени58, 59, 60, был исключен (log2 (кратное изменение) <-0,58, p-значение <0,05), что подтверждает надежность этого скрининга (рис . 1b). LINC00205, yav dhau los tau tshaj tawm los txhawb lub ntsws thiab lub siab mob qog noj ntshav58,59,60, raug cais tawm (log2 (fold hloov) <-0.58, p-value <0.05), lees paub qhov muaj zog ntawm qhov kev tshuaj ntsuam no (Fig. .1b).
Ntawm tag nrho cov lncRNAs kuaj, DARS-AS1 kuj tau tshuaj xyuas, nrog rau peb cognate sgRNA oligonucleotides txo qis tom qab 18 hnub ntawm kab lis kev cai, qhia tias knockdown ntawm lncRNA no ua rau txo qis kev loj hlob ntawm qog nqaij hlav (Fig. 1b).Qhov txiaj ntsig no tau txhawb ntxiv los ntawm MTS kev tshuaj xyuas hauv cov qog nqaij hlav qog nqaij hlav hauv plab uas qhia tau tias qhov kev loj hlob ntawm DARS-AS1 knockdown hlwb tsuas yog halved piv rau cov tswj hlwb (Daim duab 1c) thiab tau ua raws li cov ntawv tshaj tawm yav dhau los ntawm ntau lwm hom mob qog noj ntshav.: Clear cell mob cancer, thyroid cancer thiab non-small cell lung cancer51,52,53,55.Txawm li cas los xij, nws txoj haujlwm thiab cov txheej txheem molecular hauv cov qog nqaij hlav hauv plab tseem tsis tau tshawb nrhiav.Yog li ntawd, peb xaiv qhov no lncRNA rau kev kawm ntxiv.
Txhawm rau kawm DARS-AS1 cov lus qhia hauv cov neeg mob, peb tau txheeb xyuas 10,327 cov qog qog los ntawm Cancer Genome Atlas (TCGA) qhov project.Peb cov txiaj ntsig tau pom tias DARS-AS1 tau nthuav tawm dav dav thiab txhim kho hauv cov hlwb noj qab haus huv hauv ntau cov qog nqaij hlav, suav nrog cov hnyuv adenocarcinoma (COAD), lub raum ntshiab cell carcinoma (KIRC), thiab lub raum papillary cell carcinoma (KIRP)..Tsawg heev (Fig. 1d thiab Ntxiv Fig. 1a, b). Kev soj ntsuam ntawm kev noj qab haus huv / qog cov qauv txuas ntxiv tau lees paub qhov kev qhia ntau dua ntawm DARS-AS1 hauv cov qog ntawm lub zais zis urothelial carcinoma (BLCA), lub raum lub raum ntshiab cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), ntsws squamous cell carcinoma (LUSC) , uterine corpus endometrial carcinoma (UCEC), ntsws adenocarcinoma (LUAD), lub siab hepatocellular carcinoma (LIHC), raum raum papillary cell carcinoma (KIRP), thiab colon adenocarcinoma (COAD) (p tus nqi <0.05) (Fig. 1e–m) . Kev soj ntsuam ntawm kev noj qab haus huv / qog cov qauv txuas ntxiv tau lees paub qhov kev qhia ntau dua ntawm DARS-AS1 hauv cov qog ntawm lub zais zis urothelial carcinoma (BLCA), lub raum lub raum ntshiab cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), ntsws squamous cell carcinoma (LUSC) , uterine corpus endometrial carcinoma (UCEC), ntsws adenocarcinoma (LUAD), lub siab hepatocellular carcinoma (LIHC), raum raum papillary cell carcinoma (KIRP), thiab colon adenocarcinoma (COAD) (p tus nqi <0.05) (Fig. 1e–m) .Kev soj ntsuam ntawm kev noj qab haus huv / qog qog kuj tau lees paub qhov kev qhia ntau dua ntawm DARS-AS1 hauv zais zis urothelial carcinoma (BLCA), lub raum ntshiab thiab lub raum cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), ntsws squamous cell carcinoma (LUSC) qog., карцинома эндометрия тела матки (UCEC), аденокарцинома легкого (LUAD), гепатоцеллюлярная карцинома печени (LIHC), папиллярно-клеточная карцинома почки (KIRP) и аденокарцинома толстой кишки (COAD) (значение p <0,05) (рис. 1e– m). , endometrial carcinoma ntawm corpus uteri (UCEC), adenocarcinoma ntawm lub ntsws (LUAD), hepatocellular carcinoma ntawm lub siab (LIHC), papillary cell carcinoma ntawm lub raum (KIRP), thiab adenocarcinoma ntawm txoj hnyuv (COAD) (p tus nqi < 0.05) (Fig. 1e– m).健康 健康 / 肿瘤样本 的 进一步 dars-as1 在在 膀胱膀胱膀胱) as as as as as1111111111111皮癌皮癌皮癌皮癌皮癌皮癌皮癌皮癌皮癌皮癌皮癌皮癌皮癌皮癌皮癌皮癌皮癌皮癌皮癌皮癌皮癌皮癌皮癌皮癌皮癌皮癌 (kirc), 肺鳞状 腺腺癌 (Prad), 肺鳞状肺鳞状 (Lusc) 肿瘤 的显着更高表达，子宫体子宫内膜癌（UCEC），肺腺癌（LUAD），肝肝细胞癌（LIHC），肾肾乳头瞶癌癌<0.05) (图1e-m).配 对 健康 健康 / 肿瘤样本 的 分析 了 dars-OS1 在 上 上 皮癌 皮癌 皮癌 皮癌 皮癌 细胞癌 细胞癌细胞癌 细胞癌细胞癌 细胞癌细胞癌 细胞癌细胞癌 细胞癌腺腺癌 细胞癌腺腺癌 细胞癌腺腺癌腺腺癌 细胞癌腺腺癌 前列腺腺癌 腺腺癌腺腺癌腺腺癌腺腺癌腺腺癌 (Prad), 细胞癌 细胞癌(Lusc) Koj cia li tso koj tseg ((u) 肺腺癌 (lHC) 肾 肾 肾 肾癌 (kirp) (coad) (p 值 <0.05) (图1e-m).Kev soj ntsuam kev noj qab haus huv / qog ua piv txwv ntxiv txhawb nqa lub luag haujlwm ntawm DARS-AS1 hauv zais zis urothelial carcinoma (BLCA), ntshiab cell renal cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), thiab ntsws squamous cell carcinoma (LUSC) qog.экспрессия при карциноме тела матки (UCEC), аденокарциноме легкого (LUAD), гепатоцеллюлярной карциноме (LIHC), почечно-почечной папиллярно-клеточной карциноме (KIRP) и аденокарциноме толстой кишки (COAD) (значение p <0,05) (рис. 1e -m). kev qhia hauv corpus uterine carcinoma (UCEC), ntsws adenocarcinoma (LUAD), hepatocellular carcinoma (LIHC), lub raum papillary cell carcinoma (KIRP), thiab colon adenocarcinoma (COAD) (p tus nqi <0.05) (Daim duab 1e -m).Ua ke, cov txiaj ntsig no qhia tau tias DARS-AS1 tau dav thiab nthuav tawm ntau yam mob qog noj ntshav.
