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Kwayoyin ciwon daji sun haifar da hanyoyi daban-daban don shawo kan damuwa ta salula kuma su ci gaba da ci gaba.Protein kinase R (PKR) da furotin mai kunnawa (PACT) sune masu amsawa na farko waɗanda ke lura da alamun damuwa daban-daban waɗanda ke haifar da hana yaduwar kwayar halitta da apoptosis.Koyaya, ƙa'idar hanyar PACT-PKR a cikin ƙwayoyin cutar kansa ya kasance ba a san shi sosai ba.Anan, mun gano cewa doguwar RNA (lncRNA) mara coding RNA (lncRNA) aspartyl tRNA synthetase antisense RNA 1 (DARS-AS1) yana da hannu kai tsaye a cikin hana hanyar PACT-PKR kuma yana haɓaka haɓakar ƙwayoyin kansa.Yin amfani da babban aikin nunawa na CRISPRi 971 mai alaƙa da ciwon daji lncRNA, mun gano cewa DARS-AS1 yana da alaƙa da haɓakar ƙwayar cutar kansa sosai.Sabili da haka, DARS-AS1 knockout yana hana yaduwar kwayar halitta kuma yana inganta apoptosis cell ciwon daji a cikin nau'o'in kwayoyin cutar kansa a cikin vitro kuma yana rage girman ci gaba a cikin vivo.Mechanically, DARS-AS1 yana ɗaure kai tsaye zuwa yankin kunnawa PACT kuma yana hana hulɗar PACT-PKR, ta haka yana rage kunna PKR, eIF2a phosphorylation, da hana mutuwar kwayar cutar apoptotic.A asibiti, DARS-AS1 an bayyana shi sosai a cikin cututtukan daji da yawa, kuma wuce gona da iri na wannan lncRNA na nuni da rashin hangen nesa.Wannan binciken yana bayyana ƙayyadaddun ƙa'idodin ciwon daji na hanyar PACT-PKR ta DARS-AS1 lncRNA kuma yana ba da wata manufa don hasashen cutar kansa da jiyya.
Ƙarfin daidaitawa da damuwa shine muhimmiyar halayyar rayuwa ta kwayar cutar kansa da yaduwa.Saurin yaduwa da alamomin rayuwa na ciwon daji a cikin ƙananan ƙananan ƙananan ƙwayoyin cuta-rashin abinci mai gina jiki, hypoxia, da ƙananan pH-wanda zai iya haifar da alamun mutuwar kwayar halitta.Dysregulation na kwayoyin halitta masu damuwa irin su p535, sunadaran zafi mai zafi 6, 7, KRAS8, 9, da HIF-110, 11, 12, 13 ana lura da su akai-akai a cikin ciwon daji, don haka yana toshe apoptosis da inganta rayuwa.
Protein kinase R (PKR) shine muhimmin firikwensin danniya da kuma subunit kinase na eukaryotic initiation factor 2α (eIF2α), mai sarrafa fassarar da ke danganta damuwa ta salula zuwa mutuwar tantanin halitta.An fara gano PKR a matsayin furotin antiviral ta hanyar gano RNA (dsRNA) na waje.Bayan kunnawa, PKR phosphorylates eIF2a don hana ƙwayoyin cuta da furotin na salula14,15,16.PACT (Protein activator PKR) an gano shi azaman furotin mai kunnawa na farko na PKR in babu dsRNA17,18,19,20,21,22,23.Ta hanyar hulɗar kai tsaye tare da PKR, PACT tana canza damuwa daban-daban (yunwa, peroxide ko maganin arsenite) zuwa PKR da hanyoyin sigina na ƙasa.Bugu da ƙari ga eIF2a phosphorylation, PACT-matsakaici PKR kunnawa yana haifar da abubuwa daban-daban da ke hade da amsawar danniya, ciki har da canza yanayin redox ta hanyar PI3K / Akt24, inganta lalacewar DNA ta hanyar p5325,26 da NF-κB27,28 Yana daidaita rubutun, 29. Ganin muhimmancin rawar da suke takawa wajen mayar da martani ga danniya, yaduwa, apoptosis da sauran mahimman hanyoyin salula, PKR da PACT suna ba da alamar maganin warkewa ga cututtuka da yawa, musamman ciwon daji30,31,32,33.Koyaya, duk da wannan aikin pleiotropic da mahimmancin ilimin halitta, ƙa'idodin ayyukan PACT/PKR a cikin ƙwayoyin kansa ya kasance mai wuya.
lncRNAs rubuce-rubuce ne da suka fi girma fiye da nucleotides 200 ba tare da yuwuwar rikodin furotin ba.Tun da manyan ayyukan jerin kwayoyin halitta sun gano dubunnan lncRNAs, 35,36 an yi ƙoƙari da yawa don fayyace ayyukan nazarin halittu.Wani ci gaba na bincike ya nuna cewa lncRNAs suna da hannu a yawancin hanyoyin nazarin halittu37 ciki har da ka'idar rashin kunnawa X-chromosome38,39, bugawa40, fassarar41,42, fassarar43 har ma da ci gaban ciwon daji44,45,46,47.Waɗannan binciken sun ba da rahoton cewa yawancin lncRNAs suna da hannu a cikin hanyar PACT/PKR.Ɗaya daga cikin irin wannan binciken ya nuna cewa lncRNA ASPACT ya hana rubutun PACT da kuma ƙara riƙe da makaman nukiliya na PACT mRNA.Sauran binciken sun nuna cewa lncRNA nc886 yana ɗaure zuwa PKR kuma yana hana phosphorylation 49,50.Ya zuwa yanzu, lncRNA da ke daidaita kunna PKR mai matsakaicin PACT ba a ba da rahoton ba.
Aspartyl-tRNA synthetase antisense RNA 1 (DARS-AS1) an gano shi azaman lncRNA51,52,53,54 oncogenic.Ta hanyar ƙa'idar miP-194-5p53, miP-12952 da miP-532-3p51, an nuna DARS-AS1 don inganta haɓakar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar cuta.Tong da abokan aiki sun kuma gano cewa DARS-AS1 yana inganta ci gaban myeloma ta hanyar kiyaye kwanciyar hankali na furotin 39 (RBM39) RNA-daure motif.Duk da haka, ba a gudanar da bincike kan ko wannan lncRNA yana da hannu a cikin ƙa'idar kunnawa PACT-PKR da amsa damuwa na ƙwayoyin ciwon daji.
Anan, mun yi babban allon hasara-na aiki ta amfani da tsarin CRISPRi kuma mun ƙaddara cewa DARS-AS1 lncRNA yana haɓaka yaduwar nau'ikan ƙwayoyin cutar kansa.Bugu da ƙari, mun gano wani babban tsari: DARS-AS1 yana ɗaure kai tsaye zuwa PACT, yana hana PACT da PKR ɗaurin, yana hana phosphorylation na eIF2a, ƙananan ƙananan PKR, kuma a ƙarshe ya hana mutuwar kwayar cutar apoptotic.A ƙarshe, aikinmu yana bayyana DARS-AS1 lncRNA a matsayin mai kula da hanyar PACT-PKR da yuwuwar manufa don maganin ciwon daji da tsinkaye.
