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Ua hoʻomohala ʻia nā ʻano hana like ʻole e pale aku i ke koʻikoʻi kelepona a hoʻomau i ka holomua.ʻO ka Protein kinase R (PKR) a me kāna mea hoʻoulu i ka protein (PACT) nā mea pane mua e nānā i nā hōʻailona koʻikoʻi like ʻole e alakaʻi ana i ka pale ʻana i ka hoʻonui ʻana o ka cell a me ka apoptosis.Eia nō naʻe, ʻaʻole i ʻike nui ʻia ka hoʻoponopono ʻana o ke ala PACT-PKR i loko o nā keʻena maʻi maʻi.Maʻaneʻi, uaʻike mākou ua pili pono ka RNA non-coding RNA (lncRNA) aspartyl tRNA synthetase antisense RNA 1 (DARS-AS1) i ka pale ʻana i ke ala PACT-PKR a paipai i ka hoʻonui ʻana o ka maʻi kanesa.Ke hoʻohana nei i ka nānā ʻana i ka hana nui o CRISPRi 971 lncRNA pili i ka maʻi kanesa, ua ʻike mākou ua pili ʻo DARS-AS1 me ka hoʻonui nui ʻana o ka maʻi kanesa.No laila, ʻaʻole ʻo DARS-AS1 knockout i ka hoʻonui ʻana o ka cell a hoʻolaha i ka apoptosis cell cancer i nā laina cell cancer in vitro a hoʻemi nui i ka ulu ʻana o ka maʻi maʻi i vivo.Mechanically, DARS-AS1 paʻa pololei i ka PACT activation domain a pale i ka PACT-PKR pili, a laila e hōʻemi ana i ka PKR activation, eIF2α phosphorylation, a me ka pale ʻana i ka make cell apoptotic.ʻO ka maʻi maʻi, hōʻike nui ʻia ʻo DARS-AS1 i nā maʻi maʻi he nui, a ʻo ka overexpression o kēia lncRNA e hōʻike ana i kahi prognosis maikaʻi ʻole.Hōʻike kēia haʻawina i ka hoʻoponopono kikoʻī o ka maʻi kanesa o ke ala PACT-PKR e DARS-AS1 lncRNA a hāʻawi i kahi pahuhopu hou no ka wānana a me ka mālama ʻana.
ʻO ka hiki ke hoʻololi i ke koʻikoʻi he ʻano koʻikoʻi o ke ola a me ka hoʻonui ʻana o ka cell cancer.ʻO ka hoʻonui wikiwiki ʻana a me nā hiʻohiʻona metabolic o ka maʻi kanesa ke piʻi nei i nā microenvironment paʻakikī - ka nele i ka meaʻai, hypoxia, a me ka pH haʻahaʻa - hiki ke hoʻoulu i nā ala hōʻailona make cell.ʻO ka dysregulation o nā genes koʻikoʻi koʻikoʻi e like me ka p535, nā protein haʻalulu wela 6, 7, KRAS8, 9, a me HIF-110, 11, 12, 13 ke nānā pinepine ʻia i ka maʻi kanesa, no laila ke kāohi nei i ka apoptosis a hoʻoikaika i ke ola.
ʻO ka protein kinase R (PKR) kahi mea ʻike koʻikoʻi koʻikoʻi a me nā subunit kinase o ka eukaryotic initiation factor 2α (eIF2α), kahi mea hoʻoponopono unuhi e hoʻopili ai i ke koʻikoʻi cellular i ka make cell.Ua ʻike mua ʻia ʻo PKR he protein antiviral ma o ka ʻike ʻana i kahi RNA pālua pālua (dsRNA).Ma ka hoʻoulu ʻana, hoʻopili ʻo PKR i ka eIF2α e kāohi i ka synthesis protein viral a me cellular14,15,16.Ua ʻike ʻia ʻo PACT (PKR activator protein) ʻo ia ka mea mua PKR activator protein i ka nele o dsRNA17,18,19,20,21,22,23.Ma o ka launa pū ʻana me PKR, hoʻololi ʻo PACT i nā koʻikoʻi like ʻole (serum starvation, peroxide or arsenite treatment) i PKR a me nā ala hōʻailona o lalo.Ma waho aʻe o ka eIF2α phosphorylation, hoʻoulu ka PACT-mediated PKR activation i nā hanana like ʻole e pili ana i ka pane koʻikoʻi, me ka hoʻololi ʻana i ke kūlana redox ma o ke ala PI3K / Akt24, hoʻonui ʻia ka nānā ʻana i ka pōʻino DNA ma o p5325,26 a me NF-κB27,28 Regulates transcription, 29. Hāʻawi ʻia i kā lākou koʻikoʻi koʻikoʻi i ka pane ʻana i ke koʻikoʻi, ka hoʻonui ʻana, apoptosis a me nā kaʻina cellular koʻikoʻi, ke hoʻohiki nei ʻo PKR a me PACT i nā pahuhopu therapeutic no nā maʻi he nui, ʻoi aku ka maʻi cancer30,31,32,33.Eia nō naʻe, ʻoiai kēia pleiotropic hana a me ke koʻikoʻi koʻikoʻi, ʻo ka hoʻoponopono ʻana o ka hana PACT/PKR i nā maʻi maʻi maʻi e paʻa mau.
ʻO nā lncRNA nā transcripts ʻoi aku ka nui ma mua o 200 nucleotides me ka hiki ʻole o ka protein-coding.Ma muli o ka ʻike ʻana o nā papahana hoʻonohonoho genome holoʻokoʻa i nā tausani o nā lncRNA, 35,36 ka nui o ka hoʻoikaika ʻana e wehewehe i kā lākou mau hana olaola.Ke ulu nui nei ka noiʻi ua hōʻike ʻia ua komo nā lncRNA i nā kaʻina hana biological37 me ka hoʻoponopono ʻana i ka X-chromosome inactivation38,39, imprinting40, transcription41,42, unuhi43 a me ka ulu ʻana o ka maʻi kanesa44,45,46,47.Ua hōʻike kēia mau haʻawina he nui nā lncRNA i komo i ke ala PACT/PKR.Ua hōʻike ʻia kekahi haʻawina ʻo lncRNA ASPACT i kaohi ʻana i ka palapala PACT a hoʻonui i ka paʻa nuklea o PACT mRNA.Ua hōʻike ʻia nā haʻawina ʻē aʻe ua hoʻopaʻa ʻia ka lncRNA nc886 iā PKR a kāohi i kāna phosphorylation49,50.I kēia manawa, ʻaʻole i hōʻike ʻia ka lncRNA e hoʻoponopono ana i ka hoʻoulu ʻana o PKR-mediated PKR.
Ua ʻike ʻia ʻo Aspartyl-tRNA synthetase antisense RNA 1 (DARS-AS1) he oncogenic lncRNA51,52,53,54.Ma o ka hooponopono ana o ka miP-194-5p53, miP-12952 a me ka miP-532-3p51, ua hoikeia o DARS-AS1 e paipai i ka ulu ana o ka ma'i ma'i ma'ema'e o ka ma'i ma'i, thyroid carcinoma a me ka li'ili'i li'ili'i lung carcinoma.Ua ʻike pū ʻo Tong a me nā hoa hana ʻo DARS-AS1 e hāpai i ka holomua myeloma ma ka mālama ʻana i ka paʻa o ka protein 39 (RBM39) RNA-binding motif.Eia naʻe, ʻaʻohe haʻawina i hana ʻia inā pili kēia lncRNA i ka hoʻoponopono ʻana i ka hoʻōla ʻana o PACT-PKR a me ka pane ʻana i ke koʻikoʻi o nā maʻi kanesa.
Maʻaneʻi, ua hana mākou i kahi pale pohō o ka hana nui me ka hoʻohana ʻana i ka ʻōnaehana CRISPRi a ua hoʻoholo ʻo DARS-AS1 lncRNA e hāpai i ka hoʻonui ʻana o nā ʻano ʻano maʻi kanesa.Eia kekahi, ua ʻike mākou i kahi ʻano hana nui: ʻo DARS-AS1 e hoʻopaʻa pololei i ka PACT, kāohi i ka PACT a me PKR i ka hoʻopaʻa ʻana, pale i ka phosphorylation o eIF2α, kahi haʻahaʻa haʻahaʻa PKR, a i ka hopena ke kāohi i ka make cell apoptotic.I ka hopena, hōʻike kā mākou hana i ka DARS-AS1 lncRNA ma ke ʻano he mea hoʻoponopono o ke ala PACT-PKR a me kahi pahuhopu no ka mālama ʻana a me ka wānana.