Vim hais tias DARS-AS1 thiab DARS (cov noob encoding antisense strand) sib koom tib tus neeg txhawb nqa thiab nyob ib sab ntawm ib sab, peb tsim shRNA rau tshwj xeeb knockdown DARS-AS1 tab sis tsis yog DARS (Sab Ntxiv Fig. 2a,b thiab Supplementary Table 2) .Ntxiv nrog rau SW620, peb kuj tau siv peb lwm cov kab ntawm tes qhia DARS-AS1 los kawm txog kev ua tau zoo thiab kev ua haujlwm ntawm shRNA knockdown (Table 3).Peb cov txiaj ntsig tau qhia tias tag nrho peb lub shRNAs tsim ua tiav tsawg kawg yog 80% DARS-AS1 knockdown efficiency nrog me ntsis cuam tshuam rau tus nqi ntawm DARS mRNA (Ntxiv daim duab 2c–f).Tsis tas li ntawd, peb pom tias DARS-AS1 knockdown nrog cov shRNAs no cuam tshuam kev loj hlob ntawm tes hauv cov kab mob qog nqaij hlav hauv plab SW620 (49.7%) thiab HCT116 (27.7%), kab mob qog noj ntshav mis MBA-MD-231 (53.4%).) thiab HepG2 hepatoma cell kab (92.7% txo), nrog rau lawv lub peev xwm los tsim spheres unanchored spheres (qhov nruab nrab txo ntawm ~ 50.8%, 44.6%, 40.7% thiab 75.7% ib kab ntawm tes) (Fig. 2a, b).Nyob rau hauv SW620, cov txiaj ntsig ntawm qhov kev ntsuam xyuas colony ntxiv tau lees paub tias DARS-AS1 shRNA cuam tshuam cov cell proliferation nrog qhov nruab nrab qis ntawm kwv yees li 69.6% (Fig. 2c).
Cov nyhuv ntawm kev tswj shRNA thiab DARS-AS1 shRNA ntawm cell proliferation (a) thiab spheroid tsim (b) hauv SW620, HCT116, MBA-MD-231, thiab HepG2 hlwb.c Cov nyhuv ntawm kev tswj shRNA thiab DARS-AS1 shRNA ntawm kev tsim cov colony hauv SW620 hlwb.Cell proliferation (d), spheroid formation (e), thiab colony formation (f) ntawm SW620 cells overexpressing DARS-AS1.Cov ntaub ntawv qhia yog qhov nruab nrab ± tus qauv sib txawv ntawm peb qhov kev sim.* p ≤ 0.05, ** p ≤ 0.01, thiab *** p ≤ 0.001 los ntawm ob-tailed Student's t-test.
Txhawm rau ntxiv cov kev tshawb fawb poob-ntawm-kev ua haujlwm, peb txuas ntxiv tsim SW620 hlwb overexpressing DARS-AS1 (Ntxiv Fig. 2g).DARS-AS1 overexpression ho nce cell loj hlob (1.8-fold), unanchored spheroid tsim (1.4-fold), thiab colony tsim (3.3-fold) hauv SW620 hlwb (Fig. 2d-f).Peb tau lees paub qhov txiaj ntsig no siv lwm qhov DARS-AS1 qhia cov xov tooj ntawm tes, A549.Qhov no txhim kho cell proliferation vim DARS-AS1 overexpression tau soj ntsuam ntxiv nyob rau hauv A549 hlwb (Suab ntxiv Fig. 2h, i thiab Supplementary Table 3).Ua ke, cov kev tshawb fawb nce thiab kev poob no qhia tau tias DARS-AS1 txhawb kev mob qog noj ntshav hauv vitro.
Txhawm rau tshawb nrhiav cov txheej txheem hauv qab uas DARS-AS1 tswj hwm kev loj hlob ntawm tes, peb tau ua RNA rub-down tsom xam los txheeb xyuas nws cov neeg koom tes muaj peev xwm sib koom ua ke.RT-qPCR cov txiaj ntsig tau pom tias kwv yees li 86.2% ntawm DARS-AS1 yog nyob rau hauv cytoplasm ntawm SW620 hlwb (Cov duab ntxiv 3a).Lub hauv vitro transcribed biotinylated DARS-AS1 los yog pseudoRNA ces incubated nrog SW620 cell lysates tom qab SDS-PAGE sib cais.Tom qab cov nyiaj staining pom tau hais tias ib pawg sib txawv (~ 38 kDa) tau ua kom muaj txiaj ntsig zoo hauv DARS-AS1 rub cov qauv tab sis tsis nyob hauv dummy RNA lossis cov qauv hlaws (Fig. 3a).Cov qhab no tau raug txheeb xyuas tias yog PKR activating protein (PACT) los ntawm huab hwm coj spectrometry (MS) thiab tau lees paub ntxiv los ntawm immunoblotting hauv SW620, HCT116, thiab HepG2 cell kab (Fig. 3a,b).Kev txhawb nqa ntawm DARS thiab lwm yam PACT cov protein - PKR thiab TRBP - kuj tau tshawb xyuas siv RNA tsom xam los ntawm Western blotting (WB).Cov txiaj ntsig tau qhia tias tsis muaj kev sib cuam tshuam ncaj qha ntawm DARS-AS1 RNA thiab peb cov proteins no tau pom (Ntxiv Fig. 3b).Qhov kev cuam tshuam tshwj xeeb ntawm DARS-AS1 thiab PACT tau lees paub ntxiv los ntawm RNA immunoprecipitation (RIP) kev soj ntsuam, uas pom tau tias DARS-AS1 tau ua kom muaj txiaj ntsig zoo hauv cov tshuaj tiv thaiv PACT tab sis tsis yog lwm yam kev tswj hwm RNAs (Daim duab 3c).Txhawm rau txiav txim siab seb DARS-AS1 cuam tshuam ncaj qha nrog PACT thaum tsis muaj lwm cov khoom siv ntawm tes, ib qho kev ntsuam xyuas hauv vitro biolayer interferometry (BLI) tau ua los ntawm kev siv PACT huv.Biotin-labeled DARS-AS1 los yog dummy RNA tau immobilized ntawm streptavidin (SA) biosensors thiab ces incubated nyob rau hauv kinetic tsis muaj 1 μM PACT.Qhov tseem ceeb, PACT khi ruaj khov rau DARS-AS1 (KD tus nqi ~ 26.9 nM), tab sis tsis txhob ua raws li RNA (Daim duab 3d).Ua ke, cov txiaj ntsig no qhia tau hais tias muaj kev cuam tshuam ncaj qha thiab kev sib raug zoo ntawm DARS-AS1 thiab PACT.