Fasalin nazarin bayanan kwayoyin halitta sun gano ɗaruruwan lncRNA masu alaƙa da ciwon daji.Koyaya, aikinsu ya kasance ba a san shi ba56.Don gano 'yan takarar lncRNA masu ban sha'awa da ke da hannu a ci gaban ciwon daji, mun yi hasara-na aikin allo don rage yaduwa a cikin SW620 na ciwon daji ta hanyar amfani da tsarin CRISPRi (Fig. 1a).Siffa ta musamman na layin SW480 da SW620 ciwon daji na hanji shine cewa an samo su daga ciwace-ciwacen farko da na sakandare a cikin majiyyaci guda.Wannan yana ba da kwatanci mai mahimmanci don nazarin canje-canjen kwayoyin halitta a cikin ci gaban ciwon daji na hanji.Don haka, mun bincika kwafin layukan ƙwayoyin cutar kansar launi (SW480 da SW620) ta amfani da jerin abubuwan RNA kuma mun tattara wasu lncRNAs masu yuwuwar aiki daga littattafan da aka buga.Dangane da waɗannan sakamakon, mun ƙirƙira ɗakin karatu na sgRNA wanda ke ɗauke da 7355 sgRNA oligos wanda ke niyya 971 masu alaƙa da lncRNAs da 500 sgRNA oligos marasa niyya don sarrafawa mara kyau (Ƙarin Bayanai 1).
Wakilin tsari na nunawa ta amfani da tsarin CRISPRi.b sgRNA haɓakawa bayan dubawa.Layin da aka kwance a kwance yana wakiltar log2 (canjin ninka) = ± 0.58.Layin dige-dige na tsaye yana nuna ƙimar p = 0.05.Baƙaƙen ɗigo suna wakiltar sgRNA mara manufa (wanda aka ƙera azaman NC).Jajayen dige sgRNAs ne da ke niyya da DARS-AS1.Dige-dige masu shuɗi sune sgRNAs masu niyya da LINC00205, wanda aka bayyana a baya oncogenic lncRNA.ninka canji = (karanta al'ada, ranar 17)/(karanta al'ada, ranar 0).c DARS-AS1 sgRNA knockdown ya hana ci gaban tantanin halitta.Kuskuren sanduna suna wakiltar ± daidaitaccen karkatar da gwaje-gwajen guda uku.* p ≤ 0.05, ** p ≤ 0.01 t-gwajin ɗalibi mai wutsiya biyu.d DARS-AS1 magana a cikin ciwace-ciwacen daji (TCGA dataset).em Bayanin DARS-AS1 a cikin samfurori na al'ada da ƙari daga marasa lafiya tare da BLCA, KIRC, PRAD, LUSC, UCEC, LUAD, LIHC, KIRP, da COAD, bi da bi (TCGA dataset).An samo p-darajar ta amfani da t-gwajin ɗalibi mai wutsiya biyu.
Bayan gina plasmid da marufi da lentivirus, mun canza layin kwayar cutar kansar launin dCas9-SW620 tare da ɗakin karatu na sama a cikin gwaje-gwajen kamuwa da cuta guda huɗu masu zaman kansu.Yawan kamuwa da cuta (MOI) na waɗannan cututtuka shine 0.1-0.3, yana nuna cewa kowace tantanin halitta za a iya ɗaukarsa da sgRNA ɗaya kawai.Bayan kwanaki 18 na al'adun in vitro, bayanan haɓakar sgRNAs masu niyya ya ragu ko haɓaka bayan an gwada su, yayin da adadin oligonucleotides da ba a yi niyya ba ya kasance da ɗan canji idan aka kwatanta da bayanin martaba na farko, yana nuna cewa manufarmu tana da takamaiman takamaiman allo. ɗakin karatu.Shinkafa1b da ƙarin tebur 1). LINC00205, wanda aka ruwaito a baya don inganta ciwon huhu da ciwon hanta da ci gaban ciwon hanta58,59,60, an nuna shi (log2 (canji) <-0.58, p darajar <0.05), yana tabbatar da amincin wannan nunawa (Fig. 1b). LINC00205, wanda aka ruwaito a baya don inganta ciwon huhu da ciwon hanta da ci gaban ciwon hanta58,59,60, an nuna shi (log2 (canji) <-0.58, p darajar <0.05), yana tabbatar da amincin wannan nunawa (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) ku. LINC00205, wanda aka ruwaito a baya don inganta ci gaban ciwon huhu da ciwon hanta58,59,60, an cire shi (log2 (canjin ninka) <-0.58, p-darajar <0.05), yana tabbatar da ƙarfin wannan gwajin (Fig. .1b) . Linc00205 之前 报道 报道 可 肺癌 和 进展 进展 58,59,60, 被被 选 选 (Log2 (倍数 变化) <-0 (倍数 变化) <-0.05, p 的值 (图 1b). Linc00205 之前 报道 报道 可 肺癌 和 进展 进展 58,59,60, 被被 选 选 (Log2 (倍数 变化) <-0 (倍数 变化) <-0.05, p 的值 (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) ku. LINC00205, wanda aka ruwaito a baya don inganta ciwon huhu da ciwon hanta na ci gaba58,59,60, an cire shi (log2 (canjin ninka) <-0.58, p-darajar <0.05), yana tabbatar da ƙarfin wannan nunawa (Fig. .1b).
Daga cikin dukkanin lncRNA da aka gwada, DARS-AS1 kuma an duba shi, tare da cognate sgRNA oligonucleotides guda uku sun ragu sosai bayan kwanaki 18 na al'ada, suna nuna cewa ƙwanƙwasa wannan lncRNA ya haifar da rage yaduwar cutar kansa (Fig. 1b).Wannan sakamakon ya ci gaba da tallafawa ta hanyar bincike na MTS a cikin ƙwayoyin ciwon daji na launin launi wanda ke nuna cewa yawan ci gaban DARS-AS1 ƙwanƙwasa ƙwanƙwasa ya ragu kawai idan aka kwatanta da kwayoyin halitta (Figure 1c) kuma ya kasance daidai da rahotanni na baya na wasu nau'in ciwon daji.: bayyanannen ciwon daji na koda, ciwon daji na thyroid da ciwon huhu mara karami51,52,53,55.Duk da haka, ba a gano aikin sa da tsarin kwayoyin halitta a cikin ciwon daji na launin fata ba.Don haka, mun zaɓi wannan lncRNA don ƙarin nazari.