Ua ʻike ʻia nā haʻawina hoʻopaʻa inoa genomic he mau haneli o nā lncRNA e pili ana me ka maʻi kanesa.Eia naʻe, ʻaʻole ʻike nui ʻia kā lākou hana56.No ka ʻike ʻana i nā moho lncRNA hoʻohiki e pili ana i ka piʻi ʻana o ka maʻi maʻi, ua hana mākou i kahi pale o ka hana no ka hoʻemi ʻana i ka hoʻonui ʻana ma ka laina cell cancer SW620 colorectal cancer me ka hoʻohana ʻana i ka ʻōnaehana CRISPRi (Fig. 1a).ʻO ka hiʻohiʻona kūʻokoʻa o ka SW480 a me SW620 colon cancer cell lines i loaʻa iā lākou mai nā maʻi maʻi mua a me ke kula kiʻekiʻe i ka maʻi hoʻokahi.Hāʻawi kēia i kahi hoʻohālikelike koʻikoʻi no ke aʻo ʻana i nā loli genetic i ka holomua ʻana o ka maʻi maʻi maʻi maʻi kiʻekiʻe.No laila, ua kālailai mākou i nā transcriptomes o ka colorectal cancer cell line (SW480 a me SW620) me ka hoʻohana ʻana i ka RNA sequencing a hōʻiliʻili i kekahi mau lncRNA hana pono mai ka puke i paʻi ʻia.Ma muli o kēia mau hopena, ua hoʻolālā mākou i kahi waihona sgRNA pooled i loaʻa iā 7355 sgRNA oligos e kuhikuhi ana i ka 971 lncRNAs pili i ka maʻi kanesa a me 500 oligos sgRNA untargeted no ka mana maikaʻi ʻole (Supplementary Data 1).
Hōʻike hoʻolālā o ka nānā ʻana me ka ʻōnaehana CRISPRi.b sgRNA hoʻonui ma hope o ka nānā ʻana.Hōʻike ka laina kiko kiko i ka log2 (hoʻololi paʻi) = ±0.58.Hōʻike ka laina kiko kiko i ka p waiwai = 0.05.Hōʻike nā kiko ʻeleʻele i ka sgRNA ʻaʻole i kuhikuhi ʻia (i koho ʻia ʻo NC).ʻO nā kiko ʻulaʻula nā sgRNA e kuhikuhi ana iā DARS-AS1.ʻO nā kiko polū nā sgRNA e kuhikuhi ana i ka LINC00205, kahi lncRNA oncogenic i wehewehe mua ʻia.hoʻololi fold = (heluhelu maʻamau, lā 17)/(heluhelu maʻamau, lā 0).c DARS-AS1 sgRNA knockdown i keakea i ka ulu o ke kelepona.Hōʻike nā pahu hewa i ka ± kaʻe ʻana o nā hoʻokolohua ʻekolu.* p ≤ 0.05, ** p ≤ 0.01 ka ho'āʻo-t a ka Haumāna huelo ʻelua.d DARS-AS1 hōʻike i nā ʻōpū (TCGA dataset).em Hōʻike o DARS-AS1 i hui pū ʻia me nā hōʻailona maʻamau a me nā ʻōpū mai nā maʻi me BLCA, KIRC, PRAD, LUSC, UCEC, LUAD, LIHC, KIRP, a me COAD, kēlā me kēia (TCGA dataset).Ua loaʻa nā p-values ​​me ka hoʻohana ʻana i ka hoʻāʻo ʻana o ka haumāna ʻelua-huelo.
Ma hope o ke kūkulu ʻana i ka plasmid a me ka hoʻopaʻa ʻana i ka lentivirus, ua hoʻololi mākou i ka dCas9-SW620 colorectal cancer cell line me ka waihona ma luna i ʻehā mau hoʻokolohua maʻi kūʻokoʻa.ʻO ka nui o ka maʻi (MOI) no kēia mau maʻi he 0.1-0.3, e hōʻike ana e hiki ke hoʻololi ʻia kēlā me kēia cell me hoʻokahi sgRNA.Ma hope o 18 mau lā o ka moʻomeheu in vitro, ua emi a hoʻonui ʻia ka ʻaoʻao hoʻonui o nā sgRNAs target ma hope o ka nānā ʻana, ʻoiai ʻo ka helu o nā oligonucleotides mana ʻole i hoʻopaʻa ʻia i hoʻololi ʻole ʻia i ka hoʻohālikelike ʻana i ka ʻaoʻao pre-screening, e hōʻike ana i kā mākou pahuhopu he pale kikoʻī loa. hale waihona puke.Laiki.1b a me ka papa kuhikuhi 1). ʻO LINC00205, ka mea i hōʻike mua ʻia e hāpai i ka maʻi kanesa a me ka holomua o ka maʻi ate58,59,60, ua nānā ʻia (log2 (foldchange) <−0.58, p waiwai <0.05), e hōʻoia ana i ka hilinaʻi o kēia kānana (Fig. 1b). ʻO LINC00205, ka mea i hōʻike mua ʻia e hāpai i ka maʻi kanesa a me ka holomua o ka maʻi ate58,59,60, ua nānā ʻia (log2 (foldchange) <−0.58, p waiwai <0.05), e hōʻoia ana i ka hilinaʻi o kēia kānana (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). ʻO LINC00205, i hōʻike muaʻia e hāpai i ka holomua o ka maʻi kanesa a me ka maʻi kanesa o ka ate58,59,60, ua kāpaeʻia (log2 (fold change) <-0.58, p-value <0.05), e hōʻoia ana i ka ikaika o kēia kānana (Fig.1b) . Linc00205 之前 被 报道 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 掉 ",05 (倍数 图2) 可靠性 (图 1,5) 可靠性) 可靠性) 可靠性) 可靠性) 可靠性) 可靠性) 可靠性 (图 1,5) 可靠性)) 可靠性 (图 18 log Linc00205 之前 被 报道 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 肝癌 掉 ",05 (倍数 图2) 可靠性 (图 1,5) 可靠性) 可靠性) 可靠性) 可靠性) 可靠性) 可靠性) 可靠性 (图 1,5) 可靠性)) 可靠性 (图 18 log 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). ʻO LINC00205, i hōʻike muaʻia e hāpai i ka holomua o ka maʻi kanesa a me ka ate58,59,60, ua kāpaeʻia (log2 (hoʻololi hoʻololi) <-0.58, p-value <0.05), e hōʻoia ana i ka ikaika o kēia kānana (Fig.1b).
Ma waena o nā lncRNA a pau i ho'āʻoʻia, ua nānā pūʻiaʻo DARS-AS1, me nā oligonucleotides sgRNA cognateʻekolu i hoʻemi nuiʻia ma hope o 18 mau lā o ka moʻomeheu, e hōʻike ana i ka hoʻokuʻuʻiaʻana o kēia lncRNA i hopena i ka ho'ēmiʻana o ka maʻi kanesa (Fig. 1b).Ua kākoʻo hou ʻia kēia hopena e ka nānā ʻana o MTS i loko o nā keʻena maʻi maʻi colorectal e hōʻike ana ua ʻoki wale ʻia ka nui o ka ulu ʻana o nā cell knockdown DARS-AS1 i hoʻohālikelike ʻia me nā cell control (Figure 1c) a ua kūlike me nā hōʻike mua o kekahi mau ʻano maʻi maʻi ʻē aʻe.: ka ma'i 'a'ai ma'i 'a'ai ma'ema'e, ka ma'i 'a'ai thyroid a me ka ma'i 'a'ai māmā 'ole li'ili'i51,52,53,55.Eia nō naʻe, ʻaʻole i ʻike ʻia kāna hana a me nā ʻano molecular i ka maʻi maʻi colorectal.No laila, ua koho mākou i kēia lncRNA no ke aʻo hou ʻana.
