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Maselo a khansa asintha njira zosiyanasiyana kuti athe kuthana ndi kupsinjika kwa ma cell ndikupitilizabe kupita patsogolo.Protein kinase R (PKR) ndi protein activator (PACT) ndi omwe amayankha koyamba omwe amayang'anira zizindikiro zosiyanasiyana za kupsinjika zomwe zimalepheretsa kuchuluka kwa ma cell ndi apoptosis.Komabe, kuwongolera kwa njira ya PACT-PKR m'maselo a khansa sikudziwikabe.Apa, tapeza kuti RNA (lncRNA) yayitali yosalemba RNA (lncRNA) aspartyl tRNA synthetase antisense RNA 1 (DARS-AS1) imakhudzidwa mwachindunji ndi kuletsa njira ya PACT-PKR ndipo imalimbikitsa kuchuluka kwa maselo a khansa.Pogwiritsa ntchito kuwunika kwakukulu kwa CRISPRi 971 lncRNA yokhudzana ndi khansa, tidapeza kuti DARS-AS1 idalumikizidwa ndikukula kwambiri kwa maselo a khansa.Chifukwa chake, kugogoda kwa DARS-AS1 kumalepheretsa kuchuluka kwa ma cell ndikulimbikitsa apoptosis ya khansa m'maselo osiyanasiyana a khansa mu vitro ndipo kumachepetsa kwambiri kukula kwa chotupa mu vivo.Mwachimake, DARS-AS1 imamangiriza mwachindunji ku domain activation ya PACT ndikuletsa kuyanjana kwa PACT-PKR, motero kumachepetsa kuyambitsa kwa PKR, eIF2α phosphorylation, ndikuletsa kufa kwa cell ya apoptotic.Kachipatala, DARS-AS1 imawonetsedwa kwambiri m'makhansa angapo, ndipo kuchulukirachulukira kwa lncRNA iyi ndikuwonetsa kusazindikira bwino.Kafukufukuyu akuwunikira malamulo okhudzana ndi khansa panjira ya PACT-PKR ndi DARS-AS1 lncRNA ndipo amaperekanso chandamale china cha matenda a khansa ndi chithandizo.
Kutha kusintha kupsinjika ndikofunikira kwambiri pakupulumuka kwa maselo a khansa komanso kuchulukana.Kuchulukirachulukira komanso zizindikiro za kagayidwe kachakudya za khansa m'magawo ang'onoang'ono - kusowa kwa michere, hypoxia, ndi pH yochepa - zomwe zingayambitse kufa kwa maselo.Kuwonongeka kwa majini okhudzidwa ndi kupsinjika maganizo monga p535, mapuloteni otenthetsa kutentha 6, 7, KRAS8, 9, ndi HIF-110, 11, 12, 13 nthawi zambiri amawoneka mu khansa, motero amalepheretsa apoptosis ndi kulimbikitsa kupulumuka.
Protein kinase R (PKR) ndi gawo lofunika kwambiri la kupsinjika maganizo ndi subunit kinase ya eukaryotic initiation factor 2α (eIF2α), yomasulira yomasulira yomwe imagwirizanitsa kupsinjika kwa ma cell ku imfa ya selo.PKR poyambilira idadziwika ngati puloteni yoletsa ma virus pozindikira RNA (dsRNA) yakunja yakunja.Poyambitsa, PKR phosphorylates eIF2α kuletsa ma virus ndi ma cell protein synthesis14,15,16.PACT (PKR activator protein) yadziwika ngati puloteni yoyamba ya PKR activator pakalibe dsRNA17,18,19,20,21,22,23.Kupyolera mu kuyanjana kwachindunji ndi PKR, PACT imatulutsa zovuta zosiyanasiyana (njala ya seramu, peroxide kapena mankhwala a arsenite) ku PKR ndi njira zowonetsera pansi.Kuphatikiza pa eIF2α phosphorylation, kuyambitsa kwa PACT-mediated PKR kumayambitsa zochitika zosiyanasiyana zomwe zimagwirizanitsidwa ndi kupsinjika maganizo, kuphatikizapo kusintha kwa redox kudzera mu njira ya PI3K/Akt24, kuyang'ana kwa kuwonongeka kwa DNA kudzera p5325,26 ndi NF-κB27,28 Regulates transcript, 29. Chifukwa cha gawo lawo lofunikira pakuyankha kupsinjika, kuchulukana, apoptosis ndi njira zina zazikulu zama cell, PKR ndi PACT akulonjeza zochizira matenda ambiri, makamaka khansa30,31,32,33.Komabe, ngakhale pleiotropic imagwira ntchito komanso kufunikira kwachilengedwe, kuwongolera kwa zochitika za PACT/PKR m'maselo a khansa kumakhalabe kovuta.
Ma lncRNA ndi zolembedwa zazikulu kuposa ma nucleotide 200 opanda mphamvu zopangira mapuloteni.Popeza mapulojekiti otsogola amtundu wonse apeza ma lncRNA masauzande ambiri, 35,36 kuyesetsa kwakukulu kwachitika kuti afotokoze ntchito zawo zachilengedwe.Kafukufuku wochuluka wasonyeza kuti lncRNAs amakhudzidwa ndi njira zambiri zamoyo37 kuphatikizapo kulamulira kwa X-chromosome inactivation38,39, imprinting40, transcription41,42, translation43 komanso ngakhale kukula kwa khansa44,45,46,47.Maphunzirowa adanenanso kuti ma lncRNA ambiri akukhudzidwa ndi njira ya PACT/PKR.Kafukufuku wina wotere adawonetsa kuti lncRNA ASPACT inaletsa kulembedwa kwa PACT ndikuwonjezera kusunga kwa nyukiliya kwa PACT mRNA.Kafukufuku wina wasonyeza kuti lncRNA nc886 imamangiriza ku PKR ndikuletsa phosphorylation49,50 yake.Pakadali pano, lncRNA yowongolera kuyambitsa kwa PACT-mediated PKR sikunanenedwe.
Aspartyl-tRNA synthetase antisense RNA 1 (DARS-AS1) yadziwika kuti ndi oncogenic lncRNA51,52,53,54.Kupyolera mu lamulo la miP-194-5p53, miP-12952 ndi miP-532-3p51, DARS-AS1 yasonyezedwa kuti ikulimbikitsa kukula kwa clear cell renal cell carcinoma, thyroid carcinoma ndi non-small cell lung carcinoma, motero.Tong ndi anzake adapezanso kuti DARS-AS1 imalimbikitsa myeloma kupita patsogolo mwa kusunga bata la mapuloteni 39 (RBM39) RNA-binding motif.Komabe, palibe kafukufuku yemwe wachitikapo ngati lncRNA iyi ikukhudzidwa ndi kuwongolera kwa PACT-PKR activation ndi kupsinjika kwa ma cell a khansa.
Apa, tidachita chinsalu chachikulu chotaya ntchito pogwiritsa ntchito dongosolo la CRISPRi ndikutsimikiza kuti DARS-AS1 lncRNA imalimbikitsa kufalikira kwa mitundu ingapo ya maselo a khansa.Kuonjezera apo, tazindikira njira yaikulu: DARS-AS1 imamangiriza mwachindunji ku PACT, imalepheretsa PACT ndi PKR kumanga, imalepheretsa phosphorylation ya eIF2α, gawo laling'ono la PKR, ndipo pamapeto pake limaletsa kufa kwa maselo a apoptotic.Pomaliza, ntchito yathu ikuwulula DARS-AS1 lncRNA monga wowongolera njira ya PACT-PKR komanso chandamale chomwe chingathe kuchiza khansa ndi matenda.