Don nazarin maganganun DARS-AS1 a cikin marasa lafiya, mun yi nazari sosai game da samfuran ƙari na 10,327 daga aikin Cancer Genome Atlas (TCGA).Sakamakonmu ya nuna cewa DARS-AS1 an bayyana shi sosai kuma yana da mahimmanci a cikin sel masu lafiya a cikin nau'o'in ciwace-ciwace, ciki har da adenocarcinoma colon (COAD), carcinoma na renal clear cell carcinoma (KIRC), da renal papillary cell carcinoma (KIRP)..Kadan ne (Fig. 1d da Ƙarin Hoto 1a, b). Binciken samfurori masu lafiya / ciwon daji ya kara tabbatar da mafi girman magana na DARS-AS1 a cikin ciwace-ciwacen daji na urothelial carcinoma (BLCA), koda clear cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), huhu squamous cell carcinoma (LUSC) , Uterine corpus endometrial carcinoma (UCEC), huhu adenocarcinoma (LUAD), hanta hepatocellular carcinoma (LIHC), koda renal papillary cell carcinoma (KIRP), da colon adenocarcinoma (COAD) (p darajar <0.05) (Fig. 1e-m) . Binciken samfurori masu lafiya / ciwon daji ya kara tabbatar da mafi girman magana na DARS-AS1 a cikin ciwace-ciwacen daji na urothelial carcinoma (BLCA), koda clear cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), huhu squamous cell carcinoma (LUSC) , Uterine corpus endometrial carcinoma (UCEC), huhu adenocarcinoma (LUAD), hanta hepatocellular carcinoma (LIHC), koda renal papillary cell carcinoma (KIRP), da colon adenocarcinoma (COAD) (p darajar <0.05) (Fig. 1e-m) .Binciken samfuran lafiya da ƙari kuma sun tabbatar da mafi girman bayyanar DARS-AS1 a cikin mafitsara urothelial carcinoma (BLCA), bayyananniyar ƙwayar ƙwayar ƙwayar ƙwayar cuta da ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar cuta (KIRC), adenocarcinoma prostate (PRAD), ciwace-ciwacen huhu squamous cell carcinoma (LUSC)., карцинома эндометрия тела матки (UCEC), аденокарцинома легкого (LUAD), гепатоцеллюлярная карцинома печени (LIHC), папиллярно-клеточная карцинома почки (KIRP) и аденокарцинома толстой кишки (COAD) (значение p <0,05) (рис. 1e– m) . , endometrial carcinoma na corpus uteri (UCEC), adenocarcinoma na huhu (LUAD), ciwon hanta na hanta (LIHC), papillary cell carcinoma na koda (KIRP), da adenocarcinoma na colon (COAD) (p darajar < 0.05) (Hoto 1e- m) .配对 / 肿瘤样本 的 的 的 的 的 Dar 证实 Dars-As1 进在 Drar-Asx, 肾肾 (Prad), 肺鳞状前列 (Prad), 肺鳞状前列 (Prad), 肺鳞状前列 (Prad), 肺鳞状前列 (Prad), 肺鳞状前列 (Lusc) 的肿瘤 中, 子宫体子宫 高 表达, 子宫体子宫 内膜 癌 (Ucec), 肺腺癌 细胞癌 细胞癌 (Luhc), 和结肠腺癌 肾肾) 和结肠腺癌 (值<0.05) (1e-m) .配 健康 / 肿瘤样本分析 的 分析分析 了 Dr Rear-Os1 在 尿路 上 皮癌皮癌 皮癌皮癌, 肾肾 细胞癌 腺腺癌 (Prad), 细胞癌 细胞癌(Lusc) 的肿瘤)) 中 的 中 中 中 中 中 中 中 中 中 中 肝肝 (Luad) 肺腺癌癌 (kirp) (coad) （p 值 <0.05） (图1e-m) .Binciken samfurori masu lafiya/tumor da aka haɗe sun kara tallafawa rawar DARS-AS1 a cikin ƙwayar cutar urothelial carcinoma mafitsara (BLCA), carcinoma cell renal cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), da ciwon huhu squamous cell carcinoma (LUSC).экспрессия при карциноме тела матки (UCEC), аденокарциноме легкого (LUAD), гепатоцеллюлярной карциноме (LIHC), почечно-почечной папиллярно-клеточной карциноме (KIRP) и аденокарциноме толстой кишки (COAD) (значение p <0,05) (рис. 1e -m). magana a cikin corpus uterine carcinoma (UCEC), huhu adenocarcinoma (LUAD), hepatocellular carcinoma (LIHC), renal papillary cell carcinoma (KIRP), da colon adenocarcinoma (COAD) (p darajar <0.05) (Figure 1e -m).Idan aka haɗu, waɗannan sakamakon suna nuna cewa DARS-AS1 ya yadu kuma yana bayyana sosai a cikin nau'ikan cututtukan daji.
Saboda DARS-AS1 da DARS (jinin halittar da ke sanya madaidaicin layin antisense) suna raba mai talla iri ɗaya kuma suna kusa da juna, mun tsara shRNA don ƙaddamar da DARS-AS1 musamman amma ba DARS (Ƙarin Hoton 2a,b da Ƙari na 2) .Baya ga SW620, mun kuma yi amfani da wasu layukan tantanin halitta guda uku waɗanda ke bayyana DARS-AS1 sosai don nazarin inganci da aikin ƙwanƙwasa shRNA (Ƙarin Teburin 3).Sakamakonmu ya nuna cewa duk shRNA guda uku da aka haɓaka sun sami aƙalla 80% DARS-AS1 ingancin knockdown tare da ƙaramin tasiri akan adadin DARS mRNA (Ƙarin Hoton 2c-f).Bugu da ƙari, mun gano cewa DARS-AS1 knockdown tare da waɗannan shRNAs sun hana ci gaban kwayar halitta a cikin layin cell cancer colorectal SW620 (49.7%) da HCT116 (27.7%), layin kwayar cutar kansar nono MBA-MD-231 (53.4%).) da kuma HepG2 hepatoma cell line (92.7% ragi), da kuma ikon su na samar da sassan da ba a haɗa su ba (matsakaicin raguwa na ~ 50.8%, 44.6%, 40.7% da 75.7% ta layin salula) (Fig. 2a,b).A cikin SW620, sakamakon ƙididdigewa na ƙirar mallaka ya ƙara tabbatar da cewa DARS-AS1 shRNA ya hana yaduwar kwayar halitta tare da raguwar matsakaicin kusan 69.6% (Fig. 2c).
Tasirin sarrafa shRNA da DARS-AS1 shRNA akan yaduwar kwayar halitta (a) da kuma samuwar spheroid (b) a cikin SW620, HCT116, MBA-MD-231, da kuma kwayoyin HepG2.c Tasirin sarrafa shRNA da DARS-AS1 shRNA akan samuwar mallaka a cikin sel SW620.Yadawar salula (d), samuwar spheroid (e), da samuwar mulkin mallaka (f) na sel SW620 masu wuce gona da iri da DARS-AS1.Bayanan da aka nuna sune ma'anar ± daidaitaccen karkacewar gwaje-gwaje uku.* p ≤ 0.05, ** p ≤ 0.01, da *** p ≤ 0.001 ta t-gwajin ɗalibi mai wutsiya.
Don kammala karatun asarar-aiki, mun ƙirƙiri sel na SW620 masu wuce gona da iri da DARS-AS1 (Ƙarin Hoton 2g).DARS-AS1 overexpression yana haɓaka haɓakar ƙwayar ƙwayar cuta (1.8-ninka), haɓakar spheroid wanda ba a haɗa shi ba (1.4-fold), da tsarin mulkin mallaka (3.3-ninki) a cikin ƙwayoyin SW620 (Fig. 2d-f).Mun tabbatar da wannan sakamakon ta amfani da wani DARS-AS1 mai bayyana layin salula, A549.Wannan haɓakar haɓakar tantanin halitta saboda DARS-AS1 overexpression an ƙara gani a cikin ƙwayoyin A549 (Ƙarin Hoton 2h, i da Ƙarin Teburin 3).Haɗe tare, waɗannan riba da binciken asara sun nuna cewa DARS-AS1 yana haɓaka haɓakar ƙwayoyin cutar kansa a cikin vitro.
Don bincika tsarin da DARS-AS1 ke daidaita yaduwar kwayar halitta, mun gudanar da bincike-bincike na RNA don gano abokan haɗin gwiwar furotin.Sakamakon RT-qPCR ya nuna cewa kusan 86.2% na DARS-AS1 yana cikin cytoplasm na sel SW620 (Ƙarin Hoton 3a).In vitro da aka rubuta biotinylated DARS-AS1 ko pseudoRNA an haɗa shi tare da sel lysates na SW620 wanda ke biye da rabuwar SDS-PAGE.Ƙwararren azurfa na gaba ya nuna cewa wani nau'i na musamman (~ 38 kDa) ya sami wadata sosai a cikin samfurori na DARS-AS1 amma ba a cikin RNA ba ko kuma samfurori na beads (Fig. 3a).An gano wannan rukunin a matsayin furotin mai kunnawa PKR (PACT) ta mass spectrometry (MS) kuma an ƙara tabbatar da shi ta hanyar immunoblotting a cikin SW620, HCT116, da layin salula na HepG2 (Fig. 3a,b).An kuma bincika haɓakar DARS da furotin PACT masu alaƙa - PKR da TRBP - kuma an bincika su ta amfani da nazarin RNA ta Western blotting (WB).Sakamakon ya nuna cewa ba a sami hulɗa kai tsaye tsakanin DARS-AS1 RNA da waɗannan sunadaran guda uku ba (Ƙarin Hoton 3b).Takamaiman hulɗar tsakanin DARS-AS1 da PACT an ƙara tabbatar da su ta hanyar bincike na immunoprecipitation RNA (RIP), wanda ya nuna cewa DARS-AS1 ya wadatar sosai a cikin ƙwayoyin anti-PACT amma ba sauran RNAs masu sarrafawa ba (Hoto 3c).Don tantance idan DARS-AS1 yana hulɗa kai tsaye tare da PACT in babu sauran abubuwan salula, an yi gwajin in vitro bilayer interferometry (BLI) ta amfani da PACT mai tsafta.Biotin-labeled DARS-AS1 ko dummy RNA an yi motsi a kan streptavidin (SA) biosensors sa'an nan kuma sanya shi a cikin buffer motsi mai dauke da 1 μM PACT.Musamman, PACT ta ɗaure da ƙarfi zuwa DARS-AS1 (KD darajar ~26.9 nM), amma ba don yin kwaikwayon RNA ba (Hoto 3d).Haɗe tare, waɗannan sakamakon suna nuna hulɗar kai tsaye da babban alaƙa tsakanin DARS-AS1 da PACT.
