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Ingirabuzimafatizo za kanseri zahinduye uburyo butandukanye bwo gutsinda ibibazo bya selile kandi bikomeza gutera imbere.Protein kinase R (PKR) hamwe na poroteyine ikora (PACT) nibisubizo byambere bikurikirana ibimenyetso bitandukanye bitera guhangayikishwa no gukumira ikwirakwizwa rya selile na apoptose.Nyamara, amabwiriza yinzira ya PACT-PKR muri selile ya kanseri ntamenyekana.Hano, twasanze RNA ndende (lncRNA) aspartyl tRNA synthetase antisense RNA 1 (DARS-AS1) igira uruhare rutaziguye mukubuza inzira ya PACT-PKR kandi itera kanseri ikwirakwira.Twifashishije isuzuma rinini rikorwa rya CRISPRi 971 ifitanye isano na kanseri lncRNA, twasanze DARS-AS1 yari ifitanye isano no gukwirakwiza kanseri ikabije.Kubwibyo, DARS-AS1 knockout ibuza ikwirakwizwa ry'uturemangingo kandi itera kanseri ya kanseri apoptose mumirongo itandukanye ya kanseri muri vitro kandi igabanya cyane imikurire yibibyimba muri vivo.Muburyo bwa DARS-AS1 ihuza byimazeyo na enterineti ya PACT kandi ikabuza imikoranire ya PACT-PKR, bityo bikagabanya ibikorwa bya PKR, fosifora eIF2α, kandi bikabuza urupfu rwa selile apoptotique.Mubuvuzi, DARS-AS1 igaragara cyane muri kanseri nyinshi, kandi gukabya gukabije kwi lncRNA byerekana guhanura nabi.Ubu bushakashatsi busobanura amabwiriza yihariye ya kanseri yinzira ya PACT-PKR na DARS-AS1 lncRNA kandi itanga indi ntego yo kumenya kanseri no kuvura.
Ubushobozi bwo guhangana nihungabana nikintu cyingenzi kiranga kanseri ya kanseri kubaho no gukwirakwira.Ikwirakwizwa ryihuse hamwe n’ibimenyetso biranga kanseri yibasiwe na mikorobe ikaze - kubura intungamubiri, hypoxia, na pH nkeya - bishobora gutera urupfu rwerekana inzira.Kugabanya ingirabuzimafatizo zangiza cyane nka p535, poroteyine 6, 7, KRAS8, 9, na HIF-110, 11, 12, 13 bikunze kugaragara muri kanseri, bityo bikabuza apoptose no guteza imbere kubaho.
Protein kinase R (PKR) ni sensor yingenzi yingirakamaro hamwe na subunit kinase ya eukaryotic yo gutangiza ibintu 2α (eIF2α), igenzura ibisobanuro bihuza imihangayiko ningirabuzimafatizo.PKR yabanje kumenyekana nka poroteyine igabanya ubukana bwa virusi ya RNA (dsRNA).Iyo ukora, PKR fosifora eIF2α kugirango ibuze synthesis ya virusi na selile selile 14,15,16.PACT (poroteyine ya PKR) yamenyekanye nka poroteyine ya mbere ya PKR ikora poroteyine idahari dsRNA17,18,19,20,21,22,23.Binyuze mu mikoranire itaziguye na PKR, PACT itera impagarara zitandukanye (inzara ya serumu, peroxide cyangwa kuvura arsenite) kuri PKR no kumanuka byerekana inzira.Usibye eIF2α fosifora, ibikorwa bya PACT byunganirwa na PKR bitera ibintu bitandukanye bifitanye isano no gukemura ibibazo, harimo na redox status yahinduwe binyuze munzira ya PI3K / Akt24, igenzura ryangiza ADN ikoresheje p5325,26 na NF-κB27,28 Igenga iyandikwa, 29. Bitewe n'uruhare runini bagize mu guhangana n'imihangayiko, ikwirakwizwa, apoptose hamwe n’ibindi bikorwa byingenzi bigize selile, PKR na PACT bitanga icyizere cyo kuvura indwara nyinshi, cyane cyane kanseri30,31,32,33.Nubwo, nubwo bifite akamaro gakomeye na biologiya, kugenzura ibikorwa bya PACT / PKR mungirangingo za kanseri biracyoroshye.
lncRNAs ni inyandiko-mvugo irenga nucleotide 200 idafite proteine-code.Kuva imishinga ikurikirana ya genome yamenyekanye ibihumbi n'ibihumbi lncRNAs, 35,36 hashyizweho ingufu nyinshi kugirango basobanure imikorere y’ibinyabuzima.Ubushakashatsi bugenda bwiyongera bwerekanye ko lncRNAs igira uruhare mubikorwa byinshi byibinyabuzima37 harimo kugenga X-chromosome idakora 38,39, gucapa 40, kwandukura 41,42, guhindura43 ndetse no gukura kwa kanseri 44,45,46,47.Ubu bushakashatsi bwatangaje ko lncRNA nyinshi zifite uruhare mu nzira ya PACT / PKR.Bumwe muri ubwo bushakashatsi bwerekanye ko lncRNA ASPACT yabujije kwanduza PACT no kongera ingufu za kirimbuzi za PACT mRNA.Ubundi bushakashatsi bwerekanye ko lncRNA nc886 ihuza PKR kandi ikabuza fosifori yayo 49,50.Kugeza ubu, lncRNA igenga ibikorwa bya PKR yunganirwa na PKR ntabwo byatangajwe.
Aspartyl-tRNA synthetase antisense RNA 1 (DARS-AS1) yamenyekanye nka oncogenic lncRNA51,52,53,54.Binyuze mu mabwiriza ya miP-194-5p53, miP-12952 na miP-532-3p51, DARS-AS1 yerekanwe guteza imbere imikurire ya kanseri yimpyiko isobanutse, kanseri ya tiroyide na kanseri y'ibihaha itari ntoya.Tong na bagenzi be basanze kandi DARS-AS1 iteza imbere myeloma ikomeza umutekano wa poroteyine 39 (RBM39) RNA-ihuza motif.Icyakora, nta bushakashatsi bwakozwe ku kumenya niba iyi lncRNA igira uruhare mu kugenzura imikorere ya PACT-PKR no gukemura ibibazo bya selile kanseri.
Hano, twakoze igihombo kinini-cyo-gukora-ecran dukoresheje sisitemu ya CRISPRi maze twemeza ko DARS-AS1 lncRNA iteza ikwirakwizwa ryubwoko butandukanye bwa kanseri.Twongeyeho, twabonye uburyo bukomeye: DARS-AS1 ihuza PACT mu buryo butaziguye, ibuza guhuza PACT na PKR, ikabuza fosifora ya eIF2α, insimburangingo ya PKR yo hepfo, kandi amaherezo ikabuza urupfu rwa selile apoptotique.Mu gusoza, akazi kacu karerekana DARS-AS1 lncRNA nkumuyobozi winzira ya PACT-PKR nintego ishobora kuvura kanseri no guhanura.
Ubushakashatsi bwimbitse bwa genomic bwerekanye amagana ya lncRNAs ifitanye isano na kanseri.Ariko, imikorere yabo iracyamenyekana cyane 56.Kugirango tumenye abakandida ba lncRNA bafite uruhare mu iterambere rya kanseri, twakoze igihombo-cyo gukora kugirango tugabanye ikwirakwizwa ry’umurongo wa kanseri ya SW620 dukoresheje sisitemu ya CRISPRi (Ishusho 1a).Ikintu kidasanzwe kiranga imirongo ya kanseri ya SW480 na SW620 ni uko ikomoka ku bibyimba by'ibanze n'icya kabiri ku murwayi umwe.Ibi biratanga igereranya ryingenzi ryo kwiga impinduka zishingiye ku ngirabuzima fatizo za kanseri yateye imbere.Kubwibyo, twasesenguye inyandiko-mvugo yumurongo wa kanseri yibara (SW480 na SW620) dukoresheje RNA ikurikirana hanyuma dukusanya lncRNAs zishobora gukora mubitabo byasohotse.Dufatiye kuri ibyo bisubizo, twateguye isomero rya sgRNA ryahujwe na 7355 sgRNA oligos yibasira lncRNAs 971 zifitanye isano na kanseri na 500 oligos ya sgRNA itagamije kugenzura nabi (Data Supplementary Data 1).
