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Mkpụrụ ndụ kansa ewepụtala usoro dị iche iche iji merie nrụgide cellular wee gaa n'ihu na-aga n'ihu.Protein kinase R (PKR) na protein activator ya (PACT) bụ ndị na-aza ajụjụ mbụ na-enyocha mgbaàmà nrụgide dị iche iche na-eduga na mgbochi nke mgbasawanye cell na apoptosis.Agbanyeghị, ụkpụrụ nke ụzọ PACT-PKR na mkpụrụ ndụ kansa ka amabeghị nke ukwuu.N'ebe a, anyị chọpụtara na ogologo oge na-abụghị koodu RNA (lncRNA) aspartyl tRNA synthetase antisense RNA 1 (DARS-AS1) na-etinye aka na mgbochi nke ụzọ PACT-PKR ma na-akwalite mmụba cell cell.N'iji nyocha ọrụ dị ukwuu nke CRISPRi 971 ọrịa cancer metụtara lncRNA, anyị chọpụtara na DARS-AS1 jikọtara ya na mmụba mkpụrụ ndụ kansa nke ukwuu.Ya mere, DARS-AS1 knockout na-egbochi mgbasawanye cell ma na-akwalite apoptosis cell cancer na ahịrị mkpụrụ ndụ cancer dị iche iche na vitro ma na-ebelata ụbụrụ tumo na vivo.N'iji arụ ọrụ, DARS-AS1 na-ejikọta ozugbo na ngalaba mmeghe PACT ma gbochie mmekọrịta PACT-PKR, si otú ahụ belata ọrụ PKR, eIF2a phosphorylation, na igbochi ọnwụ cell apoptotic.N'ụlọ ọgwụ, DARS-AS1 na-egosipụta n'ọtụtụ ebe n'ọtụtụ ọrịa cancer, na ikwubiga okwu ókè nke lncRNA a bụ ihe na-egosi prognosis na-adịghị mma.Ọmụmụ ihe a na-akọwapụta usoro ọgwụgwọ ọrịa kansa-kpọmkwem nke ụzọ PACT-PKR site na DARS-AS1 lncRNA ma na-enye ebumnuche ọzọ maka prognosis na ọgwụgwọ ọrịa kansa.
Ikike ime mgbanwe na nchekasị bụ njirimara dị mkpa nke ndụ mkpụrụ ndụ kansa na mmụba.Mgbasa ngwa ngwa na njirimara metabolic nke ọrịa cancer na-arị elu na microenvironments siri ike - ụkọ nri, hypoxia, na ala pH-nke nwere ike ịkpalite ụzọ mgbaàmà ọnwụ cell.Dysregulation nke mkpụrụ ndụ ihe nketa na-enwe mmetụta nchekasị dị ka p535, protein ndị na-ekpo ọkụ ọkụ 6, 7, KRAS8, 9, na HIF-110, 11, 12, 13 na-ahụkarị na ọrịa cancer, si otú ahụ na-egbochi apoptosis na ịkwalite ndụ.
Protein kinase R (PKR) bụ ihe mmetụta nrụgide dị mkpa na subunit kinase nke eukaryotic initiation factor 2α (eIF2α), onye na-achịkwa ntụgharị asụsụ nke na-ejikọta nrụgide cellular na ọnwụ cell.Achọpụtara PKR na mbụ dị ka protein antiviral site na nchọpụta nke RNA nwere eriri abụọ (dsRNA) nke mba ofesi.Mgbe arụ ọrụ, PKR phosphorylates eIF2a na-egbochi njikọ protein viral na cellular14,15,16.Achọpụtala PACT (protein activator PKR) dị ka protin mbụ PKR activator na enweghị dsRNA17,18,19,20,21,22,23.Site na mmekorita ya na PKR, PACT na-atụgharị nrụgide dị iche iche (agụụ serum, peroxide ma ọ bụ ọgwụgwọ arsenite) gaa na PKR na ụzọ mgbama ala.Na mgbakwunye na eIF2a phosphorylation, PACT-mediated PKR activation na-akpalite ihe omume dị iche iche metụtara nzaghachi nrụgide ahụ, gụnyere ọnọdụ redox gbanwere site na ụzọ PI3K / Akt24, nyochaa mmebi DNA na-emewanye site na p5325,26 na NF-κB27,28 na-achịkwa nsụgharị, 29. Nyere ọrụ ha dị oke mkpa na nzaghachi nrụgide, mmụba, apoptosis na usoro cellular ndị ọzọ, PKR na PACT na-ekwe nkwa ọgwụgwọ ọgwụgwọ maka ọtụtụ ọrịa, karịsịa cancer30,31,32,33.Otú ọ dị, n'agbanyeghị ọrụ pleiotropic a na ihe dị ndụ pụtara, nhazi nke PACT/PKR na mkpụrụ ndụ cancer ka na-adị mfe.
lncRNA bụ transcript karịrị 200 nucleotides na-enweghị ike itinye protein.Ebe ọ bụ na ọrụ nhazi usoro genome zuru oke achọpụtala puku kwuru puku lncRNA, 35,36 emeela ọtụtụ mgbalị iji kọwapụta ọrụ ndu ha.Otu nnyocha na-eto eto egosila na lncRNA na-etinye aka n'ọtụtụ usoro ndu37 gụnyere nhazi nke X-chromosome inactivation38,39, imprinting40, transcription41,42, translation43 na ọbụna cancer growth44,45,46,47.Ọmụmụ ihe ndị a kọrọ na ọtụtụ lncRNA na-etinye aka na PACT/PKR ụzọ.Otu nnyocha dị otú ahụ gosiri na lncRNA ASPACT gbochiri transcript PACT na ụbara njigide nuklia nke PACT mRNA.Ọmụmụ ihe ndị ọzọ egosila na lncRNA nc886 jikọtara PKR ma gbochie phosphorylation ya49,50.Ka ọ dị ugbu a, akọpụtabeghị mgbake PKR na-achịkwa lncRNA na-achịkwa PACT.
Aspartyl-tRNA synthetase antisense RNA 1 (DARS-AS1) ka achọpụtara dị ka oncogenic lncRNA51,52,53,54.Site n'usoro nke miP-194-5p53, miP-12952 na miP-532-3p51, DARS-AS1 egosila na ọ na-akwalite uto nke carcinoma cell renal cell cell doro anya, carcinoma thyroid carcinoma na-abụghị obere cell lung carcinoma, n'otu n'otu.Tong na ndị ọrụ ibe ahụ chọpụtakwara na DARS-AS1 na-akwalite ọganihu myeloma site n'ịkwado nkwụsi ike nke protein 39 (RBM39) RNA-binding motif.Agbanyeghị, ọ nweghị ọmụmụ emebeghị gbasara ma lncRNA a na-etinye aka na nhazi nke ịgbalite PACT-PKR yana nzaghachi nrụgide nke mkpụrụ ndụ kansa.
N'ebe a, anyị na-eji usoro CRISPRi rụọ ọrụ nnukwu ihuenyo na-efunahụ ma kpebisie ike na DARS-AS1 lncRNA na-akwalite mgbasa nke ọtụtụ ụdị ọrịa cancer.Tụkwasị na nke ahụ, anyị achọpụtala usoro bụ isi: DARS-AS1 na-ejikọta ya na PACT, na-egbochi PACT na PKR, na-egbochi phosphorylation nke eIF2a, ihe ntinye PKR dị ala, na n'ikpeazụ na-egbochi ọnwụ apoptotic cell.N'ikpeazụ, ọrụ anyị na-ekpughe DARS-AS1 lncRNA dị ka onye na-achịkwa ụzọ PACT-PKR na ihe nwere ike ime maka ọgwụgwọ ọrịa cancer na prognosis.