Binciken ja na RNA ya gano DARS-AS1 yana hulɗa tare da PACT a cikin ƙwayoyin SW620.A sama, tabon azurfa na sunadarai masu alaƙa.An yi ƙananan immunoblots tare da anti-PACT antibody.b RNA an gudanar da bincike-ƙasa a cikin sel HCT116 (saman) da HepG2 (ƙasa).An gano wadatar PACT ta hanyar immunoblotting.An yi gwaje-gwajen immunoprecipitation na cRNA (RIP) a cikin ƙwayoyin SW620 ta amfani da ƙwayoyin rigakafi da aka nuna.d PACT daure masu lankwasa zuwa cikakken tsayin DARS-AS1 ko RNA mai sarrafawa an sami su ta amfani da interferometry na biolayer (BLI).An cire RNA akan streptavidin biosensor.An yi amfani da 1 μM PACT don auna haɗin gwiwa.e RNA ja gwajin an yi ta amfani da biotinylated cikakken tsawon DARS-AS1 ko truncated (sama).Immunoblot yana nuna PACT da aka karɓa (ƙasa).f An haɗa PACT mai tsafta tare da cikakken tsayin biotinylated DARS-AS1 ko yanke (kamar yadda yake cikin e) don gwajin RIP na in vitro.RT-qPCR ta tabbatar da RNA da aka fitar.g An sami alaƙar dangi na guntuwar RNA daban-daban na PACT ta amfani da interferometry na biolayer.Don bincike, an yi amfani da 100 nM RNA da 1 μM RAST.h An yi gwaje-gwajen RIP na in vitro ta amfani da tsattsauran ra'ayi ko yanke mai lakabin PACT.RT-qPCR ta tabbatar da RNA da aka fitar.i Ƙimar girma na sel SW620 suna wuce gona da iri da DARS-AS1, PACT, ko duka biyun.J Overexpression na cikakken tsayi ko yanke DARS-AS1 a cikin sel na SW620 yana da tasiri daban-daban akan haɓakar tantanin halitta.k An gano Apoptosis ta hanyar immunoblotting tare da anti-PARP antibody.Knockout na DARS-AS1 yana haifar da apoptosis na sel SW620 kamar yadda aka nuna ta hanyar cytometry mai gudana.Bayanan da aka nuna sune ma'anar ± daidaitaccen karkacewar gwaje-gwaje uku. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p <0.0001, ta gwajin ɗalibi mai wutsiya biyu. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p <0.0001, ta gwajin ɗalibi mai wutsiya biyu. *p ≤ 0,05, **p ≤ 0,01, ***p ≤ 0,001, ****p <0,0001 pо двустороннему критерию Стьюдента. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p <0.0001 ta T-gwajin ɗalibi mai wutsiya. *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 ta T-gwajin ɗalibi mai wutsiya.
Daga nan mun ƙirƙiri gutsuttsarin DARS-AS1 RNA biotinylated guda uku ta hanyar rubutun in vitro don gano yankin DARS-AS1 da ake buƙata don ƙungiyar PACT (Hoto 3e).Sakamakon bincike na RNA ya nuna cewa kowane guntu yana iya yin hulɗa tare da PACT, amma yankin 3'-terminal (384-768 nucleotides mai lakabi A3) ya nuna fiye da 1-384 nucleotides mai suna A1) (Fig. 3e).An lura da irin wannan sakamakon a cikin in vitro RIP assay ta amfani da recombinant PACT (Hoto 3f).Daidai da waɗannan sakamakon, gwaje-gwaje don ɗaure gutsuttsarin RNA da ba a iya motsi zuwa PACT ta amfani da BLI kuma sun nuna cewa PACT tana da alaƙa mafi girma ga A3 (384-768 nt) (Kimanin KD na kusan 94.6 nM), yayin da kusan babu alaƙa tare da wasu yankuna.(Hoto na 3d).
Mun kuma bincika yankuna masu alaƙa a cikin PACT.PACT ya ƙunshi yankuna masu aiki guda uku, biyu daga cikinsu ana kiyaye su biyu-stranded RNA-binding domains (dsRBD) da yanki na uku (wanda aka tsara D3) wanda ke aiki azaman mai kunna hulɗar furotin.Don bincika ƙarfin ɗaurin lncRNA na kowane yanki, mun ƙirƙira maye gurbi guda uku waɗanda suka cire kowane yanki guda uku kuma muka yi gwajin RIP na in vitro.Sakamakonmu ya nuna cewa shafewa na yanki na uku (D3) na PACT ya rage yawan hulɗarsa tare da DARS-AS1 (ta 0.11-ninka idan aka kwatanta da PACT maras kyau) idan aka kwatanta da sauran maye gurbi guda biyu (Fig. 3h), an nuna cewa sakin na D3 yayi hulɗa tare da DARS.-AC1.Haɗe tare, waɗannan sakamakon suna ba da shawarar cewa hulɗar tsakanin DARS-AS1 da PACT na iya faruwa da farko ta ƙarshen 3′ na DARS-AS1 da yankin D3 na PACT.
Mun lura cewa DARS-AS1 ba shi da wani tasiri akan maganganun PACT kuma PACT ba ta da tasiri akan DARS-AS1 (Ƙarin Hoton 3c).Daga nan mun yi nazarin tasirin ƙwanƙwasa PACT akan haɓakar tantanin halitta.Ya bambanta da DARS-AS1, ƙwayoyin dangi sun girma 1.5-3 sau da sauri lokacin da aka rushe PACT (Ƙarin Hoton 3d).Sakamako na ƙididdigewa na ƙirar mallaka ya nuna cewa sel sun kafa yankuna 2-3 bayan jiyya na shRNA tare da PACT (Ƙarin Hoto 3e).Don gwada ko DARS-AS1 yana daidaita yaduwar tantanin halitta ta hanyar PACT, mun haifar da ƙwayoyin SW620 masu wuce gona da iri na PACT, DARS-AS1, ko duka biyun.Ƙarfafawa na PACT ya nuna mahimmancin hana yaduwar kwayar halitta (Hoto 3i).Duk da yake DARS-AS1 overexpression per se yana inganta haɓakar ƙwayoyin sel, babu wani bambanci mai mahimmanci a cikin yawan haɓakar ƙwayoyin sel masu wuce gona da iri da DARS-AS1 da PACT.Waɗannan sakamakon suna ba da shawarar cewa PACT na iya magance karuwar yaɗuwar da DARS-AS1 ke haifarwa.
Tun da yankuna daban-daban na DARS-AS1 suna da nau'ikan ikon ɗaure PACT daban-daban, mun bincika tasirin tasirin su akan haɓakar tantanin halitta ta hanyar wuce gona da iri na gutsuttsura DARS-AS1.Idan aka kwatanta da sauran gutsuttsura guda biyu, DARS-AS1 ya yi yawa a ƙarshen 3' (384-768 nt), babban yankin da ke da alaƙa da PACT a cikin DARS-AS1, wanda ke da mafi girman ikon haɓaka haɓakar ƙwayoyin cuta (Fig. 3j).Waɗannan sakamakon suna nuna kyakkyawar alaƙa tsakanin ƙarfin ɗauri da aikin nazarin halittu na DARS-AS1.
