Iindaba

Enkosi ngokundwendwela i-Nature.com.Inguqulelo yesikhangeli oyisebenzisayo inenkxaso enyiniweyo yeCSS.Ngowona mava angcono, sicebisa ukuba usebenzise isikhangeli esihlaziyiweyo (okanye uvale iModi yokuThelela kwi-Internet Explorer).Okwangoku, ukuqinisekisa inkxaso eqhubekayo, siya kunika isayithi ngaphandle kwezitayela kunye neJavaScript.
Iiseli zomhlaza ziye zavelisa iindlela ezahlukeneyo zokoyisa uxinzelelo lweeseli kwaye ziqhubeke nokuqhubela phambili.Iprotein kinase R (PKR) kunye neprotein activator (PACT) ngabaphenduli bokuqala abajonga iimpawu ezahlukeneyo zoxinzelelo ezikhokelela ekuthinteleni ukwanda kweeseli kunye ne-apoptosis.Nangona kunjalo, ukulawulwa kwendlela ye-PACT-PKR kwiiseli zomhlaza kuhlala kungaziwa kakhulu.Apha, sifumene ukuba i-RNA ende engekho ikhowudi (lncRNA) i-aspartyl tRNA synthetase antisense i-RNA 1 (i-DARS-AS1) ibandakanyeka ngokuthe ngqo kwi-inhibition ye-PACT-PKR indlela kwaye ikhuthaza ukwanda kweeseli zomhlaza.Sisebenzisa ujongo olukhulu olusebenzayo lwe-CRISPRI 971 enxulumene nomhlaza we-lncRNA, safumanisa ukuba i-DARS-AS1 yayinxulunyaniswa nokwandisa kakhulu ukwanda kweeseli zomhlaza.Ke ngoko, i-DARS-AS1 iknockout inqanda ukwanda kweeseli kwaye ikhuthaza i-apoptosis yeseli yomhlaza kwimigca yeeseli zomhlaza kwi-vitro kwaye inciphisa kakhulu ukukhula kwethumba kwi-vivo.Ngomatshini, i-DARS-AS1 ibophelela ngokuthe ngqo kwi-domain activation ye-PACT kwaye inqanda ukusebenzisana kwe-PACT-PKR, ngaloo ndlela inciphisa ukusebenza kwe-PKR, i-eIF2α phosphorylation, kunye nokuvimbela ukufa kweeseli ze-apoptotic.Ngokonyango, i-DARS-AS1 ibonakaliswa ngokubanzi kwii-cancer ezininzi, kwaye ukugqithiswa kwale lncRNA kubonisa ukuba i-prognosis imbi.Olu pho nonongo lucacisa umgaqo okhethekileyo womhlaza wendlela ye-PACT-PKR nge-DARS-AS1 lncRNA kwaye ibonelela ngenye injongo yesifo somhlaza kunye nonyango.
Ukukwazi ukuziqhelanisa noxinzelelo luphawu olubalulekileyo lokusinda kweseli yomhlaza kunye nokwanda.Ukwanda okukhawulezayo kunye neempawu zemetabolism zencopho yomhlaza kwi-microenvironment enobunzima-ukungabikho kwezondlo, i-hypoxia, kunye ne-pH ephantsi-enokuthi iqalise iindlela zokubonisa ukufa kweeseli.I-Dysregulation ye-stress-sensitive genes efana ne-p535, iiprotheyini zokutshatyalaliswa kobushushu 6, 7, KRAS8, 9, kunye ne-HIF-110, 11, 12, 13 ibonwa rhoqo kumhlaza, ngaloo ndlela ivimbela i-apoptosis kunye nokukhuthaza ukusinda.
Iprotein kinase R (PKR) iyinzwa ebalulekileyo yoxinzelelo kunye ne-subunit kinase ye-eukaryotic initiation factor 2α (eIF2α), umlawuli wokuguqulela odibanisa uxinzelelo lwamaselula ekufeni kweseli.I-PKR yaqatshelwa njengeprotein ye-antiviral ngokufunyanwa kwe-RNA ephindwe kabini yangaphandle (dsRNA).Emva kokusebenza, i-PKR phosphorylates eIF2α ukuvimbela i-viral and cellular protein synthesis14,15,16.I-PACT (iprotheni ye-activator ye-PKR) ichongiwe njengeprotheni yokuqala ye-PKR ye-activator ngokungabikho kwe-dsRNA17,18,19,20,21,22,23.Ngokusebenzisana ngokuthe ngqo ne-PKR, i-PACT idlulisela uxinzelelo oluhlukeneyo (indlala ye-serum, i-peroxide okanye unyango lwe-arsenite) kwi-PKR kunye neendlela ezisezantsi zokubonisa.Ukongeza kwi-eIF2α phosphorylation, i-PACT-mediated PKR activation ibangela iziganeko ezahlukahlukeneyo ezinxulumene nempendulo yoxinzelelo, kubandakanywa nesimo esitshintshileyo se-redox nge-PI3K / Akt24 indlela, i-DNA ephuculweyo yokukhangela umonakalo nge-p5325,26 kunye ne-NF-κB27,28 Ilawula ukubhaliswa, i-29. Ukunikezelwa kwendima yabo ebalulekileyo ekuphenduleni uxinzelelo, ukwanda, i-apoptosis kunye nezinye iinkqubo eziphambili zeselula, i-PKR kunye ne-PACT zithembisa iinjongo zonyango kwizifo ezininzi, ngakumbi umhlaza30,31,32,33.Nangona kunjalo, ngaphandle kokubaluleka kokusebenza kwe-pleiotropic kunye nebhayoloji, ukulawulwa komsebenzi we-PACT/PKR kwiiseli zomhlaza kuhlala kunzima.
I-lncRNAs yimibhalo emikhulu kune-nucleotides ye-200 engenakho iprotein-coding.Ukusukela oko iiprojekthi zolandelelwano lwegenome ezigqibeleleyo zichonge amawaka ee-lncRNAs, 35,36 umzamo omkhulu owenziweyo wokucacisa imisebenzi yabo yebhayoloji.Umzimba okhulayo wophando ubonise ukuba i-lncRNAs ibandakanyeka kwiinkqubo ezininzi zezinto eziphilayo37 kuquka ukulawulwa kwe-X-chromosome inactivation38,39, imprinting40, transcription41,42, translation43 kunye nokukhula komhlaza44,45,46,47.Ezi zifundo zichaze ukuba ezininzi ii-lncRNA zibandakanyeka kwindlela ye-PACT / PKR.Olunye uphononongo olunjalo lubonise ukuba i-lncRNA ASPACT inqande ushicilelo lwe-PACT kunye nokwandisa ukugcinwa kwenyukliya kwe-PACT mRNA.Olunye uphando lubonise ukuba i-lncRNA nc886 ibophelela kwi-PKR kwaye inqanda i-phosphorylation49,50 yayo.Ukuza kuthi ga ngoku, i-lncRNA elawula ukusebenza kwe-PACT-mediated PKR akukabikwa.
I-Aspartyl-tRNA synthetase antisense i-RNA 1 (i-DARS-AS1) ichongiwe njenge-oncogenic lncRNA51,52,53,54.Ngokulawulwa kwe-miP-194-5p53, i-miP-12952 kunye ne-miP-532-3p51, i-DARS-AS1 iboniswe ukukhuthaza ukukhula kwe-cell cell renal cell carcinoma, i-thyroid carcinoma kunye ne-non-small cell lung carcinoma, ngokulandelanayo.I-Tong kunye noogxa nabo bafumanisa ukuba i-DARS-AS1 ikhuthaza ukuqhubela phambili kwe-myeloma ngokugcina uzinzo lwe-protein 39 (RBM39) i-RNA-binding motif.Nangona kunjalo, akukho zifundo ezenziweyo malunga nokuba le lncRNA ibandakanyeka ekulawuleni ukusebenza kwe-PACT-PKR kunye nokuphendula koxinzelelo lweeseli zomhlaza.
Apha, senze isikrini esikhulu sokuphulukana nomsebenzi sisebenzisa inkqubo ye-CRISPRi kwaye sizimisele ukuba i-DARS-AS1 lncRNA ikhuthaza ukwanda kweentlobo ezininzi zeeseli zomhlaza.Ukongezelela, sichonge indlela enkulu: I-DARS-AS1 ibophelela ngokuthe ngqo kwi-PACT, inqanda i-PACT kunye ne-PKR yokubopha, ikhusela i-phosphorylation ye-eIF2α, i-substrate ye-PKR ephantsi, kwaye ekugqibeleni inqanda ukufa kwe-apoptotic cell.Ukuqukumbela, umsebenzi wethu ubonisa i-DARS-AS1 lncRNA njengomlawuli wendlela ye-PACT-PKR kunye nenjongo enokuthi ibe khona yonyango lomhlaza kunye nokuxilongwa.