No ke aʻo ʻana i ka ʻōlelo DARS-AS1 i ka poʻe maʻi, ua loiloi piha mākou i 10,327 mau hōʻailona maʻi mai ka papahana Cancer Genome Atlas (TCGA).Hōʻike kā mākou mau hopena i hōʻike nui ʻia ʻo DARS-AS1 a hoʻonui nui ʻia i nā cell olakino i nā ʻano maʻi like ʻole, me ka colon adenocarcinoma (COAD), renal clear cell carcinoma (KIRC), a me renal papillary cell carcinoma (KIRP)..Kakaikahi loa (Fig. 1d a me ka Fig. 1a, b). Ua hōʻoia hou ʻia ka ʻike ʻana o nā laʻana olakino / tumor i kahi hōʻike kiʻekiʻe loa o DARS-AS1 i nā ʻōpū o ka maʻi maʻi maʻi urothelial carcinoma (BLCA), ka maʻi maʻi maʻi maʻi maʻi maʻi maʻi (KIRC), prostate adenocarcinoma (PRAD), lung squamous cell carcinoma (LUSC) , uterine corpus endometrial carcinoma (UCEC), lung adenocarcinoma (LUAD), ate hepatocellular carcinoma (LIHC), kidney renal papillary cell carcinoma (KIRP), and colon adenocarcinoma (COAD) (p waiwai <0.05) (Fig. 1e–m) . Ua hōʻoia hou ʻia ka ʻike ʻana o nā laʻana olakino / tumor i kahi hōʻike kiʻekiʻe loa o DARS-AS1 i nā ʻōpū o ka maʻi maʻi maʻi urothelial carcinoma (BLCA), ka maʻi maʻi maʻi maʻi maʻi maʻi maʻi (KIRC), prostate adenocarcinoma (PRAD), lung squamous cell carcinoma (LUSC) , uterine corpus endometrial carcinoma (UCEC), lung adenocarcinoma (LUAD), ate hepatocellular carcinoma (LIHC), kidney renal papillary cell carcinoma (KIRP), and colon adenocarcinoma (COAD) (p waiwai <0.05) (Fig. 1e–m) .Ua hōʻoia ʻia ka ʻike ʻana o nā laʻana olakino/tumor i hoʻopaʻa ʻia i ka hōʻike kiʻekiʻe loa o DARS-AS1 i loko o ka maʻi maʻi urothelial carcinoma (BLCA), ka maʻi maʻi maʻi maʻemaʻe a me ka maʻi maʻi 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 of the corpus uteri (UCEC), adenocarcinoma of the lung (LUAD), hepatocellular carcinoma of the liver (LIHC), papillary cell carcinoma of the kidney (KIRP), and adenocarcinoma of the colon (COAD) (p waiwai < 0.05) (Fig. 1e– m) .配对 健康 / 肿瘤样本 的 分析 in分析 证实证实 证实证实 u "a" 腺腺癌 腺腺癌 上尿路), 肿瘤 中)显着显着更更高表达, 子宫体子宫 子宫体子宫 Isn (UCAC), 肺腺癌 (luad), 和结肠腺癌 (coad) (P 值<0.05）（图1e-m） .配对 健康/肿瘤样本 的 分析 证实 了 dars-os1 在 尿路 上 皮癌 皮癌 皮癌 皮癌 、 肾 肾 细胞癌 细胞癌 细胞癌 细胞癌 前列 腺腺癌 腺腺癌 (prad) 、 细胞癌 细胞癌(lusic) 0 的 中 中 中 肺腺癌 中 肺腺癌表达 肺腺癌 肺腺癌, 肾 (lih 细胞 肾 (Ul (UL-(UHc) 肾 肾 (UL癌 （kirp） （coad） （p 值<0.05）（图1e-m） .Kākoʻo hou ʻia ka ʻike ʻana o nā laʻana pili ola olakino / tumor i ke kuleana o DARS-AS1 i ka maʻi maʻi urothelial carcinoma (BLCA), ka maʻi maʻi maʻi maʻi maʻemaʻe cell renal (KIRC), prostate adenocarcinoma (PRAD), a me nā maʻi maʻi lung squamous cell carcinoma (LUSC).экспрессия при карциноме тела матки (UCEC), аденокарциноме легкого (LUAD), гепатоцеллюлярной карциноме (LIHC), почечно-почечной папиллярно-клеточной карциноме (KIRP) и аденокарциноме толстой кишки (COAD) (значение p <0,05) (рис. 1e -m). hōʻike i loko o ka corpus uterine carcinoma (UCEC), lung adenocarcinoma (LUAD), hepatocellular carcinoma (LIHC), renal papillary cell carcinoma (KIRP), a me colon adenocarcinoma (COAD) (p waiwai <0.05) (Figure 1e -m).Hoʻohui pū ʻia, hōʻike kēia mau hopena he ākea a hōʻike nui ʻia ʻo DARS-AS1 i nā maʻi maʻi like ʻole.
No ka mea ʻo DARS-AS1 a me DARS (ka gene e hoʻopili ana i ka antisense strand) kaʻana like i ka mea hoʻolaha a aia ma kahi kokoke i kekahi, ua hoʻolālā mākou i ka shRNA e hoʻokuʻu i ka DARS-AS1 akā ʻaʻole ʻo DARS (Supplementary Fig. 2a, b a me ka Papa Hoʻohui 2). .Ma kahi o SW620, ua hoʻohana pū mākou i ʻekolu mau laina kelepona e hōʻike nui ana iā DARS-AS1 e aʻo i ka pono a me ka hana o ka shRNA knockdown (Supplementary Table 3).Ua hōʻike ʻia kā mākou hopena i loaʻa i nā shRNA ʻekolu a pau ma kahi o 80% DARS-AS1 knockdown pono me ka liʻiliʻi o ka hopena i ka nui o DARS mRNA (Supplementary Fig. 2c-f).Eia kekahi, ua ʻike mākou ʻo DARS-AS1 knockdown me kēia mau shRNAs i kaohi nui ʻana i ka ulu ʻana o ka cell i loko o nā laina maʻi maʻi colorectal SW620 (49.7%) a me HCT116 (27.7%), ka laina maʻi maʻi maʻi umauma MBA-MD-231 (53.4%).) a me ka HepG2 hepatoma cell line (92.7% reduction), a me ko lākou hiki ke hana i nā spheres unchored (hoʻemi awelika o ~ 50.8%, 44.6%, 40.7% a me 75.7% no ka laina cell) (Fig. 2a, b).Ma SW620, ua hōʻoia hou nā hopena o ka hoʻokumu ʻana o ka colony i ka DARS-AS1 shRNA i keakea nui i ka hoʻonui ʻana o ka cell me ka emi ʻana o ka 69.6% (Fig. 2c).
Ka hopena o ka mana shRNA a me DARS-AS1 shRNA ma ka hoʻonui ʻana o ke kelepona (a) a me ka hoʻokumu ʻana o ka spheroid (b) ma SW620, HCT116, MBA-MD-231, a me nā pūnaewele HepG2.c Ka hopena o ka mana shRNA a me DARS-AS1 shRNA ma ka hoʻokumu ʻana i nā koloni ma nā pūnaewele SW620.ʻO ka hoʻonui ʻana o ke kelepona (d), ka hoʻokumu ʻana o ka spheroid (e), a me ka hoʻokumu ʻana o ka colony (f) o nā cell SW620 overexpressing DARS-AS1.ʻO ka ʻikepili i hōʻike ʻia, ʻo ia ka mean ± deviation maʻamau o nā hoʻokolohua ʻekolu.* p ≤ 0.05, ** p ≤ 0.01, a me *** p ≤ 0.001 e ka ho'āʻo-t a ka Haumāna huelo ʻelua.
No ka hoʻokō ʻana i nā haʻawina nalowale o ka hana, hana mākou i nā cell SW620 overexpressing DARS-AS1 (Supplementary Fig. 2g).ʻO ka overexpression DARS-AS1 i hoʻonui nui i ka ulu ʻana o ka cell (1.8-fold), ka hoʻokumu ʻana o ka spheroid unanchored (1.4-fold), a me ka hoʻokumu ʻana o ka colony (3.3-fold) i nā cell SW620 (Fig. 2d-f).Ua hōʻoia mākou i kēia hopena me ka hoʻohana ʻana i kahi laina kelepona hou DARS-AS1, A549.Ua ʻike hou ʻia kēia hoʻonui ʻia o ka cell cell ma muli o ka overexpression DARS-AS1 i nā cell A549 (Supplementary Fig. 2h, i a me Supplementary Table 3).Hoʻohui pū ʻia, hōʻike kēia mau haʻawina loaʻa a me ka nalowale e hoʻolaha ana ʻo DARS-AS1 i ka hoʻonui ʻana o ka maʻi maʻi maʻi in vitro.
No ka ʻimi ʻana i ke ʻano kumu e hoʻoponopono ai ʻo DARS-AS1 i ka hoʻonui ʻana o ka cell, ua hana mākou i kahi loiloi huki huki RNA e ʻike ai i kāna mau hoa pili pūmua.Hōʻike nā hualoaʻa RT-qPCR e pili ana i ka 86.2% o DARS-AS1 i loko o ka cytoplasm o nā pūnaewele SW620 (Supplementary Fig. 3a).ʻO ka in vitro transcribed biotinylated DARS-AS1 a i ʻole pseudoRNA i hoʻopili ʻia me SW620 cell lysates a ukali ʻia e ka SDS-PAGE kaʻawale.Ua hōʻike ʻia ka ʻili kālā ma hope mai ua hoʻonui nui ʻia kahi hui kūʻokoʻa (~ 38 kDa) i nā laʻana huki huki DARS-AS1 akā ʻaʻole i loko o ka dummy RNA a i ʻole nā ​​​​mea hoʻohālike (Fig. 3a).Ua ʻike ʻia kēia hui ma ke ʻano he PKR activating protein (PACT) e ka mass spectrometry (MS) a hōʻoia hou ʻia e ka immunoblotting ma SW620, HCT116, a me nā laina kelepona HepG2 (Fig. 3a,b).ʻO ka hoʻonui ʻana i ka DARS a me nā protein PACT pili - PKR a me TRBP - ua noiʻi pū ʻia me ka hoʻohana ʻana i ka loiloi RNA e Western blotting (WB).Ua hōʻike ʻia nā hopena ʻaʻole i loaʻa kahi pilina pololei ma waena o DARS-AS1 RNA a me kēia mau protein ʻekolu (Supplementary Fig. 3b).ʻO ka pilina kūikawā ma waena o DARS-AS1 a me PACT ua hōʻoia hou ʻia e ka RNA immunoprecipitation (RIP) ka nānā ʻana, i hōʻike ʻia ua hoʻonui nui ʻia ʻo DARS-AS1 i nā antibodies anti-PACT akā ʻaʻole nā ​​​​RNA mana ʻē aʻe (Figure 3c).No ka hoʻoholo inā pili pololei ʻo DARS-AS1 me PACT me ka loaʻa ʻole o nā mea kelepona ʻē aʻe, ua hana ʻia kahi in vitro biolayer interferometry (BLI) assay me ka hoʻohana ʻana i ka PACT hoʻomaʻemaʻe.Ua hoʻopaʻa ʻia ʻo Biotin-labeled DARS-AS1 a i ʻole dummy RNA i hoʻopaʻa ʻia ma nā biosensors streptavidin (SA) a laila hoʻokomo ʻia i loko o ka kinetic buffer i loaʻa iā 1 μM PACT.ʻO ka mea nui, paʻa paʻa ʻo PACT iā DARS-AS1 (KD waiwai ~ 26.9 nM), akā ʻaʻole e mimic RNA (Figure 3d).Hoʻohui pū ʻia, hōʻike kēia mau hopena i kahi pilina pololei a me ka pilina kiʻekiʻe ma waena o DARS-AS1 a me PACT.