An ba da rahoton PACT a matsayin furotin mai haɓakawa.Saboda haka, mun bincika tasirin DARS-AS1 akan apoptosis.Kamar yadda aka sa ran, DARS-AS1 knockdown ya karu sosai ya karu da raguwa na PARP a cikin sel na SW620 kuma ya karu da adadin annexin V-positive sel a cikin SW620, HCT116, HepG2, da MBA-MD-231 layin salula (Fig. 3k).3).3f-h), yana nuna cewa tasirin anti-apoptotic na DARS-AS1 a cikin ƙwayoyin kansa ya saba wa aikin apoptosis-inducing na PACT.Haɗe tare, waɗannan sakamakon sun nuna cewa tsarin aikin DARS-AS1 oncogenic na iya zama ta hanyar hana ayyukan PACT.
Na gaba, mun bincika abubuwan da ake amfani da su na ƙungiyar DARS-AS1-PACT.An ba da rahoton PACT don kunna PKR ta hanyar hulɗar kai tsaye, wanda daga baya yana haɓaka eIF2α fosforelation, yana haifar da shafewar fassarar da apoptosis17.Da farko, mun bincika ko DARS-AS1 yana shafar yanayin salon salula na PACT da PKR.Ƙwararren ƙwanƙwasa mai ƙyalli ya nuna cewa PACT da PKR sun kasance masu launi sosai a cikin sel na SW620 tare da matsakaicin haɗin haɗin Pearson na 0.72.A halin yanzu, DARS-AS1 overexpression ya rage girman PACT da PKR co-localization (ma'ana Pearson correlation coefficient 0.61) (Hoto 4a).Don bincika ko DARS-AS1 zai iya daidaita hulɗar PACT-PKR, mun yi gwajin haɗin gwiwa (co-IP) tare da anti-PACT antibody a cikin SW620 cell lysates.PKR an wadatar da shi sosai a cikin anti-PACT a cikin sel masu sarrafawa, yayin da PKR farfadowa ya ragu sosai a cikin lysates daga sel masu wuce gona da iri da DARS-AS1 (Fig. 4b).An yi amfani da PKR da aka tsarkake don daurin furotin a cikin vitro.Saboda haka, waɗanda suka ba da DARS-AS1 amma babu RNA mai sarrafawa ya nuna hulɗar PACT-PKR (Hoto 4c).Duk sakamakon ya nuna cewa DARS-AS1 ya rushe PACT da PKR sadarwa.
An lura da haɗin gwiwa na PACT da PKR a cikin sel masu sarrafawa ko sel masu wuce gona da iri da DARS-AS1 an lura da su ta amfani da microscopy confocal fluorescence.An lalata ƙwayoyin nuclei da DAPI.An samu sakamakon kididdiga daga hotuna 16.b Co-immunoprecipitation (co-IP) ta amfani da anti-PACT antibody a cikin cell lysates na iko SW620 Kwayoyin ko sel overexpressing DARS-AS1.c Labeled PACT, PKR da aka tsarkake kuma an rubuta su a cikin vitro tare da DARS-AS1 ko RNA izgili don nazarin daurin furotin in vitro.An yi amfani da ƙwayoyin rigakafi na rigakafin tuta don yin rigakafi.d Immunoblots tare da alamun rigakafin da aka nuna an yi su a cikin SW620 da ƙwayoyin HCT116 da aka canza tare da shRNA mai sarrafawa ko DARS-AS1-shRNA wanda ke biye da yunwar jini.e DARS-AS1 matakan magana sun canza yanayin salon salula zuwa thapsigargin.An canza ƙwayoyin SW620 tare da DARS-AS1 shRNA, DARS-AS1 overexpression plasmid ko plasmid mai sarrafawa.An yi amfani da kwayoyin halitta tare da thapsigargin na tsawon sa'o'i 48 kuma an ƙayyade yiwuwar tantanin halitta ta amfani da MTS reagent.f In vitro da aka rubuta DARS-AS1 ko dummy RNA da tsaftataccen PACT an yi amfani da su don gwajin kunnawa in vitro da gano immunoblot.g Immunoblots ta amfani da waɗannan ƙwayoyin rigakafi an yi su akan sel SW620-ctrl (hagu) ko sel masu wuce gona da iri na PKR (dama).An canza waɗannan sel ɗin tare da sarrafa shRNA ko DARS-AS1-shRNA sannan kuma yunwar jini ta biyo baya.h Flow cytometry ya nuna cewa rashin kunna mutant PKR ya biya diyya ga DARS-AS1-induced apoptosis a cikin sel SW620.i Immunoblots tare da alamun rigakafi an yi su a cikin SW620 (hagu) ko HCT116 (dama).Kwayoyin da aka canza tare da shRNA mai sarrafawa ko DARS-AS1 shRNA an hana su kuma an kara su tare da 100 nM PKR C16 inhibitor ko DMSO.Ma'aunin Sikeli = 5 µm.Bayanan da aka nuna sune ma'anar ± daidaitaccen karkacewar gwaje-gwaje uku.* p ≤ 0.05 t-gwajin ɗalibi mai wutsiya biyu.
An yi imani da cewa da zarar PACT ta yi hulɗa tare da PKR17, PKR phosphorylation a Thr451 za a iya haifar da shi.Sakamakonmu ya nuna cewa matakin PKR phosphorylation ya kasance mai girma a cikin DARS-AS1 ƙwanƙwasa ƙwanƙwasa bayan yunwar jini (Fig. 4d da Ƙarin Hoto 4a).Dangane da haka, mun gano cewa phosphorylation na eIF2a, babban jigon PKR, shima ya karu sosai ta DARS-AS1 shRNA (Fig. 4d da Ƙarin Hoto 4a).Thapsigargin wani damuwa ne na ER wanda ke sa ER ya saki Ca2+.An ba da rahoton jiyya tare da thapsigargin don haifar da magana da kunnawa na PACT, wanda ya kara yin hulɗa tare da kunna PKR, yana haifar da apoptosis ta hanyar ƙara eIF2α phosphorylation 18,61.Anan, mun yi amfani da thapsigargin a matsayin mai motsa jiki na hanyar PACT/PKR don bincika ko DARS-AS1 zai iya taimakawa kwayoyin su shawo kan damuwa ta hanyar hana hanyar PACT/PKR.Mun lura cewa matakin DARS-AS1 magana yana da alaƙa da alaƙa da juriya ta cell zuwa thapsigargin.Kwayoyin SW620 masu wuce gona da iri da DARS-AS1 sun tsira da kyau lokacin da aka bi da su tare da thapsigargin, yayin da ƙwayoyin da ke tare da bugun DARS-AS1 sun zama mafi sauƙi (Fig. 4e).Daidai da waɗannan sakamakon, DARS-AS1 overexpression ya rage thapsigargin-induced PKR phosphorylation (Ƙarin Hoton 4b).Ya bambanta, bayan jiyya na thapsigargin, PKR da eIF2a sun kasance phosphorylated zuwa mafi girma a cikin DARS-AS1 ƙwanƙwasa ƙwanƙwasa idan aka kwatanta da sel masu sarrafawa (Ƙarin Hoton 4b).Abin sha'awa, thapsigargin ya haifar da maganganun DARS-AS1 a cikin hanyar dogara da kashi, wanda zai iya nuna aikin anti-danniya na DARS-AS1 (Ƙarin Hoton 4c).Bugu da kari, mun yi gwaje-gwajen kunnawa in vitro don tabbatar da waɗannan abubuwan lura.A taƙaice, an tsarkake PKR daga sel lysates ta amfani da anti-PKR antibody, sannan an haɗa shi da recombinant PACT da DARS-AS1 da aka rubuta a cikin vitro.Bayan halayen enzymatic, an gano phospho-PKR ta amfani da WB.Sakamakonmu ya nuna cewa DARS-AS1 ya hana PKR phosphorylation, amma ba ta hanyar sarrafa RNA ba (Hoto 4f).Waɗannan sakamakon in vitro da in vivo suna ba da shawarar cewa DARS-AS1 yana hana kunna PKR mai matsakaicin PACT.A lokaci guda kuma, mun kuma lura da raguwar dawo da PACT a gaban DARS-AS1 (Hoto 4f).Wannan sakamakon ya yi daidai da sakamakon gwajin daurin furotin in vitro (Hoto 4c) kuma ya sake kwatanta aikin toshewar DARS-AS1 don ƙungiyar PACT-PKR.