Igishushanyo cyerekana kwerekana ukoresheje sisitemu ya CRISPRi.b sgRNA ikungahaye nyuma yo kwerekana.Umurongo utambitse utambitse ugereranya log2 (guhinduka inshuro) = ± 0.58.Umurongo utudomo uhagaze werekana p agaciro = 0.05.Utudomo twirabura twerekana sgRNA idafite intego (yagenwe nka NC).Utudomo dutukura ni sgRNAs yibasira DARS-AS1.Utudomo twubururu ni sgRNAs yibasira LINC00205, lncRNA yasobanuwe mbere.guhinduka inshuro = (gusoma bisanzwe, umunsi wa 17) / (gusoma bisanzwe, umunsi 0).c DARS-AS1 sgRNA gukomanga byabujije imikurire ya selile.Utubari twibeshya twerekana iation gutandukana bisanzwe mubigeragezo bitatu.* p ≤ 0.05, ** p ≤ 0.01 t-ikizamini cyimirizo ibiri yumunyeshuri.d Imvugo ya DARS-AS1 mubyimba (Dataset ya TCGA).em Kugaragaza DARS-AS1 muburyo busanzwe hamwe nibibyimba byabarwayi bafite BLCA, KIRC, PRAD, LUSC, UCEC, LUAD, LIHC, KIRP, na COAD, (TCGA dataset).p-indangagaciro zabonetse hakoreshejwe t-test ebyiri zabanyeshuri.
Nyuma yo kubaka plasmid no gupakira lentivirus, twandujije umurongo wa kanseri ya dCas9-SW620 hamwe nibitabo byavuzwe haruguru mubushakashatsi bune bwigenga.Ubwinshi bwubwandu (MOI) kuri izo ndwara bwari 0.1–0.3, byerekana ko buri selile ishobora kwanduzwa gusa na sgRNA imwe.Nyuma yiminsi 18 yumuco wa vitro, umwirondoro wo gutunganyiriza intego za sgRNAs wagabanutse cyangwa wiyongereye nyuma yo kwipimisha, mugihe umubare wa oligonucleotide utagenewe kugenzura wagumye udahinduka ugereranije numwirondoro wabanjirije kwerekana, byerekana ko intego yacu ifite ecran-yihariye cyane. isomero.Umuceri.1b n'imbonerahamwe y'inyongera 1). LINC00205, byavuzwe mbere ko itera kanseri y'ibihaha na kanseri y'umwijima gutera imbere 58,59,60, yasuzumwe (log2 (foldchange) <−0.58, p agaciro <0.05), yemeza ko iri suzuma ryizewe (Ishusho 1b). LINC00205, byavuzwe mbere ko itera kanseri y'ibihaha na kanseri y'umwijima gutera imbere 58,59,60, yasuzumwe (log2 (foldchange) <−0.58, p agaciro <0.05), yemeza ko iri suzuma ryizewe (Ishusho 1b). V . 1b). LINC00205, yavuzwe mbere kugirango iteze imbere kanseri yibihaha na kanseri yumwijima 58,59,60, yarashizwemo (log2 (guhindura inshuro) <-0.58, p-agaciro <0.05), yemeza ko iri suzuma rikomeye (Ishusho .1b) . LINC00205 之前 被 报道 可 促进 肺癌 和 肝癌 进展 58,59,60 ， 被 筛选 掉 （log2 （倍数 变化） <-0.58 ， p 值 <0.05） ， 证实 了 该 筛选 的 可靠性 （1b） LINC00205 之前 被 报道 可 促进 肺癌 和 肝癌 进展 58,59,60 ， 被 筛选 掉 （log2 （倍数 变化） <-0.58 ， p 值 <0.05） ， 证实 了 该 筛选 的 可靠性 （1b） V . 1b). LINC00205, yavuzwe mbere kugirango iteze imbere kanseri y'ibihaha n'umwijima 58,59,60, yarashizwemo (log2 (guhindura inshuro) <-0.58, p-agaciro <0.05), yemeza ko iri suzuma rikomeye (Ishusho .1b).
Muri lncRNAs zose zapimwe, DARS-AS1 nayo yarasuzumwe, hamwe na cognate sgRNA oligonucleotide eshatu yagabanutse cyane nyuma yiminsi 18 yumuco, byerekana ko gukubita iyi lncRNA byatumye kugabanuka kwa kanseri kugabanuka (Ishusho 1b).Iki gisubizo cyongeye gushyigikirwa n’isesengura rya MTS mu ngirabuzimafatizo ya kanseri yerekana ko umuvuduko w’ubwiyongere bwa selile DARS-AS1 wikubye kabiri ugereranije n’utugingo ngengabuzima (Ishusho 1c) kandi wari uhuye na raporo zabanjirije ubundi bwoko bwa kanseri.: gukuramo kanseri y'impyiko, kanseri ya tiroyide na kanseri y'ibihaha itari ntoya 51,52,53,55.Nyamara, imikorere yacyo hamwe na molekuline muri kanseri yibara ikomeje kutagenzurwa.Kubwibyo, twahisemo iyi lncRNA kugirango dukomeze kwiga.
Kugira ngo twige imvugo ya DARS-AS1 mu barwayi, twasesenguye byimazeyo urugero rw'ibibyimba 10.327 biva mu mushinga wa Kanseri Genome Atlas (TCGA).Ibisubizo byacu byerekana ko DARS-AS1 igaragara cyane kandi igenzurwa cyane mu ngirabuzimafatizo nzima mu bibyimba bitandukanye, harimo colon adenocarcinoma (COAD), kanseri y'impyiko isukuye (KIRC), na kanseri y'impyiko (KIRP)..Bake cyane (Igishusho 1d hamwe ninyongera Igishusho 1a, b). Isesengura ry’ubuzima bwiza / ibibyimba byongeye kwemeza ko DARS-AS1 igaragara cyane mu bibyimba byo mu ruhago urothelial carcinoma (BLCA), impyiko zifata impyiko kanseri (KIRC), prostate adenocarcinoma (PRAD), kanseri y'ibihaha kanseri (LUSC) , uterine corpus endometrial carcinoma (UCEC), adenocarcinoma y'ibihaha (LUAD), kanseri y'umwijima hepatocellular carcinoma (LIHC), impyiko z'impyiko kanseri y'impyiko (KIRP), na colon adenocarcinoma (COAD) (p agaciro <0.05) (Ishusho 1e - m). . Isesengura ry’ubuzima bwiza / ibibyimba byongeye kwemeza ko DARS-AS1 igaragara cyane mu bibyimba byo mu ruhago urothelial carcinoma (BLCA), impyiko zifata impyiko kanseri (KIRC), prostate adenocarcinoma (PRAD), kanseri y'ibihaha kanseri (LUSC) , uterine corpus endometrial carcinoma (UCEC), adenocarcinoma y'ibihaha (LUAD), kanseri y'umwijima hepatocellular carcinoma (LIHC), impyiko z'impyiko kanseri y'impyiko (KIRP), na colon adenocarcinoma (COAD) (p agaciro <0.05) (Ishusho 1e - m). .Isesengura ry’ubuzima bwiza / ibibyimba byemejwe kandi byerekana ko DARS-AS1 igaragara cyane mu ruhago rwitwa urothelial carcinoma (BLCA), kanseri yimpyiko n’impyiko (KIRC), prostate adenocarcinoma (PRAD), ibibyimba bya kanseri ya kanseri (LUSC)., m). , kanseri ya endometrale ya corpus uteri (UCEC), adenocarcinoma yo mu bihaha (LUAD), kanseri y'umwijima y'umwijima (LIHC), kanseri y'impyiko y'impyiko (KIRP), na adenocarcinoma ya colon (COAD) (p agaciro <p 0.05) (Ishusho 1e– m).配对 健康 / 肿瘤 样本 的 分析 进一步 证实 了 DARS-AS1 在 膀胱 尿路 上皮 癌 (BLCA) 、 肾 肾 透明 细胞 K (KIRC)显 着 更高 表达 ， 子宫 体 EC EC EC EC EC EC EC EC EC I I LIHC） ， 肾 （IR IR IR IR IR IR IR <0.05） （图 1e-m）.配对 健康 / 肿瘤 样本 的 分析 证实 ars dars-os1 在 尿路 上 皮 癌 皮 癌 皮 癌 皮 细胞 p p p p p p p p p p p p p p p p p rad (lusc) 肿瘤 的 的 的 的 中 中 中 中 中 中 cel cel cel cel ucel ​​cel癌 kirp） （coad） （p 值 <0.05） （图 1e-m）.Isesengura ry’ubuzima bwiza / ibibyimba byombi byashyigikiye uruhare rwa DARS-AS1 mu ruhago rwitwa urothelial carcinoma (BLCA), kanseri yimpyiko zifata kanseri (KIRC), prostate adenocarcinoma (PRAD), hamwe na kanseri y'ibihaha ya kanseri (LUSC).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). imvugo muri corpus uterine carcinoma (UCEC), ibihaha adenocarcinoma (LUAD), kanseri y'umwijima (LIHC), kanseri y'impyiko kanseri (KIRP), na colon adenocarcinoma (COAD) (p agaciro <0.05) (Ishusho 1e -m).Ufatiye hamwe, ibisubizo byerekana ko DARS-AS1 igaragara cyane kandi igaragara cyane muri kanseri zitandukanye.
