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Lisele tsa mofets'e li fetotse mekhoa e fapaneng ea ho hlola khatello ea maikutlo le ho tsoela pele ho tsoela pele.Protein kinase R (PKR) le protheine activator (PACT) ke ba arabelang pele ba hlokomelang matšoao a fapaneng a khatello ea maikutlo a lebisang ho thibelang ho ata ha lisele le apoptosis.Leha ho le joalo, taolo ea tsela ea PACT-PKR liseleng tsa mofetše e ntse e sa tsejoe haholo.Mona, re fumane hore nako e telele e se nang khoutu ea RNA (lncRNA) aspartyl tRNA synthetase antisense RNA 1 (DARS-AS1) e ameha ka ho toba ho thibela tsela ea PACT-PKR le ho khothalletsa ho ata ha lisele tsa kankere.Re sebelisa tlhahlobo e kholo ea ts'ebetso ea CRISPRi 971 lncRNA e amanang le mofets'e, re fumane hore DARS-AS1 e amahanngoa le keketseho e matla ea lisele tsa mofetše.Ka hona, DARS-AS1 knockout e thibela ho ata ha lisele le ho khothaletsa apoptosis ea mofetše meleng e fapaneng ea lisele tsa mofetše in vitro mme e fokotsa haholo kholo ea hlahala ho vivo.Ka mokhoa, DARS-AS1 e tlama ka ho toba sebakeng sa ts'ebetso ea PACT mme e thibela ho sebelisana ha PACT-PKR, kahoo e fokotsa ts'ebetso ea PKR, eIF2α phosphorylation, le ho thibela lefu la apoptotic cell.Ho latela maemo a bophelo, DARS-AS1 e hlahisoa ka bongata mefuteng e mengata ea mofetše, 'me ho feteletsa ha lncRNA ena ke sesupo sa bofokoli bo bobe.Phuputso ena e hlakisa taolo e tobileng ea mofetše ea tsela ea PACT-PKR ka DARS-AS1 lncRNA mme e fana ka sepheo se seng bakeng sa prognoza le kalafo ea mofetše.
Bokhoni ba ho ikamahanya le khatello ea maikutlo ke tšobotsi ea bohlokoa ea ho phela ha lisele tsa mofetše le ho ata.Ho ata ka potlako le matšoao a metabolic a tlhoro ea mofets'e maemong a manyane - khaello ea limatlafatsi, hypoxia, le pH e tlase - e ka bakang litsela tsa matšoao a lefu la lisele.Ho senyeha ha liphatsa tsa lefutso tse nang le khatello ea kelello tse kang p535, liprotheine tsa mocheso oa mocheso 6, 7, KRAS8, 9, le HIF-110, 11, 12, 13 hangata li bonoa mofetšeng, kahoo li thibela apoptosis le ho khothalletsa ho pholoha.
Protein kinase R (PKR) ke sensor ea bohlokoa ea khatello ea maikutlo le subunit kinase ea eukaryotic initiation factor 2α (eIF2α), molaoli oa phetolelo o kopanyang khatello ea cellular le lefu la sele.PKR qalong e ile ea tsejoa e le protheine e thibelang likokoana-hloko ka ho fumanoa ha RNA (dsRNA) e tsoang kantle ho naha e nang le likhoele tse peli.Ha e sebelisoa, PKR phosphorylates eIF2α ho thibela likokoana-hloko le liprotheine tsa cellular synthesis14,15,16.PACT (PKR activator protein) e khethiloe e le protheine ea pele ea activator ea PKR ha ho se na dsRNA17,18,19,20,21,22,23.Ka ho sebelisana ka ho toba le PKR, PACT e fetisa likhatello tse fapa-fapaneng (tlala ea serum, phekolo ea peroxide kapa arsenite) ho PKR le litsela tsa matšoao a tlaase.Ntle le phosphorylation ea eIF2α, ts'ebetso ea PACT-mediated PKR e baka liketsahalo tse fapaneng tse amanang le karabelo ea khatello ea maikutlo, ho kenyelletsa le maemo a fetotsoeng a redox ka tsela ea PI3K/Akt24, ho hlahlojoa ha tšenyo ea DNA ka p5325,26 le NF-κB27,28 E laola ho ngoloa, 29. Ka lebaka la karolo ea bona ea bohlokoa karabelong ea khatello ea maikutlo, ho ata, apoptosis le lits'ebetso tse ling tsa bohlokoa tsa cellular, PKR le PACT li ts'episa lipheo tsa kalafo bakeng sa mafu a mangata, haholo-holo mofets'e30,31,32,33.Leha ho le joalo, leha ho na le ts'ebetso ena ea pleiotropic le bohlokoa ba tlhaho, taolo ea ts'ebetso ea PACT / PKR liseleng tsa mofetše e ntse e le thata.
Li-lncRNA ke lingoloa tse kholo ho feta li-nucleotide tse 200 tse se nang bokhoni ba ho khouta protheine.Ho tloha ha merero e tsoetseng pele ea tatellano ea liphatsa tsa lefutso e bonts'itse likete tsa li-lncRNA, 35,36 ho entsoe boiteko bo boholo ho hlakisa mesebetsi ea bona ea tlhaho.Sehlopha se ntseng se hola sa lipatlisiso se bontšitse hore li-lncRNA li ameha mekhoeng e mengata ea likokoana-hloko37 ho kenyelletsa le taolo ea X-chromosome inactivation38,39, imprinting40, transcription41,42, translation43 esita le kholo ea kankere44,45,46,47.Lithuto tsena li tlalehile hore li-lncRNA tse ngata li ameha tseleng ea PACT/PKR.Phuputso e 'ngoe e joalo e bontšitse hore lncRNA ASPACT e thibela ho ngoloa ha PACT le ho eketsa ho boloka nyutlelie ea PACT mRNA.Liphuputso tse ling li bontšitse hore lncRNA nc886 e tlama ho PKR mme e thibela phosphorylation49,50 ea eona.Ho fihla hajoale, lncRNA e laolang ts'ebetso ea PACT-mediated PKR ha e so tlalehe.
Aspartyl-tRNA synthetase antisense RNA 1 (DARS-AS1) e khethiloe e le lncRNA51,52,53,54 ea oncogenic.Ka taolo ea miP-194-5p53, miP-12952 le miP-532-3p51, DARS-AS1 e bontšitsoe ho khothalletsa kholo ea lisele tse hlakileng tsa renal cell carcinoma, thyroid carcinoma le non-small cell lung carcinoma, ka ho latellana.Tong le basebetsi-'moho le bona ba fumane hore DARS-AS1 e khothalletsa tsoelo-pele ea myeloma ka ho boloka botsitso ba protheine 39 (RBM39) RNA-binding motif.Leha ho le joalo, ha ho liphuputso tse entsoeng mabapi le hore na lncRNA ena e ameha taolong ea ts'ebetso ea PACT-PKR le karabelo ea khatello ea kelello ea lisele tsa mofetše.
Mona, re ile ra etsa skrine e kholo ea tahlehelo ea ts'ebetso re sebelisa tsamaiso ea CRISPRI mme ra etsa qeto ea hore DARS-AS1 lncRNA e khothalletsa ho ata ha mefuta e mengata ea lisele tsa kankere.Ho phaella moo, re khethile mokhoa o moholo: DARS-AS1 e tlama ka ho toba ho PACT, e thibela ho tlama PACT le PKR, ho thibela phosphorylation ea eIF2α, substrate e tlaase ea PKR, 'me qetellong e thibela lefu la apoptotic cell.Qetellong, mosebetsi oa rona o senola DARS-AS1 lncRNA e le molaoli oa tsela ea PACT-PKR le sepheo se ka 'nang sa e-ba teng bakeng sa phekolo ea kankere le prognoza.