ʻIke ʻia ka loiloi huki RNA i ka DARS-AS1 e launa pū me PACT i nā cell SW620.Ma luna aʻe, ke kala kala o nā protein pili.Ua hana ʻia nā immunoblots haʻahaʻa me ka anti-PACT antibody.b Ua hana ʻia ka loiloi huki-iho RNA ma HCT116 (luna) a me HepG2 (lalo).Ua ʻike ʻia ka hoʻonui ʻana o PACT e ka immunoblotting.Ua hana ʻia nā hoʻokolohua cRNA immunoprecipitation (RIP) i nā ʻāpana SW620 me ka hoʻohana ʻana i nā antibodies i hōʻike ʻia.d Ua loaʻa nā pihi paʻa PACT i ka DARS-AS1 piha a i ʻole ka mana RNA me ka hoʻohana ʻana i ka biolayer interferometry (BLI).Ua hoʻokuʻu ʻia ʻo RNA ma kahi biosensor streptavidin.Ua hoʻohana ʻia ʻo 1 μM PACT e ana i ka hui.Ua hoʻohana ʻia ʻo RNA pull assay me ka hoʻohana ʻana i ka biotinylated piha lōʻihi DARS-AS1 a i ʻole ʻoki ʻia (luna).Immunoblot e hōʻike ana i ka PACT i loaʻa (lalo).f Ua hoʻomaʻemaʻe ʻia ka PACT hae i hoʻomaʻemaʻe ʻia me ka biotinylated piha lōʻihi DARS-AS1 a ʻoki ʻia (e like me ka e) no ka in vitro RIP assay.Ua hōʻoia ʻia ka RNA i unuhi ʻia e RT-qPCR.g Ua loaʻa ka pilina pili o nā ʻāpana RNA like ʻole no PACT me ka hoʻohana ʻana i ka biolayer interferometry.No ka nānā ʻana, ua hoʻohana ʻia ʻo 100 nM RNA a me 1 μM RAST.h In vitro RIP assays ua hana ʻia me ka hoʻohana ʻana i ka PACT i hoʻomaʻemaʻe ʻia a ʻoki ʻia paha.Ua hōʻoia ʻia ka RNA i unuhi ʻia e RT-qPCR.i Ka nui o ka ulu ʻana o nā pūnaewele SW620 e hōʻike ana i ka DARS-AS1, PACT, a i ʻole nā ​​mea ʻelua.j He mau hopena like 'ole ko ka overexpression o DARS-AS1 holo'oko'a a i 'oki 'ia i loko o nā pūnaewele SW620 i ka ulu 'ana o ke kelepona.Ua ʻike ʻia ka Apoptosis e ka immunoblotting me ka anti-PARP antibody.l Knockout o DARS-AS1 hoʻoulu i ka apoptosis o nā pūnaewele SW620 e like me ka hōʻike ʻana e ka cytometry kahe.ʻO ka ʻikepili i hōʻike ʻia, ʻo ia ka mean ± deviation maʻamau o nā hoʻokolohua ʻekolu. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001, ma ka ho'āʻo t haumāna ʻelua-huelo. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001, ma ka ho'āʻo t haumāna ʻelua-huelo. *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 na ka ho'āʻo t a ka Haumāna huelo ʻelua. *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 na ka ho'āʻo t a ka Haumāna huelo ʻelua.
A laila ua hana mākou i ʻekolu mau ʻāpana DARS-AS1 RNA biotinylated e ka in vitro transcription e ʻike i ka ʻāina DARS-AS1 i koi ʻia no ka hui PACT (Figure 3e).Ua hōʻike nā hualoaʻa RNA i hiki i kēlā me kēia ʻāpana ke launa pū me PACT, akā ʻo ka 3′-terminal region (384-768 nucleotides labeled A3) ua hōʻike ʻoi aku ma mua o 1-384 nucleotides i kapa ʻia A1) (Fig. 3e).Ua ʻike ʻia nā hopena like i ka in vitro RIP assay me ka hoʻohana ʻana i ka PACT recombinant (Figure 3f).E like me kēia mau hopena, ua hōʻike ʻia nā hoʻokolohua e hoʻopaʻa i nā ʻāpana RNA immobilized i ka PACT me ka hoʻohana ʻana i ka BLI i hōʻike pū ʻia he pili kiʻekiʻe ka PACT no A3 (384-768 nt) (ka waiwai KD ma kahi o 94.6 nM), ʻoiai ʻaʻohe pili me nā wahi ʻē aʻe.(Fig. 3d).
Ua nānā pū mākou i nā wahi paʻa pili i PACT.Aia i loko o ka PACT ʻekolu mau kikowaena hana, ʻelua o ia mau mea i mālama ʻia i nā domain RNA-binding pālua (dsRBD) a me ke kolu o ka domain (i koho ʻia ʻo D3) e hana ma ke ʻano he mea hoʻoikaika i nā pilina protein.No ka nānā ʻana i ka mana hoʻopaʻa ʻana o ka lncRNA o kēlā me kēia kikowaena, ua hana mākou i ʻekolu mutations i wehe i kēlā me kēia o nā kāʻei kapu ʻekolu a hana i kahi in vitro RIP assay.Ua hōʻike ʻia kā mākou mau hopena i ka holoi ʻana i ke kolu o ka domain (D3) o PACT i hōʻemi nui i kāna pilina me DARS-AS1 (e 0.11-fold i hoʻohālikelike ʻia me PACT intact) i hoʻohālikelike ʻia me nā ʻano ʻelua ʻelua (Fig. 3h), ua hōʻike ʻia ka hoʻokuʻu ʻana. o D3 i launa pū me DARS.-AC1.Hoʻohui pū ʻia, hōʻike kēia mau hopena i ka launa pū ʻana ma waena o DARS-AS1 a me PACT hiki ke hana mua ma o ka hopena 3 'o DARS-AS1 a me ka D3 domain o PACT.
Ua ʻike mākou ʻaʻohe hopena o DARS-AS1 i ka ʻōlelo PACT a ʻaʻohe hopena o PACT ma DARS-AS1 (Supplementary Fig. 3c).A laila ua nānā mākou i ka hopena o PACT knockdown i ka ulu ʻana o ka cell.Ma keʻano likeʻole me DARS-AS1, ua ulu nā pūnaewele pili i ka 1.5-3 manawa wikiwiki i ka wā i hoʻokuʻuʻia ai ka PACT (Supplementary Fig. 3d).Ua hōʻike ʻia nā hopena o ka hoʻokumu ʻana o ka colony i ka hoʻokumu ʻana o nā cell 2-3-fold colonies ma hope o ka mālama ʻana shRNA me PACT (Supplementary Fig. 3e).No ka hoʻāʻo ʻana inā hoʻoponopono ʻo DARS-AS1 i ka hoʻonui ʻana o ka cell ma o PACT, ua hana mākou i nā cell SW620 overexpressing PACT, DARS-AS1, a i ʻole ʻelua.ʻO ka overexpression o PACT i hōʻike i ka inhibition koʻikoʻi o ka hoʻonui ʻana o ka cell (Figure 3i).ʻOiai ʻo DARS-AS1 overexpression i kēlā me kēia mea i hoʻolaha nui i ka hoʻonui ʻana o ka cell, ʻaʻohe ʻokoʻa koʻikoʻi i ka ulu ʻana o nā cell overexpressing DARS-AS1 a me PACT.Ke hōʻike nei kēia mau hopena e hiki i ka PACT ke kūʻē i ka hoʻonui nui ʻana o ka overexpression DARS-AS1.
No ka loaʻa ʻana o nā ʻāpana like ʻole o DARS-AS1 i nā mana paʻa PACT, ua noiʻi mākou i kā lākou pili pili i ka hoʻonui ʻana o ka cell e ka overexpression o nā ʻāpana DARS-AS1.Ke hoʻohālikelikeʻia me nā'āpana'ē aʻeʻelua, ua hoʻonuiʻia ka DARS-AS1 ma ka hopena 3' (384-768 nt), ka māhele pili PACT nui ma DARS-AS1, nona ka mana kiʻekiʻe loa e hoʻoulu i ka hoʻonuiʻana o ka cell (Fig. 3j).Hōʻike kēia mau hopena i kahi pilina maikaʻi ma waena o ka mana paʻa a me ka hana olaola o DARS-AS1.
