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Amangqamuzana omdlavuza aguqule izindlela ezihlukahlukene zokunqoba ukucindezeleka kwamaselula futhi aqhubeke nokuthuthuka.I-protein kinase R (PKR) kanye ne-protein activator (PACT) yizimpendulo zokuqala eziqapha amasiginali ahlukahlukene wokucindezeleka okuholela ekuvinjweni kokwanda kwamaseli kanye ne-apoptosis.Kodwa-ke, ukulawulwa kwendlela ye-PACT-PKR kumaseli omdlavuza kuhlala kungaziwa kakhulu.Lapha, sithole ukuthi i-RNA ende engekho ikhodi (lncRNA) aspartyl tRNA synthetase antisense RNA 1 (DARS-AS1) ihileleke ngokuqondile ekuvimbeleni indlela ye-PACT-PKR futhi ikhuthaza ukwanda kwamangqamuzana omdlavuza.Sisebenzisa ukuhlolwa kokusebenza okukhulu kwe-CRISPRI 971 i-lncRNA ehlobene nomdlavuza, sithole ukuthi i-DARS-AS1 yayihlotshaniswa nokusabalala kwamangqamuzana omdlavuza okuthuthuke kakhulu.Ngakho-ke, i-DARS-AS1 knockout ivimbela ukwanda kwamaseli futhi ikhuthaze i-apoptosis yeseli yomdlavuza emigqeni ehlukahlukene yamaseli omdlavuza ku-vitro futhi kunciphisa kakhulu ukukhula kwesimila ku-vivo.Ngomshini, i-DARS-AS1 ibophezela ngokuqondile kusizinda sokusebenza se-PACT futhi ivimbele ukusebenzisana kwe-PACT-PKR, ngaleyo ndlela yehlise ukusebenza kwe-PKR, i-eIF2α phosphorylation, futhi ivimbele ukufa kweseli ye-apoptotic.Ngokomtholampilo, i-DARS-AS1 ivezwa kabanzi emakhazeni amaningi, futhi ukuvezwa ngokweqile kwale lncRNA kuyinkomba yokubikezela okubi.Lolu cwaningo lucacisa umthethonqubo othize womdlavuza wendlela ye-PACT-PKR nge-DARS-AS1 lncRNA futhi ihlinzeka ngokunye okuhlosiwe kokubikezelwa komdlavuza kanye nokwelashwa.
Ikhono lokuzivumelanisa nokucindezeleka kuyisici esibalulekile sokusinda kwamangqamuzana omdlavuza nokwanda.Ukwanda okusheshayo kanye nezimpawu ze-metabolic zokuphakama komdlavuza ezindaweni ezincane ezinokhahlo-ukuntuleka kwezakhi, i-hypoxia, kanye ne-pH ephansi-okungabangela izindlela zokubonisa ukufa kwamaseli.Ukungasebenzi kahle kwezakhi zofuzo ezizwela ukucindezeleka njenge-p535, amaprotheni okushisa ukushisa 6, 7, KRAS8, 9, kanye ne-HIF-110, 11, 12, 13 kuvame ukubonwa kumdlavuza, ngaleyo ndlela kuvimbe i-apoptosis futhi kukhuthaze ukusinda.
I-protein kinase R (PKR) iyinzwa yokucindezeleka ebalulekile kanye ne-subunit kinase ye-eukaryotic initiation factor 2α (eIF2α), umlawuli wokuhumusha oxhumanisa ukucindezeleka kwamaselula nokufa kweseli.I-PKR ekuqaleni yahlonzwa njengeprotein evimbela amagciwane ngokutholwa kwe-RNA ephindwe kabili yangaphandle (dsRNA).Lapho kusebenze, i-PKR phosphorylates eIF2α ukuvimbela i-viral and cell protein synthesis14,15,16.I-PACT (iphrotheni ye-activator ye-PKR) ikhonjwe njengeprotheni yokuqala ye-activator ye-PKR lapho ingekho i-dsRNA17,18,19,20,21,22,23.Ngokusebenzisana okuqondile ne-PKR, i-PACT idlulisa ukucindezeleka okuhlukahlukene (indlala ye-serum, i-peroxide noma ukwelashwa kwe-arsenite) ku-PKR nezindlela zokubonisa ezingezansi.Ngokungeziwe ku-eIF2α phosphorylation, ukusebenza kwe-PACT-mediated PKR kubangela izehlakalo ezihlukahlukene ezihlobene nokusabela kokucindezeleka, okuhlanganisa isimo se-redox esishintshiwe ngendlela ye-PI3K/Akt24, ukuhlolwa komonakalo we-DNA okuthuthukisiwe nge-p5325,26 kanye ne-NF-κB27,28 Ilawula ukuloba, 29. Njengoba kunikezwe indima yabo ebalulekile ekuphenduleni ukucindezeleka, ukwanda, i-apoptosis nezinye izinqubo ezibalulekile zamaselula, i-PKR ne-PACT zithembisa izinhloso zokwelapha izifo eziningi, ikakhulukazi umdlavuza30,31,32,33.Kodwa-ke, naphezu kwalokhu kubaluleka kokusebenza kwe-pleiotropic kanye nebhayoloji, ukulawulwa komsebenzi we-PACT/PKR kumangqamuzana omdlavuza kuhlala kunzima.
Ama-lncRNA ayimibhalo emikhudlwana kunama-nucleotide angama-200 angenawo amandla okufaka ikhodi amaprotheni.Njengoba amaphrojekthi asezingeni eliphezulu okulandelana kofuzo ehlonze izinkulungwane zama-lncRNA, umzamo omkhulu we-35,36 wenziwe ukucacisa imisebenzi yawo yebhayoloji.Indikimba ekhulayo yocwaningo ibonise ukuthi ama-lncRNA abandakanyeka ezinqubweni eziningi zezinto eziphilayo37 okuhlanganisa nokulawulwa kwe-X-chromosome inactivation38,39, imprinting40, transcription41,42, translation43 ngisho nokukhula komdlavuza44,45,46,47.Lezi zifundo zibike ukuthi ama-lncRNA amaningi abandakanyeka endleleni ye-PACT/PKR.Olunye ucwaningo olunjalo lubonise ukuthi i-lncRNA ASPACT ivimbele ukulotshwa kwe-PACT kanye nokugcinwa kwe-nuclear ye-PACT mRNA.Ezinye izifundo zibonise ukuthi i-lncRNA nc886 ibophezela ku-PKR futhi ivimbele i-phosphorylation49,50 yayo.Kuze kube manje, i-lncRNA elawula ukwenziwa kusebenze kwe-PKR ye-PACT-mediated akubikwanga.
I-Aspartyl-tRNA synthetase antisense RNA 1 (DARS-AS1) ikhonjwe njenge-lncRNA51,52,53,54 ye-oncogenic.Ngokulawulwa kwe-miP-194-5p53, miP-12952 kanye ne-miP-532-3p51, i-DARS-AS1 iboniswe ukukhuthaza ukukhula kwe-cell renal cell carcinoma ecacile, i-thyroid carcinoma kanye ne-non-small cell lung carcinoma, ngokulandelanayo.U-Tong nozakwabo baphinde bathola ukuthi i-DARS-AS1 ikhuthaza ukuqhubeka kwe-myeloma ngokugcina ukuzinza kwe-protein 39 (RBM39) ye-RNA-binding motif.Kodwa-ke, azikho izifundo ezenziwe mayelana nokuthi le lncRNA iyabandakanyeka yini ekulawulweni kokusebenza kwe-PACT-PKR kanye nokuphendula kokucindezeleka kwamangqamuzana omdlavuza.
Lapha, senze isikrini esikhulu sokulahlekelwa komsebenzi sisebenzisa uhlelo lwe-CRSPRI futhi sanquma ukuthi i-DARS-AS1 lncRNA ikhuthaza ukwanda kwezinhlobo eziningana zamangqamuzana omdlavuza.Ukwengeza, sihlonze indlela enkulu: I-DARS-AS1 ibophezela ngokuqondile ku-PACT, ivimbela ukubopha kwe-PACT ne-PKR, ivimbela i-phosphorylation ye-eIF2α, i-substrate ye-PKR ephansi, futhi ekugcineni ivimbele ukufa kwe-apoptotic cell.Sengiphetha, umsebenzi wethu wembula i-DARS-AS1 lncRNA njengomlawuli wendlela ye-PACT-PKR kanye nethagethi engaba khona yokwelashwa komdlavuza kanye nokubikezelwa.