Liphuputso tse pharalletseng tsa profiling ea genomic li bonts'itse makholo a li-lncRNA tse amanang le mofetše.Leha ho le joalo, mosebetsi oa bona o lula o sa tsejoe haholo56.Ho tsebahatsa bakhethoa ba lncRNA ba ts'episang ba amehang ts'ebetsong ea mofets'e, re ile ra etsa skrine ea ho lahleheloa ke mosebetsi bakeng sa ho fokotsa ho ata ha lisele tsa kankere ea SW620 ho sebelisa tsamaiso ea CRISPRI (Fig. 1a).Tšobotsi e ikhethang ea lisele tsa mofetše oa SW480 le SW620 ke hore li tsoa ho lihlahala tsa mantlha le tsa bobeli ho mokuli a le mong.Sena se fana ka papiso ea bohlokoa bakeng sa ho ithuta liphetoho tsa liphatsa tsa lefutso tsoelo-peleng ea mofetše o tsoetseng pele oa mala.Ka hona, re ile ra hlahlobisisa li-transcriptomes tsa lisele tsa mofetše oa colorectal (SW480 le SW620) re sebelisa tatellano ea RNA mme ra bokella li-lncRNA tse ka sebetsang ho tsoa lingoliloeng tse hatisitsoeng.Ho ipapisitsoe le liphetho tsena, re thehile laeborari e kopaneng ea sgRNA e nang le 7355 sgRNA oligos e shebileng li-lncRNA tse amanang le mofets'e tse 971 le li-oligo tse 500 tse sa rereloang tsa sgRNA bakeng sa taolo e mpe (Supplementary Data 1).
Kemiso e hlophisitsoeng ea ho hlahloba ho sebelisa tsamaiso ea CRISPRI.b sgRNA ho ntlafatsa ka mor'a ho hlahlojoa.Mohala o matheba o otlolohileng o emela log2 (phetoho e mene) = ± 0.58.Mohala o matheba o otlolohileng o bontša boleng ba p = 0.05.Matheba a matšo a emela sgRNA e sa lebelloang (e khethiloeng e le NC).Matheba a mafubelu ke li-sgRNA tse lebisitseng DARS-AS1.Matheba a maputsoa ke li-sgRNA tse shebileng LINC00205, lncRNA e neng e hlalositsoe pejana.phetolo ya mena = (ho bala ka tlwaelo, letsatsi la 17)/(ho bala ka tlwaelo, letsatsi 0).c DARS-AS1 sgRNA e thibela kholo ea lisele.Melallo ea liphoso e emela ± kheloha e tloaelehileng ea liteko tse tharo.* p ≤ 0.05, ** p ≤ 0.01 teko ea t ea Moithuti ea nang le mehatla e 'meli.d Polelo ea DARS-AS1 ho li-tumor (TCGA dataset).em Pontšo ea DARS-AS1 ka lisampole tse kopantsoeng tse tloaelehileng le tsa tumor ho tsoa ho bakuli ba nang le BLCA, KIRC, PRAD, LUSC, UCEC, LUAD, LIHC, KIRP, le COAD, ka ho latellana (TCGA dataset).litekanyetso tsa p li fumanoe ho sebelisoa tlhahlobo ea t ea Moithuti e nang le mehatla e 'meli.
Kamora ho theha plasmid le ho paka lentivirus, re ile ra fetisa mohala oa sele ea mofetše oa dCas9-SW620 le laeborari e kaholimo litekong tse 'ne tse ikemetseng tsa ts'oaetso.Multiplicity of infection (MOI) bakeng sa ts'oaetso ena e ne e le 0.1–0.3, e leng se bontšang hore sele ka 'ngoe e ka fetisoa feela ka sgRNA e le 'ngoe.Kamora matsatsi a 18 a setso sa in vitro, profil ea ntlafatso ea target sgRNAs e fokotsehile kapa ea eketseha kamora ho hlahlojoa, athe palo ea li-oligonucleotide tse sa lebelloang li ile tsa lula li sa fetohe ha li bapisoa le profil ea pele ho tlhahlobo, e bonts'a hore sepheo sa rona se na le skrine e khethehileng haholo. laeborari.Raese.1b le tafole ea tlatsetso 1). LINC00205, eo pele ho neng ho tlalehoa hore e khothalletsa kankere ea matšoafo le tsoelo-pele ea kankere ea sebete58,59,60, e ile ea hlahlojoa (log2 (foldchange) <-0.58, p value <0.05), e tiisang ho tšepahala ha tlhahlobo ena (Fig. 1b). LINC00205, eo pele ho neng ho tlalehoa hore e khothalletsa kankere ea matšoafo le tsoelo-pele ea kankere ea sebete58,59,60, e ile ea hlahlojoa (log2 (foldchange) <-0.58, p value <0.05), e tiisang ho tšepahala ha tlhahlobo ena (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, e tlalehiloeng pele e khothalletsa tsoelo-pele ea kankere ea matšoafo le kankere ea sebete58,59,60, e ne e sa kenyelelitsoe (log2 (phetoho e fokolang) <-0.58, p-value <0.05), e tiisang ho tiea ha tlhahlobo ena (Fig. .1b) . Linc00205 之前 被 报道 可 促进 促进 肝癌 肝癌 进展 进展 58,59,60, 被 筛 选 (证实 变化). Linc00205 之前 被 报道 可 促进 促进 肝癌 肝癌 进展 进展 58,59,60, 被 筛 选 (证实 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, eo pele e neng e tlalehoa hore e khothalletsa tsoelo-pele ea kankere ea matšoafo le sebete58,59,60, e ne e sa kenyelletsoa (log2 (fetola phetoho) <-0.58, p-value <0.05), e tiisang ho tiea ha tlhahlobo ena (Fig. .1b).
Har'a li-lncRNA tsohle tse lekiloeng, DARS-AS1 e ile ea boela ea hlahlojoa, 'me li-oligonucleotide tse tharo tsa cognate sgRNA li fokotsehile haholo ka mor'a matsatsi a 18 a setso, ho fana ka maikutlo a hore ho kokota ha lncRNA ena ho ile ha fella ka ho fokotsa ho ata ha kankere (setšoantšo sa 1b).Sephetho sena se ile sa tšehetsoa hape ke tlhahlobo ea MTS liseleng tsa mofetše oa colorectal e bonts'ang hore sekhahla sa kholo ea lisele tsa DARS-AS1 se ne se le halofo feela ha se bapisoa le lisele tsa taolo (setšoantšo sa 1c) mme se ne se lumellana le litlaleho tse fetileng tsa mefuta e meng e mengata ea mofetše.: kankere ea liphio e hlakileng ea lisele, kankere ea qoqotho le kankere ea matšoafo ea sele e seng e nyenyane51,52,53,55.Leha ho le joalo, ts'ebetso ea eona le mekhoa ea limolek'hule ho mofetše oa colorectal li ntse li sa tsejoe.Ka hona, re khethile lncRNA ena bakeng sa boithuto bo eketsehileng.