Kafukufuku wochuluka wa genomic profiling wapeza mazana a LncRNAs okhudzana ndi khansa.Komabe, ntchito yawo imakhalabe yosadziwika56.Kuti tidziwe odalirika a lncRNA omwe akukhudzidwa ndi kukula kwa khansa, tinachita chinsalu chotaya ntchito kuti tichepetse kuchulukana kwa maselo a khansa yamtundu wa SW620 pogwiritsa ntchito CRISPRI (Mkuyu 1a).Chopadera cha mizere ya khansa ya m'matumbo a SW480 ndi SW620 ndikuti amachokera ku zotupa zoyambirira ndi zachiwiri mwa wodwala m'modzi.Izi zimapereka kufananitsa kofunikira pophunzira kusintha kwa majini pakukula kwa khansa ya m'matumbo.Chifukwa chake, tidasanthula zolemba zama cell a khansa ya colorectal (SW480 ndi SW620) pogwiritsa ntchito kutsata kwa RNA ndikusonkhanitsa ma lncRNA ena omwe angagwire ntchito kuchokera m'mabuku osindikizidwa.Kutengera zotsatira izi, tidapanga laibulale ya sgRNA yophatikizidwa yomwe ili ndi 7355 sgRNA oligos yolunjika 971 okhudzana ndi khansa lncRNAs ndi 500 osatsata sgRNA oligos kuti azitha kuwongolera (Supplementary Data 1).
Kuyimilira kwadongosolo kowunika pogwiritsa ntchito dongosolo la CRISPRI.b sgRNA kulemeretsa pambuyo powunika.Mzere wamadontho wopingasa umayimira log2 (kusintha kosintha) = ± 0.58.Mzere wamadontho woyima umasonyeza p mtengo = 0.05.Madontho akuda akuyimira sgRNA yopanda cholinga (yotchedwa NC).Madontho ofiira ndi ma sgRNA omwe akulunjika ku DARS-AS1.Madontho a buluu ndi ma sgRNA omwe akulunjika ku LINC00205, lncRNA yofotokozedwa kale.pinda kusintha = (kuwerenga mwachizolowezi, tsiku 17)/(kuwerenga mwachizolowezi, tsiku 0).c DARS-AS1 sgRNA kugwetsa kumalepheretsa kukula kwa maselo.Mipiringidzo yolakwika imayimira ± kupatuka kokhazikika kwa zoyeserera zitatuzi.* p ≤ 0.05, ** p ≤ 0.01 Mayeso a Wophunzira a michira iwiri.d DARS-AS1 mawu mu zotupa (TCGA dataset).em Kufotokozera kwa DARS-AS1 mu zitsanzo zofananira bwino ndi zotupa kuchokera kwa odwala omwe ali ndi BLCA, KIRC, PRAD, LUSC, UCEC, LUAD, LIHC, KIRP, ndi COAD, motsatana (TCGA dataset).p-values adapezedwa pogwiritsa ntchito t-test ya Student-tailed two.
Titapanga plasmid ndikuyika lentivirus, tidapanga cell cell ya khansa ya dCas9-SW620 ndi laibulale yomwe ili pamwambapa pamayesero anayi odziyimira pawokha.Kuchuluka kwa matenda (MOI) kwa matendawa kunali 0.1-0.3, kusonyeza kuti selo lililonse limatha kupatsirana ndi sgRNA imodzi.Pambuyo pa masiku a 18 a chikhalidwe cha in vitro, chiwerengero cha sgRNAs chandamale chinachepa kapena chinawonjezeka pambuyo poyang'anitsitsa, pamene chiwerengero cha oligonucleotides osayang'aniridwa sichinasinthidwe poyerekeza ndi mbiri yowonetseratu, kusonyeza kuti chandamale chathu chili ndi mawonekedwe enieni. laibulale.Mpunga.1b ndi tebulo lowonjezera 1). LINC00205, yomwe poyamba inanenedwa kuti imalimbikitsa khansa ya m'mapapo ndi khansa ya chiwindi58,59,60, inayesedwa (log2 (foldchange) <-0.58, p value <0.05), kutsimikizira kudalirika kwa kufufuza uku (mkuyu 1b). LINC00205, yomwe poyamba inanenedwa kuti imalimbikitsa khansa ya m'mapapo ndi khansa ya chiwindi58,59,60, inayesedwa (log2 (foldchange) <-0.58, p value <0.05), kutsimikizira kudalirika kwa kufufuza uku (mkuyu 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 ndi. 1b). LINC00205, yomwe idanenedwapo kale kuti ikulimbikitsa kukula kwa khansa ya m'mapapo ndi khansa ya chiwindi58,59,60, idachotsedwa (log2 (kusintha pindani) <-0.58, p-value <0.05), kutsimikizira kulimba kwa kuwunika uku (Fig. .1b) . Linc00205 之前 被 报道 报道 可 促进 肺癌 和 和 进展 58,59,65) 掉 选 变化 变化 变化 Linc00205 之前 被 报道 报道 可 促进 肺癌 和 和 进展 58,59,65) 掉 选 变化 变化 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 ndi. 1b). LINC00205, yomwe idanenedwa kale kuti imalimbikitsa kukula kwa khansa ya m'mapapo ndi chiwindi58,59,60, idachotsedwa (log2 (kusintha pindani) <-0.58, p-value <0.05), kutsimikizira kulimba kwa kuwunika uku (Mkuyu .1b).
Pakati pa ma lncRNA onse omwe anayesedwa, DARS-AS1 inayesedwanso, ndi atatu cognate sgRNA oligonucleotides yachepetsedwa kwambiri pambuyo pa masiku a 18 a chikhalidwe, kutanthauza kuti kugogoda kwa lncRNA iyi kunachititsa kuti kuchepetsa kuchuluka kwa khansa (mkuyu 1b).Chotsatirachi chinathandizidwanso ndi kusanthula kwa MTS m'maselo a khansa ya colorectal kusonyeza kuti kukula kwa maselo ogwetsa a DARS-AS1 kunali kochepa chabe poyerekeza ndi maselo olamulira (Chithunzi 1c) ndipo zinali zogwirizana ndi malipoti am'mbuyomu a mitundu ina yambiri ya khansa.: khansa ya impso yoyera, khansa ya chithokomiro komanso khansa ya m'mapapo yopanda maselo51,52,53,55.Komabe, ntchito zake ndi njira zama cell mu khansa ya colorectal sizikudziwika.Chifukwa chake, tidasankha lncRNA iyi kuti tipitirize kuphunzira.
Kuti tiphunzire mawu a DARS-AS1 mwa odwala, tidasanthula mwatsatanetsatane zitsanzo 10,327 zotupa kuchokera ku projekiti ya Cancer Genome Atlas (TCGA).Zotsatira zathu zikuwonetsa kuti DARS-AS1 imawonetsedwa kwambiri ndikuwongolera kwambiri m'maselo athanzi m'matumbo osiyanasiyana, kuphatikiza colon adenocarcinoma (COAD), renal clear cell carcinoma (KIRC), ndi renal papillary cell carcinoma (KIRP)..Ochepa kwambiri (mkuyu 1d ndi Supplementary Fig. 1a, b). Kuwunika kwa zitsanzo zathanzi / zotupa zotupa zinatsimikiziranso kuti DARS-AS1 yotupa ya chikhodzodzo urothelial carcinoma (BLCA), impso renal clear cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), lung squamous cell carcinoma (LUSC) , uterine corpus endometrial carcinoma (UCEC), lung adenocarcinoma (LUAD), chiwindi hepatocellular carcinoma (LIHC), impso aimpso papillary cell carcinoma (KIRP), ndi colon adenocarcinoma (COAD) (p mtengo <0.05) (Mkuyu 1e-m) . Kuwunika kwa zitsanzo zathanzi / zotupa zotupa zinatsimikiziranso kuti DARS-AS1 yotupa ya chikhodzodzo urothelial carcinoma (BLCA), impso renal clear cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), lung squamous cell carcinoma (LUSC) , uterine corpus endometrial carcinoma (UCEC), lung adenocarcinoma (LUAD), chiwindi hepatocellular carcinoma (LIHC), impso aimpso papillary cell carcinoma (KIRP), ndi colon adenocarcinoma (COAD) (p mtengo <0.05) (Mkuyu 1e-m) .Kufufuza kwa zitsanzo zathanzi / zotupa zotupa zinatsimikiziranso kuti DARS-AS1 mu chikhodzodzo urothelial carcinoma (BLCA), clear cell renal and renal cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), lung squamous cell carcinoma (LUSC) zotupa., 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 ya corpus uteri (UCEC), adenocarcinoma ya m'mapapo (LUAD), hepatocellular carcinoma ya chiwindi (LIHC), papillary cell carcinoma ya impso (KIRP), ndi adenocarcinoma ya m'matumbo (COAD) (p value < 0.05) (Mkuyu 1e- m) .配对 健康 / 肿瘤样本 的 进一步 分析 了 了 了 了 了 了 了 了 了 膀胱 膀胱 尿路 肾 肾 (肾 腺腺癌 (腺腺癌) 肿瘤 细胞癌 (LOSC), 细胞癌显着 更 高 高, 子宫体子宫 内膜 内膜 (ucec), 肺腺癌 (luad), 细胞癌 (lihc), 肾<0.05（图1e-m)配 对 / 肿瘤样本 的 证实 了 了 了 了 Dars-Os1 皮癌 尿路 尿路 皮癌 皮癌 肾 肾 肾(Lusc) 肿瘤 的 的 中 中 中中 中中 中显着癌 (kirp) (coad) (p 值<0.05) (图1e-m) .Kuwunika kwa zitsanzo zathanzi / zotupa zophatikizika ndi chotupa zinathandiziranso ntchito ya DARS-AS1 mu chikhodzodzo urothelial carcinoma (BLCA), clear cell renal cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), ndi lung squamous cell carcinoma (LUSC) zotupa.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). kufotokoza mu corpus uterine carcinoma (UCEC), lung adenocarcinoma (LUAD), hepatocellular carcinoma (LIHC), renal papillary cell carcinoma (KIRP), ndi colon adenocarcinoma (COAD) (p mtengo <0.05) (Chithunzi 1e -m).Kuphatikizidwa pamodzi, zotsatirazi zikuwonetsa kuti DARS-AS1 imawonetsedwa kwambiri m'makhansa osiyanasiyana.