Ua hōʻike ʻia ʻo PACT he protein pro-apoptotic19.No laila, ua noiʻi mākou i ka hopena o DARS-AS1 ma ka apoptosis.E like me ka mea i manaʻo ʻia, ua hoʻonui nui ʻo DARS-AS1 knockdown i ka cleavage PARP i nā cell SW620 a hoʻonui i ka hapa o nā cell annexin V-positive ma SW620, HCT116, HepG2, a me MBA-MD-231 mau laina kelepona (Fig. 3k).3).3f-h), e hōʻike ana i ka hopena anti-apoptotic o DARS-AS1 i nā maʻi maʻi maʻi e kūʻē i ka hana apoptosis-inducing o PACT.Hoʻohui pū ʻia, hōʻike kēia mau hopena i ke ʻano o ka hana oncogenic DARS-AS1 ma o ka pale ʻana i ka hana PACT.
A laila, ua ʻimi mākou i nā hopena hana o ka hui DARS-AS1-PACT.Ua hōʻike ʻia ʻo PACT e hoʻāla iā PKR ma o ka launa pū ʻana, kahi e hoʻonui ai i ka eIF2α phosphorylation, e hoʻopau ana i ka unuhi a me ka apoptosis17.ʻO ka mea mua, ua nānā mākou inā pili ʻo DARS-AS1 i ka cellular localization o PACT a me PKR.Ua hōʻike ʻia ka microscopy fluorescence Confocal ua hoʻopili nui ʻia ʻo PACT a me PKR i nā pūnaewele SW620 me ka awelika Pearson correlation coefficient o 0.72.I kēia manawa, ua hōʻemi nui ka overexpression DARS-AS1 i ka PACT a me PKR co-localization (mean Pearson correlation coefficient 0.61) (Figure 4a).No ka noiʻi inā hiki i ka DARS-AS1 ke hoʻololi i ka pilina PACT-PKR, ua hana mākou i kahi co-immunoprecipitation (co-IP) assay me anti-PACT antibody ma SW620 cell lysates.Ua hoʻonui nui ʻia ʻo PKR i ka anti-PACT i nā cell control, ʻoiai ua hoʻemi nui ʻia ka hoʻihoʻi ʻana o PKR i nā lysates mai nā cell overexpressing DARS-AS1 (Fig. 4b).Ua hoʻohana ʻia ka PACT a me PKR i hoʻomaʻemaʻe ʻia no ka in vitro protein binding assays.No laila, ʻo nā mea i hāʻawi iā DARS-AS1 akā ʻaʻohe mana RNA i hōʻike i ka hoʻopaʻa ʻana i ka pilina PACT-PKR (Figure 4c).Ua hōʻike ʻia nā hopena āpau ua hoʻopau ʻo DARS-AS1 i ka kamaʻilio PACT a me PKR.
Ua ʻike ʻia kahi Co-localization o PACT a me PKR i nā cell control a i ʻole nā ​​​​cell overexpressing DARS-AS1 me ka hoʻohana ʻana i ka microscopy fluorescence confocal.Ua uhi ʻia ka nuclei me DAPI.Loaʻa nā hopena helu mai nā kiʻi 16.b Co-immunoprecipitation (co-IP) me ka hoʻohana ʻana i ka anti-PACT antibody i nā cell lysates o ka mana SW620 cell a i ʻole nā ​​​​pūnaewele overexpressing DARS-AS1.c Lepili ʻia ʻo PACT, hoʻomaʻemaʻe ʻia ʻo PKR a kākau ʻia i loko o ka vitro me DARS-AS1 a i ʻole RNA hoʻohenehene i hoʻomoʻa ʻia no ka nānā ʻana i ka hoʻopaʻa ʻana i ka protein in vitro.Ua hoʻohana ʻia nā antibodies anti-flag no ka immunoprecipitation.d Ua hana ʻia nā immunoblots me nā antibodies i hōʻike ʻia ma SW620 a me HCT116 mau mea i hoʻololi ʻia me ka mana shRNA a i ʻole DARS-AS1-shRNA i ukali ʻia e ka pōloli serum.Ua hoʻololi nā pae haʻihaʻi DARS-AS1 i ka naʻau kelepona i ka thapsigargin.Ua hoʻololi ʻia nā cell SW620 me DARS-AS1 shRNA, DARS-AS1 overexpression plasmid a i ʻole control plasmid.Ua mālama ʻia nā kelepona me ka thapsigargin no nā hola 48 a ua hoʻoholo ʻia ke ola o ke kelepona me ka hoʻohana ʻana i ka reagent MTS.f In vitro transcribe DARS-AS1 a i ʻole dummy RNA a me PACT i hoʻomaʻemaʻe ʻia i hoʻohana ʻia no ka in vitro activation assay a me ka ʻike immunoblot.g Ua hana ʻia nā Immunoblots e hoʻohana ana i kēia mau antibodies ma nā keena SW620-ctrl (hema) a i ʻole nā ​​​​pūnaewele e hōʻike ana i nā mutants PKR (akau).Ua hoʻololi ʻia kēia mau cell me ka mana shRNA a i ʻole DARS-AS1-shRNA a ukali ʻia e ka pōloli serum.h Flow cytometry hōʻike i ka inactivation o ka mutant PKR i uku no DARS-AS1-induced apoptosis ma SW620 pūnaewele.Ua hana ʻia nā Immunoblots me nā antibodies i hōʻike ʻia ma SW620 (hema) a i ʻole HCT116 (ʻākau).ʻO nā pūnaewele i hoʻololi ʻia me ka mana shRNA a i ʻole DARS-AS1 shRNA i hoʻopau ʻia me ka 100 nM PKR C16 inhibitor a i ʻole DMSO.Paʻa unahi = 5 µm.ʻO ka ʻikepili i hōʻike ʻia, ʻo ia ka mean ± deviation maʻamau o nā hoʻokolohua ʻekolu.* p ≤ 0.05 ka ho'āʻo t a ka Haumāna huelo ʻelua.
Manaʻo maʻamau i ka manawa e launa pū ai ʻo PACT me PKR17, hiki ke hoʻokomo ʻia ka phosphorylation PKR ma Thr451.Ua hōʻike ʻia kā mākou mau hopena ua hoʻokiʻekiʻe nui ʻia ke kiʻekiʻe o ka phosphorylation PKR i nā cell knockdown DARS-AS1 ma hope o ka pōloli serum (Fig. 4d and Supplementary Fig. 4a).No laila, ua ʻike mākou ua hoʻonui nui ʻia ka phosphorylation o eIF2α, ka substrate PKR nui e DARS-AS1 shRNA (Fig. 4d a me Supplementary Fig. 4a).ʻO Thapsigargin kahi pilikia ER e hoʻokuʻu ai ka ER i Ca2+.Ua hōʻike ʻia ka lāʻau lapaʻau me ka thapsigargin e hoʻoulu i ka ʻōlelo a me ka hoʻoulu ʻana o PACT, ka mea e launa pū me a hoʻōla iā PKR, e alakaʻi ana i ka apoptosis ma ka hoʻonui ʻana i ka eIF2α phosphorylation 18,61.Maʻaneʻi, ua hoʻohana mākou i ka thapsigargin ma ke ʻano he mea hoʻoulu i ke ala PACT/PKR e noiʻi inā hiki i ka DARS-AS1 ke kōkua i nā cell e lanakila i ke kaumaha ma ke kāohi ʻana i ke ala PACT/PKR.Ua ʻike mākou i ke kiʻekiʻe o ka ʻōlelo DARS-AS1 i hoʻopili maikaʻi ʻia me ka pale ʻana o ka cell i ka thapsigargin.ʻOi aku ka maikaʻi o ke ola ʻana o nā cell SW620 overexpressing DARS-AS1 ke mālama ʻia me ka thapsigargin, aʻo nā cell me DARS-AS1 knockdown i ʻoi aku ka maʻalahi (Fig. 4e).E like me kēia mau hopena, ua hōʻemi ka overexpression DARS-AS1 i ka thapsigargin-induced PKR phosphorylation (Supplementary Fig. 4b).I ka hoʻohālikelike ʻana, ma hope o ka mālama ʻana i ka thapsigargin, ua phosphorylated ʻo PKR a me eIF2α i kahi kiʻekiʻe ma nā cell knockdown DARS-AS1 i hoʻohālikelike ʻia i nā cell control (Supplementary Fig. 4b).ʻO ka mea e mahalo ai, ua hoʻokomo ʻo thapsigargin i ka ʻōlelo DARS-AS1 ma kahi ʻano i hilinaʻi ʻia, e hōʻike ana i kahi hana anti-stress o DARS-AS1 (Supplementary Fig. 4c).Eia kekahi, ua hana mākou i nā ho'āʻo in vitro activation assays e hōʻoia i kēia mau ʻike.I ka pōkole, ua hoʻomaʻemaʻe ʻia ʻo PKR mai nā cell lysates me ka hoʻohana ʻana i kahi anti-PKR antibody, a laila hoʻokomo ʻia me ka recombinant PACT a me DARS-AS1 i kākau ʻia i loko o ka vitro.Ma hope o ka hopena enzymatic, ua ʻike ʻia ka phospho-PKR me ka hoʻohana ʻana iā WB.Ua hōʻike ʻia kā mākou mau hopena ua kāohi nui ʻia ka phosphorylation PKR e DARS-AS1, akā ʻaʻole ma ka mana RNA (Figure 4f).ʻO kēia mau hopena in vitro a in vivo hōʻike ʻo DARS-AS1 e kāohi i ka hoʻoulu ʻana o PKR-mediated PACT.Ma ka manawa like, ʻike pū mākou i ka emi ʻana o ka hoʻihoʻi ʻana o PACT i mua o DARS-AS1 (Figure 4f).Ua kūlike kēia hopena me nā hopena o ka in vitro protein binding assay (Figure 4c) a hōʻike hou i ka hana pale o DARS-AS1 no ka hui PACT-PKR.