Ana buƙatar Ser246 da Ser287 a cikin yankin D3 na PACT don kunna PKR a ƙarƙashin damuwa ta salula.Sauya ragowar serine guda biyu don alanine ya ba da mutant PACT (mutD), wanda ya kunna PKR a cikin rashin damuwa, da maye gurbin alanine (mutA) ya juya ka'idar.Tun da mun nuna mahimmancin wannan yanki a cikin haɗin kai kai tsaye tare da DARS-AS1, mun ƙirƙiri waɗannan mutanan PACT guda biyu don gwada ko waɗannan ragowar kuma za su iya shiga cikin hulɗa tare da DARS-AS1.Abin sha'awa, duka maye gurbi sun rasa ikon ɗaure zuwa DARS-AS1 (Ƙarin Hoton 4d), yana nuna cewa ana iya buƙatar cikakken tsarin furotin na PACT don ingantaccen hulɗa tare da DARS-AS1.
Bugu da ƙari kuma, sakamakonmu kuma yana ba da shawarar cewa DARS-AS1-shRNA-induced hanawa na yaduwa tantanin halitta za a iya mayar da wani bangare ta hanyar wuce gona da iri na PACT mutant (PACTmutA) ko kuma mummunan PKR mutant (PKRmut) (Ƙarin Hoton 4e. e).Ƙarfafawa na mutanan PKR masu rinjaye-korau sun rage PKR phosphorylation da DARS-AS1 knockdown ya haifar da eIF2a phosphorylation a cikin kwayoyin da ba su da jini (Fig. 4g).Mafi mahimmanci, rabon sel apoptotic da DARS-AS1 knockdown ya haifar kuma an rage shi a cikin sel masu wuce gona da iri na PKRMut (Fig. 4h da Ƙarin Hoto 4g).Hana aikin PKR kinase kuma yana lalata aikin DARS-AS1, kamar yadda sakamakon ya nuna cewa DARS-AS1 knockdown da wuya ya haifar da PKR da eIF2a phosphorylation lokacin da aka bi da kwayoyin halitta tare da PKR-specific C16 inhibitor (Fig. 4i da Ƙarin Fig. 4h).).Haɗe tare, sakamakonmu yana ba da shawarar cewa DARS-AS1 yana haɓaka haɓakar tantanin halitta, aƙalla a wani ɓangare, ta hana kunna PKR mai matsakaicin PACT.
Don ƙarin bincika rawar DARS-AS1 a cikin ƙwayar cuta, mun yi a cikin gwaje-gwajen vivo ta amfani da ƙirar xenograft na linzamin kwamfuta. Sakamako ya nuna cewa ƙwanƙwasa DARS-AS1 ya ragu sosai da haɓaka haɓakar ƙwayar cuta a cikin beraye (ƙimar p <0.0001) (Fig. 5a). Sakamako ya nuna cewa ƙwanƙwasa DARS-AS1 ya ragu sosai da haɓaka haɓakar ƙwayar cuta a cikin beraye (ƙimar p <0.0001) (Fig. 5a). Результаты показывают, DARS-AS1 резко снижает рост опухоли у мышей (значение p <0,0001) (рис. Sakamakon ya nuna cewa DARS-AS1 ƙwanƙwasa yana rage girman haɓakar ƙari a cikin mice (ƙimar p <0.0001) (Hoto 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) (5). Sakamakon ya nuna cewa ƙwanƙwasa DARS-AS1 ya rage girman haɓakar ƙwayar cuta a cikin mice (ƙimar p <0.0001) (Hoto 5a).Don haka, a cikin rukunin ƙwanƙwasa DARS-AS1, an sami raguwa mai yawa a cikin ƙimar ƙwayar ƙwayar cuta ta kusan 72.9% kuma yana nufin ƙwayar ƙwayar cuta ta kusan 87.8% (Hoto 5b-d).Waɗannan sakamakon suna ba da shawarar da ƙarfi cewa DARS-AS1 na iya haɓaka haɓakar ƙari sosai a cikin vivo.
Tasirin ad DARS-AS1 ƙwanƙwasawa akan oncogenesis na colorectal a cikin mice tsirara.Ana nuna maƙallan girma (a), girman ƙari (b), nauyi (c), da hotunan ƙari (d).Kuskuren sanduna suna wakiltar ±SEM. n = 10. ****p <0.0001, ta jarrabawar Student mai wutsiya biyu. n = 10. ****p <0.0001, ta jarrabawar Student mai wutsiya biyu. n = 10. ****p <0,0001 по двустороннему критерию Стьюдента. n = 10. ****p <0.0001 t-gwajin ɗalibi mai wutsiya biyu.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-gwajin ɗalibi mai wutsiya biyu.e Kaplan-Meier yayi nazarin daidaituwa tsakanin matakan magana da DARS-AS1 da kuma rayuwa gaba ɗaya a cikin marasa lafiya tare da UVM, KICH, KIRP, MESO, GBM, da LGG.Babban matakan DARS-AS1 magana a cikin marasa lafiya sun kasance a saman 50%;ƙananan matakin DARS-AS1 magana a cikin marasa lafiya yana cikin ƙasa 50%.p-values ​​an ƙaddara ta amfani da gwajin darajar log.f Samfurin da aka tsara wanda DARS-AS1 ke tsara hanyar PACT-PKR da haɓakar ƙari.
Don ƙarin fahimtar tasirin asibiti na DARS-AS1, mun bincika alaƙar da ke tsakanin maganganun sa da rayuwa mai haƙuri.Ta hanyar nazarin bayanan TCGA, mun gano cewa mafi girma DARS-AS1 magana yana hade da uveal melanoma (UVM), renal chromophobia (KICH), renal papillary cell carcinoma (KIRP), mesothelioma (MESO), multiplex.Ƙarƙashin rayuwa yana da alaƙa da mahimmanci tare da glioblastoma morphosis (GBM) da marasa lafiya tare da ƙananan glioma na kwakwalwa (LGG) (Hoto 5e).Wadannan sakamakon sun nuna cewa DARS-AS1 na iya taka muhimmiyar rawa a ci gaban ciwon daji na asibiti kuma yana iya kasancewa mai iya tsinkayar kwayoyin halitta a cikin cututtuka masu yawa.
A cikin wannan binciken, ta yin amfani da babban aikin aikin CRISPRi, mun ƙaddara cewa DARS-AS1 lncRNA ta shawo kan matsalolin ciwon daji ta hanyar daidaita ma'auni guda biyu masu amsa damuwa, PACT da PKR.Ta hanyar yin hulɗa kai tsaye tare da PACT, DARS-AS1 ya hana PACT-matsakaicin kunnawa PKR, don haka ya hana mutuwar kwayar cutar apoptotic da inganta yaduwar kwayar halitta (Fig. 5f).An lura da haɓakar DARS-AS1 a cikin nau'ikan ciwon daji da yawa, yana ba da shawarar cewa aikin sa na haɓaka rayuwar ƙwayar cutar kansa a ƙarƙashin yanayin damuwa na iya zama mai fa'ida ga nau'ikan ciwon daji da yawa.
