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Masero eCancer akagadzira nzira dzakasiyana dzekukunda kushushikana kweserura uye kuramba achifambira mberi.Protein kinase R (PKR) uye mapuroteni ayo activator (PACT) ndivo vekutanga vanopindura vanotarisisa zviratidzo zvakasiyana-siyana zvekushushikana zvinotungamira mukudzivirirwa kwekuwedzera kwesero uye apoptosis.Nekudaro, kudzorwa kwePACT-PKR nzira mumasero egomarara kunoramba kusingazivikanwe.Pano, takaona kuti yakareba isiri-coding RNA (lncRNA) aspartyl tRNA synthetase antisense RNA 1 (DARS-AS1) inobatanidzwa zvakananga mukudzivisa kwePACT-PKR nzira uye inokurudzira kenza yekenza.Tichishandisa hukuru-hukuru hunoshanda kuongorora kweCRISPRi 971 yakabatana nekenza lncRNA, takaona kuti DARS-AS1 yaisanganiswa nekuwedzerwa kwakanyanya kwegomarara masero.Naizvozvo, DARS-AS1 kugogodza inhibits kuwedzera kwesero uye inosimudzira cancer cell apoptosis mune akasiyana cancer maseru mitsara in vitro uye inoderedza zvakanyanya bundu kukura mu vivo.Mechanically, DARS-AS1 inosunga zvakananga kuPACT activation domain uye inodzivirira kupindirana kwePACT-PKR, nokudaro kuderedza PKR activation, eIF2α phosphorylation, uye inhibiting apoptotic cell death.Mukiriniki, DARS-AS1 inoratidzirwa zvakanyanya mumakenza akawanda, uye kuwandisa kweiyi lncRNA kunoratidza fungidziro yakaipa.Ichi chidzidzo chinojekesa kenza-chaiyo mutemo wePACT-PKR nzira neDARS-AS1 lncRNA uye inopa chimwe chinangwa chechirwere chegomarara uye kurapwa.
Iko kugona kujairana nekunetsekana chinhu chakakosha chekenza cell kupona uye kuwanda.Kukurumidzira kuwanda uye metabolic zviratidzo zvegomarara rinokwira munzvimbo dzakaomarara-kushaikwa kwemuviri, hypoxia, uye yakaderera pH-iyo inogona kukonzeresa kufa kwesero nzira.Dysregulation yemagetsi-anonzwa majeni akadai se p535, kupisa kutyisa mapuroteni 6, 7, KRAS8, 9, uye HIF-110, 11, 12, 13 inowanzoonekwa mugomarara, nekudaro ichivharira apoptosis uye kukurudzira kupona.
Protein kinase R (PKR) inokosha yekushungurudzika sensor uye subunit kinase ye eukaryotic initiation factor 2α (eIF2α), inoshandura regulator inobatanidza kushushikana kwesero kune kufa kwesero.PKR yakatanga kuzivikanwa seprotein yeantiviral nekuonekwa kweyekunze-yakapetwa kaviri RNA (dsRNA).Pamusoro pekushanda, PKR phosphorylates eIF2α kuti inhibit viral uye cellular protein synthesis14,15,16.PACT (PKR activator protein) yakaonekwa seyekutanga PKR activator protein pasina dsRNA17,18,19,20,21,22,23.Kuburikidza nekudyidzana kwakananga nePKR, PACT inoshandura kushushikana kwakasiyana-siyana (serum nzara, peroxide kana arsenite kurapwa) kuPKR uye kuzasi kwenzira dzechiratidzo.Mukuwedzera kune eIF2α phosphorylation, PACT-mediated PKR activation inokonzera zviitiko zvakasiyana-siyana zvine chokuita nemhinduro yekushungurudzika, kusanganisira kuchinjwa redox mamiriro kuburikidza nePI3K / Akt24 nzira, yakagadziridzwa DNA kukanganisa kutarisa kuburikidza nep5325,26 uye NF-κB27,28 Regulates transcription, 29. Zvichipa basa ravo rakakosha mukupindura kwekushushikana, kupararira, apoptosis uye mamwe maitiro akakosha emasero, PKR nePACT vari kuvimbisa zvinangwa zvekurapa kwezvirwere zvakawanda, kunyanya cancer30,31,32,33.Nekudaro, kunyangwe iyi pleiotropic inoshanda uye yakakosha kukosha, mutemo wePACT / PKR chiitiko mumaseru egomarara unoramba uripo.
lncRNAs zvinyorwa zvakakura kupfuura 200 nucleotides isina mapuroteni-coding anogona.Sezvo yekucheka-kumucheto kwese genome sequencing mapurojekiti aratidza zviuru zvelncRNAs, 35,36 yakawanda kuedza kwakaitwa kujekesa mabasa avo ebhayoloji.Boka rinokura rekutsvagisa rakaratidza kuti lncRNAs inobatanidzwa mune dzakawanda biological process37 kusanganisira kudzora kweX-chromosome inactivation38,39, imprinting40, transcription41,42, dudziro43 uye kunyange cancer kukura44,45,46,47.Zvidzidzo izvi zvakashuma kuti akawanda lncRNA anobatanidzwa muPACT/PKR nzira.Imwe ongororo yakadai yakaratidza kuti lncRNA ASPACT inhibited PACT transcription uye yakawedzera kuchengetedza kwenyukireya kwePACT mRNA.Zvimwe zvidzidzo zvakaratidza kuti lncRNA nc886 inosunga kuPKR uye inhibits phosphorylation yayo49,50.Parizvino, lncRNA inoronga PACT-mediated PKR activation haina kutaurwa.
Aspartyl-tRNA synthetase antisense RNA 1 (DARS-AS1) yakaonekwa se oncogenic lncRNA51,52,53,54.Kuburikidza nemutemo we miP-194-5p53, miP-12952 uye miP-532-3p51, DARS-AS1 yakaratidzwa kukurudzira kukura kwepachena cell renal cell carcinoma, thyroid carcinoma uye isiri-small cell lung carcinoma, maererano.Tong uye vaaishanda navo vakawanawo kuti DARS-AS1 inosimudzira myeloma kufambira mberi nekuchengetedza kugadzikana kweprotein 39 (RBM39) RNA-binding motif.Zvisinei, hapana zvidzidzo zvakaitwa pamusoro pekuti iyi lncRNA inobatanidzwa mukugadzirisa kwePACT-PKR activation uye kupindura kwekunetseka kwemasero ekenza.
Pano, takaita chidzitiro chakakura chekurasikirwa-kwe-basa tichishandisa CRISPRi system uye takasarudza kuti DARS-AS1 lncRNA inosimudzira kuwanda kwemhando dzinoverengeka dzekenza maseru.Mukuwedzera, takaona nzira huru: DARS-AS1 inosunga zvakananga kuPACT, inhibits PACT uye PKR kusunga, inodzivirira phosphorylation ye eIF2α, pasi PKR substrate, uye pakupedzisira inhibits apoptotic cell death.Mukupedzisa, basa redu rinoratidza iyo DARS-AS1 lncRNA semutongi wePACT-PKR nzira uye inogona kuve chinangwa chekurapa cancer uye kufungidzira.