Pono ʻo Ser246 a me Ser287 ma ka D3 domain o PACT no ka hoʻāla ʻana o PKR ma lalo o ke kaumaha cellular.ʻO ka hoʻololi ʻana i ʻelua koena serine no ka alanine i hāʻawi i ka mutant PACT (mutD), ka mea i hoʻāla iā PKR me ka ʻole o ke koʻikoʻi, a me ka hoʻololi ʻana i ka alanine (mutA) i hoʻohuli i ka protocol.No ka mea ua hōʻike mākou i ke koʻikoʻi o kēia kikowaena i ka hui pololei me DARS-AS1, ua hana mākou i kēia mau mutants PACT ʻelua e hoʻāʻo ai inā hiki ke komo pū kēia mau koena i ka launa pū me DARS-AS1.ʻO ka mahalo, ua nalowale nā ​​mutants ʻelua i ka hiki ke hoʻopaʻa iā DARS-AS1 (Supplementary Fig. 4d), e hōʻike ana e koi ʻia ke ʻano piha o ka protein PACT no ka launa pū ʻana me DARS-AS1.
Eia kekahi, ʻo kā mākou mau hopena e hōʻike ana e hiki ke hoʻihoʻi ʻia ka DARS-AS1-shRNA-inhibition o ka hoʻonui ʻia ʻana o ka cell ma o ka overexpressing i kahi mana PACT mutant maikaʻi ʻole (PACTmutA) a i ʻole ka mutant PKR maikaʻi ʻole (PKRmut) (Supplementary Fig. 4e. e).Overexpression o dominant-negative PKR mutants hoemi PKR phosphorylation i hoʻokomo 'ia e DARS-AS1 knockdown a me eIF2α phosphorylation i loko o serum-deprived cell (Fig. 4g).ʻO ka mea nui, ua hoʻemi ʻia ka hapa o nā cell apoptotic i hoʻoiho ʻia e DARS-AS1 knockdown i nā cell overexpressing PKRmut (Fig. 4h a me Supplementary Fig. 4g).Hoʻopilikia pū ka hana ʻana o ka PKR kinase i ka hana DARS-AS1, no ka mea, ua hōʻike ʻia nā hopena ʻaʻole hiki ke hoʻoulu ʻia ʻo DARS-AS1 knockdown i ka PKR a me ka eIF2α phosphorylation i ka wā i mālama ʻia ai nā cell me ka PKR-specific C16 inhibitor (Fig. 4i a me Supplementary Fig. 4h).).Hoʻohui pū ʻia, hōʻike kā mākou hopena e hoʻolaha ʻo DARS-AS1 i ka hoʻonui ʻana o ka cell, ma ka liʻiliʻi loa, ma ke kāohi ʻana i ka hoʻoulu ʻana o PKR-mediated PACT.
No ka ʻimi hou ʻana i ke kuleana o DARS-AS1 i ka tumorigenesis, ua hana mākou i nā hoʻokolohua vivo me ka hoʻohana ʻana i kahi kiʻi xenograft kiʻi. Hōʻike nā hopena i ka hoʻohaʻahaʻa ʻana o DARS-AS1 i ka nui o ka ulu ʻana o nā ʻiole (p waiwai <0.0001) (Fig. 5a). Hōʻike nā hopena i ka hoʻohaʻahaʻa ʻana o DARS-AS1 i ka nui o ka ulu ʻana o nā ʻiole (p waiwai <0.0001) (Fig. 5a). Результати показывают, что нокдаун DARS-AS1 резко снижает рост опухоли у мышей (значение p <0,0001) (рис. 5а). Hōʻike nā hualoaʻa i ka DARS-AS1 knockdown e hōʻemi nui i ka ulu ʻana o nā ʻiole (p waiwai <0.0001) (Figure 5a).结果表明，DARS-AS1 的敲低显着降低了小鼠的肿瘤生长（p 值< 0.0001）（图5a）。结果表明，DARS-AS1 的敲低显着降低了小鼠的肿瘤生长（p值<0.0001）（图5a）。 Результати показали, что нокдаун DARS-AS1 значительно снижает рост опухоли у мышей (значение р <0,0001) (рис. 5а). Ua hōʻike nā hopena i ka hoʻohaʻahaʻa nui ʻana o DARS-AS1 i ka ulu ʻana o nā ʻiole (p waiwai <0.0001) (Figure 5a).No laila, i loko o ka pūʻulu knockdown DARS-AS1, ua nui ka emi ʻana o ka nui o ka nui ma kahi o 72.9% a me ka nui o ka nui ma kahi o 87.8% (Figure 5b-d).Manaʻo ikaika kēia mau hopena e hiki i ka DARS-AS1 ke hoʻoikaika nui i ka ulu ʻana o ka maʻi ma vivo.
Nā hopena o ka ad DARS-AS1 knockdown ma ka colorectal oncogenesis i nā ʻiole olohelohe.Hōʻike ʻia nā pihi ulu (a), ka nui o ka ʻōpū (b), ke kaumaha (c), a me nā kiʻi ʻōpū (d).Hōʻike nā pahu hewa i ka ± SEM. n = 10. ****p < 0.0001, ma ka ho'āʻo t a ka Haumāna huelo ʻelua. n = 10. ****p < 0.0001, ma ka ho'āʻo t a ka Haumāna huelo ʻelua. n = 10. ****p < 0,0001 по двустороннему критерию Стьюдента. n = 10. ****p < 0.0001 ka ho'āʻo-t a ka Haumāna huelo ʻelua.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 ka ho'āʻo-t a ka Haumāna huelo ʻelua.e Kaplan-Meier i kālailai i ka pilina ma waena o DARS-AS1 pae haʻi'ōlelo a me ke ola holoʻokoʻa i nā maʻi me UVM, KICH, KIRP, MESO, GBM, a me LGG.ʻO nā kiʻekiʻe kiʻekiʻe o ka'ōlelo DARS-AS1 i nā maʻi ma ka 50% kiʻekiʻe;ʻO ka haʻahaʻa haʻahaʻa o ka hōʻike DARS-AS1 i nā maʻi ma lalo o 50%.Ua hoʻoholo ʻia nā p-values ​​me ka hoʻohana ʻana i ka hōʻike log rank.f Hoʻohālike i manaʻo ʻia e hoʻoponopono ai ʻo DARS-AS1 i ke ala PACT-PKR a me ka ulu ʻana o ka maʻi maʻi.
No ka hoʻomaopopo maikaʻi ʻana i ka hopena lapaʻau o DARS-AS1, ua nānā mākou i ka pilina ma waena o kāna hōʻike a me ke ola maʻi.Ma ka nānā ʻana i ka ʻikepili TCGA, ua ʻike mākou ua pili ka ʻōlelo DARS-AS1 kiʻekiʻe me ka uveal melanoma (UVM), renal chromophobia (KICH), renal papillary cell carcinoma (KIRP), mesothelioma (MESO), multiplex.Ua pili nui ke ola haʻahaʻa me ka glioblastoma morphosis (GBM) a me nā maʻi me ka glioma lolo haʻahaʻa (LGG) (Figure 5e).Hōʻike kēia mau hopena e hiki ke hana nui ʻo DARS-AS1 i ka piʻi ʻana o ka maʻi maʻi maʻi a hiki paha i kahi biomarker wānana i nā maʻi maʻi he nui.
Ma kēia noiʻi ʻana, me ka hoʻohana ʻana i ka screening functional CRISPRi nui, ua hoʻoholo mākou ua lanakila ʻo DARS-AS1 lncRNA i ke koʻikoʻi o ka maʻi kanesa ma o ka hoʻoponopono ʻana i ʻelua mau mea pane koʻikoʻi, PACT a me PKR.Ma ka launa pū ʻana me PACT, ua kāohi ʻo DARS-AS1 i ka PACT-mediated PKR activation, ma laila e pale ai i ka make cell apoptotic a me ka hoʻolaha ʻana i ka hoʻonui ʻana o ka cell (Fig. 5f).Ua ʻike ʻia ka hoʻoponopono ʻana o DARS-AS1 i nā ʻano maʻi maʻi he nui, e hōʻike ana i kāna hana o ka hoʻolaha ʻana i ke ola o ka maʻi kanesa ma lalo o nā kūlana koʻikoʻi hiki ke pili i nā ʻano maʻi maʻi he nui.