Kuberako DARS-AS1 na DARS (gene ikubiyemo umurongo wa antisense) basangiye porotokoro imwe kandi ikaba iri iruhande rwundi, twashizeho shRNA kugirango dukubite DARS-AS1 ariko ntabwo ari DARS (Ishusho yinyongera 2a, b hamwe nimbonerahamwe 2) .Usibye SW620, twakoresheje kandi indi mirongo itatu y'utugari tugaragaza cyane DARS-AS1 kugirango twige imikorere n'imikorere ya shRNA knockdown (Imbonerahamwe 3).Ibisubizo byacu byagaragaje ko shRNA zose uko ari eshatu zateye imbere zageze byibuze 80% DARS-AS1 yo gukubita hasi nta ngaruka nini ku mubare wa DARS mRNA (Ishusho yinyongera. 2c - f).Twongeyeho, twasanze DARS-AS1 gukubita hamwe na shRNAs byabujije cyane imikurire yimikorere ya selile kanseri yibara ya SW620 (49.7%) na HCT116 (27.7%), umurongo wa kanseri yamabere MBA-MD-231 (53.4%).) n'umurongo w'akagari ka HepG2 (kugabanuka kwa 92.7%), hamwe n'ubushobozi bwabo bwo gukora urwego rutemewe (kugabanuka kugabanuka ~ 50.8%, 44,6%, 40.7% na 75.7% kumurongo w'akagari) (Ishusho 2a, b).Muri SW620, ibyavuye mu bushakashatsi bwakozwe na koloni byongeye kwemeza ko DARS-AS1 shRNA yabujije cyane ikwirakwizwa ry’utugari hamwe no kugabanuka ku kigereranyo cya 69.6% (Ishusho 2c).
Ingaruka zo kugenzura shRNA na DARS-AS1 shRNA ku gukwirakwiza selile (a) no gukora spheroid (b) muri SW620, HCT116, MBA-MD-231, na HepG2.c Ingaruka zo kugenzura shRNA na DARS-AS1 shRNA kumikorere ya koloni muri selile SW620.Ikwirakwizwa ry'utugingo ngengabuzima (d), imiterere ya spheroid (e), hamwe na koloni (f) ya SW620 selile ikabije DARS-AS1.Amakuru yerekanwe nuburyo ± gutandukana bisanzwe mubigeragezo bitatu.* p ≤ 0.05, ** p ≤ 0.01, na *** p ≤ 0.001 na t-test yumurizo ibiri.
Kugirango twuzuze igihombo-cy-imikorere, ubutaha twaremye SW620 selile ikabije DARS-AS1 (Ishusho yinyongera. 2g).DARS-AS1 gukabya gukabije byongereye cyane imikurire ya selile (inshuro 1.8), spheroid idafite imiterere (inshuro 1,4), hamwe na koloni (3,3-inshuro) muri selile SW620 (Ishusho 2d - f).Twemeje iki gisubizo dukoresheje indi DARS-AS1 yerekana umurongo wa selire, A549.Uku kwiyongera kwingirabuzimafatizo kubera DARS-AS1 gukabya gukabije byagaragaye no muri selile A549 (Ishusho yinyongera. 2h, i na Imbonerahamwe 3).Ufatiye hamwe, ubushakashatsi bwunguka nigihombo byerekana ko DARS-AS1 itera kanseri ya kanseri ikwirakwizwa muri vitro.
Kugirango tumenye uburyo bwibanze DARS-AS1 igenga ikwirakwizwa ry'utugari, twakoze isesengura rya RNA kugirango tumenye abafatanyabikorwa ba poroteyine.Ibisubizo bya RT-qPCR byerekanye ko hafi 86.2% ya DARS-AS1 iherereye muri cytoplazme ya selile SW620 (Ishusho yinyongera. 3a).Muri vitro yandukuye biotinylated DARS-AS1 cyangwa pseudoRNA noneho yashizwemo na lysates ya SW620 ikurikirwa no gutandukana kwa SDS-PAGE.Irangi rya feza ryakurikiyeho ryerekanaga ko umurongo utandukanye (~ 38 kDa) wakungahajwe cyane mubikurura DARS-AS1 ariko ntibiri muri RNA yuzuye cyangwa amasaro (Ishusho 3a).Iri tsinda ryamenyekanye nka PKR ikora poroteyine (PACT) na mass spectrometrie (MS) ikanashimangirwa na immunoblotting muri SW620, HCT116, na HepG2 imirongo ya selire (Ishusho 3a, b).Gutunganyiriza DARS hamwe na poroteyine zijyanye na PACT - PKR na TRBP - na byo byakorewe iperereza hakoreshejwe isesengura rya RNA ryakozwe na Western blotting (WB).Ibisubizo byagaragaje ko nta mikoranire itaziguye hagati ya DARS-AS1 RNA na poroteyine eshatu zabonetse (Ishusho yinyongera. 3b).Imikoranire yihariye hagati ya DARS-AS1 na PACT yongeye gushimangirwa n’isesengura rya RNA immunoprecipitation (RIP), ryerekanaga ko DARS-AS1 yari ikungahaye cyane kuri antibodiyide zirwanya PACT ariko ko atari izindi RNAs (Igicapo 3c).Kugirango umenye niba DARS-AS1 ikorana na PACT mu gihe nta bindi bice bigize selile, ubushakashatsi bwakozwe na vitro biolayer interferometrie (BLI) bwakozwe hakoreshejwe PACT isukuye.Biotin yanditseho DARS-AS1 cyangwa dummy RNA yimuwe kuri biosensor ya streptavidin (SA) hanyuma ikabikwa muri buffer kinetic irimo 1 μM PACT.Ikigaragara ni uko PACT ihambiriye cyane kuri DARS-AS1 (KD agaciro ~ 26.9 nM), ariko ntabwo yigana RNA (Ishusho 3d).Ufatiye hamwe, ibisubizo byerekana imikoranire itaziguye kandi bifitanye isano iri hagati ya DARS-AS1 na PACT.