Uphononongo olubanzi lweprofayili ye-genomic ichonge amakhulukhulu e-lncRNAs ezinxulumene nomhlaza.Nangona kunjalo, umsebenzi wabo uhlala ungaziwa kakhulu56.Ukuchonga abaviwa be-lncRNA abathembisayo ababandakanyekayo ekuqhubeleni phambili komhlaza, senze isikrini sokulahlekelwa ngumsebenzi wokunciphisa ukwanda kwi-SW620 ye-cell cell cell line usebenzisa inkqubo ye-CRSPRI (Fig. 1a).Olona phawu lukhethekileyo lwe-SW480 kunye ne-SW620 yemigca yeeseli zomhlaza wekholoni kukuba zisuka kumathumba akwinqanaba lokuqala kunye nelesibini kwisigulana esinye.Oku kunika uthelekiso oluxabisekileyo lokufunda utshintsho lwemfuzo ekuqhubekeni phambili komhlaza wekholoni.Ke ngoko, siye sahlalutya i-transcriptomes yemigca yeseli yomhlaza we-colorectal (SW480 kunye ne-SW620) sisebenzisa ulandelelwano lwe-RNA kwaye saqokelela ezinye ii-lncRNA ezinokusebenza kuncwadi olupapashiweyo.Ngokusekwe kwezi ziphumo, siyile ithala leencwadi le-sgRNA elidityanisiweyo eliqulathe i-7355 sgRNA oligos ejolise kwi-971 enxulumene nomhlaza we-lncRNAs kunye ne-500 engajoliswanga ye-sgRNA oligos yolawulo olubi (iDatha eyoNgezelelweyo yoku-1).
Ukubonakaliswa kweSchematic yokuhlola usebenzisa inkqubo yeCRISPRI.b sgRNA ukutyetyiswa emva kokuhlolwa.Umgca wamachaphaza othe tye umele i-log2 (utshintsho olugobileyo) = ±0.58.Umgca wamachaphaza othe nkqo ubonisa ixabiso le-p = 0.05.Amachaphaza amnyama amele i-sgRNA engajoliswanga (echongwe njenge-NC).Amachaphaza abomvu ngama-sgRNA ajolise kwi-DARS-AS1.Amachaphaza aluhlaza zii-sgRNAs ezijolise kwi-LINC00205, i-oncogenic lncRNA echazwe ngaphambili.ukugoqa utshintsho = (ukufunda okuqhelekileyo, usuku 17)/(ukufunda okuqhelekileyo, usuku 0).c I-DARS-AS1 i-sgRNA inqande ukukhula kweeseli.Iibha zeempazamo zimele ± ukutenxa okusemgangathweni kwemifuniselo emithathu.* p ≤ 0.05, ** p ≤ 0.01 Uvavanyo lomfundi olunemisila emibini.d I-DARS-AS1 inkcazo kumathumba (i-TCGA dataset).em Ukubonakaliswa kwe-DARS-AS1 kwiisampuli eziqhelekileyo ezidibeneyo kunye ne-tumor ezivela kwizigulane ezine-BLCA, KIRC, PRAD, LUSC, UCEC, LUAD, LIHC, KIRP, kunye ne-COAD, ngokulandelanayo (i-TCGA dataset).amaxabiso e-p afunyenwe kusetyenziswa uvavanyo loMfundi olunemisila emibini edityanisiweyo.
Emva kokwakha iplasmid kunye nokupakisha i-lentivirus, sigqithise umgca weseli yomhlaza we-dCas9-SW620 kunye nethala leencwadi elingasentla kwiimvavanyo ezine ezizimeleyo zosulelo.Ubuninzi bosulelo (MOI) kolu sulelo yi-0.1–0.3, ebonisa ukuba iseli nganye inokudluliselwa kuphela nge-sgRNA enye.Emva kweentsuku ze-18 zenkcubeko ye-in vitro, iprofayili yokutyebisa ekujoliswe kuyo ye-sgRNAs yehla okanye yonyuka emva kokuhlolwa, ngelixa inani le-oligonucleotides yokulawula engajoliswanga lihlala lingatshintshi xa lithelekiswa neprofayili yokuhlola kwangaphambili, ebonisa ukuba ithagethi yethu inesikrini esithe ngqo. ithala leencwadi.Irayisi.1b kunye netheyibhile eyongezelelweyo 1). I-LINC00205, eyayixelwe ngaphambili ukuba ikhuthaze umhlaza wemiphunga kunye nokuqhubekeka komhlaza wesibindi58,59,60, ihlolwe ngaphandle (log2 (foldchange) <-0.58, p value <0.05), iqinisekisa ukuthembeka kolu vavanyo (Fig. 1b). I-LINC00205, eyayixelwe ngaphambili ukuba ikhuthaze umhlaza wemiphunga kunye nokuqhubekeka komhlaza wesibindi58,59,60, ihlolwe ngaphandle (log2 (foldchange) <-0.58, p value <0.05), iqinisekisa ukuthembeka kolu vavanyo (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, eyayixelwe ngaphambili ukukhuthaza ukuqhubela phambili komhlaza wemiphunga kunye nomhlaza wesibindi58,59,60, yayingabandakanywanga (log2 (utshintsho oluphindwe kabini) <-0.58, p-value <0.05), eqinisekisa ukuqina kolu vavanyo (Fig. .1b) . Linc00205 之前 被 可 可 和 和 和 和 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 58,59,60, 被被 选 选 选 掉 选 筛选 该 该 该 该 筛选的 筛选的 筛选的 的的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选 筛选的 筛选的 筛选的 筛选的 的的 筛选的 筛选的 的的 筛选的 的的 的的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 的的 筛选的 筛选的 筛选的的. Linc00205 之前 被 可 可 和 和 和 和 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 58,59,60, 被被 选 选 选 掉 选 筛选 该 该 该 该 筛选的 筛选的 筛选的 的的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选 筛选的 筛选的 筛选的 筛选的 的的 筛选的 筛选的 的的 筛选的 的的 的的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 筛选的 的的 筛选的 筛选的 筛选的的. 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, echazwe ngaphambili ukukhuthaza ukuqhubeka komhlaza wemiphunga kunye nesibindi58,59,60, yayingabandakanywanga (log2 (utshintsho lwe-fold) <-0.58, p-value <0.05), eqinisekisa ukuqina kolu vavanyo (Fig. .1b).
Phakathi kwazo zonke ii-lncRNA ezivavanyiweyo, i-DARS-AS1 nayo ihlolwe, kunye ne-cognate sgRNA oligonucleotides ezintathu ezincitshiswe kakhulu emva kweentsuku ze-18 zenkcubeko, ebonisa ukuba i-knockdown yale lncRNA yabangela ukunciphisa ukwanda komhlaza (Fig. 1b).Esi siphumo saye saxhaswa ngakumbi luhlalutyo lwe-MTS kwiiseli zomhlaza we-colorectal ezibonisa ukuba izinga lokukhula kweeseli ze-DARS-AS1 ze-knockdown zahlulwa ngesiqingatha kuphela xa kuthelekiswa neeseli zokulawula (Umfanekiso 1c) kwaye wawuhambelana neengxelo zangaphambili zezinye iintlobo zomhlaza.: umhlaza wesifo sezintso ezicacileyo, umhlaza we-thyroid kunye nomhlaza wemiphunga ongeyena omncinci51,52,53,55.Nangona kunjalo, umsebenzi wayo kunye neendlela zeemolekyuli kumhlaza we-colorectal zihlala zingahlolwa.Ke ngoko, sikhethe le lncRNA ukuze sifunde ngakumbi.