Ua ʻike ʻia ʻo PACT ma ke ʻano he protein activator PKR, a he kuleana koʻikoʻi ka PACT-mediated PKR activation i nā pane koʻikoʻi ma o ka hoʻoponopono ʻana i ka transcription, unuhi, apoptosis, a me nā kaʻina cellular koʻikoʻi62.No nā makahiki he ʻumi, ua hoʻāʻo ʻia e hoʻomaopopo i ka hoʻoponopono kikoʻī kikoʻī o ka PACT-PKR cascade.Maʻaneʻi, ua hōʻike kā mākou noiʻi i kahiʻano likeʻole o ka hoʻoponoponoʻana i ka PACT-PKR i loko o nā pūnaewele maʻi kanesa ma o ka cellular lncRNA DARS-AS1, e hoʻopaʻa pono ana i ka PACT, nā poloka PACT-PKR i ka pilina, ke kāohi i ka hoʻouluʻana o PKR a me ka eIF2α phosphorylation, a laila e pale ai i ka apoptosis i hoʻokomoʻia i ke kaumaha. hoʻoulu ʻana i ka hoʻonui ʻana o ka maʻi kanesa.pūnaewele.Hōʻike kēia ʻike i ka mālamalama i nā pahuhopu lncRNA no ka wānana a me ka lapaʻau.
Ua hōʻike kā mākou ʻikepili i ka hoʻopaʻa ʻana o DARS-AS1 i nā cell i ka pōloli serum me ka piʻi nui o ka phosphorylated PKR a me eIF2α.Hōʻike kēia mau hopena i ka hoʻolaha ʻana o DARS-AS1 i ke ola maʻi maʻi maʻi ma lalo o nā kūlana paʻakikī ma ke kāohi ʻana i ka hana PACT/PKR.ʻO kekahi mau RNA non-coding ʻē aʻe, e like me ASPACT a me nc886, ke komo pū nei i ka axis PACT/PKR ma ka hoʻohaʻahaʻa ʻana i ka PACT48 mRNA a i ʻole ka hoʻoponopono ʻana i ka autophosphorylation ma ka hoʻopaʻa ʻana iā PKR49,50,64.Ma waena o lākou, hana ʻo DARS-AS1 ma ke ʻano he mea hoʻohaunaele i ka hui PACT-PKR.Hoʻonui kēia haʻawina i ko mākou ʻike i ka hoʻoponopono axis PACT/PKR a me ke kuleana o nā lncRNA i nā pane koʻikoʻi.
Loaʻa i ka PACT ʻekolu mau kikowaena kaʻawale.Ua lawa kēlā me kēia o nā dsRBD mua ʻelua no ka hoʻokō ʻana i ka pilina pili kiʻekiʻe o PACT iā PKR, aʻo ke kolu o ka domain (D3) e koi ʻia no ka hoʻāla ʻana o PKR in vitro a in vivo.Ua hōʻike kā mākou haʻawina e pili ana ʻo DARS-AS1 me ka D3 domain (Fig. 3h).Hāʻawi ʻia i ka nui nui o ka lncRNA (768 nucleotides), ʻo DARS-AS1 e hoʻopaʻa ana iā D3 hiki ke pale kino i ka pilina ma waena o ka domain PACT o dsRBD a me PKR, no laila ke kāohi nei i ka hui o PACT a me PKR.ʻO nā hoʻololi helu PACT i hoʻololi iā Ser246 a me Ser287 ma D3 me ka alanine a i ʻole aspartate i hoʻopau i kona pilina paʻa no DARS-AS1, e kuhikuhi ana i ke koʻikoʻi o nā waiwai a me nā waiwai uila o D3 i kā lākou hui.E koi ʻia nā kikoʻī hou aʻe o kēia mīkini i ka wā e hiki mai ana, me ka hoʻohana ʻana i ka loiloi biochemical pololei a me ka hoʻonā kiʻekiʻe PACT structural analysis.
Ua hōʻike ʻia nā haʻawina mua e hoʻolaha ana ʻo DARS-AS1 i ka hoʻonui ʻana o ka cell ma o kekahi mau mechanical51,52,53.I kekahi laʻana, ua ʻike nā mea noiʻi ua hoʻonui ʻo DARS-AS1 i kāna gen antisense protein-encoding DARS gene ma ka huli ʻana i ka miP-194-5p i loko o nā pūnaewele maʻi kanesa.Eia nō naʻe, i ka haʻawina i kēia manawa, ʻaʻohe hopena liʻiliʻi o DARS-AS1 knockdown i ka palapala palapala DARS i nā ʻano maʻi maʻi he nui, me ka liʻiliʻi loa o ka colorectal, ka umauma, a me nā maʻi maʻi ate.Ma muli o ka hōʻike ʻana o nā lncRNA i nā hiʻohiʻona hōʻike kikoʻī cell-a me ka kiko, ʻaʻole mālama ʻia nā mīkini hana ma nā ʻano maʻi kanesa, hiki ke kōkua i kēia ʻokoʻa ma waena o kā mākou nānā ʻana a me nā loiloi mua o nā maʻi maʻi maʻi like ʻole.Pono nā haʻawina kūikawā e wehewehe i nā mīkini kiko'ī o nā kaʻina hana physiological a me nā pathological.
Ua hōʻike ʻia kahi loiloi o ka ʻikepili lapaʻau e pili ana ka ʻōlelo DARS-AS1 i nā ʻōpū me ke ola ʻana o nā maʻi maʻi maʻi, e kuhikuhi ana i ke koʻikoʻi o ka axis DARS-AS1/PACT/PKR i ka wānana maʻi.I ka hopena, hōʻike kā mākou noiʻi ʻana ʻo DARS-AS1 he mea hoʻoponopono o ka axis hōʻailona PACT/PKR, hoʻolaha i ka hoʻonui ʻana o ka maʻi maʻi maʻi, a kāohi i ka apoptosis i ka wā o ka pane ʻana i ke kaumaha, e hāʻawi ana i kahi laina o ka noiʻi ʻana a he hoihoi no ka noiʻi e hiki mai ana i nā lāʻau lapaʻau. .
Loaʻa nā laina kelepona kanaka SW620, A549, MBA-MD-231, HCT116, HepG2 a me HEK293T mai ka National Cell Line Resource Infrastructure ma Kina.Ua mālamaʻia nā pūnaewele a pau i ka DMEM medium (DMEM, Thermo Fisher Scientific, Waltham, MA) i hoʻonuiʻia me 10% FBS (Gemini, Brooklyn, NY) a me 1% penicillin-streptomycin (Thermo Fisher Scientific) ma 37 ° C, 5% CO2.incubator.
Anti-PACT, Abcam (ab31967);Anti-PKR, Abcam (ab184257);Anti-PKR (phospho-T451), Abcam (ab81303);Anti-Flag, Abcam (ab125243);Anti-eIF2α, Abcam (A0764));anti-eIF2α (phosphorus S51), Abcam (ab32157);anti-PACT (phosphorus S246), Abgent (AP7744b);anti-β-tubulin, CST (2128);IgG maʻamau, CST (5415S);lapiti maʻamau IgG, CST (2729S).Ua hoʻoheheʻe ʻia nā antibody i ka 1:1000 ma PBST no Western blotting a me 1:100 no IP.
Ua hoʻomohala ʻia nā sgRNA me ka hoʻohana ʻana i kahi mea hana i loaʻa i ka lehulehu i kapa ʻia ʻo CRISPR-ERA66.Ua hoʻohana mākou i nā ʻāpana hāmeʻa paʻamau no ka hoʻomohala ʻana i ka sgRNA a me ka algorithm i helu ʻia i nā pūnaewele hoʻopaʻa sgRNA ma ka ʻāpana 3 kb.kikowaena ma TSS.Hoʻopili ʻia nā loko o sgRNA oligonucleotides ma CustomArray, Inc. (Bothewell, WA) a hoʻopili ʻia i loko o nā plasmids pgRNA kanaka (Addgene #44248).ʻO ka huina o 12 µg o ka pgRNA plasmid kanaka, 7.2 µg o psPAX2 (Addgene #12260), a me 4.8 µg o pMD2.G (Addgene #12259) i hoʻololi pū ʻia i loko o 5 x 106 HEK293T dishes me ka hoʻohana ʻana i ka DNAagent Transfection cm 10 pūnaewele (CWBIO, Beijing, Kina) ma muli o nā ʻōlelo a ka mea hana.Ua hōʻiliʻili ʻia nā supernatants i loaʻa i ka maʻi virus i 48 a me 72 mau hola ma hope o ka hoʻololi ʻana a kānana ʻia ma kahi kānana 0.45 µm.No ka nānā ʻana, ua loaʻa nā pūnaewele SW620 e hōʻike ana i ka protein fusion dCas9/KRAB ma o ka lawe ʻana i ka maʻi virus.Ua hoʻololiʻia nā pūnaewele SW620 i hoʻololiʻia me ka waihona virus ma nā hoʻokolohua maʻi kūʻokoʻaʻehā ma kahi MOI o 0.1-0.3 a ua hoʻohālikeʻia me 2 μg / ml puromycin (Sigma, St. Louis, MO) no nā lā 2.Ma hope mai, hoʻoulu ʻia nā cell no 18 mau lā in vitro me ka liʻiliʻi o ka uhi waihona puke o 500 cell/sgRNA no ka nānā ʻana.