Isesengura rya RNA ryerekanye DARS-AS1 ikorana na PACT muri selile SW620.Hejuru, ifeza yerekana poroteyine zifitanye isano.Immunoblots zo hepfo zakozwe hamwe na antibody anti-PACT.b Isesengura rya RNA ryakorewe muri selile HCT116 (hejuru) na HepG2 (hepfo).Gutunganyiriza PACT byagaragaye na immunoblotting.cRNA immunoprecipitation (RIP) isuzuma ryakorewe muri selile SW620 hakoreshejwe antibodies zerekanwe.d PACT ihuza umurongo kugeza uburebure bwa DARS-AS1 cyangwa kugenzura RNA byabonetse hakoreshejwe biolayeri interferometrie (BLI).RNA yimuwe kuri biosensor ya streptavidin.1 μM PACT yakoreshejwe mu gupima ishyirahamwe.e RNA gukurura byakozwe hakoreshejwe biotinylated yuzuye-DARS-AS1 cyangwa yaciwe (hejuru).Immunoblot yerekana PACT yakiriwe (hepfo).f PACT isukuye ibendera yashizwemo na biotinylated yuzuye ya DARS-AS1 cyangwa yaciwe (nko muri e) kugirango muri vitro RIP.RNA yakuweho yagenzuwe na RT-qPCR.g Ugereranije isano ya RNA itandukanye ya PACT yabonetse hakoreshejwe biolayeri interferometrie.Kubisesengura, 100 nM RNA na 1 μM RAST yakoreshejwe.h Muri vitro RIP isuzuma ryakozwe hakoreshejwe isuku idahwitse cyangwa yaciwe yanditseho PACT.RNA yakuweho yagenzuwe na RT-qPCR.i Iterambere ryikura rya SW620 selile ikabije DARS-AS1, PACT, cyangwa byombi.j Gukabya gukabije k'uburebure bwuzuye cyangwa bwaciwe DARS-AS1 muri selile SW620 byagize ingaruka zitandukanye kumikurire.k Apoptose yamenyekanye na immunoblotting hamwe na antibody anti-PARP.Knockout ya DARS-AS1 itera apoptose ya selile SW620 nkuko bigaragazwa na cytometrike.Amakuru yerekanwe nuburyo ± gutandukana bisanzwe mubigeragezo bitatu. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p <0.0001, n'ikizamini cya t umurizo ibiri. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p <0.0001, n'ikizamini cya t umurizo ibiri. * p ≤ 0,05, ** p ≤ 0,01, *** p ≤ 0,001, **** p <0,0001 по дву зоннему критерию Стьюдента. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p <0.0001 na t-ikizamini cy-imirizo ibiri. * 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 дву зоннему критерию Стьюдента. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p <0.0001 na t-ikizamini cy-imirizo ibiri.
Twahise dukora ibice bitatu bya biotinylated DARS-AS1 RNA dukoresheje vitro transcription kugirango tumenye akarere ka DARS-AS1 gakenewe kugirango ishyirahamwe PACT (Ishusho 3e).Ibisubizo by'isesengura rya RNA byerekanaga ko buri gice cyashoboye gukorana na PACT, ariko akarere ka 3′-terminal (384-768 nucleotide yanditseho A3) yerekanye nucleotide zirenga 1-384 zanditseho A1) (Ishusho 3e).Ibisubizo nkibi byagaragaye muri vitro RIP assay ukoresheje recombinant PACT (Ishusho 3f).Bihuye nibi bisubizo, ubushakashatsi bwo guhuza ibice bya RNA byimuwe na PACT ukoresheje BLI byerekanaga kandi ko PACT ifitanye isano ya A3 (384–768 nt) (agaciro ka KD kangana na 94,6 nM), mugihe hafi ntaho ihuriye nibindi bice.(Isanamu 3d).
Twasuzumye kandi uduce duhuza uturere muri PACT.PACT ikubiyemo domaine eshatu zikora, ebyiri murizo zibitswemo imirongo ibiri ya RNA-ihuza domaine (dsRBD) hamwe nubutegetsi bwa gatatu (bwagenwe D3) bukora nkibikorwa bya poroteyine.Kugirango dusuzume ubushobozi bwa lncRNA bwo guhuza buri domeni, twakoze ihinduka ryimiterere itatu yakuyeho buri domaine eshatu hanyuma ikora in vitro RIP.Ibisubizo byacu byerekanaga ko gusiba domaine ya gatatu (D3) ya PACT byagabanije cyane imikoranire yayo na DARS-AS1 (inshuro 0,11 ugereranije na PACT idahwitse) ugereranije nizindi mutation ebyiri (Ishusho 3h), herekanywe ko irekurwa ya D3 yakoranye na DARS.-AC1.Ufatiye hamwe, ibisubizo byerekana ko imikoranire hagati ya DARS-AS1 na PACT ishobora kubaho cyane cyane binyuze kuri 3 ′ iherezo rya DARS-AS1 na D3 ya PACT.
Twabonye ko DARS-AS1 nta ngaruka yagize ku mvugo ya PACT kandi PACT nta ngaruka yagize kuri DARS-AS1 (Ishusho yinyongera. 3c).Twahise dusuzuma ingaruka za PACT gukubita kumikurire.Bitandukanye na DARS-AS1, selile zigereranije ziyongereye inshuro 1.5-33 mugihe PACT yakubiswe (Ishusho yinyongera. 3d).Ibisubizo byubushakashatsi bwakoronijwe bwerekanye ko ingirabuzimafatizo zagize ubukoroni 2-3 nyuma yo kuvura shRNA hamwe na PACT (Ishusho yinyongera. 3e).Kugirango tumenye niba DARS-AS1 igenga ikwirakwizwa rya selile binyuze muri PACT, twabyaye SW620 selile ikabije PACT, DARS-AS1, cyangwa byombi.Gukabya gukabije kwa PACT byagaragaje kubuza gukwirakwiza selile (Ishusho 3i).Mugihe DARS-AS1 gukabya gukabije kuri buri kintu cyateje imbere cyane ikwirakwizwa ry'utugingo ngengabuzima, nta tandukaniro rikomeye ryagaragaye mu mikurire yo gukura kw'utugingo dukabije DARS-AS1 na PACT.Ibisubizo byerekana ko PACT ishobora kurwanya ikwirakwizwa ryinshi ryatewe na DARS-AS1 gukabya.
Kubera ko uturere dutandukanye twa DARS-AS1 dufite ubushobozi butandukanye bwo guhuza PACT, twasesenguye ingaruka zijyanye no gukwirakwiza selile dukabije gukabya gutandukanya DARS-AS1.Ugereranije n'ibindi bice bibiri, DARS-AS1 yarakabije cyane kuri 3 ′ (384–768 nt), agace gakomeye kajyanye na PACT muri DARS-AS1, kari gafite ubushobozi buke bwo gukurura ikwirakwizwa ry'uturemangingo (Ishusho 3j).Ibisubizo byerekana isano iri hagati yubushobozi bwo guhuza nibikorwa bya DARS-AS1.
PACT yavuzwe ko ari poroteyine yo mu bwoko bwa apoptotique19.Kubwibyo, twasesenguye ingaruka za DARS-AS1 kuri apoptose.Nkuko byari byitezwe, DARS-AS1 gukomanga byongereye cyane PARP clavage muri selile SW620 kandi byongera umubare wutugingo ngengabuzima twa annexine V muri SW620, HCT116, HepG2, na MBA-MD-231 kumurongo (Ishusho 3k).3).3f - h), byerekana ko ingaruka zo kurwanya apoptotique ya DARS-AS1 mu ngirangingo za kanseri zinyuranye n’imikorere itera apoptose ya PACT.Ufatiye hamwe, ibisubizo byerekana ko uburyo bwimikorere ya DARS-AS1 oncogenic bushobora kuba binyuze mukubuza imikorere ya PACT.