Nnukwu ọmụmụ profaịlụ genomic achọpụtala ọtụtụ narị lncRNA metụtara ọrịa kansa.Agbanyeghị, ọrụ ha ka bụ nke amabeghị56.Iji chọpụta ndị na-ekwe nkwa lncRNA na-etinye aka na ọganihu ọrịa cancer, anyị mere ihuenyo na-efunahụ maka mbelata mgbasa na SW620 colorectal cancer cell line site na iji usoro CRISPRi (Fig. 1a).Akụkụ pụrụ iche nke ahịrị sel cancer nke SW480 na SW620 bụ na ha sitere na etuto isi na nke abụọ n'ime otu onye ọrịa.Nke a na-enye ntụnyere bara uru maka ịmụ mgbanwe mkpụrụ ndụ ihe nketa na ọganihu nke ọrịa cancer eriri afọ.Ya mere, anyị nyochara transcriptomes nke ahịrị cell cancer colorectal (SW480 na SW620) site na iji usoro RNA wee nakọta ụfọdụ lncRNA nwere ike ịrụ ọrụ na akwụkwọ ndị e bipụtara.Dabere na nsonaazụ ndị a, anyị haziri ọbá akwụkwọ sgRNA nwere 7355 sgRNA oligos na-ezubere iche lncRNAs metụtara ọrịa kansa 971 yana 500 sgRNA oligos na-achọghị maka njikwa na-adịghị mma (Data mgbakwunye 1).
Ngosipụta atụmatụ nke nyocha site na iji sistemụ CRISPRi.b sgRNA na-abawanye mgbe nyochachara.Ahịrị nwere ntụpọ kwụ ọtọ na-anọchi anya log2 (mgbanwe mpịaji) = ± 0.58.Ahịrị nwere ntụpọ kwụ ọtọ na-egosi uru p = 0.05.Ntụpọ ojii na-anọchite anya sgRNA na-abụghị ebumnuche (nke akpọrọ NC).Ntụpọ uhie bụ sgRNA na-ezubere DARS-AS1.ntụpọ na-acha anụnụ anụnụ bụ sgRNA na-ezubere LINC00205, oncogenic lncRNA akọwara na mbụ.mkpụkọ mgbanwe = (agụmagụ nkịtị, ụbọchị 17)/(ịgụ ihe, ụbọchị 0).c DARS-AS1 sgRNA knockdown gbochiri uto cell.Ogwe mperi na-anọchite anya ± ọkọlọtọ nke nnwale atọ ahụ.* p ≤ 0.05, ** p ≤ 0.01 t-ule nwata akwụkwọ nwere ọdụ abụọ.d DARS-AS1 okwu na etuto ahụ (TCGA dataset).em Ngosipụta nke DARS-AS1 na ihe atụ nkịtị na tumor sitere na ndị ọrịa nwere BLCA, KIRC, PRAD, LUSC, UCEC, LUAD, LIHC, KIRP, na COAD, n'otu n'otu (TCGA dataset).Enwetara p-ụkpụrụ site na iji t-ule nwata akwụkwọ nwere ọdụ abụọ.
Mgbe anyị rụchara plasmid na nkwakọba lentivirus, anyị jiri ọba akwụkwọ dị n'elu were were were ọbá akwụkwọ dị n'elu were were were nyocha ọrịa anọ nweere onwe ya tụgharịa.Ọnụ ọgụgụ nke ọrịa (MOI) maka ọrịa ndị a bụ 0.1-0.3, na-egosi na cell ọ bụla nwere ike ibunye naanị otu sgRNA.Mgbe ụbọchị iri na asatọ nke ọdịnala in vitro gasịrị, profaịlụ nkwalite nke sgRNA lekwasịrị anya belatara ma ọ bụ mụbaa mgbe nyochachara, ebe ọnụọgụ oligonucleotides na-abụghị nke ezubere iche agbanwebeghị ma e jiri ya tụnyere profaịlụ nyocha mbụ, na-egosi na ebumnuche anyị nwere nnukwu ihuenyo akọwapụtara. ụlọ akwụkwọ.Osikapa.1b na tebụl mgbakwunye 1). LINC00205, bụ nke a na-akọ na mbụ iji kwalite ọrịa cancer akpa ume na ọrịa imeju na-aga n'ihu58,59,60, na-ekpuchi (log2 (foldchange) <-0.58, p value <0.05), na-akwado ntụkwasị obi nke nyocha a (Fig 1b). LINC00205, bụ nke a na-akọ na mbụ iji kwalite ọrịa cancer akpa ume na ọrịa imeju na-aga n'ihu58,59,60, na-ekpuchi (log2 (foldchange) <-0.58, p value <0.05), na-akwado ntụkwasị obi nke nyocha a (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 nke 1b). LINC00205, nke a kọrọ na mbụ iji kwalite ọganihu nke ọrịa cancer akpa ume na ọrịa imeju 58,59,60, ewepụghị (log2 (mgbanwe mgbanwe) <-0.58, p-value <0.05), na-akwadosi ike nke nyocha a (Fig. .1b) . Linc0020555 之前 之前 58,59,59,60, 被 筛 筛 变化 变化 0.5,55), 证实 了 1b). Linc0020555 之前 之前 58,59,59,60, 被 筛 筛 变化 变化 0.5,55), 证实 了 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 nke 1b). LINC00205, nke a kọrọ na mbụ iji kwalite ngụgụ na ọrịa cancer imeju58,59,60, ewepụghị (log2 (mgbanwe mgbanwe) <-0.58, p-value <0.05), na-akwado ike nke nyocha a (Fig. .1b).
N'ime lncRNA niile a nwalere, a na-enyocha DARS-AS1, na cognate atọ sgRNA oligonucleotides belatara nke ukwuu mgbe ụbọchị 18 nke omenala gasịrị, na-atụ aro na ịkụ aka nke lncRNA a mere ka mbelata ọrịa cancer (Fig 1b).Nsonaazụ a kwadoro site na nyocha MTS na mkpụrụ ndụ cancer colorectal na-egosi na ọnụ ọgụgụ uto nke mkpụrụ ndụ knockdown DARS-AS1 bụ naanị ọkara ka e jiri ya tụnyere mkpụrụ ndụ nchịkwa (Njirimara 1c) ma kwekọọ na akụkọ ndị gara aga nke ọtụtụ ụdị ọrịa cancer ndị ọzọ.: ọrịa kansa akụrụ na-ekpochapụ nke ọma, ọrịa thyroid na ọrịa kansa akpa ume na-abụghị obere cell51,52,53,55.Otú ọ dị, ọrụ ya na usoro molekụla na ọrịa cancer colorectal ka bụ nke a na-achọpụtabeghị.Ya mere, anyị họọrọ lncRNA a maka ọmụmụ ihe ọzọ.