Zvakawanda genomic profiling zvidzidzo zvakaratidza mazana elncRNAs ane chekuita negomarara.Nekudaro, basa ravo rinoramba risingazivikanwe56.Kuti tizive vanovimbisa lncRNA vamiriri vanobatanidzwa mukufambira mberi kwegomarara, takaita chidzitiro chekurasikirwa-kwe-basa rekudzikisa kupararira muSW620 colorectal cancer cell line tichishandisa CRISPRi system (Fig. 1a).Chinhu chakasarudzika cheSW480 uye SW620 colon cancer cell mitsara ndechekuti ivo vanotorwa kubva kumamota ekutanga uye echipiri mumurwere mumwechete.Izvi zvinopa kuenzanisa kwakakosha pakudzidza shanduko yemajini mukufambira mberi kwekenza yekoloni.Naizvozvo, isu takaongorora zvinyorwa zve colorectal cancer cell mitsara (SW480 uye SW620) tichishandisa RNA sequencing uye takaunganidza mamwe anogona kushanda lncRNAs kubva mumabhuku akaburitswa.Zvichienderana nemhedzisiro iyi, takagadzira raibhurari yakabatanidzwa sgRNA ine 7355 sgRNA oligos yakanangana ne971 cancer-inosanganisirwa lncRNAs uye mazana mashanu isina kutariswa sgRNA oligos yekutonga kwakashata (Supplementary Data 1).
Schematic inomiririra yekuongorora uchishandisa CRISPRI system.b sgRNA kupfumisa mushure mekuongorora.Mutsetse wakatwasuka une doti unomiririra log2 (peta shanduko) = ± 0.58.Mutsetse wedoti wakatwasuka unoratidza p kukosha = 0.05.Mavara matema anomiririra asiri-chinangwa sgRNA (yakasarudzwa seNC).Madonhwe matsvuku ari sgRNA anonangana neDARS-AS1.Madonhwe ebhuruu ari sgRNAs akananga LINC00205, yakambotsanangurwa oncogenic lncRNA.kupeta shanduko = (kuverenga kwakajairwa, zuva rechi17)/(kuverenga kwakajairwa, zuva 0).c DARS-AS1 sgRNA knockdown inhibited cell growth.Mabhawa ekukanganisa anomiririra ± yakajairwa kutsauka kwezviyedzo zvitatu.* p ≤ 0.05, ** p ≤ 0.01 t-bvunzo yeMudzidzi ine miswe miviri.d DARS-AS1 kutaura mumamota (TCGA dataset).em Kuratidzwa kweDARS-AS1 mune akajairwa akajairwa uye tumor samples kubva kuvarwere vane BLCA, KIRC, PRAD, LUSC, UCEC, LUAD, LIHC, KIRP, uye COAD, zvichiteerana (TCGA dataset).p-tsika dzakawanikwa pachishandiswa paired-t-tailed Mudzidzi's t-bvunzo.
Mushure mekugadzira iyo plasmid uye kurongedza iyo lentivirus, takapfuudza iyo dCas9-SW620 colorectal cancer cell line neraibhurari iri pamusoro mune ina yakazvimirira yehutachiona kuyedza.Kuwanda kwehutachiona (MOI) kwezvirwere izvi kwaive 0.1-0.3, zvichiratidza kuti sero rega rega rinogona kutapurirwa ne sgRNA imwe chete.Mushure memazuva gumi nemasere emu in vitro tsika, iyo yekufumisa chimiro chechinangwa sgRNAs yakadzikira kana yakawedzera mushure mekuongororwa, nepo nhamba yeasina kutariswa yekutonga oligonucleotides yakaramba isina kuchinjika kana ichienzaniswa neiyo pre-screening chimiro, zvichiratidza kuti chinangwa chedu chine yakanyanya skrini-yakanangana. library.Mupunga.1b uye tafura yekuwedzera 1). LINC00205, iyo yakambotaurwa kuti inokurudzira kenza yemapapu uye kuwedzera kwekenza yechiropa58,59,60, yakaongororwa kunze (log2 (foldchange) <-0.58, p value <0.05), ichisimbisa kuvimbika kwekuongorora uku (Fig. 1b). LINC00205, iyo yakambotaurwa kuti inokurudzira kenza yemapapu uye kuwedzera kwekenza yechiropa58,59,60, yakaongororwa kunze (log2 (foldchange) <-0.58, p value <0.05), ichisimbisa kuvimbika kwekuongorora uku (Fig. 1b). LINC00205, о котором ранее сообщалось, что он способствует прогрессированию рака легких и рака печени58, 59, 60, был исключен (log2 (кратное изменение) <-0,58, значение p <0,05), что подтверждает надежность этого скрининга (рис 1b). LINC00205, yakambotaurwa kuti inokurudzira kufambira mberi kwekenza yemapapu uye kenza yechiropa58,59,60, yakabviswa (log2 (fold change) <-0.58, p-value <0.05), ichisimbisa kusimba kwekuongororwa uku (Fig. .1b) . Linc00205 之前 被 报道 可 可 促进 肝癌 肝癌 58 ,,5 ,,5 (倍数 变化) <-0.58, p 值 <了 <了 该 筛选 的 可靠性 (图 1b). Linc00205 之前 被 报道 可 可 促进 肝癌 肝癌 58 ,,5 ,,5 (倍数 变化) <-0.58, p 值 <了 <了 该 筛选 的 可靠性 (图 1b). LINC00205, о котором ранее сообщалось, что он способствует прогрессированию рака легких и печени58, 59, 60, был исключен (log2 (кратное изменение) <-0,58, p-значение <0,05), что подтверждает надежность этого скрининга (рис 1b). LINC00205, yakambotaurwa kuti inokurudzira kukura kwekenza yemapapu uye yechiropa58,59,60, yakabviswa (log2 (fold change) <-0.58, p-value <0.05), ichisimbisa kusimba kwekuongorora uku (Fig. .1b).
Pakati pese lncRNAs yakaedzwa, DARS-AS1 yakaongororwawo, ine matatu cognate sgRNA oligonucleotides yakaderedzwa zvakanyanya mushure memazuva 18 etsika, zvichiratidza kuti kugogodza kwelncRNA iyi kwakaguma nekuderedza kupararira kwekenza (Fig. 1b).Mhedzisiro iyi yakawedzera kutsigirwa nekuongorora kweMTS mumaseru egomarara anoratidza kuti kukura kweDARS-AS1 masero ekugogodza akangoita nepakati kana zvichienzaniswa nemasero ekudzora (Mufananidzo 1c) uye zvaienderana nemishumo yapfuura yemamwe marudzi akati wandei ekenza.: kenza yakajeka yekenza yeitsvo, kenza yethyroid uye isiri-diki yekenza yemapapu yemapapu51,52,53,55.Nekudaro, basa rayo uye nzira dzemamorekuru mune colorectal cancer inoramba isina kuongororwa.Naizvozvo, takasarudza iyi lncRNA kuti tiwedzere kudzidza.