Ukufunda ukubonakaliswa kwe-DARS-AS1 kwizigulana, sihlalutye ngokubanzi iisampulu zethumba ezili-10,327 kwiprojekthi yeCancer Genome Atlas (TCGA).Iziphumo zethu zibonisa ukuba i-DARS-AS1 ibonakaliswe ngokubanzi kwaye ilawulwa kakhulu kwiiseli eziphilileyo kwiintlobo ezahlukeneyo ze-tumor, kubandakanywa ne-colon adenocarcinoma (COAD), i-renal clear cell carcinoma (KIRC), kunye ne-renal papillary cell carcinoma (KIRP)..Bambalwa kakhulu (umzobo 1d kunye neFig. 1a, b). Uhlalutyo lweesampulu ezidityanisiweyo ezisempilweni / zethumba ziqinisekisa ngakumbi ukubonakaliswa okuphezulu kakhulu kwe-DARS-AS1 kumathumba e-bladder urothelial carcinoma (BLCA), i-renal renal clear cell carcinoma (KIRC), i-prostate adenocarcinoma (PRAD), ilung squamous cell carcinoma (LUSC) , i-uterine corpus endometrial carcinoma (UCEC), i-lung adenocarcinoma (LUAD), i-hepatocellular carcinoma yesibindi (LIHC), i-renal renal papillary cell carcinoma (KIRP), kunye ne-colon adenocarcinoma (COAD) (ixabiso le-p <0.05) (Umfanekiso 1e-m) . Uhlalutyo lweesampulu ezidityanisiweyo ezisempilweni / zethumba ziqinisekisa ngakumbi ukubonakaliswa okuphezulu kakhulu kwe-DARS-AS1 kumathumba e-bladder urothelial carcinoma (BLCA), i-renal renal clear cell carcinoma (KIRC), i-prostate adenocarcinoma (PRAD), ilung squamous cell carcinoma (LUSC) , i-uterine corpus endometrial carcinoma (UCEC), i-lung adenocarcinoma (LUAD), i-hepatocellular carcinoma yesibindi (LIHC), i-renal renal papillary cell carcinoma (KIRP), kunye ne-colon adenocarcinoma (COAD) (ixabiso le-p <0.05) (Umfanekiso 1e-m) .Uhlalutyo lweesampulu ezidibeneyo eziphilileyo / ze-tumor nazo ziqinisekisile ukubonakaliswa okuphezulu kakhulu kwe-DARS-AS1 kwi-bladder urothelial carcinoma (BLCA), i-cell cell renal kunye ne-renal cell carcinoma (KIRC), i-prostate adenocarcinoma (PRAD), i-lung squamous cell carcinoma (LUSC) izicubu., 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 ye-lung (LUAD), i-hepatocellular carcinoma yesibindi (LIHC), i-papillary cell carcinoma yezintso (KIRP), kunye ne-adenocarcinoma yekholoni (COAD) (ixabiso le-p. 0.05) (Umfanekiso 1e- m) .配对 / 肿瘤样本 分析 进一步 证实 了 了 了 膀胱 上 上 皮癌 皮癌 皮癌 透明 透明 透明 (kirc), 前列前列显着 更更 高高 表达, 子宫体子宫子宫体子宫 癌 (i-UCC), 肺腺癌<0.05（图1e-m） .配配 健康 健康 健康 证实 了 了 了 了 了 在 上 皮癌 皮癌 皮癌 皮癌 皮癌 细胞癌 细胞癌 细胞癌 细胞癌 细胞癌 前列 腺腺癌 腺腺癌 腺腺癌 腺腺癌 腺腺癌 腺腺癌 腺腺癌 腺腺癌(LUSC) 肿瘤 的 的 的 中 中 中 中 中 中 中 中 中 中 中 中 中 显着 显着 更 显着 高 更 高 高 高 高, 内膜内膜内膜 癌 ((i-iCEL) 肝肝癌 (kirp) (icoad) (p 值<0.05) (图1e-m) .Uhlalutyo lweesampulu ezidibeneyo ezinempilo / ze-tumor zixhasa ngakumbi indima ye-DARS-AS1 kwi-bladder urothelial carcinoma (BLCA), i-cell cell renal cell carcinoma (KIRC), i-prostate adenocarcinoma (PRAD), kunye ne-lung squamous cell carcinoma (LUSC) izicubu.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). ukubonakaliswa kwi-corpus uterine carcinoma (UCEC), i-lung adenocarcinoma (LUAD), i-hepatocellular carcinoma (LIHC), i-renal papillary cell carcinoma (KIRP), kunye ne-colon adenocarcinoma (COAD) (ixabiso le-p <0.05) (Umfanekiso 1e -m).Xa zidibene, ezi ziphumo zibonisa ukuba i-DARS-AS1 ibonakaliswe ngokubanzi kwaye ibonakaliswe kakhulu kwiintlobo zomhlaza.
Ngenxa yokuba i-DARS-AS1 kunye ne-DARS (i-gene encoding i-antisense strand) yabelana ngomgqugquzeli ofanayo kwaye ibekwe ecaleni komnye nomnye, siye sayila i-shRNA ukuba ibethe ngokuthe ngqo i-DARS-AS1 kodwa ingabi yi-DARS (iFig. .Ukongeza kwi-SW620, siphinde sasebenzisa ezinye iiseli zeeseli ezintathu ezichaza kakhulu i-DARS-AS1 ukufunda ukusebenza kunye nokusebenza kwe-shRNA knockdown (iTheyibhile eyoNgezelelweyo yesi-3).Iziphumo zethu zibonise ukuba zontathu ii-shRNAs eziphuhliswe zifezekiseke ubuncinci be-80% ye-DARS-AS1 ye-knockdown ye-knockdown enesiphumo esincinci kwisixa se-DARS mRNA (Supplementary Fig. 2c–f).Ukongeza, sifumene ukuba i-DARS-AS1 i-knockdown ngezi shRNAs ithintele kakhulu ukukhula kweeseli kwimigca yeseli yomhlaza we-colorectal SW620 (49.7%) kunye ne-HCT116 (27.7%), umgca weseli yomhlaza webele MBA-MD-231 (53.4%).) kunye ne-HepG2 i-hepatoma cell line (ukunciphisa i-92.7%), kunye nokukwazi kwabo ukwenza ii-spheres ezingabanjwanga (ukunciphisa umyinge we ~ 50.8%, 44.6%, 40.7% kunye ne-75.7% ngomgca weseli) (Umfanekiso 2a, b).Kwi-SW620, iziphumo zovavanyo lokwenziwa kwekholoni ziphinde zaqinisekisa ukuba i-DARS-AS1 shRNA ithintele kakhulu ukwanda kweeseli ngokuncipha komndilili malunga ne-69.6% (Fig. 2c).
Impembelelo yokulawula i-shRNA kunye ne-DARS-AS1 shRNA kwi-cell proliferation (a) kunye nokwakheka kwe-spheroid (b) kwi-SW620, HCT116, MBA-MD-231, kunye neeseli ze-HepG2.c Impembelelo yolawulo lwe-shRNA kunye ne-DARS-AS1 shRNA ekubunjweni kwekholoni kwiiseli ze-SW620.Ukwanda kweeseli (d), ukubunjwa kwe-spheroid (e), kunye nokwakhiwa kwekholoni (f) yeeseli ze-SW620 ezigqithise ngokugqithiseleyo i-DARS-AS1.Idatha ebonisiweyo yintsingiselo ± ukutenxa okusemgangathweni kwemifuniselo emithathu.* p ≤ 0.05, ** p ≤ 0.01, kunye *** p ≤ 0.001 ngovavanyo lwe-t yoMfundi enemisila emibini.
Ukuncedisana nezifundo ezilahlekileyo-zokusebenza, ngokulandelayo senze iiseli ze-SW620 ezigqithise kakhulu i-DARS-AS1 (i-Supplementary Fig. 2g).I-DARS-AS1 i-overexpression ikhulise kakhulu ukukhula kweeseli (i-1.8-fold), ukubunjwa kwe-spheroid engavumelekanga (i-1.4-fold), kunye nokwakhiwa kwekholoni (3.3-fold) kwiiseli ze-SW620 (Fig. 2d-f).Siye saqinisekisa esi siphumo sisebenzisa enye i-DARS-AS1 echaza umgca weseli, i-A549.Oku kwandiswa kweeseli eziphuculweyo ngenxa ye-DARS-AS1 overexpression yaqwalaselwa ngakumbi kwiiseli ze-A549 (i-Supplementary Fig. 2h, i kunye ne-Supplementary Table 3).Zithathiwe kunye, ezi zifundo zenzuzo kunye nelahleko zibonisa ukuba i-DARS-AS1 ikhuthaza ukwanda kweeseli zomhlaza kwi-vitro.
Ukuphonononga indlela esisiseko apho i-DARS-AS1 ilawula ukwanda kweeseli, senze uhlalutyo lokutsalela phantsi kwe-RNA ukuchonga amaqabane ayo anokubakho kwiprotheyini.Iziphumo ze-RT-qPCR zibonise ukuba malunga ne-86.2% ye-DARS-AS1 ifumaneka kwi-cytoplasm yeeseli ze-SW620 (i-Supplementary Fig. 3a).I-in vitro iguqulelwe nge-biotinylated i-DARS-AS1 okanye i-pseudoRNA emva koko yafakwa kunye ne-SW620 ye-lysates yeeseli ilandelwa kukwahlulwa kwe-SDS-PAGE.I-staining yesilivere elandelayo ibonise ukuba ibhendi eyahlukileyo (~ 38 kDa) yayityetyiswe kakhulu kwi-DARS-AS1 iisampulu zokutsala kodwa kungekhona kwi-dummy RNA okanye iisampuli ze-beads (Fig. 3a).Eli qela lachongwa njenge-PKR esebenzayo iprotheni (i-PACT) nge-mass spectrometry (MS) kwaye yaqinisekiswa ngakumbi nge-immunoblotting kwi-SW620, i-HCT116, kunye nemigca yeseli ye-HepG2 (Umfanekiso 3a, b).Ukutyetyiswa kwe-DARS kunye neeprotheyini ze-PACT ezihambelanayo - i-PKR kunye ne-TRBP - nazo zaphandwa kusetyenziswa uhlalutyo lwe-RNA nge-Western blotting (WB).Iziphumo zibonise ukuba akukho ukusebenzisana ngokuthe ngqo phakathi kwe-DARS-AS1 RNA kunye nezi ziprotheni ezintathu zifunyenwe (i-Supplementary Fig. 3b).Ukusebenzisana okuthe ngqo phakathi kwe-DARS-AS1 kunye ne-PACT kwaqinisekiswa ngakumbi ngohlalutyo lwe-RNA immunoprecipitation (RIP), olubonisa ukuba i-DARS-AS1 yayityetyiswe kakhulu kwi-anti-PACT antibodies kodwa kungekhona enye i-RNAs yokulawula (Umfanekiso 3c).Ukufumanisa ukuba i-DARS-AS1 isebenzisana ngokuthe ngqo ne-PACT ngokungabikho kwamanye amacandelo eselula, uvavanyo lwe-in vitro biolayer interferometry (BLI) lwenziwa kusetyenziswa i-PACT ecocekileyo.I-Biotin ebhalwe nge-DARS-AS1 okanye i-dummy RNA yayingashukumi kwii-biosensors ze-streptavidin (SA) kwaye emva koko yafakwa kwi-kinetic buffer equlethe i-1 μM PACT.Ngokucacileyo, i-PACT ibotshelelwe ngamandla kwi-DARS-AS1 (ixabiso le-KD ~26.9 nM), kodwa hayi ukulinganisa i-RNA (Umfanekiso 3d).Zithathiwe kunye, ezi ziphumo zibonisa ukusebenzisana ngokuthe ngqo kunye nobudlelwane obuphezulu phakathi kwe-DARS-AS1 kunye ne-PACT.