Iji mụọ okwu DARS-AS1 n'ime ndị ọrịa, anyị nyochara nke ọma ihe atụ etuto 10,327 sitere na ọrụ Cancer Genome Atlas (TCGA).Nsonaazụ anyị na-egosi na DARS-AS1 na-egosipụta nke ọma ma na-edozi ya na mkpụrụ ndụ ahụike dị iche iche, gụnyere colon adenocarcinoma (COAD), renal clear cell carcinoma (KIRC), na renal papillary cell carcinoma (KIRP)..Obere ole na ole (Fig 1d na ihe mgbakwunye 1a, b). Nyocha nke ụdị ahụike / tumor jikọtara ọnụ na-egosikwa na nkwupụta DARS-AS1 dị elu dị elu na etuto nke eriri afo urothelial carcinoma (BLCA), akụrụ na-egbuke egbuke cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), carcinoma squamous cell (LUSC). , Uterine corpus endometrial carcinoma (UCEC), lung adenocarcinoma (LUAD), imeju hepatocellular carcinoma (LIHC), akụrụ akụrụ papillary cell carcinoma (KIRP), na colon adenocarcinoma (COAD) (p uru <0.05) (Fig. 1e-m) . Nyocha nke ụdị ahụike / tumor jikọtara ọnụ na-egosikwa na nkwupụta DARS-AS1 dị elu dị elu na etuto nke eriri afo urothelial carcinoma (BLCA), akụrụ na-egbuke egbuke cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), carcinoma squamous cell (LUSC). , Uterine corpus endometrial carcinoma (UCEC), lung adenocarcinoma (LUAD), imeju hepatocellular carcinoma (LIHC), akụrụ akụrụ papillary cell carcinoma (KIRP), na colon adenocarcinoma (COAD) (p uru <0.05) (Fig. 1e-m) .Nyocha nke ụdị ahụike / tumor jikọtara ọnụ gosikwara na ngosipụta DARS-AS1 dị elu dị elu na eriri afọ urothelial carcinoma (BLCA), renal cell renal na renal cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), squamous cell carcinoma (LUSC) etuto ahụ., 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 nke corpus uteri (UCEC), adenocarcinoma nke ngụgụ (LUAD), hepatocellular carcinoma imeju (LIHC), papillary cell carcinoma nke akụrụ (KIRP), na adenocarcinoma nke colon (COAD) (p uru < 0.05) (Fig 1e- m) .配对 / 肿瘤样本 了 的 了 了 了 了 了 了 膀胱ars 尿路 (BLCA), K肾 肾 透明 (Kirc), Prad), 肺鳞状 (Lusc) 肿瘤 中 的更 更 (UCEC), 肺腺癌 (Luad), 肝肝 (lihc), 肾 肾 乳头状 (Kirp) (p 值<0.05) (1e-m) .对 健康 健康 分析 的 的 OS1 在 OS1 在 尿路 上 肾 肾 肾 细胞癌 细胞癌 细胞癌 细胞癌 腺腺癌 腺腺癌 腺腺癌 腺腺癌 腺腺癌 腺腺癌 腺腺癌 腺腺癌 腺腺癌 腺腺癌 腺腺癌 细胞癌 细胞癌(Lusc) 肿瘤 的 的 的 中 中 中 中 中 中 癌 癌 癌 ((uplet) 肝肝 (Luhc) 肾 肾 乳头状 细胞癌 (kirp) (coad) (p 值 <0.05) (图1e-m) .Nyocha nke ụdị ahụike / tumor jikọtara ọnụ na-akwado ọrụ DARS-AS1 na eriri afo urothelial carcinoma (BLCA), cell renal cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), na ụbụrụ 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). okwu na corpus uterine carcinoma (UCEC), adenocarcinoma akpa ume (LUAD), hepatocellular carcinoma (LIHC), renal papillary cell carcinoma (KIRP), na colon adenocarcinoma (COAD) (p value <0.05) (Figure 1e -m).N'ịchịkọta ọnụ, nsonaazụ ndị a na-egosi na DARS-AS1 na-egosipụtakarị na nke ukwuu na ọrịa cancer dị iche iche.
N'ihi na DARS-AS1 na DARS (gene encoding the antisense strand) na-ekerịta otu onye na-akwalite ma dị n'akụkụ ibe anyị, anyị mere shRNA ka ọ kụọ DARS-AS1 kpọmkwem ma ọ bụghị DARS (Mgbakwunye 2a,b na Tebụl mgbakwunye 2) .Na mgbakwunye na SW620, anyị jikwa ahịrị mkpụrụ ndụ atọ ndị ọzọ na-egosipụta nke ọma DARS-AS1 iji mụọ arụmọrụ na ọrụ nke knockdown shRNA (Ntinye mgbakwunye 3).Nsonaazụ anyị gosiri na shRNA atọ ahụ mepụtara nwetara ma ọ dịkarịa ala 80% DARS-AS1 knockdown arụmọrụ na-enwe mmetụta dị nta na ọnụọgụ nke DARS mRNA (Mgbakwunye 2c-f).Na mgbakwunye, anyị chọpụtara na DARS-AS1 knockdown na ndị a shRNAs gbochiri nke ukwuu cell uto na colorectal cancer cell ahịrị SW620 (49.7%) na HCT116 (27.7%), ara cancer cell line MBA-MD-231 (53.4%).) na HepG2 hepatoma cell line (92.7% Mbelata), yana ikike ha nwere ịmepụta oghere na-enweghị atụ (nkezi mbelata nke ~ 50.8%, 44.6%, 40.7% na 75.7% kwa cell line) (Fig. 2a,b).Na SW620, nsonaazụ nke nhazi nhazi nke colony gara n'ihu gosi na DARS-AS1 shRNA gbochiri mmụba nke cell na nkezi mbelata nke ihe dịka 69.6% (Fig. 2c).
Mmetụta nke njikwa shRNA na DARS-AS1 shRNA na mgbasawanye cell (a) na nhazi spheroid (b) na SW620, HCT116, MBA-MD-231, na sel HepG2.c Mmetụta njikwa shRNA na DARS-AS1 shRNA na nhazi colony na sel SW620.Mmụba cell (d), nhazi nke spheroid (e), na nhazi colony (f) nke sel SW620 na-egosipụta oke DARS-AS1.Data egosiri bụ ndapụta ± ọkọlọtọ nke nnwale atọ.* p ≤ 0.05, ** p ≤ 0.01, na *** p ≤ 0.001 site na t-ule nwata akwụkwọ nwere ọdụ abụọ.
Iji mejupụta ọmụmụ ihe na-abaghị uru, anyị na-ekepụta sel SW620 na-emebiga ihe ókè nke DARS-AS1 (Fig. 2g mgbakwunye).DARS-AS1 overexpression budata ụba cell (1.8-fold), nhazi spheroid na-adịghị edozi (1.4-fold), na nhazi colony (3.3-fold) na sel SW620 (Fig. 2d-f).Anyị kwadoro nsonaazụ a site na iji DARS-AS1 ọzọ na-ekwupụta ahịrị cell, A549.Mmụba cell a na-eme ka ọ dịkwuo mma n'ihi oke DARS-AS1 ka a na-ahụkwa na sel A549 (Fig. 2h, i na Mgbakwunye 3).N'ịchịkọta ọnụ, ihe ọmụmụ uru na ọnwụ ndị a na-egosi na DARS-AS1 na-akwalite mgbasa mkpụrụ ndụ cancer na vitro.