Kudzidza kutaura kweDARS-AS1 muvarwere, takaongorora zvizere zviuru gumi nemazana matatu nemakumi matatu nematatu emasamples kubva kuCancer Genome Atlas (TCGA) chirongwa.Mhedzisiro yedu inoratidza kuti DARS-AS1 inotaridzwa zvakanyanya uye inokwidziridzwa zvakanyanya mumaseru ane hutano mumhando dzakasiyana dzemamota, kusanganisira colon adenocarcinoma (COAD), renal clear cell carcinoma (KIRC), uye renal papillary cell carcinoma (KIRP)..Vashoma kwazvo (Fig. 1d uye Supplementary Fig. 1a, b). Ongororo yeaviri ane hutano / bundu samples yakawedzera kusimbisa kutaura kwakanyanya kweDARS-AS1 mumamota edundira urothelial carcinoma (BLCA), itsvo renal clear cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), lung squamous cell carcinoma (LUSC) , uterine corpus endometrial carcinoma (UCEC), lung adenocarcinoma (LUAD), chiropa hepatocellular carcinoma (LIHC), itsvo renal papillary cell carcinoma (KIRP), uye colon adenocarcinoma (COAD) (p kukosha <0.05) (Fig. 1e-m) . Ongororo yeaviri ane hutano / bundu samples yakawedzera kusimbisa kutaura kwakanyanya kweDARS-AS1 mumamota edundira urothelial carcinoma (BLCA), itsvo renal clear cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), lung squamous cell carcinoma (LUSC) , uterine corpus endometrial carcinoma (UCEC), lung adenocarcinoma (LUAD), chiropa hepatocellular carcinoma (LIHC), itsvo renal papillary cell carcinoma (KIRP), uye colon adenocarcinoma (COAD) (p kukosha <0.05) (Fig. 1e-m) .Ongororo yeaviri ane hutano / bundu samples yakasimbisawo yakakwira zvakanyanya kutaura kweDARS-AS1 mudundira urothelial carcinoma (BLCA), clear cell renal uye renal cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), lung squamous cell carcinoma (LUSC) tumors., карцинома эндометрия тела матки (UCEC), аденокарцинома легкого (LUAD), гепатоцеллюлярная карцинома печени (LIHC), папиллярно-клеточная карцинома почки (KIRP) и аденокарцинома толстой кишки (COAD) (значение p <0,05) (рис. 1e– m) . , endometrial carcinoma ye corpus uteri (UCEC), adenocarcinoma yemapapu (LUAD), hepatocellular carcinoma yechiropa (LIHC), papillary cell carcinoma yeitsvo (KIRP), uye adenocarcinoma yekoloni (COAD) (p kukosha < 0.05) (Mufananidzo 1e– m) .配对 / 肿瘤样本 肿瘤样本 的 分析 分析 了 了 了 Dars-As1显着 更 高 表达, 子宫体子宫 内膜 癌 (UCEC), 肺腺癌 (Luad), 肝肝 细胞癌 (Lihc), 肾 肾 乳头状 细胞癌 (kirp) 和结肠腺癌 (coad) (p 值<0.05（图1e-m) .配 对 健康 / 肿瘤样本 的 的 分析 了 了 Dars-Os1 在 皮癌 皮癌 皮癌 皮癌, 肾 肾 细胞癌 细胞癌 细胞癌(Lusc) 肿瘤 的 的 的 的 中 中 中 中 中 中 中 显着 显着 显着 显着 显着 显着 癌 ((luad)癌 (kirp) (coad) (p 值<0.05) (图1e-m) .Ongororo yehutano / bundu paired samples yakawedzera kutsigira basa reDARS-AS1 mudundira urothelial carcinoma (BLCA), clear cell renal cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), uye lung squamous cell carcinoma (LUSC) tumors.экспрессия при карциноме тела матки (UCEC), аденокарциноме легкого (LUAD), гепатоцеллюлярной карциноме (LIHC), почечно-почечной папиллярно-клеточной карциноме (KIRP) и аденокарциноме толстой кишки (COAD) (значение p <0,05) (рис. 1e -m). kutaura mu corpus uterine carcinoma (UCEC), lung adenocarcinoma (LUAD), hepatocellular carcinoma (LIHC), renal papillary cell carcinoma (KIRP), uye colon adenocarcinoma (COAD) (p kukosha <0.05) (Mufananidzo 1e -m).Zvakatorwa pamwechete, mhedzisiro iyi inoratidza kuti DARS-AS1 inoratidzirwa zvakanyanya uye zvakanyanya mumhando dzakasiyana dzegomarara.
Nekuti DARS-AS1 uye DARS (iyo gene encoding iyo antisense strand) inogovera imwechete inosimudzira uye iri padivi peumwe neumwe, isu takagadzira shRNA kuti inyatso kugogodza DARS-AS1 asi kwete DARS (Supplementary Fig. 2a,b uye Supplementary Tafura 2) .Pamusoro peSW620, takashandisawo mamwe mitsara mitatu yesero inonyatsoratidza DARS-AS1 kudzidza kushanda uye kushanda kweshRNA knockdown (Supplementary Table 3).Zvigumisiro zvedu zvakaratidza kuti shRNAs yose mitatu yakagadziridzwa yakawana kanenge 80% DARS-AS1 kugogodza kushanda nekuita zvishoma pahuwandu hweDARS mRNA (Supplementary Fig. 2c-f).Pamusoro pezvo, takaona kuti DARS-AS1 kugogodza neaya shRNAs zvakanyanya kutadzisa kukura kwesero mu colorectal cancer cell mitsara SW620 (49.7%) uye HCT116 (27.7%), kenza yemazamu cell line MBA-MD-231 (53.4%).) uye HepG2 hepatoma cell line (92.7% kuderedza), pamwe nekukwanisa kwavo kuumba mativi asina kuvharwa (avhareji kuderedza ~ 50.8%, 44.6%, 40.7% uye 75.7% pamutsetse wesero) (Fig. 2a, b).Muna SW620, mhedzisiro yekuumbwa kwekoloni yakasimbisa zvakare kuti DARS-AS1 shRNA yakadzivirira zvakanyanya kuwanda kwesero nekudzikira kweavhareji inosvika 69.6% (Fig. 2c).
Mhedzisiro yekudzora shRNA uye DARS-AS1 shRNA pakuwedzera kwesero (a) uye spheroid kugadzirwa (b) muSW620, HCT116, MBA-MD-231, uye HepG2 masero.c Mhedzisiro yekudzora shRNA uye DARS-AS1 shRNA pakuumbwa kwekoloni mumasero eSW620.Kuwedzera kwesero (d), spheroid formation (e), uye kuumbwa kwekoloni (f) yemasero eSW620 akanyanyisa DARS-AS1.Data inoratidzwa ndiyo inoreve ± yakajairwa kutsauka kwezviyedzo zvitatu.* p ≤ 0.05, ** p ≤ 0.01, uye *** p ≤ 0.001 ne-t-test yeMudzidzi ine miswe miviri.
Kuzadzisa zvidzidzo zvekurasikirwa-kwe-kushanda, isu takazogadzira SW620 masero akawandisa DARS-AS1 (Supplementary Fig. 2g).DARS-AS1 overexpression yakawedzera zvakanyanya kukura kwesero (1.8-fold), isina anchored spheroid formation (1.4-fold), uye kuumbwa kwekoloni (3.3-fold) mumasero eSW620 (Fig. 2d-f).Isu takasimbisa mhedzisiro iyi tichishandisa imwe DARS-AS1 inoratidzira cell line, A549.Izvi zvakawedzera kuwedzera kwesero nekuda kweDARS-AS1 kuwedzeredza kwakawedzera kucherechedzwa mumasero eA549 (Supplementary Fig. 2h, i uye Supplementary Table 3).Zvakatorwa pamwechete, izvi zvidzidzo zvekuwana uye kurasikirwa zvinoratidza kuti DARS-AS1 inosimudzira cancer cell kuwanda mu vitro.
Kuti tiongorore maitiro ari pasi peiyo DARS-AS1 inogadzirisa kuwanda kwesero, takaita ongororo yekudonhedza pasi yeRNA kuti tizive vangangove vanosunga mapuroteni.RT-qPCR zvigumisiro zvakaratidza kuti inenge 86.2% yeDARS-AS1 iri mu cytoplasm yemasero eSW620 (Supplementary Fig. 3a).Iyo in vitro yakanyorwa biotinylated DARS-AS1 kana pseudoRNA yakabva yaiswa neSW620 cell lysates ichiteverwa neSDS-PAGE kupatsanurwa.Yakazotevera tsvina yesirivha yakaratidza kuti bhendi rakasiyana (~ 38 kDa) rakanyanya kupfumiswa muDARS-AS1 kudhonza samples asi kwete mu dummy RNA kana mabhesi samples (Fig. 3a).Iri bhendi rakaonekwa sePKR activating protein (PACT) ne mass spectrometry (MS) uye yakasimbiswa ne immunoblotting mu SW620, HCT116, uye HepG2 cell mitsara (Fig. 3a, b).Kufumiswa kweDARS uye ane hukama PACT mapuroteni - PKR uye TRBP - yakaongororwawo uchishandisa RNA kuongororwa neWestern blotting (WB).Zvigumisiro zvakaratidza kuti hapana kuwirirana kwakananga pakati peDARS-AS1 RNA uye mapuroteni matatu aya akawanikwa (Supplementary Fig. 3b).Kudyidzana kwakananga pakati peDARS-AS1 nePACT kwakawedzera kusimbiswa neRNA immunoprecipitation (RIP) kuongororwa, iyo yakaratidza kuti DARS-AS1 yakafumiswa zvakanyanya muanti-PACT antibodies asi kwete mamwe maRNAs ekutonga (Mufananidzo 3c).Kuti uone kana DARS-AS1 inopindirana zvakananga nePACT mukusavapo kwezvimwe zvikamu zvemasero, in vitro biolayer interferometry (BLI) assay yakaitwa uchishandisa PACT yakacheneswa.Biotin-yakanyorwa DARS-AS1 kana dummy RNA yakanga isingafambiswi pa streptavidin (SA) biosensors uye yakabva yaiswa mu kinetic buffer ine 1 μM PACT.Zvinoshamisa, PACT yakasungwa zvakasimba kuDARS-AS1 (KD kukosha ~26.9 nM), asi kwete kutevedzera RNA (Mufananidzo 3d).Zvakatorwa pamwechete, izvi mhedzisiro inoratidza kudyidzana kwakananga uye hukama hwepamusoro pakati peDARS-AS1 nePACT.