Ucwaningo olunzulu lwe-genomic profiling lukhombe amakhulukhulu ama-lncRNA ahlotshaniswa nomdlavuza.Nokho, umsebenzi wabo uhlala ungaziwa kakhulu56.Ukuze sihlonze abantu abathembisayo be-lncRNA abathintekayo ekuqhubekeleni phambili komdlavuza, senze isikrini sokulahlekelwa umsebenzi sokunciphisa ukusabalala kumugqa weseli womdlavuza we-SW620 we-colorectal usebenzisa uhlelo lwe-CRSPRI (Fig. 1a).Isici esiyingqayizivele semigqa yamaseli omdlavuza we-colon SW480 kanye ne-SW620 ukuthi atholakala kumathumba aphansi nakwesibili esigulini esisodwa.Lokhu kunikeza isiqhathaniso esibalulekile sokutadisha izinguquko zofuzo ekuqhubekeleni phambili komdlavuza wekoloni.Ngakho-ke, sihlaziye imibhalo yemigqa yeseli yomdlavuza we-colorectal (SW480 kanye ne-SW620) sisebenzisa ukulandelana kwe-RNA futhi saqoqa amanye ama-lncRNA angasebenza ezincwadini ezishicilelwe.Ngokusekelwe kule miphumela, siklame umtapo wezincwadi we-sgRNA ohlanganisiwe oqukethe ama-7355 sgRNA oligos aqondise ama-lncRNA ahlobene nomdlavuza angu-971 kanye nama-oligo angama-sgRNA angahlosiwe angu-500 ukuze alawule okungalungile (Idatha Eyengeziwe 1).
Ukumelwa okuhleliwe kokuhlolwa kusetshenziswa uhlelo lwe-CRSPRI.b sgRNA ukunothisa ngemva kokuhlolwa.Umugqa wamachashazi ovundlile umele log2 (ushintsho olugoqiwe) = ±0.58.Umugqa wamachashazi ome mpo ubonisa inani lika-p = 0.05.Amachashazi amnyama amele i-sgRNA engahlosiwe (eqokwe njenge-NC).Amachashazi abomvu ama-sgRNA aqondise i-DARS-AS1.Amachashazi aluhlaza angama-sgRNA aqondise ku-LINC00205, i-oncogenic lncRNA echazwe ngaphambilini.ukugoqa ushintsho = (ukufunda okuvamile, usuku 17)/(ukufunda okuvamile, usuku 0).c I-DARS-AS1 i-sgRNA ivimbe ukukhula kwamaseli.Amabha wephutha amele ± ukuchezuka okujwayelekile kokuhlolwa okuthathu.* p ≤ 0.05, ** p ≤ 0.01 Ukuhlolwa kuka-t koMfundi okunemisila emibili.d Isisho se-DARS-AS1 kumathumba (idathasethi ye-TCGA).em Ukuvezwa kwe-DARS-AS1 kumasampula avamile namathumba abhanqiwe avela ezigulini ezine-BLCA, KIRC, PRAD, LUSC, UCEC, LUAD, LIHC, KIRP, kanye ne-COAD, ngokulandelana (TCGA dataset).ama-p-values ​​atholwe kusetshenziswa ukuhlolwa kuka-t koMfundi okubhanqiwe okubili okubili.
Ngemuva kokwakha i-plasmid nokupakisha i-lentivirus, sadlulisela ulayini weseli yomdlavuza we-dCas9-SW620 nomtapo wezincwadi ongenhla ezivivinyweni ezine ezizimele zokutheleleka.I-multiplicity of infection (MOI) yalezi zifo yayingu-0.1–0.3, okubonisa ukuthi iseli ngalinye lingadluliselwa kuphela nge-sgRNA eyodwa.Ngemuva kwezinsuku eziyi-18 zesiko le-in vitro, iphrofayili yokucebisa ye-target sgRNAs yehla noma yenyuka ngemuva kokuhlolwa, kuyilapho inani lama-oligonucleotides okulawula okungahlosiwe lahlala lingashintshile uma liqhathaniswa nephrofayili yokuhlolwa kwangaphambili, okubonisa ukuthi okuhlosiwe kwethu kunesikrini esicaciswe kakhulu. umtapo wolwazi.Ilayisi.1b kanye nethebula lokwengeza 1). I-LINC00205, okwabikwa ngaphambilini ukuthi ikhuthaze umdlavuza wamaphaphu nokuqhubekela phambili komdlavuza wesibindi58,59,60, yahlolwa (log2 (foldchange) <-0.58, p value <0.05), eqinisekisa ukwethembeka kwalokhu kuhlola (Fig. 1b). I-LINC00205, okwabikwa ngaphambilini ukuthi ikhuthaze umdlavuza wamaphaphu nokuqhubekela phambili komdlavuza wesibindi58,59,60, yahlolwa (log2 (foldchange) <-0.58, p value <0.05), eqinisekisa ukwethembeka kwalokhu kuhlola (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). I-LINC00205, ngaphambilini ebikwe ukuthi ikhuthaze ukuqhubeka komdlavuza wamaphaphu kanye nomdlavuza wesibindi58,59,60, yayingafakwanga (log2 (ukushintsha kokugoqa) <-0.58, p-value <0.05), eqinisekisa ukuqina kwalokhu kuhlolwa (Fig. .1b) . I-Linc00205 之前之前 이 可 促进 促进 和 肝癌 肝癌 进展 和 进展 进展 进展 进展 进展 进展 进展 进展 进展 选 选 掉 (log2 (倍数 变化) <-0.58, P 值 值 I-Linc00205 之前之前 이 可 促进 促进 和 肝癌 肝癌 进展 和 进展 进展 进展 进展 进展 进展 进展 进展 进展 选 选 掉 (log2 (倍数 变化) <-0.58, P 值 值 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). I-LINC00205, ngaphambilini ebikwe ukuthi ikhuthaze ukuqhubeka komdlavuza wamaphaphu nesibindi58,59,60, yayingafakwanga (log2 (ushintsho lokugoqa) <-0.58, p-value <0.05), eqinisekisa ukuqina kwalokhu kuhlolwa (Fig. .1b).
Phakathi kwawo wonke ama-lncRNA ahloliwe, i-DARS-AS1 nayo yahlolwa, ne-cognate sgRNA oligonucleotides emithathu encishisiwe kakhulu ngemva kwezinsuku ze-18 zesiko, okuphakamisa ukuthi i-knockdown yale lncRNA ibangele ukunciphisa ukwanda komdlavuza (Fig. 1b).Lo mphumela ubuye wasekelwa ukuhlaziya kwe-MTS kumaseli omdlavuza we-colorectal okubonisa ukuthi izinga lokukhula lamaseli e-DARS-AS1 anqotshwayo lancipha kuphela uma kuqhathaniswa namaseli okulawula (Umfanekiso 1c) futhi lalihambisana nemibiko yangaphambilini yezinye izinhlobo zomdlavuza eziningana.: sula umdlavuza wezinso zamangqamuzana, umdlavuza wegilo kanye nomdlavuza wamaphaphu ongewona omncane51,52,53,55.Kodwa-ke, umsebenzi wayo kanye nezindlela zamangqamuzana kumdlavuza we-colorectal zihlala zingahloliwe.Ngakho-ke, sikhethe le lncRNA ukuze siqhubeke nokufunda.