An gano PACT a matsayin furotin mai kunnawa PKR, kuma kunna PKR mai matsakaicin matsakaicin PACT yana taka muhimmiyar rawa a cikin martanin damuwa ta hanyar daidaita rubutun, fassarar, apoptosis, da sauran mahimman hanyoyin salon salula62.Shekaru da yawa, an yi ƙoƙari don fahimtar ƙayyadaddun ƙa'idodin ciwon daji na PACT-PKR cascade.Anan, bincikenmu ya bayyana wani nau'in tsari daban-daban na tsarin PACT-PKR a cikin ƙwayoyin kansa ta hanyar salula lncRNA DARS-AS1, wanda ke ɗaure kai tsaye zuwa PACT, yana toshe hulɗar PACT-PKR, yana hana kunna PKR da eIF2a phosphorylation, ta haka yana hana apoptosis da ke haifar da damuwa. stimulating ƙarshe ciwon daji yaduwa.Kwayoyin.Wannan binciken yana ba da haske akan yuwuwar lncRNA makasudin hasashen cutar kansa da jiyya.
Bayananmu sun nuna cewa DARS-AS1 knockdown yana wayar da kan sel zuwa yunwar jini tare da haɓaka mai girma a cikin PKR phosphorylated da eIF2a.Waɗannan sakamakon suna ba da shawarar cewa DARS-AS1 yana haɓaka rayuwar ƙwayar cutar kansa a ƙarƙashin yanayi mara kyau ta hana ayyukan PACT/PKR.Wasu RNA da ba sa coding, kamar ASPACT da nc886, suma suna da hannu a cikin PACT/PKR axis ta hanyar rage daidaita PACT48 mRNA ko daidaita autophosphorylation ta ɗaure zuwa PKR49,50,64.Daga cikinsu, DARS-AS1 yana aiki azaman mai rushe ƙungiyar PACT-PKR.Wannan binciken yana haɓaka fahimtarmu game da ka'idojin axis PACT/PKR da rawar lncRNAs a cikin martanin damuwa.
PACT ya ƙunshi yankuna daban-daban guda uku.Kowane ɗayan dsRBD guda biyu na farko ya wadatar don cimma babban haɗin kai na PACT zuwa PKR, yayin da yanki na uku (D3) ake buƙata don kunna PKR a cikin vitro da in vivo.Bincikenmu ya nuna cewa DARS-AS1 ya fi dacewa yana hulɗa tare da yankin D3 (Fig. 3h).Ganin girman girman lncRNA (768 nucleotides), DARS-AS1 daure zuwa D3 na iya hana mu'amala ta zahiri tsakanin yankin PACT na dsRBD da PKR, don haka toshe ƙungiyar PACT da PKR.Maye gurbi na PACT wanda ya maye gurbin Ser246 da Ser287 a cikin D3 tare da alanine ko aspartate ya ɓata dangantakarta da DARS-AS1, yana nuna mahimmancin D3's gabaɗayan kayan gini da lantarki a cikin ƙungiyar su.Ana buƙatar ƙarin cikakkun bayanai game da wannan hanyar a nan gaba, ta yin amfani da ƙarin madaidaicin bincike na biochemical da babban ƙuduri na tsarin PACT.
Nazarin da suka gabata sun ba da rahoton cewa DARS-AS1 yana haɓaka haɓakar ƙwayoyin cuta ta hanyar hanyoyin da yawa51,52,53.A cikin misali ɗaya, masu bincike sun lura cewa DARS-AS1 ya inganta furotin ta antisense-encoding DARS gene ta hanyar niyya miP-194-5p a cikin ƙwayoyin ciwon daji na koda.Koyaya, a cikin binciken da aka yi yanzu, ƙwanƙwasa DARS-AS1 ba ta da tasiri kan rubutun DARS a cikin nau'ikan ciwon daji da yawa, gami da aƙalla launin launi, nono, da cututtukan hanta.Saboda lncRNAs suna nuna ƙayyadaddun salon magana ta tantanin halitta da nama, ƙila ba za a iya kiyaye hanyoyin aiki a cikin nau'ikan ciwon daji ba, wanda zai iya ba da gudummawa ga wannan saɓani tsakanin abubuwan da muka lura da kuma kimantawar da suka gabata na cututtukan daji daban-daban.Ana buƙatar karatu na musamman don bayyana ƙayyadaddun ƙayyadaddun ƙayyadaddun hanyoyin hanyoyin ilimin halittar jiki da na ƙwayoyin cuta.
Binciken bayanan asibiti ya nuna cewa bayanin DARS-AS1 a cikin ciwace-ciwacen daji yana da alaƙa da alaƙa da rayuwa na marasa lafiya na ciwon daji, wanda ke nuna mahimmancin tsarin DARS-AS1 / PACT / PKR a cikin ciwon daji.A ƙarshe, bincikenmu ya nuna cewa DARS-AS1 shine mai kula da tsarin siginar PACT / PKR, yana inganta yaduwar kwayar cutar kansa, kuma yana hana apoptosis yayin amsawar damuwa, wanda ke ba da wani layi na bincike kuma yana da sha'awar bincike na gaba a cikin yiwuwar jiyya. .
Lines cell cell SW620, A549, MBA-MD-231, HCT116, HepG2 da HEK293T an samu daga National Cell Line Resource kayayyakin more rayuwa a kasar Sin.An kiyaye dukkanin kwayoyin halitta a cikin DMEM matsakaici (DMEM, Thermo Fisher Scientific, Waltham, MA) tare da 10% FBS (Gemini, Brooklyn, NY) da 1% penicillin-streptomycin (Thermo Fisher Scientific) a 37 ° C, 5% CO2.incubator.
Anti-PACT, Abcam (ab31967);Anti-PKR, Abcam (ab184257);Anti-PKR (phospho-T451), Abcam (ab81303);Anti-Ttata, Abcam (ab125243);Anti-eIF2α, Abcam (A0764));anti-eIF2a (phosphorus S51), Abcam (ab32157);anti-PACT (phosphorus S246), Abgent (AP7744b);anti-β-tubulin, CST (2128);linzamin kwamfuta na al'ada IgG, CST (5415S);na al'ada zomo IgG, CST (2729S).An narkar da ƙwayoyin rigakafi 1:1000 a cikin PBST don lalatawar Yamma da 1:100 don IP.
sgRNAs an ƙirƙira su ta amfani da kayan aiki na jama'a da ake kira CRISPR-ERA66.Mun yi amfani da tsoffin sigogin kayan aiki don haɓaka sgRNA da algorithm ɗin da aka lissafta wuraren ɗaurin sgRNA a cikin yankin 3 kb.a tsakiya a TSS.An haɗa tafkunan sgRNA oligonucleotides a CustomArray, Inc. (Bothewell, WA) kuma an haɗa su zuwa pgRNA plasmids na ɗan adam (Addgene #44248).Jimlar 12 µg na pgRNA plasmid, 7.2 µg na psPAX2 (Addgene #12260), da 4.8 µg na pMD2.G (Addgene #12259) an haɗa su cikin 5 x 106 HEK293T ta amfani da jita-jita na DNA na Rediyon cm 10. Kwayoyin (CWBIO, Beijing, China) suna bin umarnin masana'anta.An tattara abubuwan da ke ɗauke da ƙwayoyin cuta sa'o'i 48 da 72 bayan an canza su kuma an tace su ta hanyar tacewa 0.45 µm.Don dubawa, ƙwayoyin SW620 da ke bayyana furotin fusion dCas9/KRAB an samu su ta hanyar kwayar cuta.Kwayoyin SW620 da aka gyara sun kamu da ɗakin karatu na ƙwayar cuta a cikin gwaje-gwajen kamuwa da cuta guda huɗu masu zaman kansu a MOI na 0.1-0.3 kuma an gwada su tare da 2 μg / ml puromycin (Sigma, St. Louis, MO) na kwanaki 2.Bayan haka, an haɓaka sel na kwanaki 18 a cikin vitro tare da ƙaramin ɗaukar hoto na sel 500 / sgRNA don dubawa.