RNA kudhonza ongororo yakaratidza DARS-AS1 ichidyidzana nePACT muSW620 maseru.Pamusoro, kusvibiswa kwesirivha kwemapuroteni ane hukama.Lower immunoblots akaitwa ne anti-PACT antibody.b RNA kudonha-pasi kuongororwa kwakaitwa muHCT116 (kumusoro) uye HepG2 (pasi) masero.PACT hupfumi hwakaonekwa ne immunoblotting.cRNA immunoprecipitation (RIP) assays akaitwa mumaseru eSW620 achishandisa masoja ekudzivirira chirwere.d PACT inosunga macurves kusvika kuzere-refu DARS-AS1 kana kutonga RNA yakawanikwa uchishandisa biolayer interferometry (BLI).RNA yakavharwa pane streptavidin biosensor.1 μM PACT yakashandiswa kuyera kushamwaridzana.e RNA kudhonza assay yakaitwa uchishandisa biotinylated yakazara-kureba DARS-AS1 kana truncated (kumusoro).Immunoblot inoratidza PACT yakagamuchirwa (pasi).f Yakanatswa inocherekedzwa PACT yakaiswa ne biotinylated yakazara-kureba DARS-AS1 kana kuderedzwa (as in e) ye in vitro RIP assay.Iyo RNA yakabviswa yakasimbiswa neRT-qPCR.g Hukama hwehukama hwezvikamu zvakasiyana zveRNA zvePACT zvakawanikwa uchishandisa biolayer interferometry.Kuongorora, 100 nM RNA uye 1 μM RAST yakashandiswa.h In vitro RIP assays akaitwa pachishandiswa purified intact kana truncated yakanyorwa kuti PACT.Iyo RNA yakabviswa yakasimbiswa neRT-qPCR.i Kukura kwemazinga eSW620 maseru anowedzeredza DARS-AS1, PACT, kana zvese.j Kuwedzeredza kwehurefu hwakazara kana kuderedzwa DARS-AS1 mumasero eSW620 aive nemhedzisiro yakasiyana pakukura kwesero.k Apoptosis yakaonekwa nekuvharika nekudzivirira-PARP antibody.l Knockout yeDARS-AS1 inokonzera apoptosis yemasero eSW620 sezvinoratidzwa nekuyerera kwe cytometry.Data inoratidzwa ndiyo inoreve ± yakajairwa kutsauka kwezviyedzo zvitatu. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p <0.0001, ne-t bvunzo yeMudzidzi ane miswe miviri. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p <0.0001, ne-t bvunzo yeMudzidzi ane miswe miviri. *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 ne-t-test yeMudzidzi ine miswe miviri. *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 ne-t-test yeMudzidzi ine miswe miviri.
Takazogadzira zvitatu zvebiotinylated DARS-AS1 RNA zvimedu ne in vitro transcription kuti tione dunhu reDARS-AS1 rinodiwa kusangano rePACT (Mufananidzo 3e).Mhedzisiro yeRNA yekuongorora yakaratidza kuti chidimbu chimwe nechimwe chakakwanisa kuwirirana nePACT, asi 3'-terminal region (384-768 nucleotides yakanyorwa A3) yakaratidza kupfuura 1-384 nucleotides yakanyorwa A1) (Fig. 3e).Migumisiro yakafanana yakaonekwa mune in vitro RIP assay uchishandisa recombinant PACT (Mufananidzo 3f).Zvichienderana nemhedzisiro iyi, zviedzo zvekusunga zvimedu zveRNA zvisina kufambiswa kuPACT uchishandisa BLI zvakaratidzawo kuti PACT ine hukama hwepamusoro hweA3 (384–768 nt) (KD kukosha kweanosvika 94.6 nM), nepo pasina chinongedzo nedzimwe nzvimbo.(Mufananidzo 3d).
Isu takaongorora zvakare matunhu anosungirirana muPACT.PACT ine matatu anoshanda madomasi, maviri ayo akachengetwa kaviri-stranded RNA-binding domains (dsRBD) uye yechitatu domain (yakasarudzwa D3) inoshanda se activator yeprotein interactions.Kuti tiongorore iyo lncRNA inosunga simba redunhu rega rega, takagadzira matatu shanduko yakabvisa imwe neimwe yemadomasi matatu uye kuita in vitro RIP assay.Mhedzisiro yedu yakaratidza kuti kudzima kwechitatu domain (D3) yePACT kwakaderedza zvakanyanya kudyidzana kwayo neDARS-AS1 (ne0.11-fold zvichienzaniswa neiyo intact PACT) zvichienzaniswa nemamwe maviri shanduko (Fig. 3h), yakaratidzwa kuti kuburitswa. yeD3 yakabatana neDARS.-AC1.Zvakatorwa pamwechete, izvi zvawanikwa zvinoratidza kuti kudyidzana pakati peDARS-AS1 nePACT kunogona kuitika zvakanyanya kuburikidza ne3 ′ kupera kweDARS-AS1 uye D3 domain yePACT.
Takacherechedza kuti DARS-AS1 yakanga isina simba pakutaura kwePACT uye PACT yakanga isina simba paDARS-AS1 (Supplementary Fig. 3c).Takazoongorora maitiro ePACT kugogodza pakukura kwesero.Kusiyana neDARS-AS1, masero ehama akakura 1.5-3 nguva nokukurumidza apo PACT yakagumburwa pasi (Supplementary Fig. 3d).Migumisiro yekuumbwa kwekoloni yakaratidza kuti masero akaumba 2-3-fold colonies mushure mekurapa shRNA nePACT (Supplementary Fig. 3e).Kuongorora kana DARS-AS1 inogadzirisa kuwanda kwesero kuburikidza nePACT, takagadzira SW620 masero anowedzeredza PACT, DARS-AS1, kana zvese.Kuwedzeredza kwePACT kwakaratidza kudziviswa kukuru kwekuwedzera kwesero (Mufananidzo 3i).Nepo DARS-AS1 overexpression per se yakanyanya kukurudzira kuwanda kwesero, pakanga pasina misiyano yakakura mukukura kwemasero akawandisa DARS-AS1 uye PACT.Mhedzisiro iyi inoratidza kuti PACT inogona kupokana nekuwedzera kuwanda kunokonzerwa neDARS-AS1 overexpression.