Uhlalutyo lwe-RNA lokutsala luchongwe i-DARS-AS1 isebenzisana ne-PACT kwiiseli ze-SW620.Ngaphezulu, ukungcoliswa kwesilivere yeeprotheni ezinxulumene.Ii-immunoblots ezisezantsi zenziwe nge-anti-PACT antibody.b Uhlalutyo lokutsalwa kwe-RNA lwenziwa kwiiseli ze-HCT116 (phezulu) kunye ne-HepG2 (ezantsi).Ukutyetyiswa kwe-PACT kwafunyaniswa nge-immunoblotting.Uvavanyo lwe-cRNA immunoprecipitation (RIP) lwenziwa kwiiseli ze-SW620 kusetyenziswa izilwa-buhlungu ezibonisiweyo.d I-PACT yokubopha i-curves kubude obupheleleyo be-DARS-AS1 okanye i-RNA yokulawula ifunyenwe ngokusebenzisa i-biolayer interferometry (BLI).I-RNA yayingashukumi kwi-biosensor ye-streptavidin.I-1 μM PACT yayisetyenziselwa ukulinganisa unxulumano.i-RNA yokutsala uvavanyo lwenziwe kusetyenziswa i-biotinylated yobude obugcweleyo be-DARS-AS1 okanye inqunyulwe (phezulu).Immunoblot ebonisa i-PACT efunyenweyo (ezantsi).f I-PACT eneflegi ecocekileyo yafakwa kwi-biotinylated yobude obugcweleyo be-DARS-AS1 okanye yanqunyulwa (njengaku-e) kuvavanyo lwe-RIP ye-in vitro.I-RNA ekhutshiweyo yaqinisekiswa yi-RT-qPCR.g Unxulumano olunxulumeneyo lwamaqhekeza e-RNA ahlukeneyo e-PACT afunyenwe kusetyenziswa i-biolayer interferometry.Ukuhlalutya, i-100 nM RNA kunye ne-1 μM RAST yasetyenziswa.h Iimvavanyo ze-In vitro ze-RIP zenziwe kusetyenziswa i-purified intact okanye i-truncated ibhalwe PACT.I-RNA ekhutshiweyo yaqinisekiswa yi-RT-qPCR.i Izinga lokukhula kweeseli ze-SW620 ezigqithise kakhulu i-DARS-AS1, i-PACT, okanye zombini.j Ukuchazwa ngokugqithisileyo kobude obugcweleyo okanye obuncitshisiweyo be-DARS-AS1 kwiiseli ze-SW620 zineziphumo ezahlukileyo ekukhuleni kweeseli.k I-Apoptosis yabhaqwa ngokuvala amajoni omzimba nge-anti-PARP.l I-Knockout ye-DARS-AS1 yenza i-apoptosis yeeseli ze-SW620 njengoko kuboniswe yi-cytometry yokuhamba.Idatha ebonisiweyo yintsingiselo ± ukutenxa okusemgangathweni kwemifuniselo emithathu. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p <0.0001, ngovavanyo loMfundi olunemisila emibini. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p <0.0001, ngovavanyo loMfundi olunemisila emibini. *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 ngovavanyo loMfundi olunemisila emibini. *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 ngovavanyo loMfundi olunemisila emibini.
Emva koko savelisa iinqununu ezintathu ze-biotinylated ze-DARS-AS1 RNA nge-in vitro transcription ukuchonga ummandla we-DARS-AS1 ofunekayo kumbutho we-PACT (Umfanekiso 3e).Iziphumo zohlalutyo lwe-RNA zibonise ukuba isiqwenga ngasinye sikwazi ukusebenzisana ne-PACT, kodwa ummandla we-3'-terminal (384-768 nucleotides ebhalwe A3) ubonise ngaphezu kwe-1-384 nucleotides ebhalwe A1) (Umfanekiso 3e).Iziphumo ezifanayo zibonwe kwi-in vitro i-RIP assay usebenzisa i-PACT edibeneyo (Umfanekiso 3f).Ngokungqinelana nezi ziphumo, imifuniselo yokubophelela amaqhekeza e-RNA angashukumiyo kwi-PACT esebenzisa i-BLI ikwabonise ukuba i-PACT inobudlelwane obuphezulu be-A3 (384-768 nt) (ixabiso le-KD elimalunga ne-94.6 nM), ngelixa phantse akukho nxibelelwano kunye nezinye iindawo.(Umfanekiso 3d).
Sikwavavanye imimandla ebophelelayo enxulumeneyo kwi-PACT.I-PACT iqulethe imimandla emithathu esebenzayo, ezimbini zazo ezilondolozwe kabini i-RNA-binding domains (dsRBD) kunye ne-domain yesithathu (i-D3 ekhethiweyo) esebenza njenge-activator ye-protein interactions.Ukuphonononga amandla okubopha i-lncRNA kwi-domain nganye, senze iinguqu ezintathu eziye zasusa i-domain nganye kwezithathu kwaye zenza uvavanyo lwe-RIP ye-in vitro.Iziphumo zethu zibonise ukuba ukucinywa kwe-domain yesithathu (D3) ye-PACT kunciphisa kakhulu ukusebenzisana kwayo ne-DARS-AS1 (ngo-0.11-fold xa kuthelekiswa ne-PACT engaguqukiyo) xa kuthelekiswa nezinye iinguqu ezimbini (umzobo 3h), kwaboniswa ukuba ukukhululwa. ye-D3 inxibelelane ne-DARS.-AC1.Kuthatyathwe kunye, ezi ziphumo zibonisa ukuba ukusebenzisana phakathi kwe-DARS-AS1 kunye ne-PACT kungenzeka ngokuyinhloko nge-3 'isiphelo se-DARS-AS1 kunye ne-D3 domain ye-PACT.
Siqaphele ukuba i-DARS-AS1 ayinayo impembelelo kwintetho ye-PACT kwaye i-PACT ayinayo impembelelo kwi-DARS-AS1 (i-Supplementary Fig. 3c).Emva koko siye savavanya umphumo we-PACT knockdown ekukhuleni kweeseli.Ngokwahlukileyo kwi-DARS-AS1, iiseli ezihambelanayo zakhula amaxesha e-1.5-3 ngokukhawuleza xa i-PACT yachithwa (i-Supplementary Fig. 3d).Iziphumo zovavanyo lokubunjwa kwekholoni zibonise ukuba iiseli zenze ii-2-3-fold colonies emva konyango lwe-shRNA kunye ne-PACT (i-Supplementary Fig. 3e).Ukuvavanya ukuba i-DARS-AS1 ilawula ukwanda kweeseli nge-PACT, senze iiseli ze-SW620 ezigqithise i-PACT, i-DARS-AS1, okanye zombini.Ukugqithiswa kwe-PACT kubonise ukuvinjelwa okubalulekileyo kokunyuka kweeseli (Umfanekiso 3i).Nangona i-DARS-AS1 i-overexpression per se ikhuthaze kakhulu ukwanda kweeseli, kwakungekho mmahluko obalulekileyo kwizinga lokukhula kweeseli ezigqithise ngokugqithiseleyo i-DARS-AS1 kunye ne-PACT.Ezi ziphumo zibonisa ukuba i-PACT inokuchasana nokunyuka okwandisiweyo okubangelwa yi-DARS-AS1 overexpression.