Ua unuhi ʻia ka DNA Genomic e like me nā ʻōlelo a ka QIAamp DNA Blood Midi Kit (QIAGEN, Düsseldorf, Kelemānia; 51183).Ma ka huina, ua hoʻohana ʻia ka 100 μg o genomic DNA i kēlā me kēia hoʻihoʻi biological e kūkulu i ka waihona.Ua hoʻonui ʻia ka ʻāina sgRNA e ʻelua pōʻai o PCR a pili i kahi barcode.
Ua hoʻomaʻemaʻe ʻia nā huahana PCR me ka hoʻohana ʻana i ka pahu hoʻomaʻemaʻe ʻo NucleoSpin® gel a me ka pahu hoʻomaʻemaʻe PCR (MACHEREY-NAGEL, Düren, Kelemānia; 740609.250) a ua helu ʻia me ka hoʻohana ʻana i ka pahu ʻike DNA pālua ʻo Qubit™ HS (Thermo Fisher Scientific; Q32854).
Ua hoʻohana ʻia ka MTS assay no ke ana ʻana i ka hoʻonui cell.Ua kanu ʻia nā ʻāpana i loko o nā pā 96-well ma kahi ʻano mua o 2000 cell/well.Ua ana ʻia ka helu pili o nā cell i kēlā me kēia lā i ka manawa i hōʻike ʻia no ka huina o 4-6 mau lā.No kēlā me kēia pūnāwai, ua hoʻoheheʻe ʻia ʻo 20 μl o MTS reagent (Promega) me 100 μl o DMEM, incubated me nā cell no 4 h ma 37 ° C, a laila ua ana ʻia ʻo OD490.
Ua ʻike ʻia ka hiki ke ulu ʻole ma ka nānā ʻana i ka hoʻokumu ʻana o nā spheres.ʻO ka pōkole, 2000 mau pūnaewele i hoʻololi ʻia me shRNA DARS-AS1 a i ʻole control shRNA i moʻomeheu ʻia i nā microplates hoʻopili haʻahaʻa haʻahaʻa (Corning) me ka loli waena i kēlā me kēia 4 lā.Ua helu ʻia nā spheroids ma hope o 14 mau lā.Ua hoʻohana ʻia nā cell 500 i hoʻololi ʻia me ka DARS-AS1 overexpression plasmid a i ʻole kahi control plasmid no ka hoʻonui ʻana i ka assay, inā ʻaʻole i loli ke ʻano.
Ua kākau ʻia ʻo RNA me ka hoʻohana ʻana i ka T7 RNA polymerase a me ka biotin-16-UTP (Roche 1138908910) e like me nā kuhikuhi a Riboprobe® Combination Systems (Promega P1440).Ua helu ʻia nā kumu mua i hoʻohana ʻia ma ʻaneʻi ma ka Papa Pākuʻi 4.
Ua hoʻopili ʻia nā ʻāpana Protein-coding PACT a i ʻole PKR i loko o ka pET15b (Addgene #73619) a hoʻololi ʻia i BL21 (DE3).Hoʻokomo ʻia ka bacteria i ka pō i loko o ka LB i hoʻolako ʻia me ka ampicillin a laila hoʻoheheʻe ʻia he 100-fold me ka LB hou.I ka hiki ʻana o ka OD600 o ke kikowaena i ka 0.8, ua hoʻohui ʻia ka 1 mM IPTG e hoʻoulu i ka ʻōlelo protein.Ma hope o ka incubation i ka pō me ka haʻalulu mālie (250 rpm ma 20 ° C), ua hōʻiliʻili ʻia ka pellet cell e ka centrifugation (4000 rpm, 10 min, 4 ° C).E hoʻokuʻu hou i ka pellet cell i loko o ka lysis buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 1 mM PMSF) a incubate ma ka hau no 30 min, a laila sonicate (15 min, 5 s on/off, ma ka hau) a me ka centrifuge (13,000). rpm)., 30 min, 4°C).A laila ua hoʻouka ʻia ka supernatant ma luna o ka resin Ni-NTA (QIAGEN) 3 mau manawa ma 4 ° C, holoi ʻia 4 mau manawa me ka holoi holoi (50 mM Tris, pH 8.0, 40 mM imidazole, 250 mM NaCl) a eluted 3 manawa, me ka huina. o 10 ml eluent buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 300 mM imidazole).Ua hoʻoholo ʻia ka protein i hoʻomaʻemaʻe ʻia me ka hoʻohana ʻana i ka WB a ua hoʻoholo ʻia ka ʻike ʻana me ka hoʻohana ʻana i ka kit assay protein Qubit™ (Thermo Fisher Scientific; Q 33212).
Ua hana ʻia nā hoʻokolohua RIP e like me ka mea i wehewehe mua ʻia, me nā hoʻololi.ʻO ka pōkole, 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 107 cocktail (Roche, 1 mM DTT) a me ka centrifuge ma 13,000 rpm no 15 min ma 4 °C.Ua hoʻokomo ʻia ka supernatant me ka protein A + G magnetic beads (Millipore) i hui pū ʻia me 5 μg o anti-PACT antibody (Abeam) a i ʻole IgG (CST).Holoi ʻia nā pahu i 5 mau manawa me 5x RIP buffer, a laila ʻeli ʻia me ka proteinase K (NEB).Lawe ʻia ʻo RNA me Trizol a hoʻoholo ʻia e RT-qPCR.Hōʻike ʻia nā kumu mua ma ka Papa Pākuʻi 5.
Ua hana ʻia ka in vitro RIP assay e like me kahi protocol assay RIP maʻamau i hoʻololi ʻia.ʻO ka huina o 5 pmol o in vitro transcribed RNA i hoʻoheheʻe ʻia 1x me ka RIP buffer a hoʻopili ʻia e ka incubation ma 65 ° C no 5 mau minuke i ukali ʻia e ka hoʻomaha mālie i ka lumi wela.Ua hoʻomaʻemaʻe ʻia he 5 pmol o nā protein PACT i hoʻopaʻa ʻia a i ʻole i hoʻololi ʻia i ka hae mai E. coli.Hoʻopili me ka RNA i hoʻoponopono hou ʻia no 2 mau hola ma 4 ° C a hahai i ke kaʻina hana i luna no ka nānā ʻana RIP no ka anti-flag IP.
No ka nānā ʻana i ka hoʻonui ʻana o RNA, ua lysed ʻia nā cell 1 × 107 me 1xRIP buffer.Ma hope o ka centrifugation ma 13,000 rpm no 15 min ma 4 ° C, ua hana mua ʻia ka supernatant me 30 μl o streptavidin magnetic beads (Beckman) no 2 h ma 4 ° C.Hāʻawi ʻia ka lysate i hoʻomaʻemaʻe ʻia me ka tRNA hū a hoʻomoʻi ʻia me 40 pmol o RNA i hoʻoponopono hou ʻia i ka pō ma 4 ° C, a laila hoʻohui ʻia no kekahi mau hola 2 a me 20 μl o nā streptavidin magnetic beads hou i kāohi ʻia me BSA.He 4 mau manawa ka holoi ʻana me 5x RIP buffer a me 4 manawa me 1x RIP buffer.Hoʻopili ʻia nā protein e like me ka biotin elution buffer (25 mM Tris-HCl, pH 7.5, 12.5 mM D-biotin, PMSF) a hoʻokaʻawale ʻia ma NuPAGE 4-12% Bis-Tris Gel (Invitrogen).Ma hope o ka ʻili kālā (Beyotime Biotechnology), ua ʻoki ʻia a nānā ʻia kekahi mau pūʻali e MS.
Ua hana ʻia ka loiloi Co-IP e hoʻāʻo i ka pilina ma waena o PACT a me PKR.ʻO ka pōkole, ua hoʻomākaukau ʻia nā lysate supernatant ma o ka hoʻoulu ʻana i 1 x 107 lysed cell i 1 x RIP buffer a ukali ʻia e ka centrifugation ma 13,000 rpm no 15 mau minuke ma 4 ° C.Hoʻopiha ʻia nā Lysates me ka protein A + G magnetic beads, conjugated me 5 µg o anti-PACT antibody, a hoʻololi mālie i ka pō ma 4 ° C.Ua holoi ʻia nā pahu i 3 mau manawa me 5 × RIP buffer, ʻelua ʻelua me 1 × RIP buffer a eluted me 1 × SDS buffer.Ua nānā ʻia ka protein i hoʻihoʻi ʻia e ka gel SDS-PAGE a ʻike ʻia e WB.
Ua hoʻomaʻemaʻe ʻia ʻelua pmol o ka PACT hae a me 1 pmol o PKR mai E. coli.E hoʻoheheʻe ʻia i loko o 1× RIP buffer a hoʻomohu me 10 pmol o RNA i hoʻoponopono hou ʻia no 2 mau hola ma 4 °C.Ma hope o kēlā, hoʻokomo ʻia lākou me ka protein A+G magnetic bead-conjugated anti-labeled antibody no ʻelua hola hou.Holoi ʻia nā pahu i ʻehā manawa me 1x RIP buffer a hoʻoheheʻe ʻia me 1x SDS buffer.Ua ʻike ʻia ka hopena PACT a me PKR e WB.