Sezvo matunhu akasiyana eDARS-AS1 aine akasiyana PACT-anosunga hunyanzvi, takaongorora hukama hwavo pakuwanda kwesero nekuwedzeredza kwakasiyana kwezvidimbu zveDARS-AS1.Zvichienzaniswa nezvimwe zvimedu zviviri, DARS-AS1 yakanga yakanyanyisa pakuguma kwe3 (384-768 nt), nzvimbo huru yakabatana nePACT muDARS-AS1, iyo yakanga ine simba guru rekukurudzira kuwedzera kwesero (Fig. 3j).Iyi mhedzisiro inoratidza kuwirirana kwakanaka pakati pesimba rekusunga uye biological basa reDARS-AS1.
PACT yakanzi ipro-apoptotic protein19.Naizvozvo, isu takaongorora maitiro eDARS-AS1 pane apoptosis.Sezvaitarisirwa, DARS-AS1 knockdown yakawedzera zvakanyanya PARP cleavage muSW620 maseru uye yakawedzera chikamu cheannexin V-positive masero muSW620, HCT116, HepG2, uye MBA-MD-231 cell mitsara (Fig. 3k).3).3f–h), zvichiratidza kuti anti-apoptotic mhedzisiro yeDARS-AS1 mumasero egomarara inopesana neapoptosis-inducing basa rePACT.Kutorwa pamwe chete, mhedzisiro iyi inoratidza kuti maitiro eDARS-AS1 oncogenic basa anogona kunge ari kuburikidza nekuvharirwa kwePACT basa.
Tevere, takaongorora mashandiro emubatanidzwa weDARS-AS1-PACT.PACT yakashumwa kuti iite PKR kuburikidza nekudyidzana kwakananga, iyo inozosimudzira eIF2α phosphorylation, zvichikonzera kubviswa kwekushandura uye apoptosis17.Chekutanga, takaongorora kana DARS-AS1 inokanganisa cellular localization yePACT nePKR.Confocal fluorescence microscopy yakaratidza kuti PACT nePKR dzakanyanya kurongeka mumasero eSW620 ane avhareji Pearson correlation coefficient ye0.72.Zvichakadaro, DARS-AS1 overexpression yakaderedzwa zvakanyanya PACT uye PKR co-localization (zvinoreva Pearson correlation coefficient 0.61) (Mufananidzo 4a).Kuti tiongorore kana DARS-AS1 ichikwanisa kushandura kupindirana kwePACT-PKR, takaita co-immunoprecipitation (co-IP) assay ine anti-PACT antibody muSW620 cell lysates.PKR yakagadziridzwa zvakanyanya mu-anti-PACT mumasero ekudzora, nepo PKR kupora yakaderedzwa zvakanyanya mu lysates kubva kumasero overexpressing DARS-AS1 (Fig. 4b).Yakanatswa yakanyorwa kuti PACT uye PKR yakashandiswa kune in vitro protein inosunga assays.Saizvozvo, avo vakapa DARS-AS1 asi pasina kudzora RNA yakaratidza kudzvanywa PACT-PKR kupindirana (Mufananidzo 4c).Zvese zvabuda zvakaratidza kuti DARS-AS1 yakakanganisa PACT uye PKR kutaurirana.
a Co-localization yePACT uye PKR mukudzora maseru kana masero akawandisa DARS-AS1 akaonekwa achishandisa confocal fluorescence microscopy.Iyo nuclei yakasvibiswa neDAPI.Nhamba dzehuwandu dzakawanikwa kubva pamifananidzo gumi nematanhatu.b Co-immunoprecipitation (co-IP) kushandisa anti-PACT antibody muchitokisi lysates ekutonga SW620 masero kana masero akawandisa DARS-AS1.c Yakanyorwa PACT, yakacheneswa PKR uye yakanyorwa mu vitro ine DARS-AS1 kana yekunyomba RNA yakaiswa incubated ye in vitro protein binding analysis.Anti-flag masoja ekudzivirira chirwere akashandiswa kudzivirira immunoprecipitation.d Immunoblots ine masoja akaratidzwa akaitwa muSW620 uye HCT116 masero akatapurirwa nekudzora shRNA kana DARS-AS1-shRNA ichiteverwa neserum nzara.e DARS-AS1 mazinga ekutaura akachinja maseru senzwi kune thapsigargin.SW620 masero akachinjwa neDARS-AS1 shRNA, DARS-AS1 overexpression plasmid kana kudzora plasmid.Masero akabatwa nethapsigargin kwemaawa makumi mana nemasere uye kushanda kwesero kwakatemwa uchishandisa iyo MTS reagent.f In vitro yakanyorwa DARS-AS1 kana dummy RNA uye yakacheneswa PACT yakashandiswa in vitro activation assay uye immunoblot kuonekwa.g maImmunoblots achishandisa masoja ekudzivirira chirwere aya akaitwa pamaseru eSW620-ctrl (kuruboshwe) kana maseru anowedzeredza PKR mutants (kurudyi).Aya maseru akazotapurwa nekutonga shRNA kana DARS-AS1-shRNA ichiteverwa neserum nzara.h Flow cytometry yakaratidza kuti kusashanda kweiyo mutant PKR kwakabhadhara DARS-AS1-induced apoptosis mumaseru eSW620.i Immunoblots ane masoja akaratidzwa akaitwa mumasero eSW620 (kuruboshwe) kana HCT116 (kurudyi).Masero anotapurirwa ane control shRNA kana DARS-AS1 shRNA ari serum-inonyimwa uye anowedzerwa ne100 nM PKR C16 inhibitor kana DMSO.Scale bar = 5 µm.Data inoratidzwa ndiyo inoreve ± yakajairwa kutsauka kwezviyedzo zvitatu.* p ≤ 0.05 t-bvunzo yeMudzidzi ine miswe miviri.
Zvinowanzotendwa kuti kana PACT ichinge yadyidzana nePKR17, PKR phosphorylation paThr451 inogona kunyengedzwa.Zvigumisiro zvedu zvakaratidza kuti chiyero chePKR phosphorylation chakanyanya kukwidziridzwa muDARS-AS1 masero ekugogodza mushure mekuita serum nzara (Fig. 4d uye Supplementary Fig. 4a).Saizvozvowo, takaona kuti phosphorylation ye eIF2α, iyo huru yePKR substrate, yakawedzerawo zvakanyanya neDARS-AS1 shRNA (Fig. 4d uye Supplementary Fig. 4a).Thapsigargin is ER stressor inoita kuti ER isunungure Ca2 +.Kurapa ne thapsigargin kwakashumwa kuti iite kutaura uye kushandiswa kwePACT, iyo inowedzera kupindirana nekuita PKR, zvichiita kuti apoptosis nekuwedzera eIF2α phosphorylation 18,61.Pano, takashandisa thapsigargin sekusimudzira kwePACT/PKR nzira yekuongorora kana DARS-AS1 inogona kubatsira maseru kukurira kushushikana nekudzivisa iyo PACT/PKR nzira.Takaona kuti nhanho yeDARS-AS1 kutaura kwakanaka kwakabatana nesero kuramba kune thapsigargin.SW620 maseru ainyanya kudzvokora DARS-AS1 akararama zviri nani paakarapwa nethapsigargin, nepo maseru ane DARS-AS1 knockdown akawedzera kubatwa (Fig. 4e).Inopindirana nemigumisiro iyi, DARS-AS1 overexpression yakaderedza thapsigargin-induced PKR phosphorylation (Supplementary Fig. 4b).Kusiyana neizvi, mushure mekurapa kwethapsigargin, PKR uye eIF2α vakanga vari phosphorylated kusvika pamwero wakakwirira muDARS-AS1 masero ekugogodza achienzaniswa nemasero ekudzora (Supplementary Fig. 4b).Sezvineiwo, thapsigargin induced DARS-AS1 kutaura nenzira inotsamira muyero, iyo inogona kuratidza anti-stress basa reDARS-AS1 (Supplementary Fig. 4c).Uye zvakare, isu takaita in vitro activation assays kuratidza izvi zvinoonekwa.Nenguva pfupi, PKR yakacheneswa kubva kuma cell lysates ichishandisa anti-PKR antibody, ndokuzoiswa nerecombinant PACT uye DARS-AS1 yakanyorwa in vitro.Mushure mekuita enzymatic, phospho-PKR yakaonekwa ichishandisa WB.Zvigumisiro zvedu zvakaratidza kuti PKR phosphorylation yakanga yakanyanya kudziviswa neDARS-AS1, asi kwete nekutonga RNA (Mufananidzo 4f).Izvi mu vitro uye mu vivo mhedzisiro inoratidza kuti DARS-AS1 inhibits PACT-mediated PKR activation.Panguva imwecheteyo, takaonawo kuderera kwePACT kupora pamberi peDARS-AS1 (Mufananidzo 4f).Ichi chigumisiro chinopindirana nemigumisiro ye in vitro protein binding assay (Mufananidzo 4c) uye zvakare inoratidza kuvhara basa reDARS-AS1 ye PACT-PKR association.