Ekubeni imimandla eyahlukeneyo ye-DARS-AS1 inobuchule obahlukeneyo bokubopha i-PACT, siphande impembelelo yabo enxulumene nokwanda kweeseli ngokuchazwa ngokugqithisileyo okwahlukileyo kwamaqhekeza e-DARS-AS1.Xa kuthelekiswa namanye amaqhekeza amabini, i-DARS-AS1 yayigqithise ngokugqithiseleyo ekupheleni kwe-3 (384-768 nt), ummandla oyintloko we-PACT enxulumene ne-DARS-AS1, eyayinamandla aphezulu okukhuthaza ukwanda kweeseli (Umfanekiso 3j).Ezi ziphumo zibonisa ulungelelwaniso oluhle phakathi kwamandla okubopha kunye nomsebenzi webhayoloji we-DARS-AS1.
IPACT iye yaxelwa njengepro-apoptotic protein19.Ke ngoko, siye saphanda isiphumo se-DARS-AS1 kwi-apoptosis.Njengoko bekulindelekile, i-DARS-AS1 i-knockdown yandisa kakhulu i-PARP yokuqhawula kwiiseli ze-SW620 kwaye yandisa umlinganiselo weeseli ze-annexin V-positive kwi-SW620, HCT116, HepG2, kunye ne-MBA-MD-231 imigca yeseli (Umfanekiso 3k).3).I-3f-h), ebonisa ukuba i-anti-apoptotic effect ye-DARS-AS1 kwiiseli zomhlaza zichasene nomsebenzi we-apoptosis-inducing we-PACT.Kuthatyathwe kunye, ezi ziphumo zibonisa ukuba indlela ye-DARS-AS1 umsebenzi we-oncogenic ingaba ngokuthintela umsebenzi we-PACT.
Emva koko, sihlolisise iimpembelelo zokusebenza kombutho we-DARS-AS1-PACT.I-PACT iye yaxelwa ukuba isebenze i-PKR ngokusebenzisana ngokuthe ngqo, ethi emva koko ikhulise i-eIF2α phosphorylation, ibangele ukucima ukuguqulela kunye ne-apoptosis17.Okokuqala, sivavanye ukuba i-DARS-AS1 ichaphazela indawo yeselula ye-PACT kunye ne-PKR.I-Confocal fluorescence microscopy ibonise ukuba i-PACT kunye ne-PKR zadityaniswa kakhulu kwiiseli ze-SW620 ezinomndilili wolungelelwaniso lwe-Pearson coefficient ye-0.72.Okwangoku, i-DARS-AS1 i-overexpression iyancipha kakhulu i-PACT kunye ne-PKR co-localization (ithetha i-Pearson coefficient coefficient 0.61) (Figure 4a).Ukuphanda ukuba i-DARS-AS1 inokulungelelanisa ukusebenzisana kwe-PACT-PKR, senze uvavanyo lwe-co-immunoprecipitation (co-IP) kunye ne-anti-PACT antibody kwi-SW620 cell lysates.I-PKR yayityetyiswe kakhulu kwi-anti-PACT kwiiseli zokulawula, ngelixa ukubuyiswa kwe-PKR kwancitshiswa kakhulu kwii-lysates ezivela kwiiseli ezigqithise i-DARS-AS1 (umzobo 4b).I-PACT ecocekileyo ebhalwe nge-PACT kunye ne-PKR yayisetyenziselwa i-in vitro protein binding assays.Ngokufanelekileyo, abo banikezela nge-DARS-AS1 kodwa akukho lawulo lwe-RNA lubonise ukusebenzisana kwe-PACT-PKR ecinezelweyo (Umfanekiso 4c).Zonke iziphumo zibonise ukuba i-DARS-AS1 iphazamise i-PACT kunye ne-PKR unxibelelwano.
i-Co-localization ye-PACT kunye ne-PKR kwiiseli zolawulo okanye iiseli ezigqithise kakhulu i-DARS-AS1 zabonwa kusetyenziswa i-confocal fluorescence microscopy.Ii-nuclei zadyojwa nge-DAPI.Iziphumo zobalo zifunyenwe kwiifoto ezili-16.b I-Co-immunoprecipitation (co-IP) isebenzisa i-anti-PACT antibody kwi-cell lysates yokulawula iiseli ze-SW620 okanye iiseli ezigqithisa ngokugqithiseleyo i-DARS-AS1.c Ilebhile i-PACT, i-PKR esulungekileyo kwaye ibhalwe kwi-vitro nge-DARS-AS1 okanye i-RNA ehlekisayo yafakelwa uhlalutyo lwe-in vitro protein.Izilwa-buhlungu ezichasene neflegi zazisetyenziselwa ukugonyamela.d I-Immunoblots enee-antibodies ezibonakalisiweyo zenziwa kwi-SW620 kunye neeseli ze-HCT116 ezidluliselwe ngolawulo lwe-shRNA okanye i-DARS-AS1-shRNA elandelwa yindlala ye-serum.e-DARS-AS1 amanqanaba okuchaza atshintshe ubuntununtunu beselula kwi-thapsigargin.Iiseli ze-SW620 zasulelwa nge-DARS-AS1 shRNA, i-DARS-AS1 i-plasmid engaphezulu okanye i-plasmid yokulawula.Iiseli zaphathwa nge-thapsigargin kwiiyure ze-48 kwaye ukusebenza kweeseli kwagqitywa kusetyenziswa i-reagent ye-MTS.f I-In vitro ebhalwe i-DARS-AS1 okanye i-RNA ye-dummy kunye ne-PACT ecocekileyo yayisetyenziselwa i-in vitro activation assay kunye nokufunyanwa kwe-immunoblot.g Ii-Immunoblots zisebenzisa ezi zilwa-buhlungu zenziwa kwiiseli ze-SW620-ctrl (ekhohlo) okanye iiseli ezibonisa ngokugqithisileyo ii-PKR mutants (ekunene).Ezi seli zaye zadluliselwa ngolawulo lwe-shRNA okanye i-DARS-AS1-shRNA elandelwa yindlala ye-serum.h I-cytometry egelezayo ibonise ukuba ukungasebenzi kwe-PKR eguqukayo kuhlawulelwe i-DARS-AS1-induced apoptosis kwiiseli ze-SW620.ii-Immunoblots ezine-antibodies ezibonisiweyo zenziwa kwiiseli ze-SW620 (ekhohlo) okanye ze-HCT116 (ekunene).Iiseli ezidluliselwe ngokulawula i-shRNA okanye i-DARS-AS1 shRNA i-serum-deprived kwaye yongezwa nge-100 nM PKR C16 inhibitor okanye i-DMSO.Ibar yesikali = 5 µm.Idatha ebonisiweyo yintsingiselo ± ukutenxa okusemgangathweni kwemifuniselo emithathu.* p ≤ 0.05 Uvavanyo lomfundi olunemisila emibini.
Kukholelwa ngokubanzi ukuba nje ukuba i-PACT inxibelelane ne-PKR17, i-PKR phosphorylation e-Thr451 inokuqhutywa.Iziphumo zethu zibonise ukuba izinga le-PKR phosphorylation liphakanyiswe kakhulu kwiiseli ze-DARS-AS1 ze-knockdown emva kokulamba kwe-serum (Umfanekiso we-4d kunye ne-Supplementary Fig. 4a).Ngokufanelekileyo, sifumene ukuba i-phosphorylation ye-eIF2α, i-substrate ye-PKR engundoqo, nayo yanda kakhulu nge-DARS-AS1 shRNA (Umfanekiso we-4d kunye ne-Supplementary Fig. 4a).I-Thapsigargin yi-ER stressor eyenza i-ER ikhulule i-Ca2 +.Unyango kunye ne-thapsigargin kuye kwabikwa ukuba kubangele ukubonakaliswa kunye nokusebenza kwe-PACT, eqhubeka nokusebenzisana kunye nokusebenza kwe-PKR, okukhokelela kwi-apoptosis ngokunyusa i-eIF2α phosphorylation 18,61.Apha, sisebenzise i-thapsigargin njenge-stimulator ye-PACT/PKR indlela yokuphanda ukuba i-DARS-AS1 inokunceda iiseli zoyise uxinzelelo ngokuthintela indlela ye-PACT/PKR.Siye saqaphela ukuba inqanaba lenkcazo ye-DARS-AS1 ihambelana ngokufanelekileyo nokuchasana kweeseli kwi-thapsigargin.Iiseli ze-SW620 ezigqithise kakhulu i-DARS-AS1 zasinda bhetele xa ziphathwa ngethapsigargin, ngelixa iiseli ezine-DARS-AS1 knockdown ziye zachaphazeleka ngakumbi (Fig. 4e).Ngokuhambelana nezi ziphumo, i-DARS-AS1 i-overexpression iyancipha i-thapsigargin-induced PKR phosphorylation (i-Supplementary Fig. 4b).Ngokwahlukileyo, emva konyango lwe-thapsigargin, i-PKR kunye ne-eIF2α yayiyi-phosphorylated ukuya kwinqanaba eliphezulu kwiiseli ze-DARS-AS1 xa kuthelekiswa neeseli zokulawula (i-Supplementary Fig. 4b).Okubangel 'umdla kukuba, i-thapsigargin yenza i-DARS-AS1 ibonakaliso ngendlela exhomekeke kwi-dose, enokuthi ibonise umsebenzi wokulwa noxinzelelo lwe-DARS-AS1 (i-Supplementary Fig. 4c).Ukongeza, senze uvavanyo lwe-in vitro activation ukuze siqinisekise olu qwalaselo.Ngokufutshane, i-PKR yahlanjululwa kwii-lysates zeseli kusetyenziswa i-anti-PKR antibody, emva koko yafakwa kwi-PACT yokuphinda iphinde iphinde yenzeke kunye ne-DARS-AS1 ebhalwe kwi-vitro.Emva kokusabela kwe-enzymatic, i-phospho-PKR yafunyanwa kusetyenziswa i-WB.Iziphumo zethu zibonise ukuba i-PKR phosphorylation yayivinjelwe kakhulu yi-DARS-AS1, kodwa kungekhona ngolawulo lwe-RNA (Umfanekiso 4f).Ezi ziphumo ze-vitro kunye ne-vivo zibonisa ukuba i-DARS-AS1 inqanda ukusebenza kwe-PACT-mediated PKR.Ngelo xesha, siye sabona ukuhla kwe-PACT yokubuyisela phambi kwe-DARS-AS1 (Umfanekiso 4f).Esi siphumo sihambelana neziphumo ze-in vitro protein binding assay (Figure 4c) kwaye iphinda ibonise umsebenzi wokuthintela we-DARS-AS1 wombutho we-PACT-PKR.