Iji nyochaa usoro dị n'okpuru nke DARS-AS1 na-achịkwa mgbasawanye cell, anyị mere nyocha RNA na-agbada iji chọpụta ndị mmekọ nwere ike ime protein.Nsonaazụ RT-qPCR gosiri na ihe dị ka 86.2% nke DARS-AS1 dị na cytoplasm nke sel SW620 (Fig 3a mgbakwunye).Ihe in vitro degharịrị biotinylated DARS-AS1 ma ọ bụ pseudoRNA ka ejiri lysates cell SW620 wee sochie nkewa SDS-PAGE.Ncha ọlaọcha sochirinụ gosiri na otu ihe dị iche iche (~ 38 kDa) abawanyela nke ukwuu na DARS-AS1 sere sample mana ọ bụghị na RNA dummy ma ọ bụ beads sample (Fig 3a).Achọpụtara band a dị ka PKR na-arụ ọrụ protein (PACT) site na mass spectrometry (MS) wee kwadokwuo site na immunoblotting na SW620, HCT116, na HepG2 cell line (Fig. 3a,b).A na-enyochakwa mmụba nke DARS na protein PACT ndị metụtara ya - PKR na TRBP - site na iji nyocha RNA site na Western blotting (WB).Nsonaazụ gosiri na ọnweghị mmekọrịta dị n'etiti DARS-AS1 RNA na protein atọ ndị a ka achọtara (Foto mgbakwunye 3b).Mmekọrịta dị n'etiti DARS-AS1 na PACT kwadoro site na nyocha nke RNA immunoprecipitation (RIP), nke gosipụtara na DARS-AS1 bara ụba nke ukwuu na ọgwụ mgbochi PACT mana ọ bụghị RNA ndị ọzọ na-achịkwa (Nyocha 3c).Iji chọpụta ma DARS-AS1 na PACT na-emekọrịta ihe ozugbo na enweghị akụrụngwa cellular ọ bụla ọzọ, a na-eme nyocha in vitro bilayer interferometry (BLI) site na iji PACT dị ọcha.Biotin nke akpọrọ DARS-AS1 ma ọ bụ dummy RNA bụ nke enweghị ike imegharị na streptavidin (SA) biosensors wee tinye ya na nchekwa kinetic nwere 1 μM PACT.N'ụzọ doro anya, PACT jikọtara ike na DARS-AS1 (Uru KD ~ 26.9 nM), mana ọ bụghị iji ṅomie RNA (Ọnọdụ 3d).Ejikọtara ọnụ, nsonaazụ ndị a na-egosipụta mmekọrịta kpọmkwem na mmekọrịta dị elu n'etiti DARS-AS1 na PACT.
Ntụle ndọtị RNA chọpụtara DARS-AS1 na-emekọrịta ihe na PACT na sel SW620.N'elu, ncha ọlaọcha nke protein ndị metụtara ya.Ejiri mgbochi mgbochi PACT mee obere immunoblots.A na-eme nyocha mgbada b RNA na HCT116 (n'elu) na sel HepG2 (ala).Achọpụtara nkwalite PACT site na immunoblotting.A na-eme nyocha immunoprecipitation cRNA (RIP) na mkpụrụ ndụ SW620 site na iji ọgwụ mgbochi egosipụtara.d PACT ejikọtara akụkụ na DARS-AS1 zuru oke ma ọ bụ njikwa RNA nwetara site na iji bilayer interferometry (BLI).Akwụsịghị RNA na biosensor streptavidin.Ejiri 1 μM PACT tụọ njikọ.A na-eme nyocha RNA pull site na iji biotinylated zuru oke DARS-AS1 ma ọ bụ truncated (n'elu).Immunoblot na-egosi PACT natara (ala).f PACT chara acha ọcha ka ejikọtara ya na DARS-AS1 ogologo biotinylated zuru oke ma ọ bụ gbarie (dị ka ọ dị na e) maka nyocha RIP in vitro.RT-qPCR kwadoro RNA ahụ ewepụtara.g E nwetara mmekọ nke iberibe RNA dị iche iche maka PACT site na iji interferometry bilayer.Maka nyocha, ejiri 100 nM RNA na 1 μM RAST mee ihe.h A na-eme nyocha in vitro RIP site na iji PACT emechara emecha ma ọ bụ nke akpọchiri akpọchi.RT-qPCR kwadoro RNA ahụ ewepụtara.i Ọnụego uto nke mkpụrụ ndụ SW620 na-egosipụta oke DARS-AS1, PACT, ma ọ bụ ha abụọ.Nkwusa oke nke ogologo zuru oke ma ọ bụ mkpọda DARS-AS1 na sel SW620 nwere mmetụta dị iche iche na uto cell.k Achọpụtara apoptosis site na immunoblotting nwere mgbochi PARP.l Knockout nke DARS-AS1 na-ebute apoptosis nke mkpụrụ ndụ SW620 dị ka cytometry na-asọpụta gosipụtara.Data egosiri bụ ndapụta ± ọkọlọtọ nke nnwale atọ. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p <0.0001, site n'ule nke ụmụ akwụkwọ nwere ọdụ abụọ. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p <0.0001, site n'ule nke ụmụ akwụkwọ nwere ọdụ abụọ. *p ≤ 0,05, **p ≤ 0,01, ***p ≤ 0,001, ****p <0,0001 site n'ike mmụọ nsọ. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p <0.0001 site na t-ule nwata akwụkwọ nwere ọdụ abụọ. *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 na двустороннему критерию Стьюдента. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p <0.0001 site na t-ule nwata akwụkwọ nwere ọdụ abụọ.
Anyị meziri iberibe atọ biotinylated DARS-AS1 RNA site na ntụgharị in vitro iji mata mpaghara DARS-AS1 achọrọ maka mkpakọrịta PACT (Nyocha 3e).Nsonaazụ nyocha nke RNA gosiri na iberibe ọ bụla nwere ike imekọrịta ihe na PACT, mana mpaghara 3′-terminal (384-768 nucleotides akpọrọ A3) gosipụtara karịa 1-384 nucleotides akpọrọ A1) (Fig 3e).Ahụrụ nsonaazụ ndị yiri ya na in vitro RIP assay site na iji recombinant PACT (Foto 3f).N'ikwekọ na nsonaazụ ndị a, nnwale iji kechie iberibe RNA na-enweghị ike na PACT site na iji BLI gosikwara na PACT nwere mmekọrịta dị elu maka A3 (384-768 nt) (Uru KD nke ihe dịka 94.6 nM), ebe ọ fọrọ nke nta ka ọ bụrụ njikọ na mpaghara ndị ọzọ.(Foto 3d).
Anyị nyochakwara mpaghara njikọ ejikọta na PACT.PACT nwere ngalaba na-arụ ọrụ atọ, abụọ n'ime ha bụ nke echekwara RNA-binding ngalaba (dsRBD) na ngalaba nke atọ (nke akpọrọ D3) nke na-arụ ọrụ dị ka onye na-eme ka mmekọrịta protein rụọ ọrụ.Iji nyochaa ikike njide lncRNA nke ngalaba ọ bụla, anyị rụpụtara mmụgharị atọ wepụrụ ngalaba atọ nke ọ bụla wee mee nyocha in vitro RIP.Nsonaazụ anyị gosiri na ihichapụ ngalaba nke atọ (D3) nke PACT belatara mmekọrịta ya na DARS-AS1 (site na 0.11-fold ma e jiri ya tụnyere PACT na-emebibeghị) ma e jiri ya tụnyere mgbanwe abụọ ọzọ (Fig 3h), e gosipụtara na ntọhapụ ahụ. nke D3 nwere mmekọrịta ya na DARS.-AC1.Ejikọtara ọnụ, nsonaazụ ndị a na-atụ aro na mmekọrịta dị n'etiti DARS-AS1 na PACT nwere ike ịmalite site na njedebe 3′ nke DARS-AS1 na ngalaba D3 nke PACT.