RNA rub tsom xam pom DARS-AS1 cuam tshuam nrog PACT hauv SW620 hlwb.Saum toj no, nyiaj staining ntawm cov proteins uas cuam tshuam.Cov tshuaj tiv thaiv qis dua tau ua nrog cov tshuaj tiv thaiv PACT.b RNA rub-down tsom xam tau ua nyob rau hauv HCT116 (sab saum toj) thiab HepG2 (hauv qab) hlwb.PACT enrichment tau kuaj pom los ntawm immunoblotting.cRNA immunoprecipitation (RIP) kev soj ntsuam tau ua nyob rau hauv SW620 hlwb siv cov tshuaj tiv thaiv.d PACT binding curves rau tag nrho-ntev DARS-AS1 lossis tswj RNA tau txais siv biolayer interferometry (BLI).RNA tau immobilized ntawm streptavidin biosensor.1 μM PACT tau siv los ntsuas kev sib koom ua ke.e RNA rub kev soj ntsuam tau ua tiav siv biotinylated full-length DARS-AS1 lossis truncated (sab saum toj).Immunoblot qhia PACT tau txais (hauv qab).f Purified flagged PACT tau incubated nrog biotinylated tag nrho-ntev DARS-AS1 los yog truncated (raws li nyob rau hauv e) rau hauv vitro RIP xeem.Cov RNA rho tawm tau txheeb xyuas los ntawm RT-qPCR.g Cov txheeb ze sib txawv ntawm cov khoom sib txawv RNA rau PACT tau siv biolayer interferometry.Rau kev tsom xam, 100 nM RNA thiab 1 μM RAST tau siv.h Kev ntsuam xyuas hauv vitro RIP tau ua los ntawm kev ua kom huv si tsis zoo lossis raug txiav sau npe PACT.Cov RNA rho tawm tau txheeb xyuas los ntawm RT-qPCR.Kuv loj hlob tus nqi ntawm SW620 hlwb overexpressing DARS-AS1, PACT, los yog ob qho tib si.j Overexpression of full-length or truncated DARS-AS1 in SW620 cells have different effects on cell growth.k Apoptosis tau kuaj pom los ntawm immunoblotting nrog anti-PARP antibody.l Knockout ntawm DARS-AS1 induces apoptosis ntawm SW620 hlwb raws li qhia los ntawm flow cytometry.Cov ntaub ntawv qhia yog qhov nruab nrab ± tus qauv sib txawv ntawm peb qhov kev sim. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001, los ntawm ob-tailed Student's t test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001, los ntawm ob-tailed Student's t test. *p ≤ 0,05, **p ≤ 0,01, ***p ≤ 0,001, ****p < 0,0001 по двустороннему критерию Стьюдента. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001 los ntawm ob-tailed Student's t-test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001, 通过双尾学生t 检验. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001, 通过双尾学生t 检验. *p ≤ 0,05, **p ≤ 0,01, ***p ≤ 0,001, ****p <0,0001 по двустороннему критерию Стьюдента. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001 los ntawm ob-tailed Student's t-test.
Tom qab ntawd peb tau tsim peb biotinylated DARS-AS1 RNA fragments los ntawm hauv vitro transcription los txheeb xyuas thaj tsam DARS-AS1 xav tau rau PACT koom haum (Daim duab 3e).Cov txiaj ntsig ntawm RNA tsom xam pom tau tias txhua qhov tawg tuaj yeem cuam tshuam nrog PACT, tab sis thaj tsam 3′-terminal (384–768 nucleotides sau A3) pom ntau dua 1–384 nucleotides sau A1) (Fig. 3e).Cov txiaj ntsig zoo sib xws tau pom nyob rau hauv vitro RIP kev soj ntsuam siv cov PACT rov ua dua (Daim duab 3f).Raws li cov txiaj ntsig no, kev sim los khi immobilized RNA fragments rau PACT siv BLI kuj pom tias PACT muaj kev sib raug zoo dua rau A3 (384-768 nt) (KD tus nqi kwv yees li 94.6 nM), thaum yuav luag tsis muaj kev sib txuas nrog lwm thaj chaw.(Daim duab 3d).
Peb kuj tau tshuaj xyuas cov cheeb tsam cuam tshuam hauv PACT.PACT muaj peb lub luag haujlwm ua haujlwm, ob qho ntawm cov uas tau khaws cia ob-stranded RNA-binding domains (dsRBD) thiab ib qho thib peb (tsim D3) uas ua haujlwm ua haujlwm ntawm cov protein sib cuam tshuam.Txhawm rau tshuaj xyuas lncRNA binding peev ntawm txhua tus sau, peb tau tsim peb qhov kev hloov pauv uas tshem tawm txhua qhov ntawm peb lub npe thiab ua qhov kev ntsuam xyuas hauv vitro RIP.Peb cov txiaj ntsig tau pom tias kev tshem tawm ntawm qhov thib peb lub npe (D3) ntawm PACT txo nws txoj kev cuam tshuam nrog DARS-AS1 (los ntawm 0.11-fold piv nrog cov PACT tsis zoo) piv rau lwm qhov kev hloov pauv (Daim duab 3h), nws tau pom tias qhov kev tso tawm. ntawm D3 cuam tshuam nrog DARS.-AC1.Ua ke, cov txiaj ntsig no qhia tias kev sib cuam tshuam ntawm DARS-AS1 thiab PACT tuaj yeem tshwm sim feem ntau los ntawm 3' kawg ntawm DARS-AS1 thiab D3 ntawm PACT.
Peb tau sau tseg tias DARS-AS1 tsis muaj kev cuam tshuam rau PACT qhia thiab PACT tsis muaj kev cuam tshuam rau DARS-AS1 (Cov Lus Qhia Ntxiv 3c).Peb mam li tshuaj xyuas cov txiaj ntsig ntawm PACT knockdown ntawm kev loj hlob ntawm tes.Nyob rau hauv sib piv rau DARS-AS1, cov txheeb ze cov hlwb loj hlob 1.5–3 lub sij hawm sai dua thaum PACT raug tsoo (Ntxiv Fig. 3d).Cov txiaj ntsig ntawm qhov kev ntsuam xyuas colony tsim tau qhia tias cov hlwb tsim 2-3-fold colonies tom qab shRNA kho nrog PACT (Ntxiv daim duab 3e).Txhawm rau kuaj seb DARS-AS1 tswj cov cell proliferation los ntawm PACT, peb tsim SW620 hlwb overexpressing PACT, DARS-AS1, los yog ob qho tib si.Overexpression ntawm PACT pom qhov tseem ceeb inhibition ntawm cell proliferation (Daim duab 3i).Thaum DARS-AS1 overexpression per se tseem ceeb txhawb kev loj hlob ntawm tes, tsis muaj qhov sib txawv tseem ceeb hauv kev loj hlob ntawm cov hlwb overexpressing DARS-AS1 thiab PACT.Cov txiaj ntsig no qhia tias PACT tuaj yeem cuam tshuam qhov kev loj hlob ntxiv los ntawm DARS-AS1 overexpression.