Ibikurikira, twasesenguye ingaruka zimikorere yishyirahamwe DARS-AS1-PACT.PACT yavuzwe ko ikora PKR ikoresheje imikoranire itaziguye, nyuma ikazamura fosifori ya eIF2α, igatera gusiba hamwe na apoptose17.Ubwa mbere, twasuzumye niba DARS-AS1 igira ingaruka kuri selire ya selile ya PACT na PKR.Microscopi ya fluorescence yerekanaga ko PACT na PKR byakoronijwe cyane muri selile SW620 hamwe nimpuzandengo ya Pearson ihuza 0,72.Hagati aho, gukabya gukabije kwa DARS-AS1 byagabanije cyane PACT na PKR gufatanya (bivuze ko coeffisiyoneri ya Pearson 0.61) (Ishusho 4a).Kugira ngo dukore iperereza niba DARS-AS1 ishobora guhindura imikoranire ya PACT-PKR, twakoze ubushakashatsi bwa immunoprecipitation (co-IP) hamwe na antibody anti-PACT muri lysates ya SW620.PKR yari ikungahaye cyane muri anti-PACT mu ngirabuzimafatizo, mu gihe gukira kwa PKR byagabanutse cyane muri lysates ziva mu ngirabuzimafatizo zikabije DARS-AS1 (Ishusho 4b).Isuku yanditseho PACT na PKR yakoreshejwe muri vitro protein ihuza ibipimo.Kubera iyo mpamvu, abatanze DARS-AS1 ariko nta kugenzura RNA yerekanaga imikoranire ya PACT-PKR yahagaritswe (Ishusho 4c).Ibisubizo byose byerekanaga ko DARS-AS1 yahagaritse itumanaho rya PACT na PKR.
Kwishyira hamwe kwa PACT na PKR mu ngirabuzimafatizo cyangwa selile bikabije DARS-AS1 byagaragaye hakoreshejwe microscopi ya fluorescence.Nuclei yandujwe na DAPI.Ibisubizo by'ibarurishamibare byabonetse ku mafoto 16.b Co-immunoprecipitation (co-IP) ukoresheje antibody anti-PACT muri selile lysates yo kugenzura selile SW620 cyangwa selile bikabije DARS-AS1.c Yanditseho PACT, isukuye PKR kandi yandukurwa muri vitro hamwe na DARS-AS1 cyangwa urw'agashinyaguro RNA yashizwemo kugirango isesengurwe rya vitro protein.Antibodiyite zirwanya ibendera zakoreshejwe mu gukingira indwara.d Immunoblots hamwe na antibodies zerekanwe zakozwe muri selile SW620 na HCT116 zandujwe no kugenzura shRNA cyangwa DARS-AS1-shRNA ikurikirwa ninzara ya serumu.e DARS-AS1 urwego rwimvugo rwahinduye selile sensitivite kuri thapsigargin.SW620 selile zahinduwe hamwe na DARS-AS1 shRNA, DARS-AS1 plasmid ikabije cyangwa igenzura plasmid.Ingirabuzimafatizo zavuwe na thapsigargin mu masaha 48 kandi ubushobozi bwa selile bwagenwe hakoreshejwe reagent ya MTS.f Muri vitro yandukuwe DARS-AS1 cyangwa dummy RNA hamwe na PACT isukuye yakoreshejwe mugukora vitro vitro no kumenya immunoblot.g Immunoblots ukoresheje izo antibodies zakozwe kuri selile SW620-ctrl (ibumoso) cyangwa selile ikabije cyane ihinduka rya PKR (iburyo).Izi selile zahinduwe hamwe no kugenzura shRNA cyangwa DARS-AS1-shRNA ikurikirwa ninzara ya serumu.h Flow cytometrie yerekanaga ko kudakora kwa mutant PKR byishyuye DARS-AS1 iterwa na apoptose muri selile SW620.i Immunoblots hamwe na antibodies zerekanwe zakozwe muri selile SW620 (ibumoso) cyangwa HCT116 (iburyo).Ingirabuzimafatizo zandujwe no kugenzura shRNA cyangwa DARS-AS1 shRNA zabuze serumu kandi zunganirwa na 100 nM PKR C16 inhibitor cyangwa DMSO.Umwanya munini = 5 µm.Amakuru yerekanwe nuburyo ± gutandukana bisanzwe mubigeragezo bitatu.* p ≤ 0.05 umurizo ibiri-t-ikizamini cyabanyeshuri.
Muri rusange abantu bemeza ko PACT imaze gukorana na PKR17, fosifora ya PKR kuri Thr451 irashobora guterwa.Ibisubizo byacu byagaragaje ko urwego rwa fosifora ya PKR rwazamutse cyane mu ngirabuzimafatizo za DARS-AS1 nyuma yo kwicwa n'inzara ya serumu (Ishusho 4d hamwe n’inyongera ya 4a).Kubera iyo mpamvu, twasanze fosifora ya eIF2α, insimburangingo nyamukuru ya PKR, nayo yariyongereye cyane na DARS-AS1 shRNA (Ishusho 4d hamwe n’inyongera ya 4a).Thapsigargin ni ER ihangayikishije itera ER kurekura Ca2 +.Kuvura hamwe na thapsigargin byavuzwe ko bitera imvugo nogukora kwa PACT, ikomeza gukorana no gukora PKR, biganisha kuri apoptose mukongera eIF2α fosifora 18,61.Hano, twakoresheje thapsigargin nkikangura inzira ya PACT / PKR kugirango tumenye niba DARS-AS1 ishobora gufasha selile gutsinda imihangayiko muguhagarika inzira ya PACT / PKR.Twabonye ko urwego rwa DARS-AS1 imvugo ifitanye isano neza no kurwanya selile kuri thapsigargin.SW620 selile ikabije DARS-AS1 yarokotse neza iyo ivuwe na thapsigargin, mugihe selile zifite DARS-AS1 gukomanga zarushijeho kwibasirwa (Ishusho 4e).Bihuye nibi bisubizo, DARS-AS1 gukabya gukabije byagabanije thapsigargin-iterwa na fosifori ya PKR (Ishusho yinyongera. 4b).Ibinyuranye, nyuma yo kuvura thapsigargin, PKR na eIF2α bya fosifora ku rugero rwo hejuru muri selile DARS-AS1 yo gukomanga ugereranije na selile igenzura (Ishusho yinyongera. 4b).Igishimishije, thapsigargin yateje imvugo ya DARS-AS1 muburyo buterwa na dose, ishobora kwerekana imikorere yo kurwanya stress ya DARS-AS1 (Ishusho yinyongera. 4c).Mubyongeyeho, twakoze muri vitro activation gusuzuma kugirango twemeze ibyo twabonye.Muri make, PKR yasukuwe muri lysates ya selile ikoresheje antibody anti-PKR, hanyuma yinjizwamo recombinant PACT na DARS-AS1 yanditswe muri vitro.Nyuma yo gukora enzymatique, fosifore-PKR yagaragaye ikoresheje WB.Ibisubizo byacu byerekanaga ko fosifora ya PKR yabujijwe cyane na DARS-AS1, ariko ntabwo yagenzuwe na RNA (Ishusho 4f).Ibi muri vitro no mubisubizo bya vivo byerekana ko DARS-AS1 ibuza gukora PKR yunganirwa na PKR.Muri icyo gihe, twabonye kandi kugabanuka kwa PACT gukira imbere ya DARS-AS1 (Ishusho 4f).Igisubizo kirahuye nibisubizo bya vitro protein ihuza assay (Igicapo 4c) kandi irongera igaragaza imikorere yo guhagarika DARS-AS1 kumuryango wa PACT-PKR.