Ho ithuta polelo ea DARS-AS1 ho bakuli, re hlahlobile ka botlalo lisampole tse 10,327 tsa tumor ho tsoa morerong oa Cancer Genome Atlas (TCGA).Liphetho tsa rona li bonts'a hore DARS-AS1 e hlahisoa ka bongata ebile e laoloa haholo liseleng tse phetseng hantle ka har'a lihlahala tse fapaneng, ho kenyeletsoa colon adenocarcinoma (COAD), renal clear cell carcinoma (KIRC), le renal papillary cell carcinoma (KIRP)..E fokolang haholo (setšoantšo sa 1d le sa Tlatsetso sa 1a, b). Tlhahlobo ea lisampole tse pentiloeng tse phetseng hantle / tsa hlahala li netefalitse polelo e phahameng haholo ea DARS-AS1 ka har'a lihlahala tsa senya urothelial carcinoma (BLCA), renal clear cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), lung squamous cell carcinoma (LUSC) , uterine corpus endometrial carcinoma (UCEC), lung adenocarcinoma (LUAD), sebete sa hepatocellular carcinoma (LIHC), renal renal papillary cell carcinoma (KIRP), le colon adenocarcinoma (COAD) (p value <0.05) (Fig. 1e-m) . Tlhahlobo ea lisampole tse pentiloeng tse phetseng hantle / tsa hlahala li netefalitse polelo e phahameng haholo ea DARS-AS1 ka har'a lihlahala tsa senya urothelial carcinoma (BLCA), renal clear cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), lung squamous cell carcinoma (LUSC) , uterine corpus endometrial carcinoma (UCEC), lung adenocarcinoma (LUAD), sebete sa hepatocellular carcinoma (LIHC), renal renal papillary cell carcinoma (KIRP), le colon adenocarcinoma (COAD) (p value <0.05) (Fig. 1e-m) .Litlhahlobo tsa lisampole tse pentiloeng tse phetseng hantle / tsa hlahala li boetse li netefalitse polelo e phahameng haholo ea DARS-AS1 ho bladder urothelial carcinoma (BLCA), clear cell renal le renal cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), lung squamous cell carcinoma (LUSC) lihlahala., 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 ea corpus uteri (UCEC), adenocarcinoma ea matšoafo (LUAD), hepatocellular carcinoma ea sebete (LIHC), papillary cell carcinoma ea liphio (KIRP), le adenocarcinoma ea colon (COAD) (p value < 0.05) (Setšoantšo sa 1e- m) .配对 健康 / 的 的 分析 了 Dars-as1 在 皮癌 尿路 皮癌 皮癌 皮癌 皮癌 皮癌 皮癌 皮癌显着显着 高 表达, 子宫体子宫子宫体子宫 内膜 (Ucec), 肺腺癌 (luad), 肝肝 细胞癌 细胞癌 乳头状<0.05（图1e-m)配配 健康 健康 健康 / T »Os1 了 了 了 了皮癌 上(lusc) 癌 癌 癌 肺腺癌 肺腺癌 细胞癌 细胞癌 细胞癌, lihc) 肾 肾 肾 肾 肾癌 (kirp) (coad) (p 值<0.05) (图1e-m) .Litlhahlobo tsa lisampole tse kopantsoeng hantle le hlahala li ile tsa tšehetsa karolo ea DARS-AS1 ho bladder urothelial carcinoma (BLCA), clear cell renal cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), le lung squamous cell carcinoma (LUSC) tumors.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). polelo ka corpus uterine carcinoma (UCEC), lung adenocarcinoma (LUAD), hepatocellular carcinoma (LIHC), renal papillary cell carcinoma (KIRP), le colon adenocarcinoma (COAD) (p boleng <0.05) (Setšoantšo sa 1e -m).Ha li kopane, liphetho tsena li bonts'a hore DARS-AS1 e hlahisoa haholo mefuteng e fapaneng ea mofetše.
Hobane DARS-AS1 le DARS (mofuta o kenyang khoele ea antisense) li arolelana motšehetsi ea tšoanang 'me li haufi le tse ling, re thehile shRNA ho kokota ka ho khetheha DARS-AS1 empa eseng DARS (Supplementary Fig. 2a,b le Supplementary Table 2) .Ntle le SW620, re sebelisitse mela e meng e meraro ea lisele tse hlalosang haholo DARS-AS1 ho ithuta ka katleho le ts'ebetso ea shRNA knockdown (Letlapa la Tlatsetso 3).Liphetho tsa rona li bonts'itse hore li-shRNA tse tharo tse ntlafalitsoeng li fihletse bonyane 80% DARS-AS1 katleho ea ho kokota ka phello e nyane ho palo ea DARS mRNA (Supplementary Fig. 2c–f).Ntle le moo, re fumane hore DARS-AS1 knockdown ka li-shRNA tsena e ile ea thibela kholo ea lisele ho mela ea lisele tsa colorectal SW620 (49.7%) le HCT116 (27.7%), mohala oa sele ea mofets'e MBA-MD-231 (53.4%).) le HepG2 hepatoma cell line (ho fokotseha ha 92.7%), hammoho le bokhoni ba bona ba ho theha likarolo tse sa tsitsitseng (phokotso e tloaelehileng ea ~ 50.8%, 44.6%, 40.7% le 75.7% ka selefouno) (Setšoantšo sa 2a, b).Ho SW620, liphetho tsa tlhahlobo ea sebopeho sa kolone li netefalitse hape hore DARS-AS1 shRNA e thibetse haholo ho ata ha lisele ka phokotseho e ka bang 69.6% (Fig. 2c).
Phello ea taolo ea shRNA le DARS-AS1 shRNA ho ata ha lisele (a) le sebopeho sa spheroid (b) ho SW620, HCT116, MBA-MD-231, le lisele tsa HepG2.c Phello ea taolo ea shRNA le DARS-AS1 shRNA ho thehoa ha kolone liseleng tsa SW620.Ho ata ha lisele (d), sebopeho sa spheroid (e), le sebopeho sa kolone (f) ea lisele tsa SW620 tse fetelletseng DARS-AS1.Lintlha tse bontšitsoeng ke moelelo ± kheloha e tloaelehileng ea liteko tse tharo.* p ≤ 0.05, ** p ≤ 0.01, le *** p ≤ 0.001 ka tlhahlobo ea t ea Moithuti ea nang le mehatla e 'meli.
Ho tlatsana le lithuto tsa tahlehelo ea ts'ebetso, ka mor'a moo re thehile lisele tsa SW620 tse fetang DARS-AS1 (Supplementary Fig. 2g).DARS-AS1 overexpression e ile ea eketsa haholo kholo ea lisele (1.8-fold), sebopeho sa spheroid se sa tsitsitseng (1.4-fold), le sebopeho sa kolone (3.3-fold) ka lisele tsa SW620 (Fig. 2d-f).Re netefalitse sephetho sena re sebelisa mohala o mong oa DARS-AS1 o hlalosang mohala, A549.Keketseho ena e ntlafetseng ea lisele ka lebaka la DARS-AS1 overexpression e ile ea boela ea hlokomeloa liseleng tsa A549 (Supplementary Fig. 2h, i le Supplementary Table 3).Ha li kopane, lithuto tsena tsa phaello le tahlehelo li bonts'a hore DARS-AS1 e khothalletsa ho ata ha lisele tsa mofetše ka har'a vitro.
Ho hlahloba mokhoa oa motheo oo DARS-AS1 e laolang ho ata ha lisele, re entse tlhahlobo ea ho theola RNA ho hlwaya balekane ba eona ba ka tlamang liprotheine.Liphetho tsa RT-qPCR li bontšitse hore hoo e ka bang 86.2% ea DARS-AS1 e fumaneha ka har'a cytoplasm ea lisele tsa SW620 (Supplementary Fig. 3a).The in vitro transcribed biotinylated DARS-AS1 kapa pseudoRNA e ile ea kenngoa ka SW620 cell lysates e lateloang ke karohano ea SDS-PAGE.Ho silafatsa silevera ka mor'a moo ho bontšitse hore sehlopha se ikhethileng (~ 38 kDa) se ruisitsoe haholo ho DARS-AS1 hula disampole empa eseng ka dummy RNA kapa lifaha disampole (Fig. 3a).Sehlopha sena se ile sa tsejoa e le PKR activating protein (PACT) by mass spectrometry (MS) 'me e netefalitsoe ka ho eketsehileng ke immunoblotting ka SW620, HCT116, le HepG2 lisele tsa lisele (Fig. 3a,b).Ho ntlafatsa DARS le liprotheine tse amanang le PACT - PKR le TRBP - le tsona li ile tsa fuputsoa ho sebelisoa tlhahlobo ea RNA ka Western blotting (WB).Liphetho li bontšitse hore ha ho na tšebelisano e tobileng pakeng tsa DARS-AS1 RNA le liprotheine tsena tse tharo tse fumanoeng (Supplementary Fig. 3b).Tšebelisano e khethehileng pakeng tsa DARS-AS1 le PACT e ile ea tiisoa ka ho eketsehileng ke tlhahlobo ea RNA immunoprecipitation (RIP), e bontšitseng hore DARS-AS1 e ne e ruisitsoe haholo ho li-anti-PACT antibodies empa eseng tse ling tsa RNAs tsa taolo (Setšoantšo sa 3c).Ho fumana hore na DARS-AS1 e sebelisana ka ho toba le PACT ha ho se na likarolo tse ling tsa cellular, tlhahlobo ea in vitro biolayer interferometry (BLI) e ile ea etsoa ho sebelisoa PACT e hloekisitsoeng.Biotin e ngotsoeng DARS-AS1 kapa dummy RNA e ne e sa sebetse ho li-biosensors tsa streptavidin (SA) ebe e kenngoa ka har'a kinetic buffer e nang le 1 μM PACT.Haholo-holo, PACT e hokahane ka matla ho DARS-AS1 (Boleng ba KD ~ 26.9 nM), empa eseng ho etsisa RNA (Setšoantšo sa 3d).Ha li kopane, liphetho tsena li bonts'a tšebelisano e tobileng le kamano e phahameng lipakeng tsa DARS-AS1 le PACT.