Txij li cov cheeb tsam sib txawv ntawm DARS-AS1 muaj qhov sib txawv ntawm PACT-binding peev xwm, peb tau tshawb xyuas lawv cov txheeb ze cuam tshuam ntawm kev loj hlob ntawm tes los ntawm kev sib txawv ntawm DARS-AS1 tawg.Piv rau lwm qhov ob feem, DARS-AS1 tau tshaj tawm ntawm 3′ kawg (384–768 nt), lub ntsiab PACT-txog thaj tsam hauv DARS-AS1, uas muaj lub peev xwm siab tshaj plaws los txhawb kev loj hlob ntawm tes (Fig. 3j).Cov txiaj ntsig no qhia tau hais tias muaj kev sib raug zoo ntawm kev sib koom ua ke thiab kev ua haujlwm lom neeg ntawm DARS-AS1.
PACT tau tshaj tawm tias yog ib qho pro-apoptotic protein19.Yog li, peb tau tshawb xyuas cov txiaj ntsig ntawm DARS-AS1 ntawm apoptosis.Raws li kev cia siab, DARS-AS1 knockdown tau nce PARP cleavage hauv SW620 hlwb thiab nce qhov kev faib ua feem ntawm annexin V-zoo hlwb hauv SW620, HCT116, HepG2, thiab MBA-MD-231 cell kab (Fig. 3k).3).3f–h), qhia tias cov nyhuv anti-apoptotic ntawm DARS-AS1 hauv cov qog nqaij hlav cancer yog qhov cuam tshuam rau apoptosis-inducing muaj nuj nqi ntawm PACT.Ua ke, cov txiaj ntsig no qhia tau hais tias cov txheej txheem ntawm DARS-AS1 oncogenic muaj nuj nqi yuav yog los ntawm inhibition ntawm PACT muaj nuj nqi.
Tom ntej no, peb tshawb nrhiav qhov cuam tshuam rau kev ua haujlwm ntawm DARS-AS1-PACT lub koom haum.PACT tau tshaj tawm kom qhib PKR los ntawm kev sib cuam tshuam ncaj qha, uas tom qab ntawd txhim kho eIF2α phosphorylation, ua rau kev hloov pauv thiab apoptosis17.Ua ntej, peb tau tshuaj xyuas seb DARS-AS1 puas cuam tshuam rau cellular localization ntawm PACT thiab PKR.Confocal fluorescence microscopy pom tias PACT thiab PKR tau colocalized hauv SW620 hlwb nrog qhov nruab nrab Pearson correlation coefficient ntawm 0.72.Lub caij no, DARS-AS1 overexpression txo qis PACT thiab PKR co-localization (txhais tau tias Pearson correlation coefficient 0.61) (Daim duab 4a).Txhawm rau tshawb xyuas seb DARS-AS1 puas tuaj yeem hloov kho PACT-PKR kev sib cuam tshuam, peb tau ua qhov kev ntsuam xyuas co-immunoprecipitation (co-IP) nrog anti-PACT antibody hauv SW620 cell lysates.PKR tau ua kom muaj txiaj ntsig zoo hauv kev tiv thaiv PACT hauv cov hlwb tswj, thaum PKR rov qab tau txo qis hauv lysates los ntawm cov cell overexpressing DARS-AS1 (Fig. 4b).Purified sau npe PACT thiab PKR tau siv rau hauv vitro protein khi kev soj ntsuam.Raws li, cov uas tau muab DARS-AS1 tab sis tsis muaj kev tswj hwm RNA tau pom tias muaj kev cuam tshuam PACT-PKR (Daim duab 4c).Tag nrho cov txiaj ntsig tau pom tias DARS-AS1 cuam tshuam PACT thiab PKR kev sib txuas lus.
Co-localization ntawm PACT thiab PKR hauv kev tswj cov hlwb lossis cov hlwb overexpressing DARS-AS1 tau pom siv confocal fluorescence microscopy.Lub nuclei tau stained nrog DAPI.Cov qhabnias tau los ntawm 16 daim duab.b Co-immunoprecipitation (co-IP) siv cov tshuaj tiv thaiv PACT hauv cell lysates ntawm kev tswj SW620 hlwb los yog cov hlwb overexpressing DARS-AS1.c Labeled PACT, purified PKR thiab transcribed nyob rau hauv vitro nrog DARS-AS1 los yog mock RNA tau incubated rau hauv vitro protein binding tsom xam.Anti-flag antibodies tau siv rau immunoprecipitation.d Immunoblots nrog cov tshuaj tiv thaiv tau ua nyob rau hauv SW620 thiab HCT116 hlwb hloov nrog kev tswj shRNA lossis DARS-AS1-shRNA ua raws li kev tshaib plab.e DARS-AS1 qhia theem hloov pauv cellular rhiab heev rau thapsigargin.SW620 hlwb tau hloov nrog DARS-AS1 shRNA, DARS-AS1 overexpression plasmid lossis tswj plasmid.Cells tau kho nrog thapsigargin rau 48 teev thiab kev muaj peev xwm ntawm tes tau txiav txim siab siv MTS reagent.f Hauv vitro transcribed DARS-AS1 los yog dummy RNA thiab purified PACT tau siv rau hauv vitro activation assay thiab immunoblot detection.g Immunoblots siv cov tshuaj tiv thaiv kab mob no tau ua rau ntawm SW620-ctrl hlwb (sab laug) lossis cov cell overexpressing PKR mutants (txoj cai).Cov hlwb no tom qab ntawd tau hloov pauv nrog kev tswj hwm shRNA lossis DARS-AS1-shRNA ua raws li kev tshaib plab.h Flow cytometry tau pom tias kev tsis ua haujlwm ntawm mutant PKR tau them nyiaj rau DARS-AS1-induced apoptosis hauv SW620 hlwb.immunoblots nrog cov tshuaj tiv thaiv tau ua hauv SW620 (sab laug) lossis HCT116 (txoj cai) hlwb.Cells transfected nrog tswj shRNA los yog DARS-AS1 shRNA yog cov ntshav-deprived thiab ntxiv nrog 100 nM PKR C16 inhibitor los yog DMSO.Luas bar = 5 µm.Cov ntaub ntawv qhia yog qhov nruab nrab ± tus qauv sib txawv ntawm peb qhov kev sim.* p ≤ 0.05 Ob tug tub-ntxhais kawm ntawv t-test.