Ukuze sifunde isisho se-DARS-AS1 ezigulini, sihlaziye kabanzi amasampula esimila angu-10,327 avela kuphrojekthi yeCancer Genome Atlas (TCGA).Imiphumela yethu ibonisa ukuthi i-DARS-AS1 ivezwa kabanzi futhi ilawulwa kakhulu kumaseli anempilo kumathumba ahlukahlukene, okuhlanganisa icolon adenocarcinoma (COAD), renal clear cell carcinoma (KIRC), kanye ne-renal papillary cell carcinoma (KIRP)..Bambalwa kakhulu (Fig. 1d kanye Fig. 1a, b). Ukuhlaziywa kwamasampula anempilo/esimila abhanqiwe kuphinde kwaqinisekisa ukubonakaliswa okuphezulu kakhulu kwe-DARS-AS1 ezimila zesinye i-urothelial carcinoma (BLCA), i-renal clear cell carcinoma (KIRC), i-prostate adenocarcinoma (PRAD), i-lung squamous cell carcinoma (LUSC) , uterine corpus endometrial carcinoma (UCEC), lung adenocarcinoma (LUAD), isibindi hepatocellular carcinoma (LIHC), renal papillary cell carcinoma (KIRP), kanye colon adenocarcinoma (COAD) (p value <0.05) (Fig. 1e-m) . Ukuhlaziywa kwamasampula anempilo/esimila abhanqiwe kuphinde kwaqinisekisa ukubonakaliswa okuphezulu kakhulu kwe-DARS-AS1 ezimila zesinye i-urothelial carcinoma (BLCA), i-renal clear cell carcinoma (KIRC), i-prostate adenocarcinoma (PRAD), i-lung squamous cell carcinoma (LUSC) , uterine corpus endometrial carcinoma (UCEC), lung adenocarcinoma (LUAD), isibindi hepatocellular carcinoma (LIHC), renal papillary cell carcinoma (KIRP), kanye colon adenocarcinoma (COAD) (p value <0.05) (Fig. 1e-m) .Ukuhlaziywa kwamasampula anempilo/esimila abhanqiwe kuphinde kwaqinisekisa ukubonakaliswa okuphezulu kakhulu kwe-DARS-AS1 ku-bladder urothelial carcinoma (BLCA), i-clear cell renal kanye ne-renal cell carcinoma (KIRC), i-prostate adenocarcinoma (PRAD), izicubu ze-lung squamous cell carcinoma (LUSC)., 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) . , i-endometrial carcinoma ye-corpus uteri (UCEC), i-adenocarcinoma yamaphaphu (LUAD), i-hepatocellular carcinoma yesibindi (LIHC), i-papillary cell carcinoma yezinso (KIRP), ne-adenocarcinoma yekholoni (COAD) (p value < 0.05) (Umdwebo 1e– m) .配对 健康 / 肿瘤样本 的 分析 进一步 证实 了 DARS-AS1 在在 尿路 上 皮癌 (BLCA), 肾肾 肾肾显着 更 高 表达, 子宫体子宫子宫体子宫 癌 (Ucec), 肺腺癌 (Luad), 肝肝 细胞癌 (lihc), 肾 肾 乳头状 细胞癌 (kirp) 乳头状 细胞癌 (kirp) 乳头状<0.05) (图1e-m) .配 对 健康 / 肿瘤样本 的 分析 证实 了 Dars-OS1 在在 上尿路 皮癌皮癌 皮癌皮癌, 肾肾 细胞癌细胞癌(LUSC) 肿瘤 的 的 的 的 中 中 中 中 中 中 中 中 中 中 中 中 中 中 中 高 更 高更癌 (kirp) (i-coad) (p 值<0.05) (图1e-m) .Ukuhlaziywa kwamasampuli abhanqiwe anempilo/esimila kuqhubeke kwasekela indima ye-DARS-AS1 ku-bladder urothelial carcinoma (BLCA), i-clear cell renal cell carcinoma (KIRC), i-prostate adenocarcinoma (PRAD), kanye nezimila ze-lung squamous cell carcinoma (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). inkulumo ku-corpus uterine carcinoma (UCEC), i-lung adenocarcinoma (LUAD), i-hepatocellular carcinoma (LIHC), i-renal papillary cell carcinoma (KIRP), kanye ne-colon adenocarcinoma (COAD) (inani le-p <0.05) (Umfanekiso 1e -m).Ihlanganiswe ndawonye, ​​le miphumela ikhombisa ukuthi i-DARS-AS1 ivezwa kabanzi futhi kakhulu ezinhlobonhlobo zomdlavuza.
Ngenxa yokuthi i-DARS-AS1 ne-DARS (ufuzo olufaka amakhodi e-antisense strand) babelana ngomgqugquzeli ofanayo futhi baseduze kwenye, siklame i-shRNA ukuthi iwise phansi ngokuqondile i-DARS-AS1 kodwa hhayi i-DARS (I-Supplementary Fig. 2a,b kanye neThebula Lokwengeza 2) .Ngokungeziwe ku-SW620, siphinde sasebenzisa abanye olayini bamaseli abathathu abaveza kakhulu i-DARS-AS1 ukuze sifunde ukusebenza kahle nomsebenzi we-shRNA knockdown (Ithebula Lokwengeza 3).Imiphumela yethu ibonise ukuthi womathathu ama-shRNA athuthukisiwe azuze okungenani u-80% we-DARS-AS1 wokugoqa kahle ngomphumela omncane enanini le-DARS mRNA (Supplementary Fig. 2c–f).Ukwengeza, sithole ukuthi i-DARS-AS1 knockdown ngalawa ma-shRNAs ivimbele kakhulu ukukhula kwamaseli kolayini bomdlavuza we-colorectal SW620 (49.7%) kanye ne-HCT116 (27.7%), umugqa weseli lomdlavuza webele MBA-MD-231 (53.4%).) kanye ne-HepG2 i-hepatoma cell line (ukunciphisa i-92.7%), kanye nekhono labo lokwenza ama-spheres angenalutho (ukunciphisa isilinganiso se-~ 50.8%, 44.6%, 40.7% kanye ne-75.7% ngomugqa weseli) (Fig. 2a, b).Ku-SW620, imiphumela yokuhlolwa kokwakheka kwekholoni yaqinisekisa futhi ukuthi i-DARS-AS1 shRNA ivimbele kakhulu ukwanda kwamaseli ngokuncipha okumaphakathi okucishe kube ngu-69.6% (Fig. 2c).
Umthelela wokulawula i-shRNA ne-DARS-AS1 shRNA ekwandeni kweseli (a) nokwakheka kwe-spheroid (b) kumaseli e-SW620, HCT116, MBA-MD-231, kanye ne-HepG2.c Umphumela wokulawula i-shRNA ne-DARS-AS1 shRNA ekwakhekeni kwekholoni kumaseli e-SW620.Ukwanda kweseli (d), ukwakheka kwe-spheroid (e), kanye nokwakheka kwekholoni (f) yamaseli angu-SW620 aveza ngokweqile i-DARS-AS1.Idatha ebonisiwe imaphakathi ± ukuchezuka okujwayelekile kokuhlolwa okuthathu.* p ≤ 0.05, ** p ≤ 0.01, futhi *** p ≤ 0.001 ngokuhlolwa kuka-t koMfundi okunemisila emibili.
Ukuhambisana nezifundo zokulahleka komsebenzi, ngokulandelayo sidale amaseli e-SW620 egcizelela ngokweqile i-DARS-AS1 (I-Supplementary Fig. 2g).I-DARS-AS1 i-expression overexpression ikhulise kakhulu ukukhula kweseli (1.8-fold), ukwakheka kwe-spheroid engagxilile (1.4-fold), nokwakheka kwekholoni (3.3-fold) kumaseli e-SW620 (Fig. 2d–f).Siqinisekise lo mphumela sisebenzisa omunye ulayini weselula oveza i-DARS-AS1, A549.Lokhu kwanda kwamaseli okuthuthukisiwe ngenxa yokuvezwa ngokweqile kwe-DARS-AS1 kwaphinde kwaqashelwa kumaseli e-A549 (I-Supplementary Fig. 2h, i kanye ne-Supplementary Table 3).Zihlanganiswe ndawonye, ​​lezi zifundo zenzuzo nokulahlekelwa zibonisa ukuthi i-DARS-AS1 ikhuthaza ukwanda kwamangqamuzana omdlavuza ku-vitro.
Ukuhlola indlela eyisisekelo i-DARS-AS1 elawula ngayo ukwanda kwamaseli, senze ukuhlaziya okudonsela phansi kwe-RNA ukuze sihlonze ozakwethu abangaba khona abahlanganisa amaprotheni.Imiphumela ye-RT-qPCR ibonise ukuthi cishe i-86.2% ye-DARS-AS1 itholakala ku-cytoplasm yamaseli e-SW620 (Supplementary Fig. 3a).I-in vitro ebhalwe nge-biotinylated biotinylated DARS-AS1 noma i-pseudoRNA yabe isifakwe ngamaseli amaseli angu-SW620 alandelwa ukuhlukaniswa kwe-SDS-PAGE.Ukungcoliswa kwesiliva okwalandela kubonise ukuthi ibhendi ehlukile (~38 kDa) yacetshiswa kakhulu kumasampula wokudonsa we-DARS-AS1 kodwa hhayi ku-dummy RNA noma amasampula obuhlalu (Fig. 3a).Leli bhendi lihlonzwe njengephrotheni esebenzayo ye-PKR (i-PACT) nge-mass spectrometry (MS) futhi yaqinisekiswa ngokungeziwe nge-immunoblotting kumigqa yeseli ye-SW620, HCT116, ne-HepG2 (Fig. 3a,b).Ukunothiswa kwe-DARS kanye namaprotheni e-PACT ahlobene - i-PKR ne-TRBP - kuphinde kwaphenywa kusetshenziswa ukuhlaziywa kwe-RNA nge-Western blotting (WB).Imiphumela ibonise ukuthi akukho ukusebenzisana okuqondile phakathi kwe-DARS-AS1 RNA kanye nalawa maprotheni amathathu atholakele (I-Supplementary Fig. 3b).Ukusebenzisana okuqondile phakathi kwe-DARS-AS1 ne-PACT kwaqinisekiswa ngokuqhubekayo ukuhlaziya kwe-RNA immunoprecipitation (RIP), okubonise ukuthi i-DARS-AS1 yayicetshiswe kakhulu kuma-anti-PACT antibodies kodwa hhayi amanye ama-RNA okulawula (Umfanekiso 3c).Ukuze unqume ukuthi ingabe i-DARS-AS1 isebenzisana ngokuqondile ne-PACT lapho zingekho ezinye izingxenye zeselula, ukuhlolwa kwe-in vitro biolayer interferometry (BLI) kwenziwa kusetshenziswa i-PACT ehlanzekile.I-Biotin enelebuli ye-DARS-AS1 noma i-dummy RNA ayizange inyakaze kuzinzwa ze-streptavidin (SA) zase zifakwe ku-kinetic buffer equkethe i-1 μM PACT.Ngokuphawulekayo, i-PACT iboshelwe ngokuqinile ku-DARS-AS1 (inani le-KD ~26.9 nM), kodwa hhayi ukulingisa i-RNA (Umfanekiso 3d).Ihlanganiswe ndawonye, ​​le miphumela ibonisa ukusebenzisana okuqondile nokuhlobana okuphezulu phakathi kwe-DARS-AS1 ne-PACT.