I-Ser246 kunye ne-Ser287 kwi-D3 domain ye-PACT iyadingeka ukuze kusebenze i-PKR phantsi koxinzelelo lweselula.Ukutshintshwa kweentsalela ze-serine ezimbini ze-alanine zinike i-PACT eguqukayo (i-mutD), eyenza i-PKR isebenze ngokungabikho koxinzelelo, kunye nokutshintshwa kwe-alanine (i-mutA) iguqule i-protocol.Ekubeni siye sabonisa ukubaluleka kwesi sizinda ngokubambisana ngokuthe ngqo ne-DARS-AS1, siye savelisa ezi zimbini eziguqukayo ze-PACT ukuvavanya ukuba ezi ntsalela zinokubandakanyeka ekusebenzisaneni ne-DARS-AS1.Kuyathakazelisa ukuba zombini iinguqu eziguquguqukayo zalahlekelwa amandla okuzibophelela kwi-DARS-AS1 (i-Supplementary Fig. 4d), ebonisa ukuba isakhiwo esipheleleyo seprotheni ye-PACT sinokufuneka ekusebenzisaneni ngokufanelekileyo kunye ne-DARS-AS1.
Ngaphaya koko, iziphumo zethu zikwacebisa ukuba i-DARS-AS1-shRNA-i-inhibition ye-cell proliferation inokubuyiselwa ngokuyinxenye ngokugqithisela ngokugqithisileyo i-PACT mutant (PACTmutA) okanye i-PKR eguquguqukayo elawulayo (PKRmut) (Fig. 4e. e).Ukugqithiswa kwezinto eziguquguqukayo ze-PKR eziphambili-negative zanciphisa i-phosphorylation ye-PKR eyenziwa yi-DARS-AS1 knockdown kunye ne-eIF2α phosphorylation kwiiseli ze-serum-deprived (Fig. 4g).Okubaluleke ngakumbi, umlinganiselo weeseli ze-apoptotic ezibangelwa yi-DARS-AS1 knockdown nayo yancitshiswa kwiiseli ezigqithisa ngokugqithiseleyo i-PKRmut (Umfanekiso we-4h kunye ne-Supplementary Fig. 4g).Ukuvinjelwa komsebenzi we-PKR kinase nako kuphazamisa umsebenzi we-DARS-AS1, njengoko iziphumo zibonise ukuba i-DARS-AS1 i-knockdown ayizange ibangele i-PKR kunye ne-eIF2α phosphorylation xa iiseli ziphathwa nge-PKR-specific C16 inhibitor (Umfanekiso 4i kunye ne-Supplementary Fig. 4h).).Kuthatyathwe kunye, iziphumo zethu zibonisa ukuba i-DARS-AS1 ikhuthaza ukwanda kweeseli, ubuncinci ngokuyinxenye, ngokuthintela ukusebenza kwe-PACT-mediated PKR.
Ukuphonononga ngakumbi indima ye-DARS-AS1 kwi-tumorigenesis, senze iimvavanyo ze-vivo sisebenzisa imodeli ye-mouse xenograft. Iziphumo zibonisa ukuba i-knockdown ye-DARS-AS1 yehla kakhulu ukukhula kwe-tumor kwiigundane (ixabiso le-p <0.0001) (Umfanekiso 5a). Iziphumo zibonisa ukuba i-knockdown ye-DARS-AS1 yehla kakhulu ukukhula kwe-tumor kwiigundane (ixabiso le-p <0.0001) (Umfanekiso 5a). Результаты показывают, что нокдаун DARS-AS1 резко снижает рост опухоли у мышей (значение p <0,0001) (рис. 5а). Iziphumo zibonisa ukuba i-DARS-AS1 i-knockdown inciphisa kakhulu ukukhula kwe-tumor kwiigundane (ixabiso 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). Iziphumo zibonise ukuba i-DARS-AS1 i-knockdown yanciphisa kakhulu ukukhula kwe-tumor kwiigundane (ixabiso le-p <0.0001) (Umfanekiso 5a).Ngaloo ndlela, kwiqela le-DARS-AS1 le-knockdown, kukho ukuhla okukhulu kwi-tumor volume volume malunga ne-72.9% kunye ne-tumor mass mass malunga ne-87.8% (Figure 5b-d).Ezi ziphumo zibonisa ngamandla ukuba i-DARS-AS1 inokukhuthaza kakhulu ukukhula kwethumba kwi-vivo.
Iziphumo ze-ad DARS-AS1 knockdown kwi-colorectal oncogenesis kwiimpuku ezihamba ze.Amagophe okukhula (a), ubungakanani bethumba (b), ubunzima (c), kunye nemifanekiso yethumba (d) iyaboniswa.Iibar zemposiso zimele ±SEM. n = 10. ****p <0.0001, ngovavanyo loMfundi olunemisila emibini. n = 10. ****p <0.0001, ngovavanyo loMfundi olunemisila emibini. n = 10. ****p < 0,0001 по двустороннему критерию Стьюдента. n = 10. ****p <0.0001 Uvavanyo loMfundi olunemisila emibini.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 Uvavanyo loMfundi olunemisila emibini.e-Kaplan-Meier uhlalutye ulungelelwaniso phakathi kwamanqanaba okuchazwa kwe-DARS-AS1 kunye nokusinda ngokubanzi kwizigulane ezine-UVM, KICH, KIRP, MESO, GBM, kunye ne-LGG.Amanqanaba aphezulu enkcazo ye-DARS-AS1 kwizigulane ayephezulu kwi-50%;inqanaba eliphantsi le-DARS-AS1 intetho kwizigulane yayingaphantsi kwe-50%.amaxabiso e-p aye amiselwa kusetyenziswa uvavanyo lwenqanaba lelog.f Imodeli ecetywayo apho i-DARS-AS1 ilawula indlela ye-PACT-PKR kunye nokukhula kwe-tumor.
Ukuqonda ngcono impembelelo yeklinikhi ye-DARS-AS1, sivavanye unxibelelwano phakathi kwentetho yayo kunye nokusinda kwesigulane.Ngokuhlalutya i-dataset ye-TCGA, sifumene ukuba inkcazo ephezulu ye-DARS-AS1 idibaniswe ne-uveal melanoma (UVM), i-renal chromophobia (KICH), i-renal papillary cell carcinoma (KIRP), i-mesothelioma (MESO), i-multiplex.Ukusinda okuphantsi kwakudityaniswa kakhulu ne-glioblastoma morphosis (GBM) kunye nezigulane ezine-low-grade brain glioma (LGG) (Figure 5e).Ezi ziphumo zibonisa ukuba i-DARS-AS1 inokudlala indima ebalulekileyo ekuqhubekeni kwethumba leklinikhi kwaye inokuba yinto enokuqikelela biomarker kwimihlaza emininzi.
Kolu phononongo, sisebenzisa i-CRISPRi enkulu yokuhlola ukusebenza, sinqume ukuba i-DARS-AS1 lncRNA inqoba uxinzelelo lweeseli zomhlaza ngokulawula abaphenduli ababini abaphambili bexinzelelo, i-PACT kunye ne-PKR.Ngokusebenzisana ngokuthe ngqo ne-PACT, i-DARS-AS1 inqanda ukusebenza kwe-PACT-mediated PKR, ngaloo ndlela ikhusela ukufa kweeseli ze-apoptotic kunye nokukhuthaza ukwanda kweeseli (umzobo 5f).Ukunyuswa kwe-DARS-AS1 kuye kwabonwa kwiintlobo ezininzi zomhlaza, ephakamisa ukuba umsebenzi wayo wokukhuthaza ukusinda kweeseli zomhlaza phantsi kweemeko ezixinzelelekileyo unokusebenza ngokubanzi kwiintlobo ezininzi zomhlaza.