Chifukwa DARS-AS1 ndi DARS (gene encoding the antisense strand) amagawana wothandizira yemweyo ndipo ali pafupi wina ndi mzake, tinapanga shRNA kuti igwetse mwachindunji DARS-AS1 koma osati DARS (Supplementary Fig. 2a,b ndi Supplementary Table 2) .Kuphatikiza pa SW620, tidagwiritsanso ntchito ma cell ena atatu ofotokoza kwambiri DARS-AS1 kuti tiphunzire momwe shRNA knockdown imagwirira ntchito (Table Supplementary Table 3).Zotsatira zathu zimasonyeza kuti ma shRNA onse atatu adapangidwa kuti apindule osachepera 80% DARS-AS1 kugogoda bwino ndi zotsatira zochepa pa kuchuluka kwa DARS mRNA (Supplementary Fig. 2c-f).Kuphatikiza apo, tapeza kuti kugogoda kwa DARS-AS1 ndi ma shRNA awa kumalepheretsa kwambiri kukula kwa ma cell mu mizere ya khansa ya colorectal SW620 (49.7%) ndi HCT116 (27.7%), cell cancer cell line MBA-MD-231 (53.4%).) ndi HepG2 hepatoma cell line (92.7% kuchepetsa), komanso kuthekera kwawo kupanga magawo osasunthika (kuchepa kwapakati kwa ~ 50.8%, 44.6%, 40.7% ndi 75.7% pa mzere wa selo) (Mkuyu 2a, b).Mu SW620, zotsatira za kuyesa kwa mapangidwe a koloni zinatsimikiziranso kuti DARS-AS1 shRNA inaletsa kwambiri kuchuluka kwa maselo ndi kuchepa kwapakati pafupifupi 69.6% (mkuyu 2c).
Zotsatira za kuwongolera shRNA ndi DARS-AS1 shRNA pakukula kwa ma cell (a) ndi mapangidwe a spheroid (b) mu SW620, HCT116, MBA-MD-231, ndi ma cell a HepG2.c Zotsatira zakuwongolera shRNA ndi DARS-AS1 shRNA pakupanga koloni m'maselo a SW620.Kuchulukitsa kwa ma cell (d), mapangidwe a spheroid (e), ndi mapangidwe a koloni (f) a SW620 cell expressing DARS-AS1.Deta yomwe yawonetsedwa ndi ± kupatuka koyenera kwa zoyeserera zitatu.* p ≤ 0.05, ** p ≤ 0.01, ndi *** p ≤ 0.001 ndi t-mayeso awiri a Wophunzira.
Kuti tithandizire maphunziro otayika, tidapanga ma cell a SW620 mopitilira muyeso DARS-AS1 (Supplementary Fig. 2g).DARS-AS1 kufotokoza mopitirira muyeso kumawonjezera kukula kwa maselo (1.8-fold), mapangidwe osasunthika a spheroid (1.4-fold), ndi mapangidwe a colony (3.3-fold) mu maselo a SW620 (Mkuyu 2d-f).Tidatsimikizira chotsatirachi pogwiritsa ntchito mzere wina wa foni wa DARS-AS1, A549.Kuchulukirachulukira kwa maselo chifukwa cha DARS-AS1 kuchulukirachulukira kunawonedwanso m'maselo a A549 (Supplementary Fig. 2h, i ndi Supplementary Table 3).Kuphatikizidwa pamodzi, maphunziro opindula ndi otayikawa akuwonetsa kuti DARS-AS1 imalimbikitsa kuchuluka kwa maselo a khansa mu vitro.
Kuti tifufuze momwe DARS-AS1 imayendera kuchuluka kwa maselo, tidachita kafukufuku wotsitsa wa RNA kuti tidziwe omwe angagwirizane ndi mapuloteni.Zotsatira za RT-qPCR zimasonyeza kuti pafupifupi 86.2% ya DARS-AS1 ili mu cytoplasm ya maselo a SW620 (Supplementary Fig. 3a).In vitro yolembedwa biotinylated DARS-AS1 kapena pseudoRNA ndiye idalumikizidwa ndi ma cell a SW620 otsatiridwa ndi kupatukana kwa SDS-PAGE.Kupaka siliva wotsatira kunasonyeza kuti gulu lodziwika bwino (~ 38 kDa) linalemeretsedwa kwambiri mu zitsanzo za DARS-AS1 zokoka koma osati mu RNA ya dummy kapena mikanda (mkuyu 3a).Gululi linadziwika kuti PKR activating protein (PACT) ndi mass spectrometry (MS) ndipo inatsimikiziridwa ndi immunoblotting mu SW620, HCT116, ndi HepG2 cell mizere (Mkuyu 3a, b).Kulemera kwa DARS ndi mapuloteni okhudzana ndi PACT - PKR ndi TRBP - adafufuzidwanso pogwiritsa ntchito RNA kusanthula ndi Western blotting (WB).Zotsatira zinasonyeza kuti palibe kuyanjana kwachindunji pakati pa DARS-AS1 RNA ndi mapuloteni atatuwa anapezeka (Supplementary Fig. 3b).Kuyanjana kwapadera pakati pa DARS-AS1 ndi PACT kunatsimikiziridwanso ndi kusanthula kwa RNA immunoprecipitation (RIP), komwe kunasonyeza kuti DARS-AS1 inalemeretsedwa kwambiri mu anti-PACT antibodies koma osati ma RNA ena olamulira (Chithunzi 3c).Kuti mudziwe ngati DARS-AS1 imalumikizana mwachindunji ndi PACT popanda zigawo zina zama cell, kuyesa kwa in vitro biolayer interferometry (BLI) kunachitika pogwiritsa ntchito PACT yoyeretsedwa.Biotin-yolemba DARS-AS1 kapena dummy RNA idasinthidwa pa streptavidin (SA) biosensors ndiyeno imayikidwa mu kinetic buffer yomwe ili ndi 1 μM PACT.Makamaka, PACT imagwirizana kwambiri ndi DARS-AS1 (mtengo wa KD ~ 26.9 nM), koma osati kutsanzira RNA (Chithunzi 3d).Kuphatikizidwa, zotsatirazi zikuwonetsa kuyanjana kwachindunji ndi kuyanjana kwakukulu pakati pa DARS-AS1 ndi PACT.