Ukuhlaziywa kokudonsa kwe-RNA kukhonjwe ukuthi i-DARS-AS1 isebenzisana ne-PACT kumaseli e-SW620.Ngaphezulu, ukungcoliswa kwesiliva kwamaprotheni ahlobene.Ama-immunoblots aphansi ayenziwa nge-anti-PACT antibody.b Ukuhlaziywa kokudonsela phansi kwe-RNA kwenziwa kumaseli e-HCT116 (phezulu) kanye ne-HepG2 (phansi).Ukunothiswa kwe-PACT kutholwe nge-immunoblotting.Ukuhlolwa kwe-cRNA immunoprecipitation (RIP) kwenziwa kumaseli e-SW620 kusetshenziswa amasosha omzimba abonisiwe.I-PACT ebopha amajika kubude obugcwele be-DARS-AS1 noma i-RNA yokulawula itholwe kusetshenziswa i-biolayer interferometry (BLI).I-RNA yayinganyakazi ku-streptavidin biosensor.I-1 μM PACT isetshenziselwe ukukala ukuhlangana.I-e RNA pull assay yenziwa kusetshenziswa i-biotinylated yobude obugcwele be-DARS-AS1 noma incishisiwe (phezulu).I-Immunoblot ebonisa i-PACT eyamukelwe (ngezansi).f I-PACT ehlatshwe umkhosi ifakwe i-biotinylated yobude obugcwele be-DARS-AS1 noma yancishiswa (njengaku-e) ukuze kuhlolwe i-in vitro RIP.I-RNA ekhishiwe iqinisekiswe yi-RT-qPCR.g Ukuhlobana okuhlobene kwezingcezu ezihlukene ze-RNA ze-PACT kutholwe kusetshenziswa i-biolayer interferometry.Ukuze kuhlaziywe, kusetshenziswe i-100 nM RNA kanye ne-1 μM RAST.h Ukuhlolwa kwe-RIP kwe-in vitro kwenziwa kusetshenziswa i-purified intact noma encishisiwe enelebula ethi PACT.I-RNA ekhishiwe iqinisekiswe yi-RT-qPCR.i Izinga lokukhula lamaseli e-SW620 aveza ngokweqile i-DARS-AS1, i-PACT, noma kokubili.j Ukuvezwa ngokweqile kobude obugcwele noma okuncishisiwe kwe-DARS-AS1 kumaseli e-SW620 kube nemiphumela ehlukile ekukhuleni kweseli.k I-Apoptosis itholwe ngokuvinjwa kwamasosha omzimba nge-anti-PARP.l Ukukhishwa kwe-DARS-AS1 kudala i-apoptosis yamaseli e-SW620 njengoba kuboniswa yi-flow cytometry.Idatha ebonisiwe imaphakathi ± ukuchezuka okujwayelekile kokuhlolwa okuthathu. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p <0.0001, ngokuhlolwa kwe-t yoMfundi enemisila emibili. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p <0.0001, ngokuhlolwa kwe-t yoMfundi enemisila emibili. *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 ngokuhlolwa kuka-t koMfundi okunemisila emibili. *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 ngokuhlolwa kuka-t koMfundi okunemisila emibili.
Sibe sesikhiqiza izingcezu ezintathu ze-DARS-AS1 RNA ze-biotinylated ngokulotshwa kwe-in vitro ukuze sihlonze isifunda se-DARS-AS1 esidingekayo kunhlangano ye-PACT (Umfanekiso 3e).Imiphumela yokuhlaziywa kwe-RNA ibonise ukuthi ucezu ngalunye lukwazile ukusebenzisana ne-PACT, kodwa isifunda se-3'-terminal (384-768 nucleotides ebhalwe A3) sibonise ngaphezu kwe-1-384 nucleotide ebhalwe A1) (Fig. 3e).Imiphumela efanayo yabonwa ekuhlolweni kwe-RIP ye-in vitro kusetshenziswa i-PACT ye-recombinant (Umfanekiso 3f).Ngokuvumelana nale miphumela, ukuhlolwa kokubopha izingcezu ze-RNA ezinganyakazi ku-PACT kusetshenziswa i-BLI kuphinde kwabonisa ukuthi i-PACT inokuhlobana okuphezulu kwe-A3 (384–768 nt) (inani le-KD elicishe libe ngu-94.6 nM), kuyilapho cishe ingekho izixhumanisi nezinye izindawo.(Umdwebo 3d).
Siphinde sahlola izifunda ezibophezelayo ezihlobene ku-PACT.I-PACT iqukethe izizinda ezintathu ezisebenzayo, ezimbili zazo okuyizizinda ezibophezela kabili ze-RNA (dsRBD) nesizinda sesithathu (esiqokiwe i-D3) esisebenza njengesivusi sokusebenzisana kwamaprotheni.Ukuhlola umthamo wokubopha we-lncRNA wesizinda ngasinye, senze izinguquko ezintathu ezisuse isizinda ngasinye kwezintathu futhi senza ukuhlolwa kwe-RIP ye-in vitro.Imiphumela yethu ibonise ukuthi ukususwa kwesizinda sesithathu (D3) se-PACT kunciphise kakhulu ukusebenzisana kwayo ne-DARS-AS1 (ngo-0.11-fold uma kuqhathaniswa ne-PACT eqinile) uma kuqhathaniswa nezinye izinguquko ezimbili (Fig. 3h), kwaboniswa ukuthi ukukhululwa ye-D3 ihlanganyele ne-DARS.-AC1.Ihlanganiswe ndawonye, ​​le miphumela iphakamisa ukuthi ukusebenzisana phakathi kwe-DARS-AS1 ne-PACT kungase kwenzeke ngokuyinhloko ekupheleni kwe-3′ ye-DARS-AS1 kanye nesizinda se-D3 se-PACT.
Siqaphele ukuthi i-DARS-AS1 ayizange ibe nomthelela ekukhulumeni kwe-PACT futhi i-PACT ayizange ibe nomthelela ku-DARS-AS1 (I-Supplementary Fig. 3c).Sibe sesihlola umthelela we-PACT knockdown ekukhuleni kwamaseli.Ngokuphambene ne-DARS-AS1, amaseli ahlobene akhula izikhathi ezingu-1.5-3 ngokushesha lapho i-PACT idilizwa phansi (I-Supplementary Fig. 3d).Imiphumela yokuhlolwa kokubunjwa kwekholoni ibonise ukuthi amangqamuzana akha amakholoni angu-2-3 ngemva kokwelashwa kwe-shRNA nge-PACT (Supplementary Fig. 3e).Ukuhlola ukuthi ingabe i-DARS-AS1 ilawula ukwanda kwamaseli nge-PACT, sikhiqize amaseli e-SW620 aveza ngokweqile i-PACT, i-DARS-AS1, noma kokubili.I-overexpression ye-PACT ibonise ukuvinjelwa okuphawulekayo kokwanda kwamaseli (Umfanekiso we-3i).Nakuba i-DARS-AS1 i-overexpression per se ikhuthaze ngokuphawulekayo ukwanda kwamaseli, awukho umehluko ophawulekayo esilinganisweni sokukhula samaseli aveza ngokweqile i-DARS-AS1 ne-PACT.Le miphumela iphakamisa ukuthi i-PACT ingase imelane nokwanda okubangelwa ukucindezeleka ngokweqile kwe-DARS-AS1.
Njengoba izifunda ezihlukene ze-DARS-AS1 zinamakhono ahlukene okubopha i-PACT, siphenye umthelela wazo ohlobene ekwandeni kwamaseli ngokuveza okuhlukile okuhlukile kwezingcezu ze-DARS-AS1.Uma kuqhathaniswa nezinye izingcezu ezimbili, i-DARS-AS1 yayigxilile kakhulu ekupheleni kwe-3 (384-768 nt), isifunda esihlobene ne-PACT esiyinhloko ku-DARS-AS1, esasinamandla aphezulu okugqugquzela ukwanda kwamaseli (Fig. 3j).Le miphumela ibonisa ukuhlobana okuhle phakathi kwamandla okubopha kanye nomsebenzi webhayoloji we-DARS-AS1.