Ser246 na Ser287 muri D3 domaine ya PACT birasabwa kugirango PKR ikoreshwe mukibazo cya selile.Gusimbuza ibisigisigi bibiri bya serine kuri alanine byatanze mutant PACT (mutD), yakoraga PKR mugihe nta mpungenge, kandi gusimbuza alanine (mutA) byahinduye protocole.Kubera ko twerekanye akamaro k'uru rubuga dufatanije na DARS-AS1, twabyaye mutant ebyiri za PACT kugirango tumenye niba ibyo bisigazwa nabyo bishobora kugira uruhare mu mikoranire na DARS-AS1.Igishimishije, ihinduka ryombi ryatakaje ubushobozi bwo guhuza DARS-AS1 (Ishusho yinyongera. 4d), byerekana ko imiterere yuzuye ya poroteyine ya PACT ishobora gukenerwa kugirango imikoranire myiza na DARS-AS1.
Ikigeretse kuri ibyo, ibisubizo byacu byerekana kandi ko DARS-AS1-shRNA iterwa no kubuza ikwirakwizwa ry’uturemangingo ishobora kugarurwa igice bitewe no gukabya gukabya kwangirika kwinshi kwa PACT (PACTmutA) cyangwa ihinduka rikomeye rya PKR (PKRmut) (Ishusho yinyongera. 4e. E).Gukabya gukabije kwimiterere ya PKR yiganje yagabanije fosifora ya PKR iterwa na DARS-AS1 gukomanga kimwe na eIF2α fosifora mu ngirabuzimafatizo zabuze serumu (Ishusho 4g).Icy'ingenzi cyane, igipimo cyingirabuzimafatizo za apoptotique zatewe na DARS-AS1 gukomanga nacyo cyaragabanutse mu ngirabuzimafatizo zikabije PKRmut (Ishusho 4h hamwe n’inyongera ya 4g).Kubuza ibikorwa bya PKR kinase binabangamira imikorere ya DARS-AS1, kuko ibisubizo byerekanaga ko gukubita DARS-AS1 bidakunze gukurura PKR na eIF2α fosifora mugihe ingirabuzimafatizo zavuwe na PKR yihariye ya C16 (Igicapo cya 4i nigishushanyo cya 4h).).Dufatiye hamwe, ibisubizo byacu byerekana ko DARS-AS1 iteza imbere gukwirakwiza selile, byibuze igice, muguhagarika ibikorwa bya PKR byunganirwa na PKR.
Kugirango turusheho gucukumbura uruhare rwa DARS-AS1 muri tumorigenez, twakoze mubushakashatsi bwa vivo dukoresheje imbeba xenograft. Ibisubizo byerekana ko gukomanga kwa DARS-AS1 byagabanutse cyane gukura kwikibyimba ku mbeba (p agaciro <0.0001) (Ishusho 5a). Ibisubizo byerekana ko gukomanga kwa DARS-AS1 byagabanutse cyane gukura kwikibyimba ku mbeba (p agaciro <0.0001) (Ishusho 5a). Результатя показывают, что удаунун DARS-AS1 резко снижает рост опухоли у мшей (значение p <0,0001) (рис. 5а). Ibisubizo byerekana ko DARS-AS1 gukomanga bigabanya cyane imikurire yikibyimba mu mbeba (p agaciro <0.0001) (Ishusho 5a).结果 表明 ， DARS-AS1 的 敲 低 显 着 降低 小鼠 的 0. 生长 （p 值 <0.0001） （图 5a）。结果 表明 ， DARS-AS1 的 敲 低 显 着 降低 小鼠 的 0. 生长 （p 值 <0.0001） （图 5a）。 Результаты показали, что Кдадаун DARS-AS1 значительно снижает рост опухоли у мшей (значение р <0,0001) (рис. 5а). Ibisubizo byerekanaga ko DARS-AS1 gukubita byagabanije cyane imikurire yikibyimba ku mbeba (p agaciro <0.0001) (Ishusho 5a).Rero, mu itsinda rya DARS-AS1 ryakomanze, habayeho kugabanuka gukabije kw’ibibyimba bigera kuri 72.9% kandi bivuze ko ibibyimba byiyongereyeho 87.8% (Ishusho 5b-d).Ibisubizo birerekana neza ko DARS-AS1 ishobora guteza imbere cyane ikibyimba muri vivo.
Ingaruka zamamaza DARS-AS1 gukomanga kuri oncogenezi yibara mu mbeba zambaye ubusa.Imikurire yo gukura (a), ingano y'ibibyimba (b), uburemere (c), n'amashusho y'ibibyimba (d) irerekanwa.Utubari twibeshya twerekana ± SEM. n = 10. **** p <0.0001, nikizamini cyumurizo wumunyeshuri ibiri. n = 10. **** p <0.0001, nikizamini cyumurizo wumunyeshuri ibiri. n = 10. **** p <0,0001 по дву зоннему критерию Стьюдента. n = 10. **** p <0.0001 umurizo ibiri-t-Ikizamini cyabanyeshuri.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 дву зоннему критерию Стьюдента. **** p <0.0001 umurizo ibiri-t-ikizamini cyabanyeshuri.e Kaplan-Meier yasesenguye isano iri hagati yimvugo ya DARS-AS1 nubuzima muri rusange kubarwayi bafite UVM, KICH, KIRP, MESO, GBM, na LGG.Urwego rwo hejuru rwimvugo ya DARS-AS1 mubarwayi bari hejuru ya 50%;urwego rwo hasi rwimvugo ya DARS-AS1 mubarwayi yari munsi ya 50%.p-indangagaciro zagenwe hakoreshejwe ikizamini cyurwego.f Icyitegererezo cyatanzwe aho DARS-AS1 igenga inzira ya PACT-PKR no gukura kwikibyimba.
Kugirango twumve neza ingaruka zamavuriro ya DARS-AS1, twasuzumye isano iri hagati yimvugo yayo no kubaho kwabarwayi.Mu gusesengura imibare ya TCGA, twasanze imvugo yo hejuru ya DARS-AS1 yari ifitanye isano na uveal melanoma (UVM), chromophobia yimpyiko (KICH), kanseri yimpyiko kanseri (KIRP), mesothelioma (MESO), multiplex.Kubaho hasi byari bifitanye isano cyane na glioblastoma morphose (GBM) hamwe n’abarwayi bafite ubwonko bwo mu rwego rwo hasi (LGG) (Ishusho 5e).Ibisubizo byerekana ko DARS-AS1 ishobora kugira uruhare runini mu gutera ikibyimba kivura kandi ishobora kuba biomarker ishobora guhanura kanseri nyinshi.
Muri ubu bushakashatsi, dukoresheje isuzuma rinini rya CRISPRi ryerekana imikorere, twahisemo ko DARS-AS1 lncRNA itsinze imihangayiko ya kanseri igenga ibibazo bibiri byingenzi byita kubibazo, PACT na PKR.Mu gukorana mu buryo butaziguye na PACT, DARS-AS1 yabujije gukora PKR yunganirwa na PKR, bityo ikumira urupfu rwa selile apoptotique no guteza imbere ikwirakwizwa ry'uturemangingo (Ishusho 5f).Kwiyongera kwa DARS-AS1 byagaragaye mu bwoko butandukanye bwa kanseri, byerekana ko umurimo wacyo wo guteza imbere ingirabuzimafatizo za kanseri mu bihe bigoye ushobora gukoreshwa cyane mu bwoko bwa kanseri.