RNA kukoka kusanthula kwazindikira DARS-AS1 yolumikizana ndi PACT m'maselo a SW620.Pamwambapa, madontho a siliva a mapuloteni okhudzana.Ma immunoblots otsika adachitidwa ndi anti-PACT antibody.b RNA kusanthula-pansi kunachitika mu HCT116 (pamwamba) ndi HepG2 (pansi) maselo.Kuchuluka kwa PACT kunadziwika ndi immunoblotting.Kuyesa kwa cRNA immunoprecipitation (RIP) kunachitika m'maselo a SW620 pogwiritsa ntchito ma antibodies owonetsedwa.d PACT yomangira ma curve ku DARS-AS1 yayitali kapena control RNA idapezedwa pogwiritsa ntchito biolayer interferometry (BLI).RNA inali yosasunthika pa streptavidin biosensor.1 μM PACT idagwiritsidwa ntchito kuyeza mayanjano.e RNA kukoka kuyesa kunachitika pogwiritsa ntchito biotinylated utali wathunthu DARS-AS1 kapena truncated (pamwamba).Immunoblot yowonetsa PACT yolandiridwa (pansi).f Purified flagged PACT idalumikizidwa ndi biotinylated utali wathunthu DARS-AS1 kapena kudulidwa (monga mu e) kwa in vitro RIP assay.RNA yochotsedwa idatsimikiziridwa ndi RT-qPCR.g Kugwirizana kwa zidutswa zosiyanasiyana za RNA za PACT zidapezedwa pogwiritsa ntchito biolayer interferometry.Kusanthula, 100 nM RNA ndi 1 μM RAST zidagwiritsidwa ntchito.h Kuyesa kwa in vitro RIP kunachitika pogwiritsa ntchito purified intact kapena truncated yotchedwa PACT.RNA yochotsedwa idatsimikiziridwa ndi RT-qPCR.i Kukula kwa ma cell a SW620 mopitilira muyeso DARS-AS1, PACT, kapena onse awiri.j Kuwonetsa kwautali wathunthu kapena kuchepetsedwa kwa DARS-AS1 m'maselo a SW620 kunali ndi zotsatira zosiyana pakukula kwa maselo.k Apoptosis idazindikirika ndi immunoblotting ndi anti-PARP antibody.l Knockout ya DARS-AS1 imapangitsa apoptosis ya ma cell a SW620 monga momwe akuwonetsera ndi flow cytometry.Deta yomwe yawonetsedwa ndi ± kupatuka koyenera kwa zoyeserera zitatu. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p <0.0001, ndi mayeso awiri a Wophunzira a t. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p <0.0001, ndi mayeso awiri a Wophunzira a t. *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 ndi t-mayeso a Wophunzira amichira iwiri. *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 ndi t-mayeso a Wophunzira amichira iwiri.
Kenako tidapanga zidutswa zitatu za biotinylated DARS-AS1 RNA polemba mu vitro kuti tizindikire chigawo cha DARS-AS1 chofunikira pa mgwirizano wa PACT (Chithunzi 3e).Zotsatira za kusanthula kwa RNA zinasonyeza kuti chidutswa chilichonse chinatha kuyanjana ndi PACT, koma chigawo cha 3'-terminal (384-768 nucleotides chotchedwa A3) chinasonyeza zoposa 1-384 nucleotides zolembedwa A1) (Mkuyu 3e).Zotsatira zofananazi zidawonedwa mu in vitro RIP assay pogwiritsa ntchito recombinant PACT (Chithunzi 3f).Mogwirizana ndi zotsatira izi, kuyesa kumanga zidutswa za RNA zosasunthika ku PACT pogwiritsa ntchito BLI kunasonyezanso kuti PACT ili ndi chiyanjano chapamwamba cha A3 (384-768 nt) (mtengo wa KD pafupifupi 94.6 nM), pamene pafupifupi palibe maulalo ndi madera ena.(mkuyu 3d).
Tidawunikanso madera ogwirizana nawo mu PACT.PACT ili ndi madera atatu ogwira ntchito, awiri mwa omwe amasungidwa a RNA-binding domains (dsRBD) ndi dera lachitatu (losankhidwa D3) lomwe limagwira ntchito ngati choyambitsa kuyanjana kwa mapuloteni.Kuti tiwone momwe lncRNA imamangirira gawo lililonse, tidapanga masinthidwe atatu omwe adachotsa madera atatuwa ndikuyesa mu vitro RIP.Zotsatira zathu zasonyeza kuti kuchotsedwa kwa dera lachitatu (D3) la PACT kunachepetsa kwambiri kuyanjana kwake ndi DARS-AS1 (ndi 0.11-fold poyerekeza ndi intact PACT) poyerekeza ndi zina ziwiri zosinthika (Fig. 3h), zinasonyezedwa kuti kumasulidwa ya D3 idalumikizana ndi DARS.-AC1.Kuphatikizidwa pamodzi, zotsatirazi zikusonyeza kuti kuyanjana pakati pa DARS-AS1 ndi PACT kungachitike makamaka kupyolera mu 3′ mapeto a DARS-AS1 ndi D3 domain ya PACT.
Tinawona kuti DARS-AS1 inalibe mphamvu pa PACT kufotokozera ndipo PACT inalibe mphamvu pa DARS-AS1 (Supplementary Fig. 3c).Kenako tidawunika momwe PACT kugwetsa kumakulirakulira.Mosiyana ndi DARS-AS1, maselo achibale anakula nthawi 1.5-3 mofulumira pamene PACT inagwetsedwa (Supplementary Fig. 3d).Zotsatira za kafukufuku wa mapangidwe a coloni zimasonyeza kuti maselo amapanga 2-3-fold colonies pambuyo pa chithandizo cha shRNA ndi PACT (Supplementary Fig. 3e).Kuti tiwone ngati DARS-AS1 imayang'anira kuchuluka kwa maselo kudzera pa PACT, tidapanga ma cell a SW620 mopitilira muyeso PACT, DARS-AS1, kapena onse awiri.Kufotokozera kwakukulu kwa PACT kunawonetsa kulepheretsa kwakukulu kwa kuchuluka kwa maselo (Chithunzi 3i).Ngakhale kuti DARS-AS1 kufotokoza mopambanitsa pa se imodzi kunalimbikitsa kwambiri kuchuluka kwa maselo, panalibe kusiyana kwakukulu pa kukula kwa maselo owonjezera DARS-AS1 ndi PACT.Zotsatirazi zikusonyeza kuti PACT ikhoza kuthana ndi kuchulukana kowonjezereka komwe kumachitika chifukwa cha DARS-AS1 overexpression.
Popeza madera osiyanasiyana a DARS-AS1 ali ndi maluso osiyanasiyana omangirira PACT, tidafufuza momwe amakhudzira kuchuluka kwa ma cell pogwiritsa ntchito kufotokoza mosiyanasiyana kwa zidutswa za DARS-AS1.Poyerekeza ndi zidutswa zina ziwiri, DARS-AS1 inali yowonjezereka kwambiri pamapeto a 3 '(384-768 nt), dera lalikulu lokhudzana ndi PACT ku DARS-AS1, lomwe linali ndi mphamvu zapamwamba kwambiri zolimbikitsa kuchuluka kwa maselo (Mkuyu 3j).Zotsatira izi zikuwonetsa kulumikizana kwabwino pakati pa mphamvu yomangiriza ndi ntchito yachilengedwe ya DARS-AS1.
PACT yanenedwa kuti ndi pro-apoptotic protein19.Chifukwa chake, tidafufuza momwe DARS-AS1 imakhudzira apoptosis.Monga momwe zimayembekezeredwa, kugogoda kwa DARS-AS1 kunachulukitsa kwambiri PARP cleavage m'maselo a SW620 ndikuwonjezera chiwerengero cha maselo a annexin V-positive mu SW620, HCT116, HepG2, ndi MBA-MD-231 mizere yamaselo (Mkuyu 3k).3).3f-h), kusonyeza kuti anti-apoptotic zotsatira za DARS-AS1 m'maselo a khansa ndizosiyana ndi apoptosis-inducing ntchito ya PACT.Kutengedwa palimodzi, zotsatirazi zikusonyeza kuti njira ya DARS-AS1 oncogenic ntchito ikhoza kukhala kupyolera mu kulepheretsa ntchito ya PACT.