Tlhahlobo ea RNA e bonts'a hore DARS-AS1 e sebelisana le PACT liseleng tsa SW620.Ka holimo, litšila tsa silevera tsa liprotheine tse amanang.Li-immunoblots tse tlase li ne li etsoa ka anti-PACT antibody.b RNA tlhahlobo ea ho theola fatše e entsoe ka lisele tsa HCT116 (holimo) le HepG2 (ka tlaase).Matlafatso ea PACT e ile ea bonoa ka immunoblotting.Litlhahlobo tsa cRNA immunoprecipitation (RIP) li entsoe ka liseleng tsa SW620 ho sebelisoa li-antibodies tse bontšitsoeng.d PACT e tlamang li-curve ho bolelele bo felletseng ba DARS-AS1 kapa taolo ea RNA e fumanoe ho sebelisoa biolayer interferometry (BLI).RNA e ne e sa sebetse hantle ho streptavidin biosensor.1 μM PACT e ne e sebelisetsoa ho lekanya mokhatlo.e RNA hula assay e ne e etsoa ho sebelisoa biotinylated full-length DARS-AS1 kapa truncated (top).Immunoblot e bonts'ang PACT e amohetse (ka tlase).f Purified PACT e tšoailoeng e ne e kentsoe ka biotinylated full-length DARS-AS1 kapa e fokotsoe (joalokaha ho e) bakeng sa tlhahlobo ea in vitro RIP.RNA e nkiloeng e netefalitsoe ke RT-qPCR.g Kamano e amanang le likhechana tse fapaneng tsa RNA bakeng sa PACT e fumanoe ho sebelisoa biolayer interferometry.Bakeng sa tlhahlobo, 100 nM RNA le 1 μM RAST li sebelisitsoe.h Litlhahlobo tsa in vitro RIP li ne li etsoa ho sebelisoa purified intact kapa truncated e ngotsoeng PACT.RNA e nkiloeng e netefalitsoe ke RT-qPCR.i Sekhahla sa kholo ea lisele tsa SW620 tse fetang DARS-AS1, PACT, kapa ka bobeli.j Khatello e feteletseng ea bolelele bo felletseng kapa e fokolitsoeng DARS-AS1 ka liseleng tsa SW620 e bile le litlamorao tse fapaneng kholong ea lisele.k Apoptosis e ile ea bonoa ka ho thibela 'mele oa ho itšireletsa mafung ka anti-PARP.l Knockout ea DARS-AS1 e etsa hore ho be le apoptosis ea lisele tsa SW620 joalokaha ho bontšoa ke cytometry ea phallo.Lintlha tse bontšitsoeng ke moelelo ± kheloha e tloaelehileng ea liteko tse tharo. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p <0.0001, ka teko ea t ea Moithuti ea nang le mehatla e 'meli. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p <0.0001, ka teko ea t ea Moithuti ea nang le mehatla e 'meli. *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 ka teko ea t ea Moithuti ea nang le mehatla e 'meli. *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 ka teko ea t ea Moithuti ea nang le mehatla e 'meli.
Eaba re hlahisa likaroloana tse tharo tsa biotinylated DARS-AS1 RNA ka in vitro transcription ho khetholla sebaka sa DARS-AS1 se hlokahalang bakeng sa mokhatlo oa PACT (Setšoantšo sa 3e).Liphetho tsa tlhahlobo ea RNA li bontšitse hore sekhechana se seng le se seng se khonne ho sebelisana le PACT, empa sebaka sa 3'-terminal (384-768 nucleotides e ngotsoeng A3) se bontšitse ho feta 1-384 nucleotide e ngotsoeng A1) (Setšoantšo sa 3e).Liphetho tse ts'oanang li ile tsa bonoa tlhahlobong ea in vitro RIP ho sebelisa recombinant PACT (Setšoantšo sa 3f).Tumellanong le liphetho tsena, liteko tsa ho tlama likhechana tsa RNA tse sa sebetseng ho PACT li sebelisa BLI li boetse li bonts'itse hore PACT e na le kamano e phahameng bakeng sa A3 (384-768 nt) (Boleng ba KD bo ka bang 94.6 nM), ha e batla e se na likhokahano le libaka tse ling.(Setšoantšo sa 3d).
Hape re hlahlobile libaka tse tlamang tse amanang le PACT.PACT e na le libaka tse tharo tse sebetsang, tse peli tsa tsona e le libaka tse tlamang tsa RNA tse nang le mela e 'meli (dsRBD) le sebaka sa boraro (se khethiloeng D3) se sebetsang e le activator ea liprotheine.Ho hlahloba matla a tlamang a lncRNA sebakeng se seng le se seng, re ile ra etsa liphetoho tse tharo tse tlositseng sebaka se seng le se seng sa libaka tse tharo mme ra etsa tlhahlobo ea in vitro RIP.Liphetho tsa rona li bontšitse hore ho hlakoloa ha sebaka sa boraro (D3) sa PACT ho fokolitse haholo ho sebelisana ha eona le DARS-AS1 (ka 0.11-fold ha e bapisoa le PACT e tsitsitseng) ha e bapisoa le liphetoho tse ling tse peli (Fig. 3h), ho bontšitsoe hore tokollo. ea D3 e sebelisana le DARS.-AC1.Ha li kopane, liphetho tsena li fana ka maikutlo a hore tšebelisano pakeng tsa DARS-AS1 le PACT e ka etsahala haholo-holo ho fihlela qetellong ea 3′ ea DARS-AS1 le sebaka sa D3 sa PACT.
Re hlokometse hore DARS-AS1 ha e na phello ho polelo ea PACT le PACT ha e na phello ho DARS-AS1 (Supplementary Fig. 3c).Ka mor'a moo re ile ra hlahloba phello ea ho fokotseha ha PACT ho kholo ea lisele.Ho fapana le DARS-AS1, lisele tse amanang le tsona li ile tsa hōla ka makhetlo a 1.5-3 ka potlako ha PACT e kokota (Supplementary Fig. 3d).Liphello tsa tlhahlobo ea ho thehoa ha kolone li bontšitse hore lisele li thehile likolone tsa 2-3 ka mor'a phekolo ea shRNA le PACT (Supplementary Fig. 3e).Ho lekola hore na DARS-AS1 e laola ho ata ha lisele ka PACT, re hlahisitse lisele tsa SW620 overexpressing PACT, DARS-AS1, kapa ka bobeli.Tlhaloso e feteletseng ea PACT e bontšitse ho thibela ho hoholo ha lisele tsa lisele (Setšoantšo sa 3i).Le hoja DARS-AS1 overexpression per se e ile ea khothalletsa haholo ho ata ha lisele, ho ne ho se na phapang e khōlō ka sekhahla sa kholo ea lisele tse fetang DARS-AS1 le PACT.Liphetho tsena li fana ka maikutlo a hore PACT e ka hanana le keketseho e ntseng e eketseha e bakoang ke DARS-AS1 overexpression.
Kaha libaka tse fapaneng tsa DARS-AS1 li na le bokhoni bo fapaneng ba ho tlama PACT, re ile ra batlisisa tšusumetso ea bona e amanang le ho ata ha lisele ka litlatsetso tse fapaneng tse fapaneng tsa likhechana tsa DARS-AS1.Ha ho bapisoa le likaroloana tse ling tse peli, DARS-AS1 e ne e feteletse haholo qetellong ea 3 (384-768 nt), sebaka se ka sehloohong se amanang le PACT ho DARS-AS1, se neng se e-na le bokhoni bo phahameng ka ho fetisisa ba ho susumetsa ho ata ha lisele (Setšoantšo sa 3j).Liphetho tsena li bonts'a kamano e ntle lipakeng tsa matla a tlamang le ts'ebetso ea baeloji ea DARS-AS1.
PACT e tlalehiloe e le pro-apoptotic protein19.Ka hona, re ile ra batlisisa phello ea DARS-AS1 ho apoptosis.Joalo ka ha ho ne ho lebelletsoe, ho kokota ha DARS-AS1 ho ile ha eketsa haholo PARP cleavage ka liseleng tsa SW620 le ho eketsa karolo ea lisele tsa annexin V-positive ho SW620, HCT116, HepG2, le MBA-MD-231 lisele tsa lisele (Setšoantšo sa 3k).3).3f–h), e bontšang hore phello e khahlanong le apoptotic ea DARS-AS1 liseleng tsa mofetše e fapane le mosebetsi oa apoptosis-inducing oa PACT.Ha li kopane, liphetho tsena li fana ka maikutlo a hore mochine oa DARS-AS1 oncogenic function e ka 'na ea e-ba ka ho thibela mosebetsi oa PACT.