I-PACT kubikwe ukuthi iyiprotheyini ye-pro-apoptotic19.Ngakho-ke, siphenye umphumela we-DARS-AS1 ku-apoptosis.Njengoba bekulindelekile, i-DARS-AS1 knockdown ikhulise kakhulu i-PARP cleavage kumaseli e-SW620 futhi yandisa ingxenye yamaseli e-annexin V-positive ku-SW620, HCT116, HepG2, kanye nemigqa yeseli ye-MBA-MD-231 (Fig. 3k).3).3f–h), okubonisa ukuthi umphumela we-anti-apoptotic we-DARS-AS1 kumaseli omdlavuza uphambene nomsebenzi wokuyenga i-apoptosis we-PACT.Ihlanganiswe ndawonye, ​​le miphumela iphakamisa ukuthi indlela yokusebenza kwe-oncogenic ye-DARS-AS1 ingase ibe ngokuvinjelwa komsebenzi we-PACT.
Okulandelayo, sihlole imithelela yokusebenza yenhlangano ye-DARS-AS1-PACT.I-PACT kubikwe ukuthi ivule i-PKR ngokusebenzisana okuqondile, okubuye kuthuthukise i-eIF2α phosphorylation, okubangela ukususwa kokuhumusha kanye ne-apoptosis17.Okokuqala, sihlole ukuthi ingabe i-DARS-AS1 iyakuthinta ukwenziwa kwasendaweni kweselula kwe-PACT ne-PKR.I-Confocal fluorescence microscopy ibonise ukuthi i-PACT ne-PKR zenziwe zahlanganiswa kakhulu kumaseli e-SW620 nge-coefficient ephakathi ye-Pearson engu-0.72.Phakathi naleso sikhathi, i-DARS-AS1 overexpression yehlise kakhulu i-PACT kanye ne-PKR co-localization (isho i-coefficient yokuxhumana ye-Pearson 0.61) (Umfanekiso 4a).Ukuze siphenye ukuthi ingabe i-DARS-AS1 ingakwazi yini ukulinganisa ukusebenzisana kwe-PACT-PKR, senze uhlolo lwe-co-immunoprecipitation (co-IP) nge-anti-PACT antibody kumaseli e-SW620 lysates.I-PKR yayicetshiswe kakhulu ku-anti-PACT kumaseli okulawula, kuyilapho ukubuyiswa kwe-PKR kuncishiswe kakhulu kuma-lysates avela kumaseli e-DARS-AS1 (Fig. 4b).Okuhlanzekile okunelebuli ye-PACT kanye ne-PKR kwasetshenziselwa ukuhlolwa kokubopha amaprotheni e-in vitro.Ngokufanelekile, lezo ezinikeze i-DARS-AS1 kodwa ingekho ukulawula i-RNA zibonise ukusebenzisana kwe-PACT-PKR okucindezelwe (Figure 4c).Yonke imiphumela ibonise ukuthi i-DARS-AS1 iphazamise ukuxhumana kwe-PACT ne-PKR.
Ukwenziwa ndawonye kwe-PACT kanye ne-PKR kumaseli okulawula noma amaseli aveza ngokweqile i-DARS-AS1 kubonwe kusetshenziswa i-confocal fluorescence microscopy.Ama-nuclei ayegcotshwe nge-DAPI.Imiphumela yezibalo itholwe ezithombeni eziyi-16.b I-Co-immunoprecipitation (co-IP) isebenzisa i-anti-PACT antibody kuma-cell lysates okulawula amaseli e-SW620 noma amaseli aveza ngokweqile i-DARS-AS1.c Ilebulwe ngokuthi i-PACT, i-PKR ehlanzekile futhi ebhalwe nge-vitro nge-DARS-AS1 noma i-RNA mbumbulu zafakwa ukuze kuhlaziywe ukubopha amaprotheni e-in vitro.Ama-anti-flag antibodies asetshenziselwa ukuvimbela ukuzivikela komzimba.d Ama-Immunoblots anamasosha omzimba abonisiwe enziwa kumaseli e-SW620 kanye ne-HCT116 adluliselwa ngokulawula i-shRNA noma i-DARS-AS1-shRNA elandelwa yindlala ye-serum.e-DARS-AS1 amazinga enkulumo aguqule ukuzwela kweselula ku-thapsigargin.Amaseli e-SW620 adluliselwe nge-DARS-AS1 shRNA, i-DARS-AS1 yokuveza ngokweqile i-plasmid noma i-plasmid yokulawula.Amaseli aphathwe nge-thapsigargin amahora angu-48 futhi ukusebenza kwamaseli kwanqunywa kusetshenziswa i-reagent ye-MTS.f I-in vitro ebhalwe ukuthi i-DARS-AS1 noma i-dummy RNA kanye ne-purified PACT yasetshenziselwa ukuhlolwa kokusebenza kwe-in vitro nokutholwa kwe-immunoblot.g Ama-Immunoblots asebenzisa lawa masosha omzimba enziwe kumaseli e-SW620-ctrl (kwesokunxele) noma amaseli aveza ama-PKR aguquguqukayo ngokweqile (kwesokudla).Lawa maseli abe esedluliselwa ngokulawula i-shRNA noma i-DARS-AS1-shRNA elandelwa yindlala ye-serum.h I-cytometry egelezayo ibonise ukuthi ukungasebenzi kwe-PKR eguquguqukayo kunxeshezelwe i-apoptosis eyenziwe yi-DARS-AS1 kumaseli e-SW620.i Ama-Immunoblots anamasosha omzimba abonisiwe enziwa kumaseli e-SW620 (kwesokunxele) noma e-HCT116 (kwesokudla).Amaseli adluliselwe ngokulawula i-shRNA noma i-DARS-AS1 shRNA awanayo i-serum futhi engezwa nge-100 nM PKR C16 inhibitor noma i-DMSO.Ibha yesikali = 5 µm.Idatha ebonisiwe imaphakathi ± ukuchezuka okujwayelekile kokuhlolwa okuthathu.* p ≤ 0.05 Ukuhlolwa kuka-t koMfundi okunemisila emibili.
Ngokuvamile kukholakala ukuthi uma i-PACT isebenzisana ne-PKR17, i-PKR phosphorylation e-Thr451 ingayengwa.Imiphumela yethu ibonise ukuthi izinga le-PKR phosphorylation laliphakanyiswe kakhulu kumaseli e-DARS-AS1 wokugoqa ngemva kokulamba kwe-serum (Fig. 4d kanye ne-Supplementary Fig. 4a).Ngokuvumelana nalokho, sithole ukuthi i-phosphorylation ye-eIF2α, i-substrate eyinhloko ye-PKR, nayo yanda kakhulu nge-DARS-AS1 shRNA (I-Fig. 4d kanye ne-Supplementary Fig. 4a).I-Thapsigargin iyi-stressor ye-ER eyenza i-ER ikhulule i-Ca2+.Ukwelashwa nge-thapsigargin kuye kwabikwa ukuthi kubangele inkulumo nokusebenza kwe-PACT, eqhubeka nokusebenzisana futhi isebenze i-PKR, okuholela ku-apoptosis ngokwandisa i-eIF2α phosphorylation 18,61.Lapha, sisebenzise i-thapsigargin njengesikhuthazi sendlela ye-PACT/PKR ukuze siphenye ukuthi i-DARS-AS1 ingasiza amaseli ukunqoba ingcindezi ngokuvimbela indlela ye-PACT/PKR.Siqaphele ukuthi izinga lesisho se-DARS-AS1 lihlotshaniswa kahle nokumelana neseli ku-thapsigargin.Amaseli e-SW620 aveza ngokweqile i-DARS-AS1 asinde kangcono lapho ephathwa nge-thapsigargin, kuyilapho amaseli ane-DARS-AS1 knockdown abe sengozini enkulu (Fig. 4e).Ngokuvumelana nale miphumela, i-DARS-AS1 overexpression yehlise i-thapsigargin-induced PKR phosphorylation (I-Supplementary Fig. 4b).Ngokuphambene, ngemva kokwelashwa kwe-thapsigargin, i-PKR ne-eIF2α babeyi-phosphorylated ngezinga eliphakeme kumaseli e-Knockdown e-DARS-AS1 uma kuqhathaniswa namaseli okulawula (I-Supplementary Fig. 4b).Kuyathakazelisa ukuthi i-thapsigargin yenza isisho se-DARS-AS1 ngendlela encike kumthamo, engabonisa umsebenzi wokulwa nokucindezeleka we-DARS-AS1 (I-Supplementary Fig. 4c).Ngaphezu kwalokho, senze izivivinyo zokusebenzisa i-in vitro ukuze siqinisekise lokhu kubonwa.Kafushane, i-PKR yahlanzwa kuma-lysate eseli kusetshenziswa i-anti-PKR antibody, yase ifakwa nge-recombinant PACT kanye ne-DARS-AS1 ebhalwe nge-vitro.Ngemuva kokusabela kwe-enzymatic, i-phospho-PKR yatholwa kusetshenziswa i-WB.Imiphumela yethu ibonise ukuthi i-PKR phosphorylation yayivinjwe kakhulu yi-DARS-AS1, kodwa hhayi ngokulawula i-RNA (Umfanekiso 4f).Le miphumela ye-in vitro ne-vivo iphakamisa ukuthi i-DARS-AS1 ivimbela ukwenziwa kusebenze kwe-PACT-mediated PKR.Ngesikhathi esifanayo, siphinde sabona ukwehla kokutholwa kwe-PACT phambi kwe-DARS-AS1 (Umfanekiso 4f).Lo mphumela uhambisana nemiphumela ye-in vitro protein binding assay (Figure 4c) futhi uphinda ubonise umsebenzi wokuvimba we-DARS-AS1 we-PACT-PKR association.