Ser246 uye Ser287 muD3 domain yePACT inodiwa kuti PKR activation pasi pekunetseka kweserura.Kutsiva maviri serine masara ealanine kwakapa mutant PACT (mutD), iyo yakamisa PKR pasina kushushikana, uye kutsiva alanine (mutA) yakadzoreredza protocol.Sezvo takaratidza kukosha kwenzvimbo iyi takabatana zvakananga neDARS-AS1, takagadzira aya maviri ePACT mutants kuti tiongorore kana izvi zvakasara zvingave zvakabatanidzwa mukudyidzana neDARS-AS1.Zvinofadza kuti zvose zvakagadziriswa zvakarasikirwa nekukwanisa kusunga kuDARS-AS1 (Supplementary Fig. 4d), zvichiratidza kuti chimiro chakakwana chePACT protein chinogona kudiwa kuti chibatane zvakanaka neDARS-AS1.
Uyezve, zvigumisiro zvedu zvinoratidzawo kuti DARS-AS1-shRNA-induced inhibition yekuwedzera kwesero inogona kudzoreredzwa zvishoma nekunyanya kuwedzeredza inotonga isina kunaka PACT mutant (PACTmutA) kana inotonga yakaipa PKR mutant (PKRmut) (Supplementary Fig. 4e. e).Kuwedzeredza kwehutongi-negative PKR mutants yakaderedza PKR phosphorylation inokonzerwa neDARS-AS1 knockdown pamwe ne eIF2α phosphorylation mumasero akadzingwa serum (Fig. 4g).Zvinonyanya kukosha, chikamu chemasero epoptotic anokonzerwa neDARS-AS1 kugogodza kwakaderedzwawo mumasero akawandisa PKRmut (Fig. 4h uye Supplementary Fig. 4g).Kuvharidzirwa kwebasa rePKR kinase kunokanganisawo basa reDARS-AS1, semigumisiro yakaratidza kuti DARS-AS1 knockdown haisati yaita PKR uye eIF2α phosphorylation apo masero aibatwa nePKR-specific C16 inhibitor (Fig. 4i uye Supplementary Fig. 4h).)Takatorwa pamwechete, mhedzisiro yedu inoratidza kuti DARS-AS1 inosimudzira kuwanda kwesero, zvirinani muchikamu, nekudzivisa PACT-mediated PKR activation.
Kuenderera mberi nekuongorora basa reDARS-AS1 mu tumorigenesis, takaita mu vivo kuyedza tichishandisa mbeva xenograft modhi. Migumisiro inoratidza kuti kugogodza kweDARS-AS1 kwakaderedza zvikuru kukura kwebundu mumakonzo (p value <0.0001) (Fig. 5a). Migumisiro inoratidza kuti kugogodza kweDARS-AS1 kwakaderedza zvikuru kukura kwebundu mumakonzo (p value <0.0001) (Fig. 5a). Результаты показывают, что нокдаун DARS-AS1 резко снижает рост опухоли у мышей (значение p <0,0001) (рис. 5а). Mhedzisiro yacho inoratidza kuti DARS-AS1 kugogodza kunoderedza zvakanyanya bundu kukura mumakonzo (p kukosha <0.0001) (Mufananidzo 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). Mhedzisiro yakaratidza kuti DARS-AS1 kugogodza zvakanyanya kudzikisa bundu kukura mumakonzo (p kukosha <0.0001) (Mufananidzo 5a).Nokudaro, muboka reDARS-AS1 rekugogodza, pakanga paine kuderera kukuru kwehuwandu hwebundu hunosvika 72.9% uye zvinoreva bundu guru ne87.8% (Mufananidzo 5b-d).Mhedzisiro iyi inoratidza zvakasimba kuti DARS-AS1 inogona kukurudzira zvakanyanya bundu kukura mu vivo.
Mhedzisiro yead DARS-AS1 kugogodza pa colorectal oncogenesis mumasikirwo emakonzo.Kukura curves (a), bundu saizi (b), uremu (c), uye bundu mifananidzo (d) inoratidzwa.Mabhawa ekukanganisa anomiririra ±SEM. n = 10. ****p <0.0001, ne-t bvunzo yeMudzidzi ine miswe miviri. n = 10. ****p <0.0001, ne-t bvunzo yeMudzidzi ine miswe miviri. n = 10. ****p < 0,0001 по двустороннему критерию Стьюдента. n = 10. ****p <0.0001 t-bvunzo yeMudzidzi ine miswe miviri.n = 10. ****p <0.0001,通过双尾学生t 检验. ****p <0.0001,通过双尾学生t检验. ****p < 0,0001 по двустороннему критерию Стьюдента. ****p < 0.0001 t-bvunzo yeMudzidzi ine miswe miviri.e Kaplan-Meier akaongorora kuwirirana pakati peDARS-AS1 mazinga ekutaura uye kupona kwese kwevarwere vane UVM, KICH, KIRP, MESO, GBM, uye LGG.Mazinga akakwirira eDARS-AS1 kutaura kune varwere vaiva mu50% yepamusoro;iyo yakaderera mwero weDARS-AS1 kutaura muvarwere kwaive muzasi 50%.p-tsika dzakatemerwa uchishandisa irogi renji bvunzo.f Inotsanangurwa modhi umo DARS-AS1 inoronga iyo PACT-PKR nzira uye bundu kukura.
Kuti tinzwisise zvirinani maitiro ekiriniki eDARS-AS1, takaongorora kuwirirana pakati pekutaura kwayo uye kupona kwemurwere.Nekuongorora dataset yeTCGA, takaona kuti yakakwira DARS-AS1 kutaura kwakabatana neveveal melanoma (UVM), renal chromophobia (KICH), renal papillary cell carcinoma (KIRP), mesothelioma (MESO), multiplex.Kupona kwepasi kwakabatana zvakanyanya ne glioblastoma morphosis (GBM) uye varwere vane low-grade brain glioma (LGG) (Mufananidzo 5e).Mhedzisiro iyi inoratidza kuti DARS-AS1 inogona kuita basa rakakosha mukufambira mberi kwechirwere chekiriniki uye inogona kunge iri yekufungidzira biomarker mumakenza akawanda.
Muchidzidzo ichi, tichishandisa yakakura-chiyero CRISPRi inoshanda yekuongorora, takaona kuti DARS-AS1 lncRNA inokunda cancer maseru kushushikana nekudzora maviri makiyi ekushushikana anopindura, PACT uye PKR.Nokubatana zvakananga nePACT, DARS-AS1 inhibited PACT-mediated PKR activation, nokudaro kudzivirira apoptotic cell death uye kukurudzira kuwedzera kwesero (Fig. 5f).Kukwidziridzwa kweDARS-AS1 kwakacherechedzwa mumhando dzakawanda dzegomarara, zvichiratidza kuti basa rayo rekusimudzira cancer maseru kupona pasi pemamiriro ekudzvinyirira rinogona kushanda zvakanyanya kune akawanda marudzi egomarara.