Ka mor'a moo, re ile ra hlahloba liphello tsa ts'ebetso ea mokhatlo oa DARS-AS1-PACT.PACT e tlalehiloe ho kenya tšebetsong PKR ka tšebelisano e tobileng, eo hamorao e ntlafatsang eIF2α phosphorylation, e bakang ho hlakoloa ha phetolelo le apoptosis17.Taba ea pele, re ile ra hlahloba hore na DARS-AS1 e ama sebaka sa cellular sa PACT le PKR.Confocal fluorescence microscopy e bonts'itse hore PACT le PKR li kopantsoe haholo liseleng tsa SW620 ka karolelano ea coefficient ea khokahano ea Pearson ea 0.72.Ho sa le joalo, DARS-AS1 overexpression e fokolitse haholo PACT le PKR co-localization (ho bolela Pearson coefficient coefficient 0.61) (Figure 4a).Ho etsa lipatlisiso tsa hore na DARS-AS1 e ka fetola tšebelisano ea PACT-PKR, re entse tlhahlobo ea co-immunoprecipitation (co-IP) ka anti-PACT anti-anti-PACT ho li-lysate tsa lisele tsa SW620.PKR e ne e ruisitsoe haholo ka anti-PACT liseleng tsa taolo, ha PKR e hlaphoheloa e fokotsehile haholo ka li-lysates tse tsoang liseleng tse fetang DARS-AS1 (Fig. 4b).E hloekisitsoeng e ngotsoeng PACT le PKR li ne li sebelisetsoa litlhahlobo tse tlamang liprotheine tsa in vitro.Ka hona, tse faneng ka DARS-AS1 empa ho se na taolo ea RNA e bonts'itse tšebelisano e hatelitsoeng ea PACT-PKR (Setšoantšo sa 4c).Liphetho tsohle li bontšitse hore DARS-AS1 e sitisitse puisano ea PACT le PKR.
Co-localization ea PACT le PKR liseleng tsa taolo kapa lisele tse fetisang DARS-AS1 li ile tsa bonoa ho sebelisoa microscopy ea confocal fluorescence.Li-nuclei li ne li silafalitsoe ke DAPI.Liphetho tsa lipalo-palo li fumanoe litšoantšong tse 16.b Co-immunoprecipitation (co-IP) ho sebelisa anti-PACT antibody ka liseleng tsa lysate tsa taolo SW620 lisele kapa lisele tse overexpressing DARS-AS1.c E ngolisitsoeng ka PACT, PKR e hloekisitsoeng le e ngotsoeng ka in vitro ka DARS-AS1 kapa RNA e somang li ile tsa kenngoa bakeng sa tlhahlobo ea in vitro e tlamang protheine.Li-anti-folakha li ne li sebelisetsoa ho thibela immunoprecipitation.d Li-Immunoblots tse nang le li-antibodies tse bontšitsoeng li entsoe ka SW620 le lisele tsa HCT116 tse fetisitsoeng ka taolo ea shRNA kapa DARS-AS1-shRNA e lateloa ke tlala ea serum.Boemo ba polelo ea DARS-AS1 bo fetotse kutloisiso ea lisele ho thapsigargin.Lisele tsa SW620 li ile tsa fetisoa ka DARS-AS1 shRNA, DARS-AS1 overexpression plasmid kapa control plasmid.Lisele li ile tsa phekoloa ka thapsigargin bakeng sa lihora tse 48 'me ho sebetsa ha lisele ho ile ha khethoa ho sebelisa mochine oa MTS.f In vitro transcribed DARS-AS1 kapa dummy RNA le purified PACT li ile tsa sebelisoa bakeng sa in vitro activation assay le ho lemoha immunoblot.g Li-Immunoblots tse sebelisang lithibela-mafu tsena li entsoe liseleng tsa SW620-ctrl (ka ho le letšehali) kapa liseleng tse hlahisang liphetoho tsa PKR (ka ho le letona).Lisele tsena li ile tsa fetisoa ka taolo ea shRNA kapa DARS-AS1-shRNA e lateloa ke tlala ea serum.h Flow cytometry e bonts'itse hore ho se sebetse ha PKR e fetotsoeng ho lefelletse bakeng sa apoptosis e hlahisitsoeng ke DARS-AS1 liseleng tsa SW620.Li-Immunoblots tse nang le li-antibodies tse bontšitsoeng li entsoe ka liseleng tsa SW620 (ka ho le letšehali) kapa tsa HCT116 (ka ho le letona).Lisele tse fetisitsoeng ka taolo ea shRNA kapa DARS-AS1 shRNA ha li na serum 'me li tlatsitsoe ka 100 nM PKR C16 inhibitor kapa DMSO.Scale bar = 5 µm.Lintlha tse bontšitsoeng ke moelelo ± kheloha e tloaelehileng ea liteko tse tharo.* p ≤ 0.05 Teko ea t ea Moithuti e nang le mehatla e 'meli.
Ka kakaretso ho lumeloa hore hang ha PACT e sebelisana le PKR17, phosphorylation ea PKR ho Thr451 e ka susumetsoa.Liphetho tsa rona li bontšitse hore boemo ba PKR phosphorylation bo ne bo phahame haholo liseleng tsa DARS-AS1 ka mor'a tlala ea serum (setšoantšo sa 4d le Supplementary Fig. 4a).Ka lebaka leo, re fumane hore phosphorylation ea eIF2α, e ka sehloohong ea PKR substrate, e boetse e eketsehile haholo ke DARS-AS1 shRNA (Fig. 4d le Supplementary Fig. 4a).Thapsigargin ke khatello ea kelello ea ER e etsang hore ER e lokolle Ca2 +.Kalafo e nang le thapsigargin e tlalehiloe hore e susumetsa polelo le ts'ebetso ea PACT, e tsoelang pele e sebelisana le PKR, e lebisang ho apoptosis ka ho eketsa eIF2α phosphorylation 18,61.Mona, re sebelisitse thapsigargin e le stimulator ea tsela ea PACT/PKR ho etsa lipatlisiso hore na DARS-AS1 e ka thusa lisele ho hlola khatello ea maikutlo ka ho thibela tsela ea PACT/PKR.Re hlokometse hore boemo ba polelo ea DARS-AS1 bo amana hantle le khanyetso ea lisele ho thapsigargin.SW620 lisele tse overexpressing DARS-AS1 ba ile ba pholoha betere ha ba ne ba tšoaroa ka thapsigargin, ha lisele tse nang le DARS-AS1 knockdown li ile tsa hlaseloa habonolo (Setšoantšo sa 4e).Tumellanong le liphetho tsena, DARS-AS1 overexpression e fokolitse thapsigargin-induced PKR phosphorylation (Supplementary Fig. 4b).Ka lehlakoreng le leng, ka mor'a phekolo ea thapsigargin, PKR le eIF2α e ne e le phosphorylated ka tekanyo e phahameng ho lisele tsa DARS-AS1 tse kokotang ha li bapisoa le lisele tsa taolo (Supplementary Fig. 4b).Hoa thahasellisa hore thapsigargin e kentse polelo ea DARS-AS1 ka mokhoa o itšetlehileng ka tekanyo, e ka 'nang ea bontša mosebetsi o khahlanong le khatello ea kelello oa DARS-AS1 (Supplementary Fig. 4c).Ntle le moo, re entse liteko tsa in vitro activation ho netefatsa lipono tsena.Ka bokhutšoanyane, PKR e ile ea hloekisoa ho tloha liseleng tsa lisele ho sebelisa anti-PKR antibody, ebe e kenngoa ka recombinant PACT le DARS-AS1 e ngotsoeng ka in vitro.Kamora karabelo ea enzymatic, phospho-PKR e ile ea bonoa e sebelisa WB.Liphetho tsa rona li bontšitse hore PKR phosphorylation e ne e thibetsoe haholo ke DARS-AS1, empa eseng ka taolo ea RNA (Setšoantšo sa 4f).Liphetho tsena tsa in vitro le in vivo li fana ka maikutlo a hore DARS-AS1 e thibela ts'ebetso ea PACT-mediated PKR.Ka nako e ts'oanang, re ile ra boela ra bona ho fokotseha ha ho hlaphoheloa ha PACT ka pel'a DARS-AS1 (Setšoantšo sa 4f).Sephetho sena se lumellana le liphello tsa in vitro protheine binding assay (Setšoantšo sa 4c) hape se bontša mosebetsi oa ho thibela DARS-AS1 bakeng sa mokhatlo oa PACT-PKR.