Nws feem ntau ntseeg tias ib zaug PACT cuam tshuam nrog PKR17, PKR phosphorylation ntawm Thr451 tuaj yeem raug ntxias.Peb cov txiaj ntsig tau qhia tias theem ntawm PKR phosphorylation tau nce siab hauv DARS-AS1 lub hlwb knockdown tom qab kev tshaib plab ntshav (Fig. 4d thiab Supplementary Fig. 4a).Raws li, peb pom tias phosphorylation ntawm eIF2α, lub ntsiab PKR substrate, kuj tau nce ntxiv los ntawm DARS-AS1 shRNA (Fig. 4d thiab Supplementary Fig. 4a).Thapsigargin yog ER stressor uas ua rau ER tso tawm Ca2+.Kev kho mob nrog thapsigargin tau raug tshaj tawm los txhawb kev qhia thiab ua kom PACT, uas txuas ntxiv cuam tshuam nrog thiab ua kom PKR, ua rau apoptosis los ntawm kev nce eIF2α phosphorylation 18,61.Ntawm no, peb siv thapsigargin ua tus txhawb nqa ntawm PACT / PKR txoj hauv kev los tshawb xyuas seb DARS-AS1 puas tuaj yeem pab cov hlwb kov yeej kev ntxhov siab los ntawm inhibiting PACT / PKR txoj hauv kev.Peb tau pom tias theem ntawm DARS-AS1 qhia tau zoo cuam tshuam nrog kev ua haujlwm ntawm tes rau thapsigargin.SW620 hlwb overexpressing DARS-AS1 muaj sia nyob zoo dua thaum kho nrog thapsigargin, thaum lub hlwb nrog DARS-AS1 knockdown ua rau muaj kev cuam tshuam ntau dua (Fig. 4e).Raws li cov txiaj ntsig no, DARS-AS1 overexpression txo thapsigargin-induced PKR phosphorylation (Ntxiv daim duab 4b).Hauv qhov sib piv, tom qab kev kho thapsigargin, PKR thiab eIF2α tau phosphorylated ntau dua nyob rau hauv DARS-AS1 knockdown hlwb piv rau cov tswj cov hlwb (Supplementary Fig. 4b).Interestingly, thapsigargin induced DARS-AS1 qhia nyob rau hauv ib tug npaum li cas-nyob ntawm seb, uas yuav qhia tau hais tias ib tug anti-kev nyuaj siab muaj nuj nqi ntawm DARS-AS1 (Supplementary Fig. 4c).Tsis tas li ntawd, peb tau ua hauv vitro activation assay kom paub meej cov kev soj ntsuam no.Luv luv, PKR raug ntxuav los ntawm cov cell lysates siv cov tshuaj tiv thaiv-PKR antibody, ces incubated nrog recombinant PACT thiab DARS-AS1 transcribed hauv vitro.Tom qab cov tshuaj tiv thaiv enzymatic, phospho-PKR tau kuaj pom siv WB.Peb cov txiaj ntsig tau qhia tias PKR phosphorylation tau cuam tshuam loj heev los ntawm DARS-AS1, tab sis tsis yog los ntawm kev tswj RNA (Daim duab 4f).Cov no hauv vitro thiab hauv vivo cov txiaj ntsig qhia tias DARS-AS1 inhibits PACT-mediated PKR activation.Nyob rau tib lub sijhawm, peb kuj tau pom qhov txo qis hauv PACT rov qab los ntawm DARS-AS1 (Daim duab 4f).Qhov kev tshwm sim no zoo ib yam nrog cov txiaj ntsig ntawm kev ntsuam xyuas hauv vitro protein binding (Daim duab 4c) thiab rov qhia txog kev thaiv kev ua haujlwm ntawm DARS-AS1 rau PACT-PKR lub koom haum.
Ser246 thiab Ser287 nyob rau hauv D3 sau ntawm PACT yog yuav tsum tau rau PKR ua kom nyob rau hauv cellular stress.Kev hloov pauv ntawm ob lub serine residues rau alanine muab mutant PACT (mutD), uas ua rau PKR thaum tsis muaj kev ntxhov siab, thiab kev hloov pauv rau alanine (mutA) thim rov qab cov txheej txheem.Txij li thaum peb tau pom qhov tseem ceeb ntawm cov npe no hauv kev koom tes ncaj qha nrog DARS-AS1, peb tau tsim ob lub PACT mutants los ntsuas seb cov seem no puas tuaj yeem koom nrog kev cuam tshuam nrog DARS-AS1.Interestingly, ob leeg mutants poob lub peev xwm los khi rau DARS-AS1 (Supplementary Fig. 4d), qhia tias tag nrho cov qauv ntawm PACT protein tej zaum yuav tsum tau rau kev sib raug zoo nrog DARS-AS1.
Tsis tas li ntawd, peb cov txiaj ntsig tseem qhia tau tias DARS-AS1-shRNA-induced inhibition ntawm cell proliferation tuaj yeem rov qab los ntawm kev nthuav tawm qhov tsis zoo PACT mutant (PACTmutA) lossis qhov tseem ceeb tsis zoo PKR mutant (PKRmut) (Supplementary Fig. 4e. e).Overexpression ntawm dominant-negative PKR mutants txo PKR phosphorylation induced los ntawm DARS-AS1 knockdown nrog rau eIF2α phosphorylation nyob rau hauv cov ntshav-deprived hlwb (Fig. 4g).Qhov tseem ceeb tshaj, qhov kev faib ua feem ntawm cov hlwb apoptotic tshwm sim los ntawm DARS-AS1 knockdown kuj raug txo qis hauv cov hlwb overexpressing PKRmut (Fig. 4h thiab Supplementary Fig. 4g).Inhibition ntawm PKR kinase kev ua si kuj cuam tshuam DARS-AS1 kev ua haujlwm, raws li cov txiaj ntsig tau pom tias DARS-AS1 knockdown tsis tshua ua rau PKR thiab eIF2α phosphorylation thaum cov hlwb raug kho nrog PKR-specific C16 inhibitor (Fig. 4i thiab Supplementary Fig. 4h).).Ua ke, peb cov txiaj ntsig tau qhia tias DARS-AS1 txhawb kev loj hlob ntawm tes, tsawg kawg hauv ib feem, los ntawm inhibiting PACT-mediated PKR activation.
Txhawm rau tshawb nrhiav ntxiv lub luag haujlwm ntawm DARS-AS1 hauv cov qog nqaij hlav, peb tau ua hauv vivo kev sim siv tus qauv nas xenograft. Cov txiaj ntsig tau pom tias kev poob qis ntawm DARS-AS1 txo qis kev loj hlob ntawm cov nas hauv nas (p tus nqi <0.0001) (Fig. 5a). Cov txiaj ntsig tau pom tias kev poob qis ntawm DARS-AS1 txo qis kev loj hlob ntawm cov nas hauv nas (p tus nqi <0.0001) (Fig. 5a). Результаты показывают, что нокдаун DARS-AS1 резко снижает рост опухоли у мышей (значение p <0,0001) (рис. Cov txiaj ntsig tau pom tias DARS-AS1 knockdown ua rau txo cov qog loj hlob hauv nas (p tus nqi <0.0001) (Daim duab 5a).结果表明，DARS-AS1 的敲低显着降低了小鼠的肿瘤生长（p 值< 0.0001）（图5a）.结果表明，DARS-AS1 的敲低显着降低了小鼠的肿瘤生长（p值<0.0001）（图5a）. Результаты показали, что нокдаун DARS-AS1 значительно снижает рост опухоли у мышей (значение р <0,0001). Cov txiaj ntsig tau pom tias DARS-AS1 knockdown txo cov qog loj hlob hauv nas (p tus nqi <0.0001) (Daim duab 5a).Yog li, nyob rau hauv pawg DARS-AS1 knockdown, muaj qhov txo qis hauv qhov nruab nrab ntawm cov qog nqaij hlav los ntawm 72.9% thiab txhais tau tias cov qog loj li ntawm 87.8% (Daim duab 5b-d).Cov txiaj ntsig no qhia tau hais tias DARS-AS1 tuaj yeem txhawb nqa cov qog loj hlob hauv vivo.