PACT yakaonekwa sePKR activator protein, uye PACT-mediated PKR activation inoita basa rakakosha mumhinduro dzekushushikana nekudzora kunyorwa, kududzira, apoptosis, uye mamwe akakosha cellular maitiro62.Kwemakumi emakore, kuedza kwakaitwa kuti tinzwisise iyo cancer-yakananga mutemo wePACT-PKR cascade.Pano, chidzidzo chedu chakaratidza nzira yakasiyana yekutonga kwePACT-PKR mumasero emukenza kuburikidza neselcRNA DARS-AS1, iyo inosunga zvakananga kuPACT, inovhara PACT-PKR kuwirirana, inhibits PKR activation uye eIF2α phosphorylation, nokudaro inhibiting stress-induced apoptosis uye. kunokurudzira kuwedzera kwegomarara.masero.Kuwanikwa uku kunopa mwenje pane zvingangoita lncRNA zvinangwa zvegomarara uye kurapwa.
Yedu data yakaratidza kuti DARS-AS1 knockdown inokurudzira maseru kune serum nzara nekuwedzera kukuru kwephosphorylated PKR uye eIF2α.Mhedzisiro iyi inoratidza kuti DARS-AS1 inosimudzira kupona kwegomarara pasi pemamiriro akaomarara nekuvharisa chiitiko chePACT/PKR.Mamwe maRNA akati wandei asina kukodha, senge ASPACT uye nc886, anobatanidzwawo muPACT/PKR axis nekudzikisira PACT48 mRNA kana kudzora autophosphorylation nekusunga kuPKR49,50,64.Pakati pavo, DARS-AS1 inoita sekuvhiringidza kwesangano rePACT-PKR.Ichi chidzidzo chinowedzera kunzwisisa kwedu kwePACT/PKR axis regulation uye basa relncRNAs mukupindura kwekushushikana.
PACT ine zvikamu zvitatu zvakasiyana.Imwe neimwe yeaviri ekutanga dsRBDs inokwana kuwana yakakwirira hukama inosunga yePACT kuPKR, nepo yechitatu domain (D3) inodiwa kuti PKR activation in vitro uye in vivo.Chidzidzo chedu chakaratidza kuti DARS-AS1 inonyanya kupindirana neD3 domain (Fig. 3h).Tichifunga nezvehukuru hukuru hwelncRNA (768 nucleotides), DARS-AS1 inosunga kuD3 inogona kuvharira mumuviri kusangana pakati pePACT domain ye dsRBD nePKR, nokudaro ichivharira kushamwaridzana kwePACT nePKR.PACT point mutations yakatsiva Ser246 neSer287 muD3 nealanine kana aspartate yakakanganisa hukama hwayo hweDARS-AS1, ichiratidza kukosha kweD3's yezvese zvimiro uye zvemagetsi zvimiro mumubatanidzwa wavo.Humwe humbowo hwemuchina uyu huchadikanwa mune ramangwana, uchishandisa yakanyatsojeka biochemical ongororo uye yakakwirira resolution PACT maitiro ekuongorora.
Zvidzidzo zvekare zvakashuma kuti DARS-AS1 inokurudzira kuwedzera kwesero kuburikidza nenzira dzakawanda51,52,53.Mune mumwe muenzaniso, vaongorori vakaona kuti DARS-AS1 yakadzora antisense protein-encoding DARS gene nekunanga miP-194-5p mumasero egomarara reitsvo.Nekudaro, muchidzidzo chazvino, kugogodza kweDARS-AS1 kwakave nemhedzisiro shoma pakunyorwa kweDARS mumhando dzakawanda dzegomarara, kusanganisira angangoita colorectal, zamu, uye chiropa.Nekuti lncRNAs inoratidzira sero- uye tishu-chaiyo yekutaura maitiro, anoshanda masisitimu anogona kunge asiri kuchengetedzwa mumhando dzegomarara, izvo zvinogona kukonzera mutsauko uyu pakati pezvatinoona uye ongororo dzakapfuura dzekenza dzakasiyana.Zvidzidzo zvakakosha zvinodikanwa kujekesa maitiro chaiwo eakasiyana physiological uye pathological process.
Ongororo yedhata rekiriniki yakaratidza kuti DARS-AS1 kutaura mumamota kunopindirana zvakanyanya nekupona kwevarwere vegomarara, izvo zvinosimbisa kukosha kweiyo DARS-AS1/PACT/PKR axis mugomarara.Mukupedzisa, chidzidzo chedu chinoratidza kuti DARS-AS1 inodzora PACT/PKR inosaina axis, inosimudzira cancer maseru, uye inhibits apoptosis panguva yekushushikana kwekupindura, iyo inopa imwe mutsara wekutsvagisa uye inofarira kutsvaga mune ramangwana mune zvinogona kurapwa. .
Mitsetse yemasero evanhu SW620, A549, MBA-MD-231, HCT116, HepG2 neHEK293T yakawanikwa kubva kuNational Cell Line Resource Infrastructure kuChina.Masero ose akachengetwa muDMEM medium (DMEM, Thermo Fisher Scientific, Waltham, MA) yakawedzerwa ne10% FBS (Gemini, Brooklyn, NY) uye 1% penicillin-streptomycin (Thermo Fisher Scientific) pa 37 ° C, 5% CO2.incubator.
Anti-PACT, Abcam (ab31967);Anti-PKR, Abcam (ab184257);Anti-PKR (phospho-T451), Abcam (ab81303);Anti-Mureza, Abcam (ab125243);Anti-eIF2α, Abcam (A0764));anti-eIF2α (phosphorus S51), Abcam (ab32157);anti-PACT (phosphorus S246), Abgent (AP7744b);anti-β-tubulin, CST (2128);zvakajairika mbeva IgG, CST (5415S);zvakajairika tsuro IgG, CST (2729S).Masoja ekudzivirira chirwere akadirutswa 1:1000 muPBST yeWestern blotting uye 1:100 yeIP.
sgRNAs dzakagadzirwa pachishandiswa chishandiso chiripo pachena chinonzi CRISPR-ERA66.Isu takashandisa iyo yakasarudzika maturusi ma paramita ekusimudzira sgRNA uye algorithm yakaverengerwa sgRNA inosunga masaiti mudunhu re3 kb.yakanangana neTSS.Madziva e sgRNA oligonucleotides akagadzirwa paCustomArray, Inc. (Bothewell, WA) uye akaumbwa muvanhu pgRNA plasmids (Addgene #44248).Huwandu hwegumi nembiri µg dzepgRNA plasmid dzakabatanidzwa nevanhu, 7.2 µg dze psPAX2 (Addgene #12260), uye 4.8 µg ye pMD2.G (Addgene #12259) yakachinjirwa mu5 x 106 HEK2933 DNA difection mu Reaya maseru (CWBIO, Beijing, China) achitevera mirairo yemugadziri.Virusi-ine mashura akaunganidzwa 48 uye 72 maawa mushure mekutakurwa uye kusefa kuburikidza ne0.45 µm sefa.Kuongororwa, masero eSW620 anoratidza dCas9/KRAB fusion protein akawanikwa nekufambiswa kwehutachiona.Maseru eSW620 akagadziridzwa akatapukirwa neraibhurari yehutachiona muongororo ina yakazvimirira yehutachiona paMOI ye0.1-0.3 uye akatorwa aine 2 μg/ml puromycin (Sigma, St. Louis, MO) kwemazuva maviri.Mushure mezvo, maseru akagadzirwa kwemazuva gumi nemasere mu vitro aine hushoma raibhurari yekuvhara mazana mashanu maseru / sgRNA yekuongororwa.