Ser246 le Ser287 sebakeng sa D3 sa PACT lia hlokahala bakeng sa ts'ebetso ea PKR tlas'a khatello ea cellular.Ho nkela sebaka sa masala a mabeli a serine bakeng sa alanine ho fane ka phetoho ea PACT (mutD), e ileng ea kenya tšebetsong PKR ha ho se khatello ea maikutlo, le ho nkela alanine (mutA) sebaka sa ho khutlisa protocol.Kaha re bontšitse bohlokoa ba sebaka sena ka kamano e tobileng le DARS-AS1, re hlahisitse liphetoho tsena tse peli tsa PACT ho hlahloba hore na mesaletsa ena e ka boela ea ameha ho sebelisana le DARS-AS1.Hoa thahasellisa hore liphetoho tse peli li ile tsa lahleheloa ke bokhoni ba ho tlama DARS-AS1 (Supplementary Fig. 4d), e fana ka maikutlo a hore sebopeho se feletseng sa protheine ea PACT se ka 'na sa hlokahala bakeng sa ho sebelisana hantle le DARS-AS1.
Ho feta moo, liphetho tsa rona li boetse li fana ka maikutlo a hore thibelo e bakoang ke DARS-AS1-shRNA ea ho ata ha lisele e ka tsosolosoa ka mokhoa o feteletseng ka ho feteletsa litaba tse ngata tse mpe tsa PACT mutant (PACTmutA) kapa PKR mutant e kholo (PKRmut) (Supplementary Fig. 4e. e).Tlhaloso e feteletseng ea liphetoho tse matla tsa PKR e fokolitse phosphorylation ea PKR e bakoang ke DARS-AS1 knockdown hammoho le eIF2α phosphorylation liseleng tse senyehileng tsa serum (Fig. 4g).Habohlokoa le ho feta, karolo ea lisele tsa apoptotic e hlahisitsoeng ke DARS-AS1 knockdown e ile ea boela ea fokotsoa ka lisele tse fetisetsang PKRmut (Fig. 4h le Supplementary Fig. 4g).Ho thibela mosebetsi oa PKR kinase ho boetse ho senya ts'ebetso ea DARS-AS1, kaha liphello li bontšitse hore ho kokota ha DARS-AS1 ha hoa ka ha baka PKR le eIF2α phosphorylation ha lisele li ne li tšoaroa ka PKR-specific C16 inhibitor (Fig. 4i le Supplementary Fig. 4h).).Ha re kopane, liphetho tsa rona li fana ka maikutlo a hore DARS-AS1 e khothalletsa ho ata ha lisele, bonyane ka karolo e itseng, ka ho thibela ts'ebetso ea PACT-mediated PKR.
Ho tsoela pele ho hlahloba karolo ea DARS-AS1 ho tumorigenesis, re entse liteko tsa vivo re sebelisa mohlala oa mouse xenograft. Liphetho li bonts'a hore ho fokotseha ha DARS-AS1 ho fokolitse kholo ea tumor ho litoeba haholo (p value <0.0001) (Fig. 5a). Liphetho li bonts'a hore ho fokotseha ha DARS-AS1 ho fokolitse kholo ea tumor ho litoeba haholo (p value <0.0001) (Fig. 5a). Результаты показывают, что нокдаун DARS-AS1 резко снижает рост опухоли у мышей (значение p <0,0001) (рис. 5а). Liphetho li bontša hore ho kokota ha DARS-AS1 ho fokotsa haholo ho hōla ha hlahala ho litoeba (p value <0.0001) (Figure 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). Liphello li bontšitse hore ho kokota ha DARS-AS1 ho fokolitse haholo ho hōla ha hlahala ho litoeba (p value <0.0001) (Figure 5a).Kahoo, sehlopheng sa DARS-AS1, ho bile le ho fokotseha ho hoholo ha molumo oa tumor ka hoo e ka bang 72.9% le ho bolela boima ba tumor ka hoo e ka bang 87.8% (Setšoantšo sa 5b-d).Liphetho tsena li fana ka maikutlo a matla a hore DARS-AS1 e ka khothaletsa kholo ea hlahala ka har'a vivo.
Litlamorao tsa papatso ea DARS-AS1 ho colorectal oncogenesis ho litoeba tse hlobotseng.Mekhahlelo ea ho hōla (a), boholo ba hlahala (b), boima ba 'mele (c), le litšoantšo tsa hlahala (d) lia bontšoa.Li-barbar tsa liphoso li emela ±SEM. n = 10. ****p <0.0001, ka teko ea t ea Moithuti ea mehatla e 'meli. n = 10. ****p <0.0001, ka teko ea t ea Moithuti ea mehatla e 'meli. n = 10. ****p < 0,0001 по двустороннему критерию Стьюдента. n = 10. ****p < 0.0001 Teko ya Moithuti ya mehatla e mmedi.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 Teko ya Moithuti ya mehatla e mmedi.e Kaplan-Meier o ile a sekaseka kamano pakeng tsa maemo a polelo ea DARS-AS1 le ho phela ka kakaretso ho bakuli ba UVM, KICH, KIRP, MESO, GBM, le LGG.Maemo a phahameng a polelo ea DARS-AS1 ho bakuli e ne e le ka holimo ho 50%;boemo bo tlase ba polelo ea DARS-AS1 ho bakuli bo ne bo le tlase ho 50%.p-values ​​e ile ea khethoa ho sebelisoa tlhahlobo ea boemo ba log.f Mohlala o sisintsoeng oo DARS-AS1 e laolang tsela ea PACT-PKR le kholo ea hlahala.
Ho utloisisa hamolemo litlamorao tsa DARS-AS1, re hlahlobile kamano lipakeng tsa polelo ea eona le ho phela ha mokuli.Ka ho sekaseka datha tsa TCGA, re fumane hore polelo e phahameng ea DARS-AS1 e ne e amahanngoa le uveal melanoma (UVM), renal chromophobia (KICH), renal papillary cell carcinoma (KIRP), mesothelioma (MESO), multiplex.Ho phela ka tlaase ho ne ho amahanngoa haholo le glioblastoma morphosis (GBM) le bakuli ba nang le bokooa ba boko bo tlaase (LGG) (Setšoantšo sa 5e).Liphetho tsena li fana ka maikutlo a hore DARS-AS1 e kanna ea bapala karolo ea bohlokoa ho tsoeleng pele ha hlahala ea bongaka mme e kanna ea ba sesupo sa biomarker mefuteng e mengata ea mofetše.
Thutong ena, re sebelisa tlhahlobo e kholo ea ts'ebetso ea CRISPRi, re entse qeto ea hore DARS-AS1 lncRNA e hlola khatello ea lisele tsa mofetše ka ho laola ba arabelang khatello ea maikutlo tse peli, PACT le PKR.Ka ho sebelisana ka ho toba le PACT, DARS-AS1 e thibela ts'ebetso ea PACT-mediated PKR, ka tsela eo e thibela lefu la lisele tsa apoptotic le ho khothalletsa ho ata ha lisele (setšoantšo sa 5f).Phatlalatso ea DARS-AS1 e 'nile ea hlokomeloa mefuteng e mengata ea mofetše, e fana ka maikutlo a hore mosebetsi oa eona oa ho khothalletsa ho phela ha lisele tsa mofetše tlas'a maemo a sithabetsang o ka sebetsa ka ho pharaletseng mefuteng e mengata ea mofetše.