Kenaka, tinayang'ana zotsatira za mgwirizano wa DARS-AS1-PACT.PACT yanenedwa kuti imayambitsa PKR kudzera mu kuyanjana kwachindunji, komwe kumawonjezera eIF2α phosphorylation, kuchititsa kuchotsedwa kwa kumasulira ndi apoptosis17.Choyamba, tidawona ngati DARS-AS1 imakhudza ma cell a PACT ndi PKR.Confocal fluorescence microscopy idawonetsa kuti PACT ndi PKR zidapangidwa kwambiri m'maselo a SW620 okhala ndi pafupifupi Pearson coefficient coefficient of 0.72.Panthawiyi, DARS-AS1 overexpression inachepetsa kwambiri PACT ndi PKR co-localization (kutanthauza Pearson coefficient coefficient 0.61) (Chithunzi 4a).Kuti tifufuze ngati DARS-AS1 ingasinthe kuyanjana kwa PACT-PKR, tidayesa co-immunoprecipitation (co-IP) ndi anti-PACT antibody mu SW620 cell lysates.PKR inalemeretsedwa kwambiri mu anti-PACT m'maselo olamulira, pamene PKR kuchira kunachepetsedwa kwambiri mu lysates kuchokera ku maselo overexpressing DARS-AS1 (Mkuyu 4b).Oyeretsedwa olembedwa kuti PACT ndi PKR adagwiritsidwa ntchito poyesa kuyesa kwa mapuloteni mu vitro.Chifukwa chake, omwe adapereka DARS-AS1 koma osawongolera RNA adawonetsa kusagwirizana kwa PACT-PKR (Chithunzi 4c).Zotsatira zonse zidawonetsa kuti DARS-AS1 idasokoneza kulumikizana kwa PACT ndi PKR.
Co-localization ya PACT ndi PKR m'maselo owongolera kapena ma cell owonjezera DARS-AS1 adawonedwa pogwiritsa ntchito ma microscopy a confocal fluorescence.Ma nuclei adadetsedwa ndi DAPI.Zotsatira zowerengera zidapezedwa kuchokera pazithunzi 16.b Co-immunoprecipitation (co-IP) pogwiritsa ntchito anti-PACT antibody mu cell lysates olamulira SW620 maselo kapena maselo overexpressing DARS-AS1.c Cholembedwa PACT, choyeretsedwa PKR ndi cholembedwa mu vitro ndi DARS-AS1 kapena mock RNA adakulungidwa kuti afufuze zomanga za mapuloteni mu vitro.Ma antibodies oletsa mbendera adagwiritsidwa ntchito kuti achepetse thupi.d Ma immunoblots okhala ndi ma antibodies owonetsedwa adachitidwa mu SW620 ndi ma HCT116 maselo osinthidwa ndi shRNA kapena DARS-AS1-shRNA yotsatiridwa ndi njala ya seramu.e DARS-AS1 milingo yofotokozera idasintha kukhudzika kwa ma cell ku thapsigargin.Maselo a SW620 adasinthidwa ndi DARS-AS1 shRNA, DARS-AS1 overexpression plasmid kapena control plasmid.Maselo amathandizidwa ndi thapsigargin kwa maola 48 ndipo kuthekera kwa cell kumatsimikiziridwa pogwiritsa ntchito reagent ya MTS.f In vitro yolembedwa DARS-AS1 kapena dummy RNA ndi purified PACT idagwiritsidwa ntchito poyesa mu vitro activation assay ndi kuzindikira kwa immunoblot.g Ma immunoblots omwe amagwiritsa ntchito ma antibodies awa adachitidwa pa ma cell a SW620-ctrl (kumanzere) kapena ma cell owonetsa PKR mutants (kumanja).Maselo awa adasinthidwa ndi shRNA kapena DARS-AS1-shRNA yotsatiridwa ndi njala ya seramu.h Flow cytometry inasonyeza kuti kutsegula kwa mutant PKR kulipiritsa DARS-AS1-induced apoptosis m'maselo a SW620.i Immunoblots yokhala ndi ma antibodies owonetsedwa adachitidwa mu ma cell a SW620 (kumanzere) kapena HCT116 (kumanja).Maselo opatsirana ndi control shRNA kapena DARS-AS1 shRNA amakhala opanda seramu ndipo amaphatikizidwa ndi 100 nM PKR C16 inhibitor kapena DMSO.Scale bar = 5 µm.Deta yomwe yawonetsedwa ndi ± kupatuka koyenera kwa zoyeserera zitatu.* p ≤ 0.05 Mayeso a t a michira iwiri.
Nthawi zambiri amakhulupirira kuti PACT ikangolumikizana ndi PKR17, PKR phosphorylation ku Thr451 imatha kukopeka.Zotsatira zathu zimasonyeza kuti mlingo wa PKR phosphorylation unali wokwezeka kwambiri m'maselo ogogoda a DARS-AS1 pambuyo pa njala ya seramu (Mkuyu 4d ndi Supplementary Fig. 4a).Choncho, tapeza kuti phosphorylation ya eIF2α, gawo lalikulu la PKR, linawonjezekanso kwambiri ndi DARS-AS1 shRNA (Mkuyu 4d ndi Supplementary Fig. 4a).Thapsigargin ndi ER stressor yomwe imapangitsa ER kumasula Ca2 +.Kuchiza ndi thapsigargin kwanenedwa kuti kumapangitsa kufotokozera ndi kuyambitsa kwa PACT, yomwe imagwirizananso ndi kuyambitsa PKR, zomwe zimatsogolera ku apoptosis powonjezera eIF2α phosphorylation 18,61.Apa, tidagwiritsa ntchito thapsigargin ngati cholimbikitsira njira ya PACT/PKR kuti tifufuze ngati DARS-AS1 ingathandize maselo kuthana ndi nkhawa poletsa njira ya PACT/PKR.Tidawona kuti mulingo wa mawu a DARS-AS1 umagwirizana bwino ndi kukana kwa maselo ku thapsigargin.SW620 maselo overexpressing DARS-AS1 anapulumuka bwino pamene mankhwala ndi thapsigargin, pamene maselo ndi DARS-AS1 knockdown anakhala atengeke (mkuyu 4e).Mogwirizana ndi zotsatirazi, DARS-AS1 overexpression inachepetsa thapsigargin-induced PKR phosphorylation (Supplementary Fig. 4b).Mosiyana ndi zimenezi, pambuyo pa chithandizo cha thapsigargin, PKR ndi eIF2α zinali phosphorylated kumtunda wapamwamba mu maselo ogogoda a DARS-AS1 poyerekeza ndi maselo olamulira (Supplementary Fig. 4b).Chochititsa chidwi n'chakuti, thapsigargin inachititsa kuti DARS-AS1 iwonetsedwe m'njira yodalira mlingo, yomwe ingasonyeze ntchito yolimbana ndi nkhawa ya DARS-AS1 (Supplementary Fig. 4c).Kuphatikiza apo, tidachita zoyeserera za in vitro activation kuti titsimikizire izi.Mwachidule, PKR idayeretsedwa ku ma cell lysates pogwiritsa ntchito anti-PKR antibody, kenako ndikuphatikizidwa ndi recombinant PACT ndi DARS-AS1 yolembedwa mu vitro.Pambuyo pakuchita kwa enzymatic, phospho-PKR idapezeka pogwiritsa ntchito WB.Zotsatira zathu zimasonyeza kuti PKR phosphorylation inali yoletsedwa kwambiri ndi DARS-AS1, koma osati ndi ulamuliro wa RNA (Chithunzi 4f).Izi mu vitro ndi mu vivo zotsatira zikusonyeza kuti DARS-AS1 imaletsa PACT-mediated PKR activation.Panthawi imodzimodziyo, tinawonanso kuchepa kwa PACT kuchira pamaso pa DARS-AS1 (Chithunzi 4f).Chotsatirachi chikugwirizana ndi zotsatira za in vitro protein binding assay (Chithunzi 4c) ndikuwonetsanso ntchito yotsekera ya DARS-AS1 ya mgwirizano wa PACT-PKR.