Anyị chọpụtara na DARS-AS1 enweghị mmetụta na okwu PACT na PACT enweghị mmetụta na DARS-AS1 (Mgbakwunye 3c).Anyị wee nyochaa mmetụta PACT kụkwasịrị na uto cell.N'adịghị ka DARS-AS1, sel ndị ikwu na-eto 1.5-3 ugboro ngwa ngwa mgbe PACT kụdara (Mgbakwunye Fig. 3d).Nsonaazụ nke nhazi nhazi nke colony gosiri na mkpụrụ ndụ kpụrụ 2-3-fold colonies mgbe ọgwụgwọ shRNA na PACT (Mgbakwunye Fig. 3e).Iji nwalee ma DARS-AS1 na-achịkwa mmụba cell site na PACT, anyị mepụtara mkpụrụ ndụ SW620 na-egosipụta PACT, DARS-AS1, ma ọ bụ ha abụọ.Ngosipụta nke PACT gosipụtara oke mgbochi nke mmụba cell (Nyocha 3i).Ọ bụ ezie na DARS-AS1 overexpression kwa se kwalitere mmụba nke mkpụrụ ndụ, ọ nweghị nnukwu ọdịiche dị na uto nke mkpụrụ ndụ na-emebiga ihe ókè DARS-AS1 na PACT.Nsonaazụ ndị a na-egosi na PACT nwere ike igbochi mmụba mmụba nke DARS-AS1 kpatara.
Ebe ọ bụ na mpaghara dị iche iche nke DARS-AS1 nwere ikike dị iche iche na-ejikọta PACT, anyị nyochara mmetụta ha nwere na mmụba cell site na oke dị iche iche nke DARS-AS1 iberibe.N'iji ya tụnyere iberibe abụọ ndị ọzọ, DARS-AS1 na-emebiga ihe ókè na njedebe 3' (384-768 nt), isi mpaghara metụtara PACT na DARS-AS1, bụ nke nwere ikike kachasị elu iji kpalite mmụba cell (Fig 3j).Nsonaazụ ndị a na-egosi njikọ dị mma n'etiti ikike njide na ọrụ ndu nke DARS-AS1.
A kọwo PACT ka ọ bụrụ protein pro-apoptotic19.Ya mere, anyị nyochara mmetụta DARS-AS1 na apoptosis.Dị ka a na-atụ anya ya, DARS-AS1 knockdown budata mụbara nkwụsị nke PARP na sel SW620 wee mee ka ọnụọgụ nke annexin V-positive cell na SW620, HCT116, HepG2, na MBA-MD-231 cell line (Fig 3k).3).3f-h), na-egosi na mmetụta mgbochi apoptotic nke DARS-AS1 na mkpụrụ ndụ kansa na-emegide ọrụ apoptosis na-ebute PACT.Ejikọtara ọnụ, nsonaazụ ndị a na-egosi na usoro nke DARS-AS1 oncogenic ọrụ nwere ike ịbụ site na igbochi ọrụ PACT.
Na-esote, anyị nyochara mmetụta arụ ọrụ nke mkpakọrịta DARS-AS1-PACT.A kọwala PACT ka ọ rụọ ọrụ PKR site na mmekọrịta ozugbo, nke mechaa welie eIF2a phosphorylation, na-eme ka mkpochapụ ntụgharị asụsụ na apoptosis17.Nke mbụ, anyị nyochara ma DARS-AS1 ọ na-emetụta mkpokọta cellular PACT na PKR.Igwe nyocha nke confocal fluorescence gosiri na PACT na PKR bụ nke ukwuu na-agbakọta na sel SW620 yana nkezi njikọ njikọ Pearson nke 0.72.Ka ọ dị ugbu a, DARS-AS1 overexpression belatara nke ukwuu PACT na PKR co-localization (pụtara Pearson correlation coefficient 0.61) (Figure 4a).Iji nyochaa ma DARS-AS1 nwere ike gbanwee mmekọrịta PACT-PKR, anyị mere nyocha co-immunoprecipitation (co-IP) na mgbochi PACT na SW620 cell lysates.PKR nwere ụbara nke ukwuu na mgbochi PACT na sel njikwa, ebe mgbake PKR belatara nke ukwuu na lysates sitere na sel na-egosipụta DARS-AS1 (Fig 4b).Ejiri PACT na PKR dị ọcha mee ihe maka nyocha njikọ protein in vitro.N'ihi ya, ndị nyere DARS-AS1 ma ọ nweghị njikwa RNA gosipụtara mmekọrịta PACT-PKR kwụsịrị (Njirimara 4c).Nsonaazụ niile gosiri na DARS-AS1 mebiri nkwukọrịta PACT na PKR.
A na-ahụta ngalaba nke PACT na PKR na sel njikwa ma ọ bụ sel na-egosipụta oke DARS-AS1 site na iji microscopy confocal fluorescence.E jiri DAPI mee ntụpọ ahụ.E nwetara nsonaazụ ọnụ ọgụgụ site na foto iri na isii.b Co-immunoprecipitation (co-IP) iji mgbochi PACT antibody na cell lysates nke njikwa sel SW620 ma ọ bụ sel na-egosipụta DARS-AS1.c akpọrọ PACT, PKR dị ọcha ma degharịa ya na vitro na DARS-AS1 ma ọ bụ RNA mock ka etinyere maka nyocha njikọ protein in vitro.A na-eji ọgwụ mgbochi ọkọlọtọ mee ihe maka mgbochi mgbochi.d Immunoblots nwere ọgwụ mgbochi egosiputara na SW620 na sel HCT116 bufere shRNA njikwa ma ọ bụ DARS-AS1-shRNA sochiri agụụ serum.e DARS-AS1 ọkwa ọkwa gbanwere nghọta ekwentị na thapsigargin.Ejiri DARS-AS1 shRNA, DARS-AS1 overexpression plasmid ma ọ bụ akara plasmid bufere mkpụrụ ndụ SW620.A na-agwọ mkpụrụ ndụ na thapsigargin maka awa 48 ma kpebie ike ndụ cell site na iji MTS reagent.f In vitro edegharịrị DARS-AS1 ma ọ bụ dummy RNA na PACT dị ọcha ka ejiri mee nyocha ọrụ in vitro na nchọpụta immunoblot.g Immunoblots na-eji ọgwụ mgbochi ndị a emebere na sel SW620-ctrl (aka ekpe) ma ọ bụ sel na-egosipụta oke PKR mutant (n'aka nri).Ejiri shRNA njikwa ma ọ bụ DARS-AS1-shRNA bugharịa mkpụrụ ndụ ndị a site na agụụ serum.h Flow cytometry gosiri na arụghị ọrụ nke mutant PKR kwụrụ ụgwọ maka apoptosis DARS-AS1 kpatara na sel SW620.A na-eme Immunoblots nwere ọgwụ mgbochi egosipụtara na sel SW620 (aka ekpe) ma ọ bụ HCT116 (n'aka nri).Selụ nke ejiri njikwa shRNA ma ọ bụ DARS-AS1 shRNA na-anapụ serum ma gbakwunye ya na 100 nM PKR C16 inhibitor ma ọ bụ DMSO.Ogwe ọnụ ọgụgụ = 5µm.Data egosiri bụ ndapụta ± ọkọlọtọ nke nnwale atọ.* p ≤ 0.05 t-ule nwata akwụkwọ nwere ọdụ abụọ.