I-Ser246 ne-Ser287 kusizinda se-D3 se-PACT ziyadingeka ukuze kusebenze i-PKR ngaphansi kwengcindezi yeselula.Ukushintshanisa izinsalela ze-serine ezimbili esikhundleni se-alanine kunikeze i-Mutant PACT (mutD), eyenza i-PKR isebenze lapho ingekho ingcindezi, futhi esikhundleni se-alanine (mutA) kwahlehlisa umthetho olandelwayo.Njengoba sibonise ukubaluleka kwalesi sizinda ngokuhlobana okuqondile ne-DARS-AS1, senze lezi ziguquli ezimbili ze-PACT ukuze sihlole ukuthi lezi zinsalela zingabandakanyeka yini ekusebenzelaneni ne-DARS-AS1.Kuyathakazelisa ukuthi zombili iziguquli eziguquliwe zalahlekelwa amandla okubophezela ku-DARS-AS1 (I-Supplementary Fig. 4d), iphakamisa ukuthi ukwakheka okuphelele kwephrotheni ye-PACT kungase kudingeke ukuze kusetshenziswe kahle i-DARS-AS1.
Ngaphezu kwalokho, imiphumela yethu iphinde iphakamise ukuthi ukuvinjelwa kwe-DARS-AS1-shRNA-okubangelwa ukwanda kwamaseli kungabuyiselwa ngokwengxenye ngokuveza ngokweqile i-PACT mutant (PACTmutA) ebusayo engalungile noma i-PKR mutant evelele (PKRmut) (I-Supplementary Fig. 4e. e).Ukuvezwa ngokweqile kweziguquguquki ze-PKR ezilawula-negative kwehlise i-phosphorylation ye-PKR edalwe ukushaya kwe-DARS-AS1 kanye ne-eIF2α phosphorylation kumaseli ancishwe i-serum (Fig. 4g).Okubaluleke nakakhulu, ingxenye yamaseli e-apoptotic eyenziwa yi-DARS-AS1 knockdown nayo yancishiswa kumaseli e-PKRmut e-overexpressing (Fig. 4h kanye ne-Supplementary Fig. 4g).Ukuvinjelwa komsebenzi we-PKR kinase kuphinde kuphazamise umsebenzi we-DARS-AS1, njengoba imiphumela ibonise ukuthi i-DARS-AS1 i-knockdown yayingavamile ukuqala i-PKR ne-eIF2α phosphorylation lapho amaseli ephathwa nge-PKR-specific C16 inhibitor (Fig. 4i kanye ne-Supplementary Fig. 4h).).Ihlanganiswe ndawonye, ​​imiphumela yethu iphakamisa ukuthi i-DARS-AS1 iphromotha ukwanda kwamaseli, okungenani ngokwengxenye, ngokuvimbela ukusebenzisa i-PACT-mediated PKR.
Ukuze siqhubeke sihlola indima ye-DARS-AS1 ku-tumorigenesis, senze ukuhlolwa kwe-vivo sisebenzisa imodeli ye-mouse xenograft. Imiphumela ibonisa ukuthi ukwehla kwe-DARS-AS1 kwehle ngokumangalisayo ukukhula kwesimila kumagundane (inani le-p <0.0001) (Fig. 5a). Imiphumela ibonisa ukuthi ukwehla kwe-DARS-AS1 kwehle ngokumangalisayo ukukhula kwesimila kumagundane (inani le-p <0.0001) (Fig. 5a). Результаты показывают, что нокдаун DARS-AS1 резко снижает рост опухоли у мышей (значение p <0,0001) (рис. 5а). Imiphumela ibonisa ukuthi ukwehla kwe-DARS-AS1 kunciphisa kakhulu ukukhula kwesimila kumagundane (inani le-p <0.0001) (Umfanekiso 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). Imiphumela ibonise ukuthi i-DARS-AS1 knockdown inciphise kakhulu ukukhula kwesimila kumagundane (inani le-p <0.0001) (Umfanekiso 5a).Ngakho-ke, eqenjini le-Knockdown le-DARS-AS1, kube nokwehla okuphawulekayo kwevolumu ye-tumor cishe ngama-72.9% kanye nesisindo se-tumor cishe ngama-87.8% (Umfanekiso 5b-d).Le miphumela iphakamisa ngokuqinile ukuthi i-DARS-AS1 ingakhuthaza kakhulu ukukhula kwesimila ku-vivo.
Imithelela yokwehla kwesikhangiso i-DARS-AS1 ku-colorectal oncogenesis kumagundane anqunu.Amajika okukhula (a), usayizi wesimila (b), isisindo (c), nemifanekiso yesimila (d) ayaboniswa.Amabha wephutha amele ±SEM. n = 10. ****p <0.0001, ngokuhlolwa kwe-t yoMfundi enemisila emibili. n = 10. ****p <0.0001, ngokuhlolwa kwe-t yoMfundi enemisila emibili. n = 10. ****p < 0,0001 по двустороннему критерию Стьюдента. n = 10. ****p < 0.0001 Ukuhlolwa kuka-t Komfundi okunemisila emibili.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 Ukuhlolwa kuka-t Komfundi okunemisila emibili.U-e Kaplan-Meier uhlaziye ukuhlobana phakathi kwamazinga enkulumo ye-DARS-AS1 kanye nokusinda sekukonke ezigulini ezine-UVM, KICH, KIRP, MESO, GBM, ne-LGG.Amazinga aphezulu wenkulumo ye-DARS-AS1 ezigulini ayephezulu ku-50%;izinga eliphansi lesisho se-DARS-AS1 ezigulini lalingaphansi kwama-50%.p-amanani anqunywa kusetshenziswa ukuhlolwa kwezinga lelogi.f Imodeli ehlongozwayo lapho i-DARS-AS1 ilawula indlela ye-PACT-PKR nokukhula kwesimila.
Ukuze siqonde kangcono umthelela womtholampilo we-DARS-AS1, sihlole ukuhlobana phakathi kokusho kwayo nokuphila kwesiguli.Ngokuhlaziya idathasethi ye-TCGA, sithole ukuthi isisho esiphezulu se-DARS-AS1 sasihlotshaniswa ne-uveal melanoma (UVM), i-renal chromophobia (KICH), i-renal papillary cell carcinoma (KIRP), i-mesothelioma (MESO), i-multiplex.Ukusinda okuphansi kwakuhlotshaniswa kakhulu ne-glioblastoma morphosis (GBM) kanye neziguli ezine-low-grade brain glioma (LGG) (Figure 5e).Le miphumela iphakamisa ukuthi i-DARS-AS1 ingase idlale indima ebalulekile ekuqhubekeleni phambili kwesimila emtholampilo futhi ingase ibe i-biomarker eqagelayo engenzeka emakhazeni amaningi.
Kulolu cwaningo, sisebenzisa ukuhlolwa kokusebenza kwe-CRISPRi okukhulu, sinqume ukuthi i-DARS-AS1 lncRNA inqoba ukucindezeleka kwamangqamuzana omdlavuza ngokulawula abaphenduli ababili bokucindezeleka ababalulekile, i-PACT ne-PKR.Ngokusebenzisana ngokuqondile ne-PACT, i-DARS-AS1 ivimbele ukusebenza kwe-PACT-mediated PKR, ngaleyo ndlela ivimbele ukufa kweseli ye-apoptotic futhi ikhuthaze ukwanda kwamaseli (Fig. 5f).Ukuphakama kwe-DARS-AS1 kuye kwabonwa ezinhlotsheni eziningi zomdlavuza, okuphakamisa ukuthi umsebenzi wayo wokukhuthaza ukusinda kwamangqamuzana omdlavuza ngaphansi kwezimo ezicindezelayo ungase usebenze kabanzi ezinhlotsheni eziningi zomdlavuza.