An fitar da DNA na genomic bisa ga umarnin QIAamp DNA Blood Midi Kit (QIAGEN, Düsseldorf, Jamus; 51183).Gabaɗaya, an yi amfani da 100 μg na genomic DNA a kowane maimaita nazarin halittu don gina ɗakin karatu.An haɓaka yankin sgRNA da zagaye biyu na PCR kuma an haɗa shi da lambar sirri.
An tsarkake samfuran PCR ta amfani da NucleoSpin® gel da PCR kit ɗin tsarkakewa (MACHEREY-NAGEL, Düren, Jamus; 740609.250) kuma an ƙididdige su ta amfani da Qubit ™ HS kayan gano DNA guda biyu (Thermo Fisher Scientific; Q32854).
An yi amfani da gwajin MTS don auna yaduwar kwayar halitta.An shuka sel a cikin faranti 96-rijiya a farkon yawa na sel 2000/riji.An auna adadin dangi kowace rana a lokacin da aka nuna don jimlar kwanaki 4-6.Ga kowace rijiya, 20 μl na MTS reagent (Promega) an diluted da 100 μl na DMEM, incubated da sel for 4 h a 37 ° C, sa'an nan OD490 aka auna.
An gano ƙarfin ci gaban da ba a haɗa shi ba ta hanyar nazarin samuwar sassa.A taƙaice, sel 2000 waɗanda aka canza tare da shRNA DARS-AS1 ko shRNA mai sarrafawa an haɓaka su a cikin ƙananan ƙananan abubuwan haɗin gwiwa (Corning) tare da matsakaicin canji kowane kwanaki 4.An ƙidaya spheroids bayan kwanaki 14.Kwayoyin 500 da aka canza tare da DARS-AS1 overexpression plasmid ko plasmid mai sarrafawa an yi amfani da su don haɓaka haɓaka, in ba haka ba hanyar ba ta canza ba.
An rubuta RNA ta amfani da T7 RNA polymerase da biotin-16-UTP (Roche 1138908910) bisa ga umarnin Riboprobe® Combination Systems (Promega P1440).An jera abubuwan da aka yi amfani da su a nan a cikin ƙarin Tebu 4.
PET15b (Addgene #73619) an canza su zuwa BL21 (DE3).An haxa kwayoyin cutar da daddare a cikin LB wanda aka ba su da ampicillin sannan a dillace su sau 100 tare da sabon LB.Lokacin da OD600 na matsakaici ya kai 0.8, an ƙara 1 mM IPTG don haifar da furcin furotin.Bayan shiryawa na dare tare da girgiza mai laushi (250 rpm a 20 ° C), pellet tantanin halitta an tattara ta ta hanyar centrifugation (4000 rpm, 10 min, 4°C).Dakatar da pellet tantanin halitta a cikin buffer lysis (50 mM Tris, pH 8.0, 250 mM NaCl, 1 mM PMSF) da kuma sanyawa akan kankara don 30 min, sannan sonicate (minti 15, 5 s kunna/kashe, akan kankara) da centrifuge (13,000) rpm)., 30 min, 4°C).Daga nan an ɗora ruwan sama a kan resin Ni-NTA (QIAGEN) sau 3 a 4°C, an wanke sau 4 tare da buffer ɗin wanka (50 mM Tris, pH 8.0, 40 mM imidazole, 250 mM NaCl) kuma an ɗora sau 3, tare da duka. na 10 ml eluent buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 300 mM imidazole).An ƙaddara furotin da aka tsarkake ta amfani da WB kuma an ƙaddara ƙaddamarwa ta amfani da kayan gwajin furotin na Qubit™ (Thermo Fisher Scientific; Q 33212).
An yi gwajin RIP kamar yadda aka bayyana a baya, tare da gyare-gyare.A taƙaice, 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 1 x hadaddiyar giyar. (Roche, 1 mM DTT) da centrifuge a 13,000 rpm na 15 min a 4 ° C.Daga nan sai aka haɗo maɗaukakan da furotin A+G magnetic beads (Millipore) wanda aka haɗa tare da 5 μg na anti-PACT antibody (Abeam) ko IgG (CST).An wanke beads sau 5 tare da buffer 5x RIP, sannan an narkar da su tare da proteinase K (NEB).An fitar da RNA tare da Trizol kuma an ƙaddara ta RT-qPCR.An gabatar da firamare a cikin ƙarin Teburi na 5.
An yi gwajin gwajin RIP na in vitro bisa ga ingantaccen ƙa'idar tantance RIP.Jimlar 5 pmol na in vitro da aka rubuta RNA an narkar da 1x tare da buffer RIP kuma an shafe ta ta shiryawa a 65°C na mintuna 5 sannan a hankali sanyaya zuwa zafin daki.An tsarkake jimlar 5 pmol na ƙayyadaddun furotin PACT masu lakabin tuta ko maye gurbinsu daga E. coli.Haɗa tare da sake fasalin RNA na sa'o'i 2 a 4 ° C kuma bi tsarin da ke sama don nazarin RIP don IP na anti-tuta.
Don nazarin tsawo na RNA, sel 1 × 107 an lalata su tare da buffer 1xRIP.Bayan centrifugation a 13,000 rpm na 15 min a 4 ° C, da supernatant aka pretreated da 30 μl na streptavidin Magnetic beads (Beckman) na 2 h a 4 ° C.An ba da lysate ɗin da aka tsarkake da tRNA yisti kuma a sanya shi da 40 pmol na RNA da aka canza a cikin dare a 4 ° C, sa'an nan kuma don ƙarin 2 hours da 20 μl na sabon streptavidin Magnetic beads da aka toshe tare da BSA.Matakin wankin ya ƙunshi sau 4 tare da buffer 5x RIP da sau 4 tare da buffer 1x RIP.Sunadaran da suka dace sun kasance tare da buffer biotin elution (25 mM Tris-HCl, pH 7.5, 12.5 mM D-biotin, PMSF) kuma an raba su akan NuPAGE 4-12% Bis-Tris Gel (Invitrogen).Bayan tabon azurfa (Beyotime Biotechnology), an cire wasu makada kuma an tantance su ta hanyar MS.
An gudanar da bincike na Co-IP don gwada hulɗar tsakanin PACT da PKR.A taƙaice, an shirya lysates masu ƙarfi ta hanyar shigar da ƙwayoyin lysed 1 x 107 a cikin buffer 1 x RIP wanda ke biye da centrifugation a 13,000 rpm na mintuna 15 a 4°C.An ɗora Lysates da furotin A + G Magnetic beads, hade tare da 5 μg na anti-PACT antibody, kuma a hankali juya dare a 4°C.An wanke beads sau 3 tare da buffer 5 × RIP, sau biyu tare da buffer 1 × RIP kuma an haɓaka tare da buffer 1 × SDS.SDS-PAGE gel ne yayi nazarin furotin da aka dawo dashi kuma WB ya gano shi.
pmol guda biyu na PACT masu tuta da 1 pmol na PKR an tsarkake su daga E. coli.Tsarma a cikin buffer 1 × RIP kuma a sanya shi tare da pmol 10 na RNA da aka sabunta don sa'o'i 2 a 4 ° C.Bayan haka, an haɗa su da sunadaran A+G magnetic bead-conjugated anti-labeled antibody na ƙarin sa'o'i biyu.An wanke beads ɗin sau huɗu tare da buffer 1x RIP kuma an lalata su da buffer 1x SDS.WB ya gano sakamakon PACT da PKR.