PACT yamenyekanye nka poroteyine ikora ya PKR, kandi ibikorwa bya PKR byunganirwa na PKR bigira uruhare runini mugukemura ibibazo muguhindura transcript, ibisobanuro, apoptose, nibindi bikorwa byingenzi bya selile62.Mu myaka ibarirwa muri za mirongo, hageragejwe gusobanukirwa amabwiriza yihariye ya kanseri ya casade ya PACT-PKR.Hano, ubushakashatsi bwacu bwerekanye ubundi buryo bwo kugenzura PACT-PKR mu ngirangingo za kanseri binyuze mu ngirabuzimafatizo ya lncRNA DARS-AS1, ihuza byimazeyo na PACT, ikabuza imikoranire ya PACT-PKR, ikabuza gukora PKR na fosifori ya eIF2α, bityo ikabuza apoptose iterwa no guhangayika na gukangura amaherezo ya kanseri.selile.Ubu buvumbuzi butanga ibisobanuro kuri lncRNA yibasirwa na kanseri yo kuvura no kuvura.
Amakuru yacu yerekanaga ko DARS-AS1 knockdown ikangurira selile inzara ya serumu hamwe no kwiyongera cyane kwa fosifori PKR na eIF2α.Ibisubizo byerekana ko DARS-AS1 iteza kanseri ya kanseri kubaho mubihe bibi muguhagarika ibikorwa bya PACT / PKR.Izindi RNA nyinshi zidafite code, nka ASPACT na nc886, nazo zigira uruhare mu murongo wa PACT / PKR mu kugabanya PACT48 mRNA cyangwa kugenzura autofosifora ihuza PKR 49,50,64.Muri bo, DARS-AS1 ikora nk'ihungabanya ishyirahamwe PACT-PKR.Ubu bushakashatsi butwongerera gusobanukirwa amabwiriza ya PACT / PKR hamwe nuruhare rwa lncRNAs mugusubiza ibibazo.
PACT ikubiyemo domaine eshatu zitandukanye.Buri kimwe muri bibiri bya mbere dsRBDs birahagije kugirango umuntu agere kuri PACT kuri PKR, mugihe domaine ya gatatu (D3) isabwa kugirango PKR ikore muri vitro no muri vivo.Ubushakashatsi bwacu bwerekanye ko DARS-AS1 ikunda gukorana na D3 (Ishusho 3h).Urebye ubunini bunini bwa lncRNA (768 nucleotide), DARS-AS1 ihuza D3 irashobora kubuza umubiri guhuza imikoranire hagati ya PACT ya dsRBD na PKR, bityo bikabuza ishyirahamwe rya PACT na PKR.Ihinduka rya PACT ryasimbuye Ser246 na Ser287 muri D3 hamwe na alanine cyangwa aspartate byahagaritse guhuza kwa DARS-AS1, byerekana akamaro ka D3′s muri rusange imiterere n’amashanyarazi mu ishyirahamwe ryabo.Ibisobanuro birambuye kuri ubu buryo bizakenerwa mugihe kizaza, ukoresheje isesengura ryibinyabuzima risobanutse neza hamwe n’ibisubizo bihanitse bya PACT.
Ubushakashatsi bwibanze bwerekanye ko DARS-AS1 iteza ikwirakwizwa ry'utugingo dukoresheje uburyo bwinshi 51,52,53.Urugero rumwe, abashakashatsi babonye ko DARS-AS1 yagenzuye poroteyine ya antisense ikubiyemo DARS gene yibasira miP-194-5p mu ngirabuzimafatizo za kanseri y'impyiko.Nyamara, muri ubu bushakashatsi, gukubita DARS-AS1 ntacyo byagize ku kwanduza DARS mu bwoko butandukanye bwa kanseri, harimo byibura kanseri yibara, amabere, n'umwijima.Kuberako lncRNAs yerekana uburyo bwihariye bwo kwerekana imiterere ya selile na tissue, uburyo bwimikorere ntibushobora kubikwa muburyo bwa kanseri, bushobora kugira uruhare muri uku kunyuranya hagati yibyo twabonye hamwe nisuzuma ryambere rya kanseri zitandukanye.Ubushakashatsi bwihariye burakenewe kugirango dusobanure uburyo bwihariye bwimikorere itandukanye ya physiologique na patologi.
Isesengura ry’amakuru y’ubuvuzi ryerekanye ko imvugo ya DARS-AS1 mu bibyimba ifitanye isano ridasanzwe n’ubuzima bw’abarwayi ba kanseri, ibyo bikaba bishimangira akamaro ka DARS-AS1 / PACT / PKR mu kumenyekanisha kanseri.Mu gusoza, ubushakashatsi bwacu bwerekana ko DARS-AS1 igenzura umurongo wa signal ya PACT / PKR, igatera ikwirakwizwa rya kanseri, kandi ikabuza apoptose mugihe cyo gukemura ibibazo, itanga undi murongo wubushakashatsi kandi ishishikajwe nubushakashatsi buzaza kubuvuzi bushobora kuvurwa .
Imirongo y'utugari ya muntu SW620, A549, MBA-MD-231, HCT116, HepG2 na HEK293T yakuwe mu bikorwa remezo by'imitungo y'igihugu mu Bushinwa.Ingirabuzimafatizo zose zabungabunzwe mu buryo bwa DMEM (DMEM, Thermo Fisher Scientific, Waltham, MA) hiyongeraho 10% FBS (Gemini, Brooklyn, NY) na 1% penisiline-streptomycine (Thermo Fisher Scientific) kuri 37 ° C, 5% CO2.incubator.
Kurwanya PACT, Abcam (ab31967);Kurwanya PKR, Abcam (ab184257);Kurwanya PKR (phospho-T451), Abcam (ab81303);Kurwanya Ibendera, Abcam (ab125243);Kurwanya eIF2α, Abcam (A0764));anti-eIF2α (fosifore S51), Abcam (ab32157);kurwanya PACT (fosifore S246), Abgent (AP7744b);anti-tub-tubuline, CST (2128);imbeba isanzwe IgG, CST (5415S);urukwavu rusanzwe IgG, CST (2729S).Antibodies zavomwe 1: 1000 muri PBST kugirango blotting yiburengerazuba na 1: 100 kuri IP.
sgRNAs yakozwe hifashishijwe igikoresho kiboneka kumugaragaro cyitwa CRISPR-ERA66.Twakoresheje ibipimo byabikoresho bisanzwe kugirango iterambere rya sgRNA hamwe na algorithm yabazwe sgRNA ihuza imbuga mukarere ka 3 kb.Hagati kuri TSS.Ibidengeri bya sgRNA oligonucleotide byakoreshwaga muri CustomArray, Inc.Igiteranyo cya 12 µg cyahujwe na pgRNA plasmid yumuntu, 7.2 µg ya psPAX2 (Addgene # 12260), na 4.8 µg ya pMD2.G (Addgene # 12259) bahujwe muri 5 x 106 HEK293T mumasahani ya cm 10 bakoresheje ADN ya Transfect Reagent. selile (CWBIO, Beijing, Ubushinwa) ukurikiza amabwiriza yabakozwe.Indwara ndengakamere zirimo virusi zegeranijwe nyuma yamasaha 48 na 72 nyuma yo kwandura hanyuma zungururwa binyuze muri 0.45 µm muyunguruzi.Kugenzura, selile SW620 yerekana proteine ​​ya dCas9 / KRAB yabonetse hakoreshejwe virusi.Ingirabuzimafatizo za SW620 zahinduwe n’isomero rya virusi mu bushakashatsi bune bwigenga bwanduye kuri MOI ya 0.1-0.3 hanyuma bapimwa na 2 μg / ml puromycine (Sigma, St. Louis, MO) iminsi 2.Nyuma yaho, selile zimaze iminsi 18 zimico muri vitro zifite byibuze isomero rifite selile 500 / sgRNA yo gusuzuma.
ADN yakuweho hakurikijwe amabwiriza ya QIAamp DNA Blood Midi Kit (QIAGEN, Düsseldorf, Ubudage; 51183).Muri rusange, 100 μg za ADN ya genomic kuri buri biologiya yakoreshejwe mu kubaka isomero.Agace ka sgRNA kongerewe ibyiciro bibiri bya PCR kandi bihuzwa na barcode.