A na-ekwenyekarị na ozugbo PACT na PKR17 na-emekọrịta ihe, enwere ike ịmalite PKR phosphorylation na Thr451.Nsonaazụ anyị gosiri na ọkwa PKR phosphorylation dị elu nke ukwuu na DARS-AS1 knockdown sel mgbe agụụ serum (Fig. 4d na mgbakwunye Fig. 4a).N'ihi nke a, anyị chọpụtara na phosphorylation nke eIF2a, isi ihe nke PKR, ka DARS-AS1 shRNA mụbara nke ukwuu (Fig. 4d na Fig. 4a).Thapsigargin bụ ihe nrụgide ER na-eme ka ER hapụ Ca2+.A kọwala ọgwụgwọ na thapsigargin iji mee ka nkwupụta na ịgbalite PACT, nke na-emekọ ihe ọzọ na-eme ka PKR rụọ ọrụ, na-eduga na apoptosis site n'ịba ụba eIF2α phosphorylation 18,61.N'ebe a, anyị na-eji thapsigargin dị ka onye na-akpali akpali nke ụzọ PACT / PKR iji nyochaa ma DARS-AS1 nwere ike inyere mkpụrụ ndụ aka imeri nrụgide site na igbochi ụzọ PACT / PKR.Anyị chọpụtara na ọkwa DARS-AS1 nwere njikọ chiri anya na nguzogide cell na thapsigargin.Mkpụrụ ndụ SW620 na-emebiga ihe ókè DARS-AS1 dị ndụ nke ọma mgbe a na-agwọ ya na thapsigargin, ebe sel ndị nwere DARS-AS1 knockdown bịara dịkwuo mfe (Fig 4e).N'ikwekọ na nsonaazụ ndị a, DARS-AS1 overexpression belatara thapsigargin-induced PKR phosphorylation (Mgbakwunye Fig. 4b).N'ụzọ dị iche, mgbe ọgwụgwọ thapsigargin gasịrị, PKR na eIF2a bụ phosphorylated ruo n'ókè dị elu na DARS-AS1 knockdown sel ma e jiri ya tụnyere sel njikwa (Mgbakwunye 4b).N'ụzọ na-akpali mmasị, thapsigargin kpalitere okwu DARS-AS1 n'ụzọ na-adabere na dose, nke nwere ike igosi ọrụ mgbochi nchekasị nke DARS-AS1 (Mgbakwunye 4c).Na mgbakwunye, anyị mere nyocha ịgbalite in vitro iji kwado nlebanya ndị a.Na nkenke, a sachara PKR na cell lysates site na iji ọgwụ mgbochi PKR, wee tinye ya na PACT recombinant na DARS-AS1 edegharịrị na vitro.Mgbe mmeghachi omume enzymatic ahụ gasịrị, ahụrụ phospho-PKR site na iji WB.Nsonaazụ anyị gosiri na DARS-AS1 gbochiri PKR phosphorylation nke ukwuu, mana ọ bụghị site na njikwa RNA (Njirimara 4f).Nsonaazụ in vitro na in vivo ndị a na-atụ aro na DARS-AS1 na-egbochi nnabata PKR na-agbado PACT.N'otu oge ahụ, anyị hụkwara mbelata na mgbake PACT n'ihu DARS-AS1 (Figure 4f).Nsonaazụ a dabara na nsonaazụ nke nyocha protein in vitro (Njirimara 4c) wee gosipụtakwa ọrụ mgbochi nke DARS-AS1 maka mkpakọrịta PACT-PKR.
Ser246 na Ser287 na ngalaba D3 nke PACT achọrọ maka ịgbalite PKR n'okpuru nrụgide cellular.Mgbanwe nke ihe fọdụrụ serine abụọ maka alanine nyere mutant PACT (mutD), nke mere PKR rụọ ọrụ na enweghị nchekasị, na nnọchi alanine (mutA) tụgharịrị ụkpụrụ ahụ.Ebe ọ bụ na anyị egosipụtala mkpa nke ngalaba a na njikọ aka kpọmkwem na DARS-AS1, anyị mepụtara mutant abụọ PACT iji nwalee ma ihe fọdụrụ ndị a nwekwara ike itinye aka na mmekọrịta na DARS-AS1.N'ụzọ na-akpali mmasị, mutant abụọ ahụ tụfuru ikike ijikọ na DARS-AS1 (Mgbakwunye 4d), na-atụ aro na a ga-achọ nhazi zuru oke nke protein PACT maka mmekọrịta dị mma na DARS-AS1.
Ọzọkwa, nsonaazụ anyị na-atụkwa aro na mgbochi DARS-AS1-shRNA na-eme ka mgbochi nke mmụba cell nwere ike weghachi nke ọma site n'ịmebiga ihe ókè PACT mutant (PACTmutA) ma ọ bụ ihe na-adịghị mma PKR mutant (PKRmut) (Mgbakwunye 4e. e).Ngosipụta nke oke-adịghị mma nke PKR mutants belatara PKR phosphorylation nke DARS-AS1 knockdown na-ebute yana eIF2a phosphorylation na mkpụrụ ndụ na-enweghị serum (Fig. 4g).Nke ka mkpa, ọnụ ọgụgụ nke mkpụrụ ndụ apoptotic kpatara site na DARS-AS1 knockdown na-ebelatakwa na sel na-egosipụta oke PKRMut (Fig. 4h na Fig. 4g).Mgbochi nke ọrụ PKR kinase na-emebikwa ọrụ DARS-AS1, dịka nsonaazụ gosiri na DARS-AS1 knockdown adịkarịghị kpalite PKR na eIF2a phosphorylation mgbe a na-emeso mkpụrụ ndụ na PKR-kpọmkwem C16 inhibitor (Fig. 4i na Fig. 4h).).Ejikọtara ọnụ, nsonaazụ anyị na-atụ aro na DARS-AS1 na-akwalite mmụba cell, opekata mpe n'akụkụ, site na igbochi PACT-mediated PKR.
Iji nyochaa ọrụ DARS-AS1 na tumorigenesis, anyị mere nnwale vivo site na iji ụdị xenograft òké. Nsonaazụ na-egosi na ịkụda DARS-AS1 n'ike n'ike belatara uto tumor na ụmụ oke (p value <0.0001) (Fig. 5a). Nsonaazụ na-egosi na ịkụda DARS-AS1 n'ike n'ike belatara uto tumor na ụmụ oke (p value <0.0001) (Fig. 5a). Результаты показывают, что нокдаун DARS-AS1 резко снижает рост опухоли у мышей (значение p <0,0001) (рис. Nsonaazụ gosiri na DARS-AS1 knockdown na-ebelata oke tumor na ụmụ oke (p value <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）。 Результаты показали, что нокдаун DARS-AS1 значительно снижает рост опухоли у мышей (значение р <0,0001) (5). Nsonaazụ gosiri na DARS-AS1 knockdown belatara uto tumor na ụmụ oke (p value <0.0001) (Figure 5a).Ya mere, na DARS-AS1 knockdown otu, e nwere nnukwu mbelata nke pụtara tumor olu ihe dị ka 72.9% na pụtara etuto uka ihe dị ka 87.8% (Nyocha 5b-d).Nsonaazụ ndị a na-ekwusi ike na DARS-AS1 nwere ike ịkwalite uto tumor na vivo.