Cov teebmeem ntawm ad DARS-AS1 knockdown ntawm colorectal oncogenesis hauv cov nas liab qab.Kev loj hlob nkhaus (a), qog loj (b), qhov hnyav (c), thiab cov duab qog (d) tau qhia.Qhov yuam kev bar sawv cev ± SEM. n = 10. ****p < 0.0001, los ntawm ob-tailed Student's t test. n = 10. ****p < 0.0001, los ntawm ob-tailed Student's t test. n = 10. ****p < 0,0001 по двустороннему критерию Стьюдента. n = 10. ****p < 0.0001 two-tailed Student's t-test.n = 10. ****p <0.0001, 通过双尾学生t 检验. ****p <0.0001, 通过双尾学生t检验. ****p < 0,0001 по двустороннему критерию Стьюдента. ****p <0.0001 Ob tug tub-ntxhais kawm ntawv t-test.e Kaplan-Meier txheeb xyuas qhov sib txheeb ntawm DARS-AS1 qhia theem thiab tag nrho cov ciaj sia nyob rau hauv cov neeg mob nrog UVM, KICH, KIRP, MESO, GBM, thiab LGG.Cov qib siab ntawm DARS-AS1 qhia hauv cov neeg mob tau nyob rau saum 50%;Cov theem qis ntawm DARS-AS1 qhia hauv cov neeg mob nyob hauv qab 50%.p-values ​​tau txiav txim siab siv qhov ntsuas ntsuas ntsuas.f Cov qauv tsim nyob rau hauv uas DARS-AS1 tswj PACT-PKR txoj hauv kev thiab cov qog loj hlob.
Txhawm rau kom nkag siab zoo dua qhov cuam tshuam ntawm kev kho mob ntawm DARS-AS1, peb tau tshuaj xyuas qhov sib cuam tshuam ntawm nws cov lus qhia thiab tus neeg mob muaj sia nyob.Los ntawm kev txheeb xyuas TCGA dataset, peb pom tias ntau dua DARS-AS1 qhia tau txuam nrog uveal melanoma (UVM), raum chromophobia (KICH), raum papillary cell carcinoma (KIRP), mesothelioma (MESO), multiplex.Kev muaj sia nyob qis dua tau cuam tshuam nrog glioblastoma morphosis (GBM) thiab cov neeg mob uas muaj qib qis hauv hlwb glioma (LGG) (Daim duab 5e).Cov txiaj ntsig no qhia tias DARS-AS1 tuaj yeem ua lub luag haujlwm tseem ceeb hauv kev kho mob qog noj ntshav thiab tej zaum yuav muaj peev xwm kwv yees biomarker hauv ntau cov qog nqaij hlav.
Hauv txoj kev tshawb no, siv qhov loj-scale CRISPRi kev tshuaj ntsuam xyuas, peb tau txiav txim siab tias DARS-AS1 lncRNA kov yeej kev mob qog noj ntshav los ntawm kev tswj hwm ob qhov kev ntxhov siab tseem ceeb, PACT thiab PKR.Los ntawm kev cuam tshuam ncaj qha nrog PACT, DARS-AS1 inhibited PACT-mediated PKR activation, yog li tiv thaiv apoptotic cell tuag thiab txhawb kev loj hlob ntawm tes (Fig. 5f).Upregulation ntawm DARS-AS1 tau pom nyob rau hauv ntau hom mob qog noj ntshav, qhia tias nws txoj haujlwm ntawm kev txhawb nqa cov qog nqaij hlav qog noj ntshav nyob rau hauv cov xwm txheej ntxhov siab tuaj yeem siv dav rau ntau hom mob qog noj ntshav.
PACT tau raug txheeb xyuas tias yog PKR activator protein, thiab PACT-mediated PKR activation plays lub luag haujlwm tseem ceeb hauv cov lus teb kev ntxhov siab los ntawm kev tswj cov ntawv sau, txhais lus, apoptosis, thiab lwm yam txheej txheem tseem ceeb ntawm tes62.Tau ntau xyoo lawm, tau sim ua kom nkag siab txog kab mob qog noj ntshav tshwj xeeb ntawm PACT-PKR cascade.Ntawm no, peb txoj kev tshawb fawb tau nthuav tawm cov txheej txheem sib txawv ntawm kev tswj hwm ntawm PACT-PKR hauv cov qog nqaij hlav qog noj ntshav los ntawm cellular lncRNA DARS-AS1, uas ncaj qha khi rau PACT, thaiv PACT-PKR kev sib cuam tshuam, inhibits PKR activation thiab eIF2α phosphorylation, yog li inhibiting kev ntxhov siab vim apoptosis thiab stimulating thaum kawg mob qog noj ntshav.hlwb.Qhov kev tshawb pom no ua rau pom qhov muaj peev xwm lncRNA lub hom phiaj rau kev mob qog noj ntshav thiab kev kho mob.
Peb cov ntaub ntawv qhia tau hais tias DARS-AS1 knockdown sensitizes hlwb rau cov ntshav qabzib nrog kev nce ntxiv hauv phosphorylated PKR thiab eIF2α.Cov txiaj ntsig no qhia tau tias DARS-AS1 txhawb nqa kev mob qog noj ntshav hauv cov xwm txheej hnyav los ntawm inhibiting PACT / PKR kev ua haujlwm.Ob peb lwm yam tsis-coding RNAs, xws li ASPACT thiab nc886, kuj tseem koom nrog PACT / PKR axis los ntawm kev txo qis PACT48 mRNA lossis tswj autophosphorylation los ntawm kev khi rau PKR49,50,64.Ntawm lawv, DARS-AS1 ua raws li kev cuam tshuam ntawm PACT-PKR lub koom haum.Txoj kev tshawb no txhawb peb txoj kev nkag siab ntawm PACT / PKR axis kev tswj hwm thiab lub luag haujlwm ntawm lncRNAs hauv cov lus teb kev ntxhov siab.
PACT muaj peb qhov sib cais.Txhua tus thawj ob dsRBDs yog txaus kom ua tiav siab affinity khi ntawm PACT rau PKR, thaum lub thib peb domain (D3) yog yuav tsum tau rau PKR activation nyob rau hauv vitro thiab vivo.Peb txoj kev tshawb fawb tau pom tias DARS-AS1 nyiam cuam tshuam nrog D3 sau (Daim duab 3h).Muab qhov loj me me ntawm lncRNA (768 nucleotides), DARS-AS1 khi rau D3 tuaj yeem cuam tshuam kev cuam tshuam ntawm PACT tus thawj ntawm dsRBD thiab PKR, yog li thaiv kev koom tes ntawm PACT thiab PKR.PACT taw tes kev hloov pauv uas hloov Ser246 thiab Ser287 hauv D3 nrog alanine lossis aspartate cuam tshuam nws txoj kev sib raug zoo rau DARS-AS1, taw qhia qhov tseem ceeb ntawm D3's tag nrho cov qauv thiab hluav taws xob hauv lawv lub koom haum.Cov ntsiab lus ntxiv ntawm cov txheej txheem no yuav xav tau yav tom ntej, siv cov kev tshuaj ntsuam xyuas biochemical ntau dua thiab cov kev daws teeb meem siab PACT cov qauv ntsuas.