I-PACT ichongiwe njengeprotheni ye-activator ye-PKR, kwaye i-PACT-mediated PKR activation idlala indima ebalulekileyo kwiimpendulo zoxinzelelo ngokulawula ukubhalwa, ukuguqulelwa, i-apoptosis, kunye nezinye iinkqubo ezibalulekileyo zeselula62.Kangangamashumi eminyaka, kuye kwenziwa iinzame zokuqonda umgaqo othe ngqo womhlaza we-PACT-PKR cascade.Apha, isifundo sethu sibonise indlela eyahlukileyo yokulawula i-PACT-PKR kwiiseli zomhlaza ngokusebenzisa i-lncRNA DARS-AS1 yeselula, ebophelela ngokuthe ngqo kwi-PACT, ivimbela ukusebenzisana kwe-PACT-PKR, inqanda ukusebenza kwe-PKR kunye ne-eIF2α phosphorylation, ngaloo ndlela inqanda i-apoptosis ebangelwa uxinzelelo kunye ukuvuselela ukwanda komhlaza ekugqibeleni.iiseli.Oku kufunyaniswe kukhanyisa kwiithagethi ezinokuthi zibe kho ze-lncRNA zesifo somhlaza kunye nonyango.
Idatha yethu ibonise ukuba i-DARS-AS1 i-knockdown ikhuthaza iiseli kwindlala ye-serum ngokunyuka okukhulu kwe-phosphorylated PKR kunye ne-eIF2α.Ezi ziphumo zibonisa ukuba i-DARS-AS1 ikhuthaza ukusinda kweseli yomhlaza phantsi kweemeko ezinzima ngokuvimbela umsebenzi we-PACT / PKR.Ezinye ii-RNA ezininzi ezingenayo ikhowudi, ezifana ne-ASPACT kunye ne-nc886, nazo zibandakanyeka kwi-PACT / PKR axis ngokunciphisa i-PACT48 mRNA okanye ukulawula i-autophosphorylation ngokuzibophelela kwi-PKR49,50,64.Phakathi kwabo, i-DARS-AS1 isebenza njengesiphazamisi sombutho we-PACT-PKR.Olu pho nonongo lucebisa ukuqonda kwethu umgaqo we-PACT/PKR axis kunye nendima ye-lncRNAs kwiimpendulo zoxinzelelo.
I-PACT iqulethe imimandla emithathu eyahlukileyo.Nganye kwii-dsRBD ezimbini zokuqala zanele ukufezekisa ukubopha okuphezulu kwe-PACT kwi-PKR, ngelixa i-domain yesithathu (D3) iyadingeka ukuze kusebenze i-PKR kwi-vitro nakwi-vivo.Uphononongo lwethu lubonise ukuba i-DARS-AS1 ngokukhethekileyo isebenzisana ne-D3 domain (Umfanekiso 3h).Ukunikezelwa kobukhulu obukhulu be-lncRNA (i-768 nucleotides), i-DARS-AS1 ebophelelayo kwi-D3 inokuthintela ngokomzimba ukusebenzisana phakathi kwe-PACT domain ye-dsRBD kunye ne-PKR, ngaloo ndlela ivimbela umbutho we-PACT kunye ne-PKR.Iinguqu zenqaku le-PACT ezithathe indawo ye-Ser246 kunye ne-Ser287 kwi-D3 nge-alanine okanye i-aspartate iphazamise ubudlelwane bayo obubophelelayo be-DARS-AS1, ebonisa ukubaluleka kwe-D3's jikelele yezakhiwo kunye neepropati zombane kumbutho wabo.Iinkcukacha ezithe vetshe ngalo matshini ziya kufuneka kwixesha elizayo, kusetyenziswa uhlalutyo oluchanekileyo ngakumbi lwe-biochemical kunye nesisombululo esiphezulu sohlalutyo lwe-PACT lwesakhiwo.
Izifundo zangaphambili ziye zaxela ukuba i-DARS-AS1 ikhuthaza ukwanda kweeseli ngeendlela ezininzi51,52,53.Kumzekelo omnye, abaphandi baqaphela ukuba i-DARS-AS1 ilawule i-antisense protein-encoding DARS gene ngokujolisa kwi-miP-194-5p kwiiseli zomhlaza wezintso.Nangona kunjalo, kolu phononongo lwangoku, ukunkqonkqozwa kwe-DARS-AS1 kwaba nefuthe elincinci kushicilelo lwe-DARS kwiintlobo ezininzi zomhlaza, kubandakanya ubuncinci be-colorectal, ibele, kunye nomhlaza wesibindi.Ngenxa yokuba ii-lncRNAs zibonisa iipateni zokubonisa iiseli kunye nezicubu ezithe ngqo, iindlela zokusebenza zisenokungalondolozwa kuzo zonke iintlobo zomhlaza, ezinokuthi zibe negalelo koku mahluko phakathi kwemigqaliselo yethu kunye novavanyo lwangaphambili lwemihlaza eyahlukeneyo.Izifundo ezikhethekileyo ziyafuneka ukucacisa iindlela ezithile zeenkqubo ezahlukeneyo ze-physiological and pathological.
Uhlalutyo lwedatha yeklinikhi lubonise ukuba ukubonakaliswa kwe-DARS-AS1 kumathumba kuhambelana ngokungafaniyo nokusinda kwezigulana ezinomhlaza, nto leyo egxininisa ukubaluleka kwe-DARS-AS1/PACT/PKR axis kwi-prognosis yomhlaza.Ukuqukumbela, uphononongo lwethu lubonisa ukuba i-DARS-AS1 ingumlawuli we-PACT/PKR yokubonisa i-axis, ikhuthaza ukwanda kweseli yomhlaza, kwaye inqanda i-apoptosis ngexesha lokuphendula koxinzelelo, olubonelela ngolunye umgca wophando kwaye lunomdla wophando lwexesha elizayo kunyango olunokubakho. .
Imigca yeeseli zabantu SW620, A549, MBA-MD-231, HCT116, HepG2 kunye ne-HEK293T zifunyenwe kwi-National Cell Line Resource Infrastructure e-China.Zonke iiseli zagcinwa kwi-DMEM medium (DMEM, Thermo Fisher Scientific, Waltham, MA) yongezwa nge-10% FBS (Gemini, Brooklyn, NY) kunye ne-1% penicillin-streptomycin (Thermo Fisher Scientific) kwi-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-Flegi, 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);imouse eqhelekileyo IgG, CST (5415S);umvundla eqhelekileyo IgG, CST (2729S).Ii-antibodies zihlanjululwe 1: 1000 kwi-PBST ye-Western blotting kunye ne-1:100 ye-IP.
Ii-sgRNA zaphuhliswa kusetyenziswa isixhobo esikhoyo esidlangalaleni esibizwa ngokuba yi-CRISPR-ERA66.Sisebenzise izixhobo ezisisiseko zeeparamitha kuphuhliso lwe-sgRNA kunye ne-algorithm ekhomputha i-sgRNA iziza ezibophelelayo kwingingqi ye-3 kb.igxile kwi-TSS.Amachibi e-sgRNA oligonucleotides aye adityaniswa eCustomArray, Inc. (Bothewell, WA) kwaye adityaniswa kwi-pgRNA plasmids yabantu (Addgene #44248).Iyonke i-12 µg ye-pgRNA plasmid edityanisiweyo edityanisiweyo, 7.2 µg ye-psPAX2 (Addgene #12260), kunye ne-4.8 µg ye-pMD2.G (i-Addgene #12259) zaye zakhutshelwa kwi-5 x 106 HEK2063cm ye-Reaction Transfection iiseli ( CWBIO, Beijing, China) ngokulandela imiyalelo yomenzi.Amandla amakhulu aqulathe iintsholongwane aqokelelwa kwiiyure ezingama-48 kunye nama-72 emva kosulelo kwaye ahluzwa ngesihluzo esiyi-0.45 µm.Ukujongwa, iiseli ze-SW620 ezibonisa iprotein ye-dCas9/KRAB edityanisiweyo zafunyanwa ngokugqithiswa kwentsholongwane.Iiseli ze-SW620 ezilungisiweyo zosulelwe yilayibrari yentsholongwane kwiimfuniselo ezine ezizimeleyo zosulelo kwi-MOI ye-0.1-0.3 kwaye zathatyathwa nge-2 μg/ml puromycin (Sigma, St. Louis, MO) iintsuku ezi-2.Emva koko, iiseli zakhuliswa kangangeentsuku ezili-18 kwi-vitro kunye nobuncinci bokugqunywa kwethala leencwadi leeseli ezingama-500/sgRNA ukuze zihlolwe.