Mmetụta mgbasa ozi DARS-AS1 knockdown na colorectal oncogenesis na ụmụ oke gba ọtọ.A na-egosi akụkụ nke uto (a), nha etuto (b), ịdị arọ (c), na onyonyo etuto (d).Ogwe mperi na-anọchi anya ±SEM. n = 10. ****p <0.0001, site n'ule nwata akwụkwọ nwere ọdụ abụọ. n = 10. ****p <0.0001, site n'ule nwata akwụkwọ nwere ọdụ abụọ. n = 10. ****p <0,0001 по двусторонему критерию Стьюдента. n = 10. ****p <0.0001 t-ule nwata akwụkwọ nwere ọdụ abụọ.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 двусторонему критерию Стьюдента. ****p <0.0001 t-ule nwata akwụkwọ nwere ọdụ abụọ.e Kaplan-Meier nyochara njikọ dị n'etiti ọkwa nkwupụta DARS-AS1 na ndụ zuru oke na ndị ọrịa nwere UVM, KICH, KIRP, MESO, GBM, na LGG.Ọnụ ọgụgụ dị elu nke okwu DARS-AS1 na ndị ọrịa nọ n'elu 50%;ọkwa dị ala nke okwu DARS-AS1 na ndị ọrịa nọ na ala 50%.A na-ekpebi p-values site na iji ule ọkwa ndekọ.f Ụdị atụpụtara nke DARS-AS1 na-ahazi ụzọ PACT-PKR na uto tumor.
Iji ghọta mmetụta ahụike nke DARS-AS1 nke ọma, anyị nyochara njikọ dị n'etiti okwu ya na ndụ onye ọrịa.Site na nyochaa dataset TCGA, anyị chọpụtara na okwu DARS-AS1 dị elu jikọtara ya na uveal melanoma (UVM), renal chromophobia (KICH), renal papillary cell carcinoma (KIRP), mesothelioma (MESO), multiplex.A na-ejikọta nlanarị dị ala na glioblastoma morphosis (GBM) na ndị ọrịa nwere glioma ụbụrụ dị ala (LGG) (Nyocha 5e).Nsonaazụ ndị a na-egosi na DARS-AS1 nwere ike ịrụ ọrụ dị mkpa na ọganihu akpụ akpụ na-agwọ ọrịa ma nwee ike bụrụ ihe na-ebu amụma biomarker na ọtụtụ ọrịa cancer.
N'ime ọmụmụ ihe a, na-eji nnukwu nyocha ọrụ CRISPRi, anyị kpebiri na DARS-AS1 lncRNA na-emeri nchekasị cell cell site na ịhazi ndị na-azaghachi nrụgide isi abụọ, PACT na PKR.Site na ịmekọrịta kpọmkwem na PACT, DARS-AS1 gbochiri PACT-mediated PKR activation, si otú ahụ gbochie ọnwụ cell apoptotic na ịkwalite mgbasawanye cell (Fig 5f).A hụla nkwalite DARS-AS1 n'ọtụtụ ụdị ọrịa kansa, na-atụ aro na ọrụ ya nke ịkwalite ndụ mkpụrụ ndụ kansa n'okpuru ọnọdụ nrụgide nwere ike itinye aka na ọtụtụ ụdị ọrịa kansa.
Achọpụtara PACT dị ka protein activator PKR, na PACT-mediated PKR activation na-arụ ọrụ dị mkpa na nzaghachi nrụgide site na ịhazi ederede, ntụgharị, apoptosis, na usoro cellular ndị ọzọ dị mkpa62.Ruo ọtụtụ iri afọ, agbaala mbọ ịghọta ụkpụrụ ọrịa kansa nke PACT-PKR cascade.N'ebe a, ọmụmụ ihe anyị kpughere usoro dị iche iche nke iwu PACT-PKR na mkpụrụ ndụ kansa site na cellular lncRNA DARS-AS1, nke na-ejikọta ya na PACT, na-egbochi mmekọrịta PACT-PKR, na-egbochi ọrụ PKR na eIF2α phosphorylation, si otú ahụ na-egbochi apoptosis na-akpata nchekasị. na-akpali akpali n'ikpeazụ ọrịa cancer.mkpụrụ ndụ.Nchọpụta a na-eme ka ìhè dị na ebumnuche lncRNA nwere ike ime maka prognosis na ọgwụgwọ ọrịa kansa.
Data anyị gosiri na DARS-AS1 knockdown na-eme ka mkpụrụ ndụ nwee agụụ agụụ na mmụba dị ukwuu na PKR phosphorylated na eIF2a.Nsonaazụ ndị a na-egosi na DARS-AS1 na-akwalite ndụ mkpụrụ ndụ kansa n'okpuru ọnọdụ siri ike site na igbochi ọrụ PACT/PKR.Ọtụtụ RNA ndị ọzọ na-abụghị koodu, dị ka ASPACT na nc886, na-etinyekwa aka na PACT/PKR axis site na iwetu PACT48 mRNA ma ọ bụ na-achịkwa autophosphorylation site na ijikọ na PKR49,50,64.N'ime ha, DARS-AS1 na-eme dị ka onye na-akpaghasị njikọ PACT-PKR.Ọmụmụ ihe a na-eme ka nghọta anyị gbasara ụkpụrụ PACT/PKR axis yana ọrụ lncRNA na nzaghachi nrụgide.
PACT nwere ngalaba atọ dị iche iche.Nke ọ bụla n'ime dsRBD abụọ mbụ zuru oke iji nweta njikọ dị elu nke PACT na PKR, ebe achọrọ ngalaba nke atọ (D3) maka ịgbalite PKR in vitro na vivo.Ọmụmụ ihe anyị gosiri na DARS-AS1 na-emekọrịta ihe na ngalaba D3 (Fig 3h).Nyere nnukwu nha nke lncRNA (768 nucleotides), DARS-AS1 na-ejikọta na D3 nwere ike igbochi mmekọrịta dị n'etiti PACT ngalaba nke dsRBD na PKR, si otú ahụ gbochie njikọ nke PACT na PKR.Mgbanwe nke PACT nke dochie Ser246 na Ser287 na D3 na alanine ma ọ bụ aspartate mebiri njikọ ya maka DARS-AS1, na-arụtụ aka na mkpa nke D3's n'ozuzu ihe nhazi na ọkụ eletrik na mkpakọrịta ha.A ga-achọrọ nkọwa ndị ọzọ gbasara usoro a n'ọdịniihu, na-eji nyocha nke biochemical karịa yana nyocha nhazi nke PACT dị elu.
Nnyocha ndị gara aga akọwo na DARS-AS1 na-akwalite mmụba cell site na ọtụtụ usoro51,52,53.N'otu ihe atụ, ndị nchọpụta chọpụtara na DARS-AS1 kwalitere mkpụrụ ndụ ihe nketa nke antisense protein-encoding DARS site n'ichekwa miP-194-5p na mkpụrụ ndụ kansa akụrụ.Otú ọ dị, na ọmụmụ ihe ugbu a, DARS-AS1 knockdown nwere mmetụta dị nta na ntinye akwụkwọ DARS n'ọtụtụ ụdị ọrịa cancer, gụnyere ma ọ dịkarịa ala colorectal, ara, na ọrịa imeju.N'ihi na lncRNA na-egosipụta usoro nkwupụta mkpụrụ ndụ na nke anụ ahụ, enweghị ike ichekwa usoro arụ ọrụ n'ofe ụdị ọrịa kansa, nke nwere ike itinye aka na nghọtahie dị n'etiti ihe anyị chọpụtara na nyocha gara aga nke ọrịa cancer dị iche iche.Ọmụmụ ihe pụrụ iche dị mkpa iji kọwapụta usoro dị iche iche nke usoro physiological na pathological dị iche iche.