I-PACT ikhonjwe njengeprotheni ye-activator ye-PKR, futhi ukusebenzisa i-PACT-mediated PKR kudlala indima ebalulekile ekuphenduleni kwengcindezi ngokulawula ukuloba, ukuhumusha, i-apoptosis, nezinye izinqubo ezibalulekile zamaselula62.Sekungamashumi eminyaka, kwenziwa imizamo yokuqonda umthethonqubo othize womdlavuza we-PACT-PKR cascade.Lapha, ucwaningo lwethu lwembula indlela ehlukile yokulawula i-PACT-PKR kumaseli omdlavuza ngokusebenzisa i-lncRNA DARS-AS1 yeselula, ebophezela ngokuqondile ku-PACT, ivimbela ukusebenzisana kwe-PACT-PKR, ivimbela ukusebenza kwe-PKR kanye ne-eIF2α phosphorylation, ngaleyo ndlela ivimbele i-apoptosis ebangelwa ukucindezeleka futhi ukukhuthaza ukwanda komdlavuza ekugcineni.amaseli.Lokhu kutholakala kukhanyisa izinhloso ze-lncRNA ezingaba khona zokubikezelwa komdlavuza kanye nokwelashwa.
Idatha yethu ibonise ukuthi i-DARS-AS1 knockdown iqwashisa amaseli endlala ye-serum ngokunyuka okukhulu kwe-phosphorylated PKR ne-eIF2α.Le miphumela iphakamisa ukuthi i-DARS-AS1 ikhuthaza ukusinda kwamangqamuzana omdlavuza ngaphansi kwezimo ezinzima ngokuvimbela umsebenzi we-PACT/PKR.Amanye ama-RNA amaningana angewona amakhodi, njenge-ASPACT ne-nc886, nawo ayabandakanyeka ku-axis ye-PACT/PKR ngokwehlisa i-PACT48 mRNA noma ngokulawula i-autophosphorylation ngokubophezela ku-PKR49,50,64.Phakathi kwazo, i-DARS-AS1 isebenza njengesiphazamisi senhlangano ye-PACT-PKR.Lolu cwaningo luthuthukisa ukuqonda kwethu ukulawulwa kwe-axis ye-PACT/PKR kanye nendima yama-lncRNA ezimpendulweni zokucindezeleka.
I-PACT iqukethe izizinda ezintathu ezihlukene.I-dsRBD ngayinye yokuqala emibili yanele ukuzuza ukubophezela okuphezulu kwe-PACT ku-PKR, kuyilapho isizinda sesithathu (D3) sidingeka ukuze kusebenze i-PKR ku-vitro kanye ne-vivo.Ucwaningo lwethu lubonise ukuthi i-DARS-AS1 isebenzisana ngokukhethekile nesizinda se-D3 (Fig. 3h).Uma kubhekwa usayizi omkhulu we-lncRNA (768 nucleotide), i-DARS-AS1 ebophezela ku-D3 inganqanda ngokoqobo ukusebenzisana phakathi kwesizinda se-PACT se-dsRBD ne-PKR, ngaleyo ndlela ivimbe inhlangano ye-PACT ne-PKR.Ukuguqulwa kwamaphoyinti e-PACT okuthathe indawo ye-Ser246 ne-Ser287 ku-D3 nge-alanine noma i-aspartate kuphazamise ukuhambisana kwayo okubophezelayo kwe-DARS-AS1, okukhomba ukubaluleka kwazo zonke izakhiwo ze-D3's zesakhiwo nogesi ekuhlotshaneni kwazo.Imininingwane eyengeziwe yale nqubo izodingeka esikhathini esizayo, kusetshenziswa ukuhlaziya okunembe kakhudlwana kwamakhemikhali ezinto eziphilayo kanye nokuhlaziywa kwesakhiwo se-PACT esinesinqumo esiphezulu.
Ucwaningo lwangaphambilini lubike ukuthi i-DARS-AS1 ikhuthaza ukwanda kwamaseli ngokusebenzisa izindlela ezimbalwa51,52,53.Kwesinye isibonelo, abaphenyi babone ukuthi i-DARS-AS1 ilawule ufuzo lwayo lwe-DARS lwe-antisense protein-encoding ngokukhomba i-miP-194-5p kumaseli omdlavuza wezinso.Kodwa-ke, ocwaningweni lwamanje, ukunqotshwa kwe-DARS-AS1 kube nomthelela omncane ekulotshweni kwe-DARS ezinhlotsheni eziningi zomdlavuza, okubandakanya okungenani umdlavuza we-colorectal, webele, nesibindi.Ngenxa yokuthi ama-lncRNA abonisa amaphethini okuchaza amaseli kanye nezicubu ezithile, izindlela zokusebenza zingahle zingalondolozwa kuzo zonke izinhlobo zomdlavuza, ezingase zibe nomthelela kulo mehluko phakathi kokuqaphela kwethu kanye nokuhlolwa kwangaphambilini komdlavuza ohlukahlukene.Kudingeka izifundo ezikhethekile ukuze kucaciswe izindlela ezithile zezinqubo ezihlukahlukene ze-physiological and pathological.
Ukuhlaziywa kwedatha yomtholampilo kubonise ukuthi inkulumo ye-DARS-AS1 kumathumba ihlotshaniswa ngokuphambene nokuphila kweziguli ezinomdlavuza, okugcizelela ukubaluleka kwe-eksisi ye-DARS-AS1/PACT/PKR ekuqaguleni komdlavuza.Sengiphetha, ucwaningo lwethu lubonisa ukuthi i-DARS-AS1 ingumlawuli we-PACT/PKR yokusayina i-axis, ikhuthaza ukwanda kwamangqamuzana omdlavuza, futhi ivimbele i-apoptosis ngesikhathi sokuphendula ukucindezeleka, ehlinzeka ngolunye umugqa wocwaningo futhi inentshisekelo yocwaningo lwesikhathi esizayo mayelana nokwelashwa okungenzeka. .
Olayini bamaseli abantu SW620, A549, MBA-MD-231, HCT116, HepG2 kanye ne-HEK293T batholwe ku-National Cell Line Resource Infrastructure e-China.Wonke amaseli ayenakekelwa ku-DMEM medium (DMEM, Thermo Fisher Scientific, Waltham, MA) engezwe ngo-10% FBS (Gemini, Brooklyn, NY) kanye no-1% penicillin-streptomycin (Thermo Fisher Scientific) ku-37°C, 5% CO2.incubator.
I-Anti-PACT, i-Abcam (ab31967);I-Anti-PKR, i-Abcam (ab184257);I-Anti-PKR (i-phospho-T451), i-Abcam (ab81303);I-Anti-Flag, i-Abcam (ab125243);I-Anti-eIF2α, i-Abcam (A0764));i-anti-eIF2α (i-phosphorus S51), i-Abcam (ab32157);i-anti-PACT (i-phosphorus S246), i-Abgent (AP7744b);i-anti-β-tubulin, i-CST (2128);igundane elijwayelekile IgG, CST (5415S);evamile unogwaja IgG, CST (2729S).Amasosha omzimba ahlanjululwe ngo-1:1000 ku-PBST ukuze kuqedwe iWestern and 1:100 ye-IP.
Ama-sgRNA athuthukiswa kusetshenziswa ithuluzi elitholakala esidlangalaleni elibizwa nge-CRISPR-ERA66.Sisebenzise amapharamitha wamathuluzi azenzakalelayo ekuthuthukisweni kwe-sgRNA kanye ne-algorithm eyenziwe ngekhompyutha amasayithi abophezelayo e-sgRNA esifundeni esingu-3 kb.igxile ku-TSS.Amachibi e-sgRNA oligonucleotides ahlanganiswa e-CustomArray, Inc. (Bothewell, WA) futhi ahlanganiswa abe ama-pgRNA plasmids (Addgene #44248).Isamba esingu-12 µg se-pgRNA plasmid ehlanganisiwe, 7.2 µg ye-psPAX2 (Addgene #12260), kanye no-4.8 µg we-pMD2.G (I-Addgene #12259) adluliselwe ku-5 x 106 HEK206 cm DNA Transfection 1 DNA dishe amaseli ( CWBIO, Beijing, China) elandela imiyalelo yomkhiqizi.Ama-supernatant aqukethe amagciwane aqoqwa emahoreni angu-48 nangama-72 ngemva kokudluliselwa futhi ahlungwa ngesihlungi esingu-0.45 µm.Ukuze kuhlolwe, amaseli e-SW620 aveza iphrotheni ehlanganisiwe ye-dCas9/KRAB atholwe ngokudluliswa kwegciwane.Amaseli e-SW620 alungisiwe atheleleke ngelabhulali yegciwane ekuhlolweni okune kokutheleleka okuzimele ku-MOI ka-0.1-0.3 futhi athathwa amasampula nge-2 μg/ml puromycin (Sigma, St. Louis, MO) izinsuku ezingu-2.Ngemuva kwalokho, amaseli akhuliswa izinsuku eziyi-18 ku-invitro enomthamo omncane womtapo wolwazi wamaseli angama-500/sgRNA ukuze ahlolwe.