Cov kev tshawb fawb yav dhau los tau tshaj tawm tias DARS-AS1 txhawb kev loj hlob ntawm tes los ntawm ntau lub tshuab 51,52,53.Hauv ib qho piv txwv, cov neeg tshawb xyuas tau pom tias DARS-AS1 tau tswj hwm nws cov tshuaj tiv thaiv protein-encoding DARS noob los ntawm kev tsom mus rau miP-194-5p hauv lub raum mob qog noj ntshav.Txawm li cas los xij, hauv txoj kev tshawb fawb tam sim no, DARS-AS1 knockdown tsis tshua muaj txiaj ntsig ntawm DARS kev hloov pauv hauv ntau hom mob qog noj ntshav, suav nrog yam tsawg kawg ntawm lub plab, lub mis, thiab mob qog noj ntshav.Vim tias lncRNAs nthuav tawm cov qauv ntawm tes- thiab cov ntaub so ntswg tshwj xeeb, cov txheej txheem ua haujlwm yuav tsis raug khaws cia thoob plaws hom mob qog noj ntshav, uas tuaj yeem ua rau muaj qhov tsis sib xws ntawm peb cov kev soj ntsuam thiab kev ntsuas yav dhau los ntawm cov qog nqaij hlav sib txawv.Cov kev tshawb fawb tshwj xeeb yog xav tau los piav qhia txog cov txheej txheem tshwj xeeb ntawm ntau yam physiological thiab pathological txheej txheem.
Kev soj ntsuam ntawm cov ntaub ntawv kho mob tau pom tias DARS-AS1 qhia hauv cov qog nqaij hlav inversely correlated nrog kev ciaj sia ntawm cov neeg mob qog noj ntshav, uas qhia txog qhov tseem ceeb ntawm DARS-AS1 / PACT / PKR axis hauv kev mob qog noj ntshav.Hauv kev xaus, peb txoj kev tshawb fawb qhia tau hais tias DARS-AS1 yog tus tswj hwm ntawm PACT / PKR signaling axis, txhawb kev mob qog noj ntshav cell proliferation, thiab inhibits apoptosis thaum lub sij hawm kev ntxhov siab, uas muab lwm txoj kev tshawb fawb thiab txaus siab rau yav tom ntej kev tshawb fawb txog kev kho mob. .
Tib neeg kab xov tooj SW620, A549, MBA-MD-231, HCT116, HepG2 thiab HEK293T tau txais los ntawm National Cell Line Resource Infrastructure hauv Suav teb.Txhua lub hlwb tau khaws cia hauv DMEM nruab nrab (DMEM, Thermo Fisher Scientific, Waltham, MA) ntxiv nrog 10% FBS (Gemini, Brooklyn, NY) thiab 1% penicillin-streptomycin (Thermo Fisher Scientific) ntawm 37 ° C, 5% CO2.incubator.
Anti-PACT, Abcam (ab31967);Anti-PKR, Abcam (ab184257);Anti-PKR (phospho-T451), Abcam (ab81303);Anti-Flag, Abcam (ab125243);Anti-eIF2α, Abcam (A0764));anti-eIF2α (phosphorus S51), Abcam (ab32157);anti-PACT (phosphorus S246), Abgent (AP7744b);anti-β-tubulin, CST (2128);ib txwm nas IgG, CST (5415S);ib txwm luav IgG, CST (2729S).Cov tshuaj tiv thaiv tau diluted 1: 1000 hauv PBST rau Western blotting thiab 1: 100 rau IP.
sgRNAs tau tsim los siv cov cuab yeej siv rau pej xeem hu ua CRISPR-ERA66.Peb siv cov cuab yeej siv tsis raug rau kev txhim kho sgRNA thiab cov txheej txheem suav sgRNA khi qhov chaw hauv thaj tsam 3 kb.centered at TSS.Cov pas dej ntawm sgRNA oligonucleotides tau tsim los ntawm CustomArray, Inc. (Bothewell, WA) thiab cloned rau hauv humanized pgRNA plasmids (Addgene #44248).Tag nrho ntawm 12 µg ntawm kev sib koom ua ke ntawm tib neeg pgRNA plasmid, 7.2 µg ntawm psPAX2 (Addgene #12260), thiab 4.8 µg ntawm pMD2.G (Addgene #12259) tau koom ua ke rau hauv 5 x 106 HEK293T hauv DNA Transfection tais diav. hlwb (CWBIO, Beijing, Suav teb) ua raws li cov chaw tsim khoom cov lus qhia.Cov kab mob uas muaj tus kab mob supernatants tau sau 48 thiab 72 teev tom qab hloov pauv thiab lim los ntawm 0.45 µm lim.Rau kev tshuaj ntsuam, SW620 hlwb qhia txog dCas9 / KRAB fusion protein tau txais los ntawm kev kis tus kab mob.Hloov kho SW620 hlwb tau kis tus kab mob hauv tsev qiv ntawv hauv plaub qhov kev sim kis kab mob ywj pheej ntawm MOI ntawm 0.1-0.3 thiab tau kuaj nrog 2 μg / ml puromycin (Sigma, St. Louis, MO) rau 2 hnub.Tom qab ntawd, cov hlwb raug coj mus kuaj rau 18 hnub hauv vitro nrog lub tsev qiv ntawv tsawg kawg nkaus ntawm 500 hlwb / sgRNA rau kev tshuaj ntsuam.
Genomic DNA tau muab rho tawm raws li cov lus qhia ntawm QIAamp DNA Blood Midi Kit (QIAGEN, Düsseldorf, Lub teb chaws Yelemees; 51183).Nyob rau hauv tag nrho, 100 μg ntawm genomic DNA ib qho kev rov ua dua tshiab tau siv los tsim lub tsev qiv ntawv.Lub cheeb tsam sgRNA tau nthuav dav los ntawm ob puag ncig ntawm PCR thiab txuas rau barcode.
Cov khoom siv PCR tau huv si siv NucleoSpin® gel thiab PCR purification cov khoom siv (MACHEREY-NAGEL, Düren, Lub teb chaws Yelemees; 740609.250) thiab suav nrog Qubit™ HS ob-stranded DNA kuaj cov khoom (Thermo Fisher Scientific; Q32854).
Kev ntsuam xyuas MTS tau siv los ntsuas kev loj hlob ntawm tes.Cells tau noob nyob rau hauv 96-zoo daim hlau ntawm qhov pib ceev ntawm 2000 hlwb/qhov zoo.Cov txheeb ze ntawm cov hlwb raug ntsuas txhua hnub ntawm lub sijhawm qhia rau tag nrho 4-6 hnub.Rau txhua qhov dej, 20 μl ntawm MTS reagent (Promega) yog diluted nrog 100 μl ntawm DMEM, incubated nrog hlwb rau 4 h ntawm 37 ° C, thiab ces OD490 yog ntsuas.