Ibicuruzwa bya PCR byahanaguwe hifashishijwe ibikoresho bya NucleoSpin® hamwe na PCR yoza (MACHEREY-NAGEL, Düren, Ubudage; 740609.250) kandi byapimwe hifashishijwe Qubit ™ HS ibikoresho bibiri byerekana ADN (Thermo Fisher Scientific; Q32854).
Isuzuma rya MTS ryakoreshejwe mu gupima ikwirakwizwa ry'utugari.Ingirabuzimafatizo zabibwe mu masahani 96-yuzuye ku ntangiriro ya selile 2000 / iriba.Umubare ugereranije w'utugingo twapimwe buri munsi mugihe cyerekanwe muminsi 4-6.Kuri buri riba, 20 μl ya reagent ya MTS (Promega) yavanze na 100 μl ya DMEM, yinjizwamo selile kuri 4 h kuri 37 ° C, hanyuma hapimwa OD490.
Ubushobozi bwo gukura budafatika bwavumbuwe no gusesengura imiterere.Muri make, selile 2000 zandujwe na shRNA DARS-AS1 cyangwa kugenzura shRNA zarezwe muri microplate ultra ultra attachment (Corning) hamwe nimpinduka ziciriritse buri minsi 4.Spheroide yabazwe nyuma yiminsi 14.Ingirabuzimafatizo 500 zahinduwe hamwe na DARS-AS1 plasmid ikabije cyangwa plasmid yo kugenzura yakoreshejwe mugutezimbere, naho ubundi uburyo ntabwo bwahindutse.
RNA yandukuwe hakoreshejwe T7 RNA polymerase na biotin-16-UTP (Roche 1138908910) ukurikije amabwiriza ya Riboprobe® Combination Systems (Promega P1440).Intangiriro zikoreshwa hano ziri kurutonde rwinyongera 4.
Intungamubiri za poroteyine PACT cyangwa PKR zashyizwe muri pET15b (Addgene # 73619) zihinduka BL21 (DE3).Indwara ya bagiteri yaraye ibaye muri LB itangwa na ampisilline hanyuma ikongerwaho inshuro 100 hamwe na LB.Iyo OD600 yikigereranyo igeze 0.8, 1 mM IPTG yongewemo kugirango itere proteine ​​imvugo.Nyuma yubushakashatsi bwijoro hamwe no kunyeganyega byoroheje (250 rpm kuri 20 ° C), pellet selile yakusanyirijwe hamwe na centrifugation (4000 rpm, 10 min, 4 ° C).Hagarika pellet selile muri lysis buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 1 mM PMSF) hanyuma ushire ku rubura kuminota 30, hanyuma sonicate (15 min, 5 s kuri / kuzimya, kurubura) na centrifuge (13,000 rpm)., Iminota 30, 4 ° С).Supernatant noneho yapakiwe kuri resin ya Ni-NTA (QIAGEN) inshuro 3 kuri 4 ° C, yozwa inshuro 4 hamwe na bffer yo gukaraba (50 mM Tris, pH 8.0, 40 mM imidazole, 250 mM NaCl) hanyuma isohoka inshuro 3, zose hamwe ya ml 10 ya eluent buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 300 mM imidazole).Poroteyine isukuye yagenwe hakoreshejwe WB kandi kwibandaho byagenwe hakoreshejwe Qubit ™ protein assay kit (Thermo Fisher Scientific; Q 33212).
RIP isuzuma ryakozwe nkuko byasobanuwe mbere, hamwe no guhindura.Muri make, buffer 1x RIP (25 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5% NP-40, inhibitor ya RNasin ribonuclease (Promega), PMSF (Beyotime Biotechnology), 1 mM DDM, protease) lyses cytostatic 1 x 107 cocktail (Roche, 1 mM DTT) na centrifuge kuri 13,000 rpm kuminota 15 kuri 4 ° C.Supernatant noneho yashizwemo proteine ​​A + G ya magnetiki (Millipore) ihujwe na 5 μg za antibody anti-PACT (Abeam) cyangwa IgG (CST).Isaro yogejwe inshuro 5 hamwe na 5x RIP buffer, hanyuma igogorwa na proteinase K (NEB).RNA yakuwe hamwe na Trizol kandi igenwa na RT-qPCR.Ibanze bitangwa mu mbonerahamwe ya 5.
Muri vitro RIP isuzuma ryakozwe ukurikije protocole isanzwe ya RIP.Igiteranyo cya saa kumi n'imwe z'umugoroba muri vitro yanditswemo RNA yavomwe 1x hamwe na RIP buffer hanyuma igashyirwa hamwe na incubation kuri 65 ° C muminota 5 ikurikirwa no gukonja buhoro kugeza ubushyuhe bwicyumba.Igiteranyo cya saa kumi n'imwe z'umugoroba za poroteyine za PACT zuzuye cyangwa zahinduwe neza zahanaguwe kuri E. coli.Ongera hamwe na RNA yahinduwe mumasaha 2 kuri 4 ° C hanyuma ukurikize inzira yavuzwe haruguru yo gusesengura RIP kuri anti-flag IP.
Kubisesengura rya RNA, selile 1 × 107 zashyizwe hamwe na 1xRIP buffer.Nyuma ya centrifugation kuri 13,000 rpm kuminota 15 kuri 4 ° C, ndengakamere yabanjirijwe hamwe na 30 μl z'amasaro ya magnetiki ya streptavidin (Beckman) kuri 2 h kuri 4 ° C.Lysate yatunganijwe noneho yahawe umusemburo tRNA hanyuma ushyirwamo 40 pmol ya RNA yahinduwe ijoro ryose kuri 4 ° C, hanyuma andi masaha 2 na 20 μl z'amasaro mashya ya streptavidin yamashanyarazi yafunzwe na BSA hiyongeraho.Intambwe yo gukaraba yari igizwe ninshuro 4 hamwe na 5x RIP buffer ninshuro 4 hamwe na 1x RIP buffer.Poroteyine zihuye nazo zashyizwe hamwe na bffer yo gukuraho biotine (25 mM Tris-HCl, pH 7.5, 12.5 mM D-biotine, PMSF) hanyuma itandukana kuri NuPAGE 4-12% Bis-Tris Gel (Invitrogen).Nyuma yo gusiga ifeza (Beyotime Biotechnology), imirwi imwe yacukuwe kandi isesengurwa na MS.
Isesengura rya IP ryakozwe kugirango hamenyekane imikoranire hagati ya PACT na PKR.Muri make, lysates ndengakamere yateguwe mugushiramo ingirabuzimafatizo 1 x 107 muri buffer 1 x RIP ikurikirwa na centrifugation saa 13,000 rpm muminota 15 kuri 4 ° C.Lysates yari yuzuye proteine ​​A + G ya magneti, ifatanije na 5 µg ya antibody anti-PACT, hanyuma izunguruka buhoro buhoro ijoro ryose kuri 4 ° C.Amasaro yogejwe inshuro 3 hamwe na 5 × RIP buffer, kabiri hamwe na 1 × RIP buffer hanyuma ushyirwa hamwe na 1 × SDS.Poroteyine yagaruwe yasesenguwe na gel SDS-PAGE hanyuma igaragazwa na WB.
Saa mbiri za mugitondo za PACT na 1 pmol ya PKR zahanaguwe kuri E. coli.Koresha muri 1 × RIP buffer hanyuma ushire hamwe na 10 pmol ya RNA yahinduwe mumasaha 2 kuri 4 ° C.Nyuma yibyo, bashizwemo poroteyine A + G ya magnetiki isaro-conjugated anti-label antibody yongeyeho amasaha abiri.Amasaro noneho yogejwe inshuro enye hamwe na 1x RIP buffer hanyuma ushyirwa hamwe na 1x SDS.Ibisubizo PACT na PKR byagaragaye na WB.