Genomic DNA yakatorwa maererano nemirairo yeQIAamp DNA Ropa Midi Kit (QIAGEN, Düsseldorf, Germany; 51183).Pakazara, 100 μg ye genomic DNA per biological repeat yakashandiswa kuvaka raibhurari.Iyo sgRNA dunhu yakakwidziridzwa nemataundi maviri ePCR uye yakabatana nebhakodhi.
Zvigadzirwa zvePCR zvakacheneswa uchishandisa NucleoSpin® gel uye PCR yekuchenesa kit (MACHHEREY-NAGEL, Düren, Germany; 740609.250) uye yakaverengerwa uchishandisa Qubit™ HS yakapetwa kaviri-stranded DNA yekuona kit (Thermo Fisher Sayenzi; Q32854).
Iyo MTS assay yakashandiswa kuyera kuwanda kwesero.Masero akadyarwa mu96-tsime mahwendefa panguva yekutanga density ye2000 maseru / tsime.Nhamba yakaenzana yemasero yakayerwa zuva nezuva panguva yakatarwa kwemazuva 4-6.Patsime rega rega, 20 μl yeMTS reagent (Promega) yakanyungudutswa ne100 μl yeDMEM, yakavharwa nemasero kwe4 h pa37 ° C, uyezve OD490 yakayerwa.
Kugona kwekukura kusina kusungirirwa kwakawanikwa nekuongorora kuumbwa kwezvikamu.Nenguva pfupi, masero e2000 akachinjirwa ne shRNA DARS-AS1 kana kudzora shRNA akagadzirwa mune ekupedzisira akaderera akabatanidzwa microplates (Corning) ane yepakati shanduko mazuva ese e4.Ma spheroid akaverengwa mushure memazuva gumi nemana.500 masero akatapurirwa neDARS-AS1 overexpression plasmid kana plasmid yekudzora yakashandiswa pakuedza kwekusimudzira, kana zvisina kudaro nzira yacho haina kuchinjwa.
RNA yakanyorwa uchishandisa T7 RNA polymerase uye biotin-16-UTP (Roche 1138908910) maererano nemirayiridzo yeRiboprobe® Combination Systems (Promega P1440).Iwo ekutanga anoshandiswa pano akanyorwa muSupplementary Tafura 4.
Mapuroteni-coding PACT kana PKR matunhu akaumbwa kuita pET15b (Addgene #73619) uye akashandurwa kuita BL21(DE3).Utachiona hwakaiswa husiku humwe muLB yakapihwa ampicillin uye ndokuzodirwa zana neLB nyowani.Kana iyo OD600 yepakati yakasvika 0.8, 1 mM IPTG yakawedzerwa kuunza mapuroteni kutaura.Mushure mekudyara kwehusiku nekubvunda zvinyoro (250 rpm pa20 ° C), cell pellet yakaunganidzwa ne centrifugation (4000 rpm, 10 min, 4 ° C).Dzorera sero pellet mu lysis buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 1 mM PMSF) woisa paaizi kwemaminetsi makumi matatu, wobva waita sonicate (15 min, 5 s on/off, pa ice) uye centrifuge (13,000). rpm)., 30 min, 4° С).Iyo supernatant yakabva yatakurwa paNi-NTA resin (QIAGEN) 3 nguva pa4 ° C, yakashambidzwa 4 nguva newash buffer (50 mM Tris, pH 8.0, 40 mM imidazole, 250 mM NaCl) uye yakanyungudutswa katatu, nehuwandu. ye10 ml eluent buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 300 mM imidazole).Mapuroteni akacheneswa akatemerwa kushandisa WB uye iko kusungwa kwakatemwa uchishandisa Qubit™ protein assay kit (Thermo Fisher Sayenzi; Q 33212).
RIP miyedzo yakaitwa sezvakatsanangurwa kare, nekugadziridzwa.Muchidimbu, 1x RIP buffer (25 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5% NP-40, RNasin ribonuclease inhibitor (Promega), PMSF (Beyotime Biotechnology), 1 mM DDM, protease) lyses cytostatic x 7 cocktail (Roche, 1 mM DTT) uye centrifuge pa13,000 rpm kwemaminetsi gumi nemashanu pa4 °C.Iyo supernatant yakabva yaiswa neprotein A + G magineti bead (Millipore) yakasanganiswa ne5 μg yeanti-PACT antibody (Abeam) kana IgG (CST).Mabhero acho akagezwa kashanu ne5x RIP buffer, ndokuzogayiwa neprotease K (NEB).RNA yakatorwa neTrizol uye yakatemwa neRT-qPCR.MaPrimes anoratidzwa muSupplementary Tafura 5.
Iyo in vitro RIP assay yakaitwa zvinoenderana neyakagadziridzwa yakajairwa RIP assay protocol.Huwandu hwe5 pmol ye in vitro yakanyorwa RNA yakadiridzwa 1x neRIP buffer uye yakanyudzwa nekufukidzira pa 65 ° C kwemaminetsi mashanu ichiteverwa nekunonoka kutonhora kusvika kune kamuri tembiricha.Huwandu hwe 5 pmol yemapuroteni asina kusimba kana akashandurwa mureza akanyorwa PACT akacheneswa kubva kuE. coli.Isai neRNA yakadzokororwa kwemaawa maviri pa4 ° C uye tevera nzira iri pamusoro yeRIP yekuongorora yeAnti-flag IP.
Kuongororwa kweRNA yekuwedzera, 1 × 107 masero akaiswa lysed ne1xRIP buffer.Mushure me centrifugation pa 13,000 rpm kwemaminetsi gumi nemashanu pa4 ° C, iyo supernatant yakagadziriswa ne 30 μl ye streptavidin magnetic beads (Beckman) kwe2 h pa4 ° C.Iyo yakacheneswa lysate yakabva yapihwa mbiriso tRNA uye yakabatanidzwa ne40 pmol yeRenatured RNA husiku humwe pa4 ° C, uye kwemamwe maawa maviri uye 20 μl yetsva streptavidin magineti bead yakavharwa neBSA yakawedzerwa.Nhanho yekugeza yaisanganisira ka4 ne5x RIP buffer uye 4 nguva ine 1x RIP buffer.Mapuroteni anoenderana akaburitswa nebiotin elution buffer (25 mM Tris-HCl, pH 7.5, 12.5 mM D-biotin, PMSF) uye akaparadzaniswa paNuPAGE 4-12% Bis-Tris Gel (Invitrogen).Mushure mekusvibiswa kwesirivha (Beyotime Biotechnology), mamwe mabhendi akabviswa uye akaongororwa neMS.
Co-IP ongororo yakaitwa kuyedza kudyidzana pakati pePACT nePKR.Nenguva pfupi, supernatant lysates yakagadzirwa nekuisa 1 x 107 lysed maseru mu1 x RIP buffer inoteverwa necentrifugation pa13,000 rpm kwemaminitsi gumi nemashanu pa4 ° C.MaLysates aive akatakurwa neprotein A + G magineti mabedhi, akasanganiswa ne5 µg yeanti-PACT antibody, uye zvinyoro nyoro aitenderedzwa husiku pa4 ° C.Mabhero acho akagezwa 3 nguva ne 5 × RIP buffer, kaviri ne 1 × RIP buffer uye eluted ne 1 × SDS buffer.Protein yakadzoserwa yakaongororwa neSDS-PAGE gel uye yakaonekwa neWB.
Mapiritsi maviri ePACT akacherechedzwa uye 1 pmol yePKR akacheneswa kubva kuE. coli.Dilute mu 1 × RIP buffer uye incubate ne 10 pmol ye renatured RNA kwemaawa maviri pa 4 °C.Mushure meizvozvo, vakaiswa neprotein A+G magnetic bead-conjugated anti-labeled antibody kwemamwe maawa maviri.Mabhero acho akazogezwa kana ne 1x RIP buffer uye akaiswa 1x SDS buffer.Izvo zvakakonzera PACT uye PKR zvakaonekwa neWB.