Lub peev xwm rau kev loj hlob unanchored tau pom los ntawm kev tshuaj xyuas qhov tsim ntawm spheres.Luv luv, 2000 hlwb hloov nrog shRNA DARS-AS1 los yog tswj shRNA tau coj mus rau hauv ultra-tsawg txuas microplates (Corning) nrog nruab nrab hloov txhua 4 hnub.Cov spheroids raug suav tom qab 14 hnub.500 hlwb hloov nrog DARS-AS1 overexpression plasmid lossis tswj plasmid tau siv rau kev ntsuas kev txhim kho, txwv tsis pub txoj kev hloov pauv.
RNA tau sau tseg siv T7 RNA polymerase thiab biotin-16-UTP (Roche 1138908910) raws li cov lus qhia ntawm Riboprobe® Combination Systems (Promega P1440).Cov primers siv ntawm no yog teev nyob rau hauv Cov Lus Qhia Ntxiv 4.
Protein-coding PACT lossis PKR cheeb tsam tau cloned rau hauv pET15b (Addgene #73619) thiab hloov mus rau BL21(DE3).Cov kab mob no tau incubated ib hmos nyob rau hauv LB muab nrog ampicillin thiab ces diluted 100-fold nrog tshiab LB.Thaum OD600 ntawm qhov nruab nrab mus txog 0.8, 1 mM IPTG tau ntxiv rau induce protein qhia.Tom qab incubation thaum hmo ntuj nrog maj mam co (250 rpm ntawm 20 ° C), lub cell pellet tau sau los ntawm centrifugation (4000 rpm, 10 min, 4 ° C).Tshem tawm cov cell pellet nyob rau hauv lysis buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 1 mM PMSF) thiab incubate on dej khov rau 30 min, ces sonicate (15 min, 5 s on / off, ice) thiab centrifuge (13,000). rpm)., 30 min, 4 ° C).Tom qab ntawd cov supernatant raug thauj mus rau Ni-NTA resin (QIAGEN) 3 zaug ntawm 4 ° C, ntxuav 4 zaug nrog ntxuav tsis (50 mM Tris, pH 8.0, 40 mM imidazole, 250 mM NaCl) thiab eluted 3 zaug, nrog rau tag nrho. 10 ml eluent buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 300 mM imidazole).Cov protein huv huv tau txiav txim siab siv WB thiab qhov siab tau txiav txim siab siv Qubit™ cov khoom siv tshuaj ntsuam xyuas protein (Thermo Fisher Scientific; Q 33212).
RIP kev ntsuam xyuas tau ua raws li tau piav qhia dhau los, nrog kev hloov kho.Luv luv, 1x RIP buffer (25 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5% NP-40, RNasin ribonuclease inhibitor (Promega), PMSF (Beyotime Biotechnology), 1 mM DDM, protease) lyses 7 x 10 cytostatic cocktail (Roche, 1 mM DTT) thiab centrifuge ntawm 13,000 rpm rau 15 min ntawm 4 ° C.Tom qab ntawd tus supernatant tau incubated nrog protein A + G sib nqus hlaws (Millipore) conjugated nrog 5 μg ntawm anti-PACT antibody (Abeam) los yog IgG (CST).Cov hlaws dai tau ntxuav 5 zaug nrog 5x RIP tsis, tom qab ntawd zom nrog proteinase K (NEB).RNA tau muab rho tawm nrog Trizol thiab txiav txim siab los ntawm RT-qPCR.Primers tau nthuav tawm hauv Cov Lus Ntxiv 5.
Qhov kev ntsuam xyuas hauv vitro RIP tau ua raws li kev hloov kho tus qauv RIP kev soj ntsuam raws tu qauv.Tag nrho ntawm 5 pmol ntawm in vitro transcribed RNA yog diluted 1x nrog RIP tsis thiab annealed los ntawm incubation ntawm 65 ° C rau 5 feeb tom qab los ntawm qeeb txias rau chav tsev kub.Tag nrho ntawm 5 pmol ntawm qhov tsis zoo lossis hloov pauv tus chij-labeled PACT cov proteins tau huv los ntawm E. coli.Incubate nrog renatured RNA rau 2 teev ntawm 4 ° C thiab ua raws li cov txheej txheem saum toj no rau RIP tsom xam rau kev tiv thaiv tus chij IP.
Rau RNA txuas ntxiv tsom xam, 1 × 107 hlwb tau lysed nrog 1xRIP tsis.Tom qab centrifugation ntawm 13,000 rpm rau 15 min ntawm 4 ° C, tus supernatant tau pretreated nrog 30 μl ntawm streptavidin magnetic hlaws (Beckman) rau 2 h ntawm 4 ° C.Cov purified lysate ces muab cov poov xab tRNA thiab incubated nrog 40 pmol ntawm reatured RNA thaum hmo ntuj ntawm 4 ° C, ces rau lwm 2 teev thiab 20 μl ntawm tshiab streptavidin magnetic hlaws dai nrog BSA tau ntxiv.Cov kauj ruam ntxuav muaj 4 zaug nrog 5x RIP tsis thiab 4 zaug nrog 1x RIP tsis.Cov proteins uas sib raug tau raug eluted nrog biotin elution buffer (25 mM Tris-HCl, pH 7.5, 12.5 mM D-biotin, PMSF) thiab sib cais ntawm NuPAGE 4-12% Bis-Tris Gel (Invitrogen).Tom qab nyiaj staining (Beyotime Biotechnology), qee cov qhab nia raug tshem tawm thiab tshuaj xyuas los ntawm MS.
Co-IP tsom xam tau ua los ntsuas kev sib cuam tshuam ntawm PACT thiab PKR.Luv luv, supernatant lysates tau npaj los ntawm incubating 1 x 107 lysed hlwb hauv 1 x RIP tsis ua raws li centrifugation ntawm 13,000 rpm rau 15 feeb ntawm 4 ° C.Lysates tau ntim nrog cov protein A + G sib nqus hlaws, sib txuas nrog 5 µg ntawm cov tshuaj tiv thaiv PACT, thiab maj mam tig ib hmos ntawm 4 ° C.Cov hlaws tau raug ntxuav 3 zaug nrog 5 × RIP tsis, ob zaug nrog 1 × RIP tsis thiab eluted nrog 1 × SDS tsis.Cov protein rov qab tau txheeb xyuas los ntawm SDS-PAGE gel thiab kuaj pom los ntawm WB.
Ob pmol ntawm flagged PACT thiab 1 pmol ntawm PKR raug purified los ntawm E. coli.Dilute hauv 1 × RIP tsis thiab incubate nrog 10 pmol ntawm renatured RNA rau 2 teev ntawm 4 ° C.Tom qab ntawd, lawv tau incubated nrog protein A + G magnetic hlaws-conjugated anti-labeled antibody rau ob teev ntxiv.Cov hlaws tau raug ntxuav plaub zaug nrog 1x RIP tsis thiab eluted nrog 1x SDS tsis.PACT thiab PKR tau pom los ntawm WB.