PACT e khethiloe e le protheine ea activator ea PKR, 'me ts'ebetso ea PACT-mediated PKR e phetha karolo ea bohlokoa likarabong tsa khatello ea kelello ka ho laola ho ngoloa, phetolelo, apoptosis, le mekhoa e meng ea bohlokoa ea cellular62.Ka lilemo tse mashome, ho entsoe liteko tsa ho utloisisa taolo e tobileng ea mofets'e ea cascade ea PACT-PKR.Mona, boithuto ba rona bo senotse mokhoa o fapaneng oa taolo ea PACT-PKR liseleng tsa mofets'e ka lncRNA DARS-AS1 ea cellular, e tlamang ka kotloloho ho PACT, e thibela ts'ebelisano ea PACT-PKR, e thibela ts'ebetso ea PKR le phosphorylation ea eIF2α, ka tsela eo e thibela apoptosis e bakoang ke khatello ea maikutlo le khatello ea maikutlo. e khothalletsa ho ata ha mofets'e qetellong.lisele.Se sibolotsoeng sena se fana ka leseli mabapi le lipheo tse ka bang teng tsa lncRNA bakeng sa ponelopele ea mofetše le kalafo.
Lintlha tsa rona li bontšitse hore DARS-AS1 knockdown e hlokomelisa lisele ho tlala ea serum ka keketseho e kholo ea phosphorylated PKR le eIF2α.Liphetho tsena li fana ka maikutlo a hore DARS-AS1 e khothalletsa ho phela ha lisele tsa mofetše tlas'a maemo a thata ka ho thibela ts'ebetso ea PACT/PKR.Li-RNA tse ling tse 'maloa tseo e seng li-coding, tse kang ASPACT le nc886, le tsona li kentse letsoho ho PACT/PKR axis ka ho theola PACT48 mRNA kapa ho laola autophosphorylation ka ho tlama PKR49,50,64.Har'a bona, DARS-AS1 e sebetsa e le moferefere oa mokhatlo oa PACT-PKR.Boithuto bona bo ntlafatsa kutloisiso ea rona ea taolo ea axis ea PACT/PKR le karolo ea li-lncRNA likarabelong tsa khatello ea maikutlo.
PACT e na le libaka tse tharo tse arohaneng.E 'ngoe le e' ngoe ea li-dsRBD tse peli tsa pele e lekane ho fihlela tlamahano e phahameng ea PACT ho PKR, ha sebaka sa boraro (D3) se hlokahala bakeng sa ts'ebetso ea PKR in vitro le vivo.Boithuto ba rona bo bontšitse hore DARS-AS1 e sebelisana ka mokhoa o ikhethileng le sebaka sa D3 (setšoantšo sa 3h).Ka lebaka la boholo bo boholo ba lncRNA (768 nucleotides), DARS-AS1 e tlamang ho D3 e ka thibela 'mele ho sebelisana pakeng tsa sebaka sa PACT sa dsRBD le PKR, kahoo e thibela mokhatlo oa PACT le PKR.Liphetoho tsa lintlha tsa PACT tse nkileng sebaka sa Ser246 le Ser287 ho D3 ka alanine kapa aspartate li ile tsa senya kamano ea eona e tlamang bakeng sa DARS-AS1, e supang bohlokoa ba thepa ea D3's ea sebopeho le motlakase ka kakaretso mokhatlong oa bona.Lintlha tse ling tsa mochini ona li tla hlokahala nakong e tlang, ho sebelisoa tlhahlobo e nepahetseng ea biochemical le tlhahlobo e phahameng ea PACT ea sebopeho.
Liphuputso tse fetileng li tlalehile hore DARS-AS1 e khothalletsa ho ata ha lisele ka mekhoa e mengata51,52,53.Mohlala o mong, bafuputsi ba hlokometse hore DARS-AS1 e laetse mofuta oa eona oa antisense-encoding DARS ka ho lebisa miP-194-5p liseleng tsa mofetše oa liphio.Leha ho le joalo, thutong ea hona joale, ho kokota ha DARS-AS1 ho bile le phello e fokolang ho phetisetso ea DARS mefuteng e mengata ea mofetše, ho kenyeletsoa bonyane mofetše oa colorectal, matsoele le sebete.Hobane li-lncRNA li bonts'a lipaterone tse ikhethileng tsa lisele le lisele, mekhoa ea ts'ebetso e kanna ea se bolokoe mefuta eohle ea mofetše, e ka tlatsetsang phapanong ena lipakeng tsa litebello tsa rona le litlhahlobo tse fetileng tsa mofetše o fapaneng.Ho hlokahala lithuto tse khethehileng ho hlakisa mekhoa e fapaneng ea mekhoa e fapaneng ea 'mele le ea pathological.
Tlhahlobo ea lintlha tsa kliniki e bonts'itse hore polelo ea DARS-AS1 ka har'a lihlahala e amana haholo le ho phela ha bakuli ba mofets'e, e totobatsang bohlokoa ba DARS-AS1/PACT/PKR axis ho prognoza ea mofets'e.Qetellong, thuto ea rona e bontša hore DARS-AS1 ke molaoli oa PACT / PKR pontšo ea axis, e khothalletsa ho ata ha lisele tsa mofetše, 'me e thibela apoptosis nakong ea karabelo ea khatello ea maikutlo, e fanang ka mokhoa o mong oa lipatlisiso 'me e thahasella lipatlisiso tsa nakong e tlang mabapi le mekhoa ea phekolo. .
Mehala ea lisele tsa batho SW620, A549, MBA-MD-231, HCT116, HepG2 le HEK293T e fumanoe ho tsoa ho National Cell Line Resource Infrastructure in China.Lisele tsohle li ne li bolokiloe DMEM medium (DMEM, Thermo Fisher Scientific, Waltham, MA) e tlatsitsoeng ka 10% FBS (Gemini, Brooklyn, NY) le 1% penicillin-streptomycin (Thermo Fisher Scientific) ho 37 ° C, 5% CO2.seqhoqhoa.
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);e tloaelehileng mouse IgG, CST (5415S);tloaelehileng mmutla IgG, CST (2729S).Lithibela-mafu li hlakotsoe 1:1000 ho PBST bakeng sa blotting ea Bophirimela le 1:100 bakeng sa IP.
Li-sgRNA li entsoe ho sebelisoa sesebelisoa se fumanehang phatlalatsa se bitsoang CRISPR-ERA66.Re sebelisitse lisebelisoa tsa kamehla bakeng sa nts'etsopele ea sgRNA le algorithm e tlamang libaka tsa sgRNA sebakeng sa 3 kb.e thehiloe ho TSS.Matamo a sgRNA oligonucleotides a ile a etsoa CustomArray, Inc. (Bothewell, WA) 'me a etsoa ho batho ba pgRNA plasmids (Addgene #44248).Kakaretso ea 12 µg ea pgRNA plasmid e kopantsoeng ea motho, 7.2 µg ea psPAX2 (Addgene #12260), le 4.8 µg ea pMD2.G (Addgene #12259) li fetiselitsoe ho 5 x 106 HEK206 DNA Transfection 106 HEK293T ho Rea lisele ( CWBIO, Beijing, China) ho latela litaelo tsa moetsi.Li-supernatant tse nang le kokoana-hloko li ile tsa bokelloa lihora tse 48 le 72 ka mor'a hore li fetisetsoe 'me li sefshoa ka sefe ea 0.45 µm.Bakeng sa tlhahlobo, lisele tsa SW620 tse hlalosang protheine ea fusion ea dCas9/KRAB li fumanoe ka phetisetso ea vaerase.Lisele tse fetotsoeng tsa SW620 li ile tsa tšoaetsoa ke laebrari ea kokoana-hloko litekong tse 'nè tse ikemetseng tsa ts'oaetso ho MOI ea 0.1-0.3 'me tsa etsoa sampole ka 2 μg/ml puromycin (Sigma, St. Louis, MO) matsatsi a 2.Kamora moo, lisele li ile tsa hlahisoa ka matsatsi a 18 ka har'a li-in vitro tse nang le bonyane laeborari ea 500 cell/sgRNA bakeng sa tlhahlobo.
Genomic DNA e ntšitsoe ho latela litaelo tsa QIAamp DNA Blood Midi Kit (QIAGEN, Düsseldorf, Germany; 51183).Ka kakaretso, 100 μg ea DNA ea genomic ho pheta-pheta ea likokoana-hloko e sebelisitsoe ho haha ​​​​laebrari.Sebaka sa sgRNA se ile sa holisoa ka mekhahlelo e 'meli ea PCR mme se hokahantsoe le barcode.