I-Genomic DNA yakhishwa ngokulandela imiyalelo ye-QIAamp DNA Blood Midi Kit (QIAGEN, Düsseldorf, Germany; 51183).Sekukonke, i-100 μg ye-genomic DNA ngokuphinda kwebhayoloji yasetshenziswa ukwakha umtapo wolwazi.Isifunda se-sgRNA sandiswa imizuliswano emibili ye-PCR futhi yaxhunyaniswa nebhakhodi.
Imikhiqizo ye-PCR yahlanzwa kusetshenziswa ijeli ye-NucleoSpin® kanye nekhithi yokuhlanza ye-PCR (MACHHEREY-NAGEL, Düren, Germany; 740609.250) futhi yalinganiswa kusetshenziswa ikhithi yokutholwa ye-DNA enezintambo ezimbili ye-Qubit™ HS (Thermo Fisher Scientific; Q32854).
Ukuhlolwa kwe-MTS kwasetshenziswa ukukala ukwanda kwamaseli.Amaseli afakwe ezitsheni zemithombo engama-96 ekumineni kokuqala kwamaseli angama-2000/emthonjeni.Inani elihlobene lamaseli likalwa nsuku zonke ngesikhathi esibonisiwe sengqikithi yezinsuku ezingu-4-6.Emthonjeni ngamunye, u-20 μl we-reagent ye-MTS (i-Promega) yahlanjululwa ngo-100 μl we-DMEM, yafakwa namaseli amahora angu-4 ku-37°C, kwase kuthi i-OD490 yalinganiswa.
Amandla okukhula okungagxilile atholwe ngokuhlaziya ukwakheka kwama-sphere.Kafushane, amaseli angu-2000 adluliselwe nge-shRNA DARS-AS1 noma i-shRNA yokulawula akhuliswe kuma-microplates aphansi okunamathiselwe aphansi (Corning) anoshintsho oluphakathi njalo ezinsukwini ezi-4.Ama-spheroids abalwa ngemva kwezinsuku ezingu-14.Amaseli angu-500 adluliselwe nge-DARS-AS1 overexpression plasmid noma i-plasmid yokulawula asetshenzisiwe ukuze kuhlolwe ukuthuthukiswa, ngaphandle kwalokho indlela ayizange ishintshwe.
I-RNA yabhalwa kusetshenziswa i-T7 RNA polymerase ne-biotin-16-UTP (Roche 1138908910) ngokuya ngemiyalelo ye-Riboprobe® Combination Systems (Promega P1440).Iziqalo ezisetshenziswe lapha zibalwe kuThebula Lokwengeza 4.
Izifunda ze-protein-coding PACT noma i-PKR zenziwe zaba i-pET15b (Addgene #73619) futhi zaguqulwa zaba i-BL21(DE3).Amagciwane afukanyelwa ubusuku bonke ku-LB enikezwe i-ampicillin yase ihlanjululwa ngokuphindwe kayi-100 nge-LB entsha.Lapho i-OD600 ye-medium ifinyelela ku-0.8, i-1 mM IPTG yengezwe ukuze kutholwe ukubonakaliswa kwamaprotheni.Ngemva kokufukamela ngobusuku ngokuzamazama okuthambile (250 rpm ku-20°C), i-cell pellet yaqoqwa nge-centrifugation (4000 rpm, 10 min, 4°C).Misa kabusha i-cell pellet ku-lysis buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 1 mM PMSF) bese ufukamela eqhweni imizuzu engama-30, bese u-sonicate (15 min, 5 s on/off, on ice) kanye ne-centrifuge (13,000 rpm)., 30 amaminithi, 4° С).I-supernatant yabe ilayishwa ku-Ni-NTA resin (QIAGEN) izikhathi ezi-3 ku-4 ° C, yagezwa izikhathi ezingu-4 ngesiphazamiso sokugeza (50 mM Tris, pH 8.0, 40 mM imidazole, 250 mM NaCl) futhi yakhishwa izikhathi ezingu-3, ​​ngenani eliphelele. ye-10 ml yebhafa eyi-eluent (50 mM Tris, pH 8.0, 250 mM NaCl, 300 mM imidazole).Iphrotheni ehlanziwe yanqunywa kusetshenziswa i-WB futhi ukugxila kwanqunywa kusetshenziswa ikhithi yokuhlola amaprotheni e-Qubit™ (Thermo Fisher Scientific; Q 33212).
Ukuhlolwa kwe-RIP kwenziwa njengoba kuchazwe ngaphambilini, kwalungiswa.Kafushane, 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 71 (Roche, 1 mM DTT) kanye ne-centrifuge ku-13,000 rpm imizuzu engu-15 ku-4 °C.I-supernatant yabe ihlanganiswa neprotheni A+G yobuhlalu kazibuthe (Millipore) ehlanganiswe no-5 μg we-anti-PACT antibody (Abeam) noma i-IgG (CST).Ubuhlalu bugezwe izikhathi ezi-5 nge-5x RIP buffer, bese bugaywa nge-proteinase K (NEB).I-RNA ikhishwe nge-Trizol futhi yanqunywa i-RT-qPCR.Ama-primers ethulwe kuThebula Lokwengeza 5.
Ukuhlolwa kwe-RIP ye-in vitro kwenziwa ngokuvumelana nephrothokholi yokuhlola ye-RIP evamile.Isamba esingu-5 pmol we-in vitro ebhaliwe ye-RNA yahlanjululwa ngo-1x nge-RIP buffer futhi yafakwa ekufukameleni ku-65°C imizuzu emi-5 okulandelwa ukupholisa okunensayo kumazinga okushisa asekamelweni.Isamba esingu-5 pmol samaprotheni e-PACT angaguquki noma ashintshiwe abhalwe ngefulegi ahlanzwa ku-E. coli.Fukamisa nge-RNA eshintshwe kabusha amahora angu-2 ku-4°C futhi ulandele inqubo engenhla yokuhlaziya i-RIP ye-anti-flag IP.
Ukuze uthole ukuhlaziywa kwesandiso se-RNA, amaseli angu-1 × 107 afakwe kubhafa we-1xRIP.Ngemuva kwe-centrifugation ku-13,000 rpm imizuzu engu-15 ku-4°C, i-supernatant yaphathwa ngaphambili ngo-30 μl wobuhlalu kazibuthe be-streptavidin (Beckman) amahora angu-2 ku-4°C.I-lysate ehlanzekile yabe ihlinzekwa ngeyeast tRNA futhi yafakwa ku-40 pmol ye-RNA evuselelwe ngobusuku obunye ku-4°C, kwase kwengezwa amanye amahora angu-2 kanye no-20 μl wobuhlalu obusha be-streptavidin obuvinjwe nge-BSA.Isinyathelo sokugeza sasihlanganisa izikhathi ezingu-4 nge-5x RIP buffer kanye nezikhathi ezingu-4 nge-1x RIP buffer.Amaprotheni ahambisanayo akhishwe nge-biotin elution buffer (25 mM Tris-HCl, pH 7.5, 12.5 mM D-biotin, PMSF) futhi ahlukaniswa ku-NuPAGE 4-12% Bis-Tris Gel (Invitrogen).Ngemuva kokungcoliswa kwesiliva (Beyotime Biotechnology), amabhendi athile asikwa futhi ahlaziywa yi-MS.
Ukuhlaziywa kwe-Co-IP kwenziwa ukuze kuhlolwe ukusebenzisana phakathi kwe-PACT ne-PKR.Kafushane, ama-lysates anamandla alungiswa ngokufukamela amaseli a-1 x 107 a-lysed ku-1 x RIP buffer elandelwa yi-centrifugation ku-13,000 rpm imizuzu engu-15 ku-4°C.Ama-Lysates ayelayishwe iphrotheni A + G yobuhlalu kazibuthe, ahlanganiswe no-5 µg we-anti-PACT antibody, futhi azungeziswa ngobumnene ngobusuku obungu-4°C.Ubuhlalu bugezwe izikhathi ezi-3 nge-5×RIP buffer, kabili nge-1×RIP buffer futhi bakhishwa nge-1×SDS buffer.Iphrotheni etholiwe yahlaziywa ijeli ye-SDS-PAGE futhi yatholwa yi-WB.
Ama-pmol amabili e-PACT ehlatshwe umkhosi kanye ne-1 pmol ye-PKR ahlanzwa ku-E. coli.Nciphisa ku-1× RIP buffer bese ufukamela nge-10 pmol ye-RNA evuselelwe kabusha amahora ama-2 ku-4 °C.Ngemva kwalokho, zafakwa iprotheni A+G yobuhlalu obuzibuthe obuhlanganiswe ne-anti-lebulad antibody amahora amabili engeziwe.Ubuhlalu bese bugezwa izikhathi ezine nge-1x RIP buffer futhi bukhishwe nge-1x SDS buffer.I-PACT ne-PKR etholakele itholwe yi-WB.