Ser246 ndi Ser287 mu D3 domain ya PACT amafunikira kuti PKR atsegule pansi pazovuta zama cell.Kulowetsa zotsalira ziwiri za serine m'malo mwa alanine kunapatsa mutant PACT (mutD), yomwe idayambitsa PKR pakalibe kupsinjika, ndikulowetsa alanine (mutA) idasintha ndondomekoyi.Popeza tawonetsa kufunikira kwa derali molumikizana mwachindunji ndi DARS-AS1, tidapanga zosinthika ziwiri za PACT kuti tiyese ngati zotsalirazi zitha kuphatikizidwanso polumikizana ndi DARS-AS1.Chochititsa chidwi n'chakuti, osinthika onsewa anataya mphamvu yomangirira ku DARS-AS1 (Supplementary Fig. 4d), kutanthauza kuti mapangidwe athunthu a mapuloteni a PACT angafunike kuti agwirizane bwino ndi DARS-AS1.
Kuphatikiza apo, zotsatira zathu zikuwonetsanso kuti kuletsa kwa DARS-AS1-shRNA-kulepheretsa kuchuluka kwa maselo kumatha kubwezeretsedwa pang'ono pofotokoza mochulukirachulukira kwambiri PACT mutant (PACTmutA) kapena PKR mutant (PKRmut) (Supplementary Fig. 4e. e).Kufotokozera mopambanitsa kwa kusintha kwakukulu kwa PKR kumachepetsa phosphorylation ya PKR yoyambitsidwa ndi kugogoda kwa DARS-AS1 komanso eIF2α phosphorylation m'maselo opanda seramu (Fig. 4g).Chofunika kwambiri, chiwerengero cha maselo a apoptotic opangidwa ndi DARS-AS1 kugogoda chinachepetsedwanso m'maselo owonjezera PKRmut (Mkuyu 4h ndi Supplementary Fig. 4g).Kuletsa ntchito ya PKR kinase kumawononganso ntchito ya DARS-AS1, monga zotsatira zimasonyeza kuti kugogoda kwa DARS-AS1 sikunayambe kuyambitsa PKR ndi eIF2α phosphorylation pamene maselo amathandizidwa ndi PKR-specific C16 inhibitor (Mkuyu 4i ndi Supplementary Fig. 4h).).Kuphatikizidwa, zotsatira zathu zikuwonetsa kuti DARS-AS1 imalimbikitsa kuchulukana kwa maselo, makamaka mwa zina, poletsa kuyambitsa kwa PACT-mediated PKR.
Kuti tifufuzenso ntchito ya DARS-AS1 mu tumorigenesis, tidayesa mu vivo pogwiritsa ntchito mbewa xenograft model. Zotsatira zikuwonetsa kuti kugwetsa kwa DARS-AS1 kunachepetsa kwambiri kukula kwa chotupa mu mbewa (p mtengo <0.0001) (mkuyu 5a). Zotsatira zikuwonetsa kuti kugwetsa kwa DARS-AS1 kunachepetsa kwambiri kukula kwa chotupa mu mbewa (p mtengo <0.0001) (mkuyu 5a). Результаты показывают, что нокдаун DARS-AS1 резко снижает рост опухоли у мышей (значение p <0,0001) (рис. 5а). Zotsatira zikuwonetsa kuti kugwetsa kwa DARS-AS1 kumachepetsa kwambiri kukula kwa chotupa mu mbewa (p mtengo <0.0001) (Chithunzi 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) (рис. 5а). Zotsatira zinawonetsa kuti kugwetsa kwa DARS-AS1 kunachepetsa kwambiri kukula kwa chotupa mu mbewa (p mtengo <0.0001) (Chithunzi 5a).Choncho, mu gulu la knockdown la DARS-AS1, panali kuchepa kwakukulu kwa voliyumu ya chotupa chapakati pafupifupi 72.9% ndikutanthauza chotupa chochuluka ndi pafupifupi 87.8% (Chithunzi 5b-d).Zotsatira izi zikuwonetsa mwamphamvu kuti DARS-AS1 imatha kulimbikitsa kukula kwa chotupa mu vivo.
Zotsatira za kugwetsa kwa ad DARS-AS1 pa colorectal oncogenesis mu mbewa zamaliseche.Zopindika (a), kukula kwa chotupa (b), kulemera (c), ndi zithunzi zotupa (d) zikuwonetsedwa.Zolemba zolakwika zikuyimira ±SEM. n = 10. ****p <0.0001, ndi mayeso a t a michira iwiri. n = 10. ****p <0.0001, ndi mayeso a t a michira iwiri. n = 10. ****p <0,0001 по двустороннему критерию Стьюдента. n = 10. ****p <0.0001 Mayeso a t a michira iwiri.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 t-mayeso a Wophunzira wa michira iwiri.e Kaplan-Meier adasanthula kulumikizana pakati pa mayendedwe a DARS-AS1 ndi kupulumuka kwathunthu kwa odwala omwe ali ndi UVM, KICH, KIRP, MESO, GBM, ndi LGG.Mawonekedwe apamwamba a DARS-AS1 kufotokozera odwala anali pamwamba pa 50%;otsika mlingo wa DARS-AS1 kufotokoza odwala anali pansi 50%.p-values adatsimikiziridwa pogwiritsa ntchito mayeso a chipika.f Chitsanzo chomwe DARS-AS1 chimawongolera njira ya PACT-PKR ndi kukula kwa chotupa.
Kuti timvetsetse bwino momwe DARS-AS1 imakhudzira, tidawona kulumikizana pakati pa mawu ake ndi kupulumuka kwa odwala.Posanthula deta ya TCGA, tidapeza kuti mawu apamwamba a DARS-AS1 adalumikizidwa ndi uveal melanoma (UVM), renal chromophobia (KICH), aimpso papillary cell carcinoma (KIRP), mesothelioma (MESO), multiplex.Kupulumuka kwapansi kunali kogwirizana kwambiri ndi glioblastoma morphosis (GBM) ndi odwala omwe ali ndi ubongo wa glioma (LGG) (Chithunzi 5e).Zotsatirazi zikuwonetsa kuti DARS-AS1 ikhoza kukhala ndi gawo lofunikira pakuchulukira kwa chotupa chachipatala ndipo ikhoza kukhala chodziwikiratu pamakhansa angapo.
Mu phunziroli, pogwiritsa ntchito kuwunika kwakukulu kwa CRISPRi, tidatsimikiza kuti DARS-AS1 lncRNA imagonjetsa kupsinjika kwa maselo a khansa mwa kuwongolera oyankha awiri ofunikira, PACT ndi PKR.Mwa kuyanjana mwachindunji ndi PACT, DARS-AS1 inaletsa PACT-mediated PKR activation, motero imalepheretsa kufa kwa maselo a apoptotic ndikulimbikitsa kukula kwa maselo (Mkuyu 5f).Kuwongolera kwa DARS-AS1 kwawonedwa m'mitundu ingapo ya khansa, ndikuwonetsa kuti ntchito yake yolimbikitsa kupulumuka kwa maselo a khansa pansi pazovuta zitha kugwiritsidwa ntchito pamitundu ingapo ya khansa.