I-Genomic DNA ikhutshwe ngokwemiyalelo ye-QIAamp DNA Blood Midi Kit (QIAGEN, Düsseldorf, Germany; 51183).Lilonke, i-100 μg ye-genomic DNA ngokuphinda kwebhayoloji yasetyenziswa ukwakha ithala leencwadi.Ummandla we-sgRNA uye wandiswa yimijikelo emibini ye-PCR kwaye yadityaniswa nebhakhowudi.
Iimveliso ze-PCR zahlanjululwa kusetyenziswa ijeli ye-NucleoSpin® kunye nekiti yokucoca i-PCR (MACHHEREY-NAGEL, Düren, Germany; 740609.250) kwaye yalinganiswa kusetyenziswa i-Qubit™ HS ephindwe kabini kwikhithi yokufumanisa i-DNA (i-Thermo Fisher Scientific; Q32854).
Uvavanyo lwe-MTS lusetyenziswe ukulinganisa ukwanda kweeseli.Iiseli zaye zafakwa kwiipleyiti ezinamaqula angama-96 ekuxinaniseni kokuqala kweeseli ezingama-2000/emthonjeni.Inani elihambelanayo leeseli lilinganiswa imihla ngemihla ngexesha elibonisiweyo leentsuku ezi-4-6.Kwiqula ngalinye, i-20 μl ye-MTS reagent (i-Promega) yaxutywa nge-100 μl ye-DMEM, ifakwe kwiiseli i-4 h ngo-37 ° C, kwaye emva koko i-OD490 yalinganiswa.
Umthamo wokukhula okungenambombo wafunyanwa ngokuhlalutya ukubunjwa kwamangqamuzana.Ngamafutshane, iiseli ezingama-2000 ezosulelwe nge-shRNA DARS-AS1 okanye i-shRNA yolawulo yakhuliswa kwii-microplates ezincanyathisiweyo ezisezantsi (iCorning) kunye notshintsho oluphakathi rhoqo ngeentsuku ze-4.I-spheroids ibalwe emva kweentsuku ezili-14.Iiseli ze-500 ezidluliselwe nge-DARS-AS1 i-plasmid yokugqithisa okanye i-plasmid yokulawula isetyenziselwe ukuvavanya ukuphucula, ngaphandle koko indlela ayizange iguqulwe.
I-RNA yabhalwa kusetyenziswa i-T7 RNA polymerase kunye ne-biotin-16-UTP (i-Roche 1138908910) ngokwemiyalelo ye-Riboprobe® Combination Systems (Promega P1440).Ii-primers ezisetyenziswe apha zidweliswe kwiTheyibhile eyoNgezelelweyo yesi-4.
Iprotein-coding PACT okanye imimandla ye-PKR yenziwa i-pET15b (Addgene #73619) kwaye yatshintshwa yaba yi-BL21(DE3).Ibhaktiriya yafukanywa ngobusuku kwi-LB yabonelelwa nge-ampicillin kwaye emva koko yaxutywa ka-100 nge-LB entsha.Xa i-OD600 ye-medium ifikelele kwi-0.8, i-1 mM IPTG yongezwa ukuze ifake iprotheni yokubonakaliswa.Emva kokufukanyelwa ngobusuku ngokungcangcazela okuthambileyo (250 rpm kwi-20 ° C), i-cell pellet yaqokelelwa nge-centrifugation (4000 rpm, 10 min, 4 ° C).Phinda umise i-cell pellet kwi-lysis buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 1 mM PMSF) kwaye ufukamele emkhenkceni imizuzu engama-30, emva koko i-sonicate (15 min, 5 s on/off, on ice) kunye ne-centrifuge (13,000). rpm)., 30 min, 4 ° С).I-supernatant yabe ilayishwa kwi-Ni-NTA resin (QIAGEN) ngamaxesha e-3 kwi-4 ° C, ihlanjwe ngamaxesha e-4 nge-buffer yokuhlamba (50 mM Tris, pH 8.0, 40 mM imidazole, 250 mM NaCl) kwaye ihlanjwe ngamaxesha e-3, kunye nenani elipheleleyo. ye-10 ml eluent buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 300 mM imidazole).Iprotheyini ehlanjululweyo yamiselwa kusetyenziswa i-WB kwaye ugxininiso lwamiselwa kusetyenziswa i-Qubit™ protein assay kit (Thermo Fisher Scientific; Q 33212).
Uvavanyo lwe-RIP lwenziwa njengoko kuchaziwe ngaphambili, ngohlengahlengiso.Ngokufutshane, 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 (I-Roche, i-1 mM DTT) kunye ne-centrifuge kwi-13,000 rpm kwi-15 min kwi-4 °C.I-supernatant yaza yafakwa kwiprotheni A + G yamaso kazibuthe (Millipore) edityaniswe ne-5 μg ye-anti-PACT antibody (Abeam) okanye i-IgG (CST).Amaso ahlanjwe amaxesha angama-5 nge-5x RIP buffer, emva koko agaywe ngeproteinase K (NEB).I-RNA ikhutshwe kunye ne-Trizol kwaye inqunywe yi-RT-qPCR.Iiprayimari zinikwe kwiTheyibhile eyoNgezelelweyo yesi-5.
Uvavanyo lwe-RIP lwe-in vitro lwenziwa ngokuhambelana neprotocol yovavanyo oluqhelekileyo lwe-RIP.Itotali ye-5 pmol ye-in vitro eshicilelweyo ye-RNA yahlanjululwa 1x nge-RIP buffer kwaye yafakwa ngokufukama kwi-65 ° C imizuzu emi-5 elandelwa kukupholisa okucothayo kwiqondo lobushushu begumbi.Iyonke i-5 pmol yeeprotheyini ze-PACT ezingaguqukiyo okanye eziguqulweyo ezibhalwe iflegi zahlanjululwa kwi-E. coli.Faka i-RNA ehlaziyiweyo kwiiyure ze-2 kwi-4 ° C kwaye ulandele inkqubo engentla yokuhlalutya kwe-RIP ye-anti-flag IP.
Uhlalutyo olongezelelweyo lwe-RNA, iiseli ze-1 × 107 zifakwe kwi-1xRIP buffer.Emva kwe-centrifugation kwi-13,000 rpm kwi-15 min kwi-4 ° C, i-supernatant yaphathwa kwangaphambili nge-30 μl ye-streptavidin amaso kazibuthe (Beckman) i-2 h kwi-4 ° C.I-lysate ecociweyo emva koko yabonelelwa ngeyeast tRNA yaza yafukanyelwa nge-40 pmol ye-RNA ehlaziyiweyo ngobusuku kwi-4 ° C, emva koko ezinye iiyure ezi-2 kunye ne-20 μl ye-streptavidin amaso azibuthe evalwe nge-BSA yongezwa.Isinyathelo sokuhlamba sasiquka amaxesha e-4 kunye ne-5x ye-RIP buffer kunye namaxesha e-4 kunye ne-1x ye-RIP buffer.Iiprotheni ezihambelanayo zaye zahlanjululwa nge-biotin elution buffer (25 mM Tris-HCl, pH 7.5, 12.5 mM D-biotin, PMSF) kwaye yahlulwe kwi-NuPAGE 4-12% Bis-Tris Gel (Invitrogen).Emva kokungcoliswa kwesilivere (Beyotime Biotechnology), amabhendi athile akhutshiwe kwaye ahlalutywa nguMS.
Uhlalutyo lwe-Co-IP lwenziwa ukuvavanya intsebenziswano phakathi kwe-PACT kunye ne-PKR.Ngokufutshane, ii-lysates ezinamandla zalungiswa ngokufukamela i-1 x 107 iiseli ze-lysed kwi-1 x i-RIP buffer elandelwa yi-centrifugation kwi-13,000 rpm imizuzu ye-15 kwi-4 ° C.IiLysates zazilayishwe ngeprotein A + G amaso kazibuthe, adityaniswe ne-5 µg ye-anti-PACT antibody, kwaye ajikeleziswa ngobunono ngobusuku kwi-4°C.Amaso ahlanjwe ngamaxesha e-3 nge-5 × i-RIP buffer, kabini nge-1 × i-RIP buffer kunye ne-1 × SDS buffer.Iprotheni efunyenweyo yahlalutywa yi-SDS-PAGE gel kwaye yafunyanwa yi-WB.
Ii-pmol ezimbini ze-PACT eziphawulweyo kunye ne-1 pmol ye-PKR zahlanjululwa kwi-E. coli.Nciphisa kwi-1× i-RIP buffer kwaye ufukame nge-10 pmol ye-RNA ehlaziyiweyo ngeeyure ezi-2 kwi-4 °C.Emva koko, zafakwa kwiprotein A+G magnetic bead-conjugated anti-labeleled antibody iiyure ezimbini ezongezelelweyo.Amaso aye ahlanjwa kane nge-1x RIP buffer kwaye eluted nge-1x SDS buffer.Iziphumo ze-PACT kunye ne-PKR zifunyenwe yi-WB.