Lihlahisoa tsa PCR li ile tsa hloekisoa ka ho sebelisa gel ea NucleoSpin® le PCR purification kit (MACHHEREY-NAGEL, Düren, Germany; 740609.250) 'me ea lekanngoa ho sebelisoa Qubit™ HS e nang le likarolo tse peli tsa ho lemoha DNA (Thermo Fisher Scientific; Q32854).
Teko ea MTS e ne e sebelisetsoa ho lekanya ho ata ha lisele.Lisele li ile tsa jaloa lipoleiting tsa liliba tse 96 ka bongata ba lisele tse 2000 / seliba.Palo e lekanyelitsoeng ea lisele e ne e lekantsoe letsatsi le letsatsi ka nako e bontšitsoeng bakeng sa kakaretso ea matsatsi a 4-6.Bakeng sa seliba se seng le se seng, 20 μl ea reagent ea MTS (Promega) e ile ea hlapolloa ka 100 μl ea DMEM, e kenngoa ka lisele tsa 4 h ho 37 ° C, 'me joale OD490 e lekantsoe.
Bokhoni ba ho hōla bo sa tsitsang bo ile ba fumanoa ka ho hlahloba sebōpeho sa lidikadikwe.Ka bokhutšoanyane, lisele tse 2000 tse fetisitsoeng ka shRNA DARS-AS1 kapa control shRNA li ile tsa hlahisoa ka li-microplates tse tlase tse tlase (Corning) ka phetoho e mahareng matsatsi a mang le a mang a 4.Li-spheroid li ne li baloa ka mor'a matsatsi a 14.Lisele tse 500 tse fetisitsoeng ka DARS-AS1 overexpression plasmid kapa plasmid ea taolo li ile tsa sebelisoa bakeng sa tlhahlobo ea ntlafatso, ho seng joalo mokhoa o ne o sa fetoha.
RNA e ngotsoe ho sebelisoa T7 RNA polymerase le biotin-16-UTP (Roche 1138908910) ho ea ka litaelo tsa Riboprobe® Combination Systems (Promega P1440).Li-primers tse sebelisitsoeng mona li thathamisitsoe ho Lethathamo la 4 la Tlatsetso.
Protein-coding PACT kapa libaka tsa PKR li ile tsa etsoa pET15b (Addgene #73619) 'me tsa fetoloa BL21(DE3).Baktheria e ile ea kenngoa ka bosiu bo le bong ho LB e fanoang ka ampicillin 'me ea hlapolloa ka makhetlo a 100 ka LB e ncha.Ha OD600 ea mahareng e fihla ho 0.8, 1 mm IPTG e ile ea eketsoa ho susumetsa polelo ea protheine.Ka mor'a ho kenngoa bosiu ka ho sisinyeha ha bonolo (250 rpm ho 20 ° C), pellet ea sele e ile ea bokelloa ka centrifugation (4000 rpm, 10 min, 4 ° C).Khutlisetsa pellet ea sele ka har'a lysis buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 1 mM PMSF) 'me u e kenye holim'a leqhoa ka metsotso e 30, ebe u sonicate (15 min, 5 s on/off, on ice) le centrifuge (13,000). rpm)., 30 mets, 4° С).The supernatant e ile ea laeloa holim'a Ni-NTA resin (QIAGEN) makhetlo a 3 ho 4 ° C, e hlatsuoa ka makhetlo a 4 ka sesepa sa ho hlatsoa (50 mM Tris, pH 8.0, 40 mM imidazole, 250 mM NaCl) 'me e hlatsuoa ka makhetlo a 3, ka kakaretso. ea 10 ml eluent buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 300 mM imidazole).Protheine e hloekisitsoeng e ile ea khethoa ho sebelisoa WB 'me khatello ea maikutlo e ile ea khethoa ho sebelisoa Qubit™ protein assay kit (Thermo Fisher Scientific; Q 33212).
Litlhahlobo tsa RIP li entsoe joalo ka ha ho hlalositsoe pejana, ka liphetoho.Ka bokhutšoanyane, 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) le centrifuge ho 13,000 rpm bakeng sa metsotso e 15 ho 4 °C.Supernatant e ile ea kenngoa ka protheine A + G magnetic beads (Millipore) e kopantsoeng le 5 μg ea anti-PACT antibody (Abeam) kapa IgG (CST).Lifaha li ne li hlatsuoa ka makhetlo a 5 ka 5x RIP buffer, ebe li cheka ka proteinase K (NEB).RNA e ile ea ntšoa ka Trizol 'me ea khethoa ke RT-qPCR.Li-primers li hlahisoa ho Lethathamo la 5 la Tlatsetso.
Teko ea in vitro RIP e entsoe ho latela protocol ea tlhahlobo ea RIP e fetotsoeng.Kakaretso ea 5 pmol ea in vitro transcribed RNA e ile ea hlapolloa 1x ka RIP buffer 'me ea kenngoa ka ho incubation ka 65 ° C metsotso e 5 e lateloa ke ho pholisa butle ho ea mocheso oa kamore.Kakaretso ea 5 pmol ea liprotheine tsa PACT tse sa fetoheng kapa tse fetotsoeng tse ngotsoeng ka folakha li ile tsa hloekisoa ho tsoa ho E. coli.Eketsa ka RNA e nchafalitsoeng bakeng sa lihora tsa 2 ho 4 ° C 'me u latele mokhoa o ka holimo bakeng sa tlhahlobo ea RIP bakeng sa anti-flag IP.
Bakeng sa tlhahlobo ea katoloso ea RNA, lisele tsa 1 × 107 li ne li kentsoe ka 1xRIP buffer.Ka mor'a centrifugation ka 13,000 rpm bakeng sa 15 min ho 4 ° C, supernatant e ile ea etsoa pele ka 30 μl ea streptavidin magnetic beads (Beckman) bakeng sa 2 h ho 4 ° C.Lysate e hloekisitsoeng e ile ea fanoa ka tomoso ea tRNA 'me ea kenngoa ka 40 pmol ea renatured RNA bosiu bo le bong ho 4 ° C, ebe lihora tse ling tsa 2 le 20 μl ea lifaha tse ncha tsa streptavidin tse koetsoeng ka BSA li ile tsa eketsoa.Mohato oa ho hlatsoa o ne o e-na le makhetlo a 4 ka 5x RIP buffer le makhetlo a 4 ka 1x RIP buffer.Liprotheine tse tsamaellanang li ile tsa hlakisoa ka biotin elution buffer (25 mM Tris-HCl, pH 7.5, 12.5 mM D-biotin, PMSF) 'me tsa aroloa ho NuPAGE 4-12% Bis-Tris Gel (Invitrogen).Kamora 'mala oa silevera (Beyotime Biotechnology), lihlopha tse ling li ile tsa khaoloa le ho hlahlojoa ke MS.
Ho ile ha etsoa tlhahlobo ea Co-IP ho leka tšebelisano lipakeng tsa PACT le PKR.Ka bokhutšoanyane, li-lysates tse matla li ile tsa lokisoa ka ho kenya lisele tse 1 x 107 tse lysed ka 1 x RIP buffer e lateloa ke centrifugation ho 13,000 rpm bakeng sa metsotso ea 15 ho 4 ° C.Li-lysate li ne li kentsoe protheine A + G lifaha tsa makenete, tse kopantsoeng le 5 µg tsa anti-PACT anti-PACT, 'me li fetotsoe ka bonolo bosiu bo le bong ho 4 ° C.Lifaha li ne li hlatsuoa ka makhetlo a 3 ka 5 × RIP buffer, habeli ka 1 × RIP buffer le e luted ka 1 × SDS buffer.Protheine e fumanoeng e ile ea hlahlojoa ke gel ea SDS-PAGE mme ea fumanoa ke WB.
Li-pmol tse peli tsa PACT e tšoailoeng le 1 pmol ea PKR li ile tsa hloekisoa ho tloha ho E. coli.Hlapolla ka har'a 1 × RIP buffer 'me u e kenye ka 10 pmol ea RNA e tsosolositsoeng ka lihora tse 2 ho 4 °C.Ka mor'a moo, li ile tsa kenngoa ka protheine A+G magnetic bead-conjugated anti-labeled anti-antibody bakeng sa lihora tse ling tse peli.Lifaha li ne li hlatsuoa ka makhetlo a mane ka 1x RIP buffer 'me li hlatsuoa ka 1x SDS buffer.PACT le PKR tse hlahisitsoeng li ile tsa fumanoa ke WB.