PACT yadziwika kuti ndi PKR activator protein, ndipo PACT-mediated PKR activation imakhala ndi gawo lofunikira pakuyankha kupsinjika pakuwongolera zolembedwa, kumasulira, apoptosis, ndi njira zina zofunika zama cell62.Kwa zaka zambiri, kuyesayesa kwapangidwa kuti amvetsetse malamulo okhudzana ndi khansa a PACT-PKR cascade.Pano, kafukufuku wathu adawulula njira yosiyana yoyendetsera PACT-PKR m'maselo a khansa kudzera m'maselo a lncRNA DARS-AS1, omwe amamangiriza mwachindunji ku PACT, amalepheretsa kuyanjana kwa PACT-PKR, amalepheretsa PKR activation ndi eIF2α phosphorylation, potero amalepheretsa apoptosis yochititsa kupsinjika maganizo komanso kulimbikitsa kukula kwa khansa.maselo.Kupeza uku kumawunikira zomwe zingachitike ndi lncRNA pazachidziwitso cha khansa ndi chithandizo.
Deta yathu idawonetsa kuti kugwetsa kwa DARS-AS1 kumathandizira ma cell ku njala ya seramu ndikuwonjezeka kwakukulu kwa phosphorylated PKR ndi eIF2α.Zotsatirazi zikusonyeza kuti DARS-AS1 imalimbikitsa kupulumuka kwa maselo a khansa pansi pa zovuta poletsa ntchito ya PACT/PKR.Ma RNA angapo osalemba zilembo, monga ASPACT ndi nc886, amakhalanso ndi gawo la PACT/PKR pochepetsa PACT48 mRNA kapena kuwongolera autophosphorylation pomanga PKR49,50,64.Pakati pawo, DARS-AS1 imakhala ngati yosokoneza mgwirizano wa PACT-PKR.Kafukufukuyu akuthandizira kumvetsetsa kwathu malamulo a PACT/PKR axis ndi gawo la lncRNAs pakuyankha kupsinjika.
PACT ili ndi madambwe atatu osiyana.Iliyonse mwa ma dsRBD awiri oyambilira ndi okwanira kukwaniritsa mgwirizano wapamwamba wa PACT ku PKR, pomwe dera lachitatu (D3) likufunika kuti PKR itsegule mu vitro ndi mu vivo.Kafukufuku wathu adawonetsa kuti DARS-AS1 imakonda kuyanjana ndi dera la D3 (mkuyu 3h).Popeza kukula kwakukulu kwa lncRNA (768 nucleotides), DARS-AS1 yomangiriza ku D3 imatha kuletsa mwakuthupi kuyanjana pakati pa dera la PACT la dsRBD ndi PKR, potero kuletsa kuyanjana kwa PACT ndi PKR.Kusintha kwa mfundo za PACT zomwe zidalowa m'malo mwa Ser246 ndi Ser287 mu D3 ndi alanine kapena aspartate zidasokoneza mgwirizano wake womangirira wa DARS-AS1, ndikulozera kufunikira kwa magwiridwe antchito a D3's zonse zamapangidwe ndi zamagetsi mu mgwirizano wawo.Zambiri zamakinawa zidzafunikanso m'tsogolomu, pogwiritsa ntchito kusanthula kwatsatanetsatane kwachilengedwe komanso kusanthula kwadongosolo kwa PACT.
Kafukufuku wam'mbuyomu adanenanso kuti DARS-AS1 imalimbikitsa kukula kwa maselo kudzera mu njira zingapo51,52,53.Mu chitsanzo chimodzi, ofufuza adawona kuti DARS-AS1 idawongolera jini yake ya antisense protein-encoding DARS polunjika miP-194-5p m'maselo a khansa ya impso.Komabe, mu kafukufuku wapano, kugwetsa kwa DARS-AS1 sikunakhudze zolembedwa za DARS mumitundu ingapo ya khansa, kuphatikiza khansa ya colorectal, m'mawere, ndi chiwindi.Chifukwa ma lncRNA amawonetsa mawonekedwe amtundu wa cell- ndi minofu, njira zogwirira ntchito sizingasungidwe pamitundu yonse ya khansa, zomwe zingapangitse kusiyana kumeneku pakati pa zomwe taziwona ndi zowunika zam'mbuyomu zamakhansa osiyanasiyana.Maphunziro apadera amafunikira kuti afotokoze njira zenizeni zamagulu osiyanasiyana amthupi ndi ma pathological.
Kuwunika kwa data yachipatala kunawonetsa kuti mawu a DARS-AS1 mu zotupa amagwirizana mosagwirizana ndi kupulumuka kwa odwala khansa, zomwe zimatsimikizira kufunikira kwa DARS-AS1/PACT/PKR axis pakuzindikira khansa.Pomaliza, kafukufuku wathu akuwonetsa kuti DARS-AS1 ndi wowongolera wa PACT/PKR signing axis, imalimbikitsa kuchuluka kwa maselo a khansa, ndikuletsa apoptosis panthawi yoyankha kupsinjika, yomwe imapereka mzere wina wa kafukufuku ndipo ili ndi chidwi pa kafukufuku wamtsogolo pazamankhwala omwe angathe. .
Maselo a anthu SW620, A549, MBA-MD-231, HCT116, HepG2 ndi HEK293T adapezedwa kuchokera ku National Cell Line Resource Infrastructure ku China.Maselo onse adasungidwa mu DMEM medium (DMEM, Thermo Fisher Scientific, Waltham, MA) yowonjezeredwa ndi 10% FBS (Gemini, Brooklyn, NY) ndi 1% penicillin-streptomycin (Thermo Fisher Scientific) pa 37 ° C, 5% CO2.chofungatira.
Anti-PACT, Abcam (ab31967);Anti-PKR, Abcam (ab184257);Anti-PKR (phospho-T451), Abcam (ab81303);Anti-Mbendera, Abcam (ab125243);Anti-eIF2α, Abcam (A0764));anti-eIF2α (phosphorous S51), Abcam (ab32157);anti-PACT (phosphorous S246), Abgent (AP7744b);anti-β-tubulin, CST (2128);mbewa yachibadwa IgG, CST (5415S);kalulu wamba IgG, CST (2729S).Ma antibodies adasungunuka 1:1000 mu PBST paza Western blotting ndi 1:100 pa IP.
Ma sgRNA adapangidwa pogwiritsa ntchito chida chopezeka pagulu chotchedwa CRISPR-ERA66.Tidagwiritsa ntchito zida zokhazikika pakukula kwa sgRNA komanso ma algorithm omwe amamangirira sgRNA mdera la 3 kb.ku TSS.Maiwe a sgRNA oligonucleotides adapangidwa ku CustomArray, Inc. (Bothewell, WA) ndikupangidwa kukhala pgRNA plasmids yamunthu (Addgene #44248).Zokwana 12 µg za pgRNA plasmid yophatikizidwa ndi munthu, 7.2 µg ya psPAX2 (Addgene #12260), ndi 4.8 µg ya pMD2.G (Addgene #12259) adapatsirana mu 5 x 106 HEK206 DNA Transfection 1 cm mu Rea maselo ( CWBIO, Beijing, China) kutsatira malangizo opanga.Ma supernatant okhala ndi ma virus adasonkhanitsidwa patatha maola 48 ndi 72 atapatsirana ndikusefa kudzera pa fyuluta ya 0.45 µm.Kuti muwunikire, ma cell a SW620 omwe akuwonetsa dCas9/KRAB fusion protein adapezedwa ndikutengera kachilombo.Maselo osinthidwa a SW620 adagwidwa ndi laibulale ya virus muzoyeserera zinayi zodziyimira pawokha pa MOI ya 0.1-0.3 ndipo adayesedwa ndi 2 μg/ml puromycin (Sigma, St. Louis, MO) kwa masiku awiri.Pambuyo pake, ma cell adakulitsidwa kwa masiku 18 mu vitro ndikuyika laibulale yochepera 500 ma cell / sgRNA kuti awonedwe.
Genomic DNA inatengedwa motsatira malangizo a QIAamp DNA Blood Midi Kit (QIAGEN, Düsseldorf, Germany; 51183).Pazonse, 100 μg ya DNA ya genomic pa kubwereza kwachilengedwe idagwiritsidwa ntchito pomanga laibulale.Dera la sgRNA lidakulitsidwa ndi mizere iwiri ya PCR ndikulumikizidwa ndi barcode.
Zogulitsa za PCR zidatsukidwa pogwiritsa ntchito zida za NucleoSpin® gel ndi PCR purification kit (MACHHEREY-NAGEL, Düren, Germany; 740609.250) ndikuyesedwa pogwiritsa ntchito zida za Qubit™ HS zozindikira za DNA ziwiri (Thermo Fisher Scientific; Q32854).
Kuyeza kwa MTS kunagwiritsidwa ntchito kuyesa kuchuluka kwa maselo.Maselo adabzalidwa m'mbale zachitsime 96 pakuchulukira koyamba kwa ma cell 2000 / chitsime.Chiwerengero cha maselo chinkayezedwa tsiku lililonse pa nthawi yosonyezedwa kwa masiku 4-6.Pa chitsime chilichonse, 20 μl ya MTS reagent (Promega) idasungunuka ndi 100 μl ya DMEM, yolumikizidwa ndi ma cell kwa 4 h pa 37 ° C, ndiyeno OD490 idayesedwa.
Kuthekera kwa kukula kosasunthika kunapezeka popenda mapangidwe a mabwalo.Mwachidule, ma cell a 2000 omwe adasinthidwa ndi shRNA DARS-AS1 kapena control shRNA adakulitsidwa mu ma ultra low attachment microplates (Corning) ndi kusintha kwapakatikati masiku 4 aliwonse.Ma spheroids adawerengedwa pambuyo pa masiku 14.Maselo a 500 opatsirana ndi DARS-AS1 overexpression plasmid kapena control plasmid anagwiritsidwa ntchito poyesa zowonjezera, mwinamwake njirayo sinasinthidwe.
RNA inalembedwa pogwiritsa ntchito T7 RNA polymerase ndi biotin-16-UTP (Roche 1138908910) malinga ndi malangizo a Riboprobe® Combination Systems (Promega P1440).Zoyambira zomwe zimagwiritsidwa ntchito pano zalembedwa mu Supplementary Table 4.
Magawo a protein-coding PACT kapena PKR adapangidwa kukhala pET15b (Addgene #73619) ndikusinthidwa kukhala BL21(DE3).Mabakiteriyawo adakulungidwa usiku wonse mu LB yoperekedwa ndi ampicillin kenako ndikusungunula ka 100 ndi LB yatsopano.Pamene OD600 ya sing'anga inafika pa 0.8, 1 mm IPTG inawonjezeredwa kuti ipangitse kufotokoza kwa mapuloteni.Pambuyo pakuyamwitsa usiku wonse ndikugwedeza mofatsa (250 rpm pa 20 ° C), cell pellet inasonkhanitsidwa ndi centrifugation (4000 rpm, 10 min, 4 ° C).Resuspending cell pellet mu lysis buffer (50 mmM Tris, pH 8.0, 250 mm NaCl, 1 mm PMSF) ndikuyalira pa ayezi kwa mphindi 30, ndiye sonicate (15 min, 5 s on/off, pa ayezi) ndi centrifuge (13,000) rpm)., 30 min, 4° С).The supernatant ndiye adakwezedwa pa Ni-NTA resin (QIAGEN) 3 nthawi pa 4 ° C, kutsukidwa 4 nthawi ndi bafa kusamba (50 mM Tris, pH 8.0, 40 mM imidazole, 250 mM NaCl) ndi eluted 3 nthawi, ndi okwana. ya 10 ml yotsekeka yotchinga (50 mM Tris, pH 8.0, 250 mM NaCl, 300 mM imidazole).Mapuloteni oyeretsedwa adatsimikiziridwa pogwiritsa ntchito WB ndipo ndendeyo idatsimikiziridwa pogwiritsa ntchito zida za Qubit ™ protein assay (Thermo Fisher Scientific; Q 33212).
Mayeso a RIP adachitidwa monga momwe tafotokozera kale, ndikusinthidwa.Mwachidule, 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 cytostatic x 7 1 (Roche, 1 mM DTT) ndi centrifuge pa 13,000 rpm kwa mphindi 15 pa 4 °C.Wopambanayo adalumikizidwa ndi mapuloteni A + G maginito mikanda (Millipore) yolumikizidwa ndi 5 μg ya anti-PACT antibody (Abeam) kapena IgG (CST).Mikanda idatsukidwa kasanu ndi 5x RIP buffer, kenako ndikugayidwa ndi proteinase K (NEB).RNA idatulutsidwa ndi Trizol ndikutsimikiziridwa ndi RT-qPCR.Zoyambira zimaperekedwa mu Supplementary Table 5.
The in vitro RIP assay idachitidwa molingana ndi ndondomeko yosinthidwa ya RIP assay protocol.Kuchuluka kwa 5 pmol ya in vitro yolembedwa RNA inachepetsedwa 1x ndi RIP buffer ndi kulowetsedwa ndi incubation pa 65 ° C kwa mphindi zisanu ndikutsatiridwa ndi kuzizira pang'onopang'ono mpaka kutentha kwa chipinda.Chiwerengero cha 5 pmol cha mapuloteni a PACT osasunthika kapena osinthika omwe amalembedwa ndi mbendera adayeretsedwa kuchokera ku E. coli.Yambani ndi RNA yosinthidwanso kwa maola a 2 pa 4 ° C ndikutsatira ndondomeko yomwe ili pamwambayi yowunikira RIP ya anti-flag IP.
Pakuwunika kowonjezera kwa RNA, ma cell a 1 × 107 adayikidwa ndi 1xRIP buffer.Pambuyo centrifugation pa 13,000 rpm kwa 15 min pa 4 ° C, supernatant anali pretreated ndi 30 μl wa streptavidin maginito mikanda (Beckman) kwa 2 h pa 4 ° C.Lysate yoyeretsedwayo idaperekedwa ndi yisiti tRNA ndikuphatikizidwa ndi 40 pmol ya RNA yosinthidwa usiku pa 4 ° C, kenako kwa maola ena a 2 ndi 20 μl ya mikanda yatsopano ya streptavidin yotsekedwa ndi BSA idawonjezedwa.Njira yotsuka inali ndi nthawi 4 ndi 5x RIP buffer ndi 4 nthawi ndi 1x RIP buffer.Mapuloteni ofananirawa adachotsedwa ndi biotin elution buffer (25 mM Tris-HCl, pH 7.5, 12.5 mM D-biotin, PMSF) ndikusiyanitsidwa pa NuPAGE 4-12% Bis-Tris Gel (Invitrogen).Pambuyo kupaka siliva (Beyotime Biotechnology), magulu ena adadulidwa ndikuwunikidwa ndi MS.
Kusanthula kwa Co-IP kunachitika kuyesa kuyanjana pakati pa PACT ndi PKR.Mwachidule, ma lysates apamwamba adakonzedwa ndikuyika ma cell a 1 x 107 mu 1 x RIP buffer yotsatiridwa ndi centrifugation pa 13,000 rpm kwa mphindi 15 pa 4 ° C.Ma Lysates anali odzaza ndi mapuloteni A + G mikanda ya maginito, yolumikizidwa ndi 5 µg ya anti-PACT antibody, ndipo imazungulira mofatsa usiku pa 4 ° C.Mikandayo idatsukidwa nthawi za 3 ndi 5 × RIP buffer, kawiri ndi 1 × RIP buffer ndipo idatulutsidwa ndi 1 × SDS buffer.Mapuloteni omwe adachira adawunikidwa ndi gel osakaniza a SDS-PAGE ndipo adapezeka ndi WB.
Ma pmol awiri a PACT odziwika ndi 1 pmol ya PKR adayeretsedwa ku E. coli.Sungunulani mu 1 × RIP buffer ndikuyikani ndi 10 pmol ya RNA yosinthidwanso kwa maola awiri pa 4 °C.Pambuyo pake, adayikidwa ndi puloteni A+G maginito bead-conjugated anti-labeled antibody kwa maola awiri owonjezera.Mikandayo idatsukidwa kanayi ndi buffer ya 1x RIP ndikutulutsidwa ndi 1x SDS buffer.Zotsatira za PACT ndi PKR zidapezeka ndi WB.
Nthawi yotumiza: Sep-23-2022