Nnyocha nke data ụlọ ọgwụ gosiri na okwu DARS-AS1 na etuto ahụ na-ejikọta ya na ndụ nke ndị ọrịa cancer, nke na-egosi mkpa nke DARS-AS1/PACT/PKR axis na ọrịa cancer prognosis.N'ikpeazụ, ọmụmụ ihe anyị na-egosi na DARS-AS1 bụ onye na-achịkwa ihe mgbaàmà nke PACT / PKR, na-akwalite mgbasa mkpụrụ ndụ cancer, ma na-egbochi apoptosis n'oge nzaghachi nrụgide, nke na-enye usoro nyocha ọzọ ma nwee mmasị maka nyocha n'ọdịnihu n'ime ọgwụgwọ ndị nwere ike. .
E nwetara ahịrị mkpụrụ ndụ mmadụ SW620, A549, MBA-MD-231, HCT116, HepG2 na HEK293T sitere na akụrụngwa akụrụngwa akụrụngwa nke National Cell Line na China.A na-echekwa mkpụrụ ndụ niile na DMEM ọkara (DMEM, Thermo Fisher Scientific, Waltham, MA) gbakwunyere na 10% FBS (Gemini, Brooklyn, NY) na 1% penicillin-streptomycin (Thermo Fisher Scientific) na 37 ° C, 5% CO2.incubator.
Anti-PACT, Abcam (ab31967);Anti-PKR, Abcam (ab184257);Anti-PKR (phospho-T451), Abcam (ab81303);Mgbochi ọkọlọtọ, Abcam (ab125243);Anti-eIF2α, Abcam (A0764));mgbochi eIF2a (phosphorus S51), Abcam (ab32157);mgbochi PACT (phosphorus S246), Abgent (AP7744b);mgbochi β-tubulin, CST (2128);òké nkịtị IgG, CST (5415S);oke bekee IgG, CST (2729S).A gbajiri ọgwụ mgbochi 1:1000 na PBST maka ihichapụ Western yana 1:100 maka IP.
Emepụtara sgRNA site na iji ngwa ọhaneze dị akpọrọ CRISPR-ERA66.Anyị na-eji paramita ngwa ndabara maka mmepe sgRNA yana algọridim gbakọọ saịtị njikọ sgRNA na mpaghara 3 kb.dabere na TSS.Ejikọtara ọdọ mmiri nke sgRNA oligonucleotides na CustomArray, Inc. (Bothewell, WA) wee mechie n'ime pgRNA plasmids (Addgene #44248).Ngụkọta 12 µg nke pgRNA plasmid nke mmadụ, 7.2 µg nke psPAX2 (Addgene #12260), na 4.8 µg nke pMD2.G (Addgene #12259) ka ebufere n'ime 5 x 106 HEK293T na DNA Transfection cm 10. sel (CWBIO, Beijing, China) na-agbaso ntuziaka onye nrụpụta.Achịkọtara ihe ndị nwere nje virus n'ime awa 48 na 72 ka ebufechara ya wee kpochaa site na nzacha 0.45µm.Maka nyocha, mkpụrụ ndụ SW620 na-ekwupụta protein fusion dCas9/KRAB nwetara site na ntụgharị nje.sel SW620 emezigharịrị butere ọbá akwụkwọ nje ahụ n'ime nnwale ọrịa anọ nọọrọ onwe ya na MOI nke 0.1-0.3 wee were 2 μg/ml puromycin (Sigma, St. Louis, MO) were were were were 2 μg/ml puromycin (Sigma, St. Louis, MO) were were ụbọchị 2.Mgbe nke ahụ gasịrị, a zụrụ mkpụrụ ndụ maka ụbọchị 18 in vitro nwere mkpuchi ọbá akwụkwọ kacha nta nke sel 500/sgRNA maka nyocha.
Ewepụtara Genomic DNA dịka ntuziaka nke QIAamp DNA Blood Midi Kit (QIAGEN, Düsseldorf, Germany; 51183).Na mkpokọta, 100 μg nke genomic DNA kwa ugboro ugboro ka ejiri wuo ọba akwụkwọ ahụ.Ejiri PCR abụọ gbasaa mpaghara sgRNA wee jikọta ya na koodu mkpọchi.
A na-asachapụ ngwaahịa PCR site na iji NucleoSpin® gel na PCR purification kit (MACHEREY-NAGEL, Düren, Germany; 740609.250) wee tụọ ya site na iji Qubit ™ HS ihe nchọpụta DNA nwere eriri abụọ (Thermo Fisher Scientific; Q32854).
A na-eji nyocha MTS iji tụọ mmụba cell.A na-akụ mkpụrụ ndụ mkpụrụ ndụ n'ime efere 96 nke ọma na njupụta mbụ nke sel 2000 / olulu mmiri.A na-atụ ọnụ ọgụgụ sel ndị ikwu kwa ụbọchị n'oge egosipụtara maka ngụkọta nke ụbọchị 4-6.Maka olulu mmiri ọ bụla, 20 μl nke MTS reagent (Promega) ka ejiri 100 μl nke DMEM tinye ya na sel maka 4 h na 37 Celsius C, wee tụọ OD490.
Achọpụtara ikike nke uto na-enweghị mgbakasị site na nyocha nhazi nke spheres.Na nkenke, sel 2000 bufere shRNA DARS-AS1 ma ọ bụ njikwa shRNA ka emebere ya na microplates mgbakwunye dị ala (Corning) na-agbanwe agbanwe kwa ụbọchị anọ.A na-agụta spheroids mgbe ụbọchị iri na anọ gachara.A na-eji mkpụrụ ndụ 500 na-ebugharị na DARS-AS1 overexpression plasmid ma ọ bụ plasmid njikwa maka nyocha nkwalite, ma ọ bụghị na usoro ahụ agbanweghị.
Edere RNA site na iji T7 RNA polymerase na biotin-16-UTP (Roche 1138908910) dịka ntuziaka nke Riboprobe® Combination Systems (Promega P1440).Edepụtara primers ndị a na-eji ebe a na tebụl mgbakwunye 4.
Edebere mpaghara PACT ma ọ bụ PKR na-edozi protein ka ọ bụrụ pET15b (Addgene #73619) wee gbanwee ghọọ BL21(DE3).A na-ebute nje bacteria n'otu abalị na LB nke a na-enye ampicillin wee were LB ọhụrụ gbanye 100-fold.Mgbe OD600 nke ọkara ruru 0.8, 1 mM IPTG agbakwunyere iji mee ka okwu protein pụta.Mgbe incubation n'abali na-ama jijiji (250 rpm na 20 Celsius C), a na-anakọta pellet cell site na centrifugation (4000 rpm, 10 min, 4°C).Weghachite pellet cell na ihe nchekwa lysis (50 mM Tris, pH 8.0, 250 mM NaCl, 1 mM PMSF) wee tinye na ice maka 30 min, wee sonicate (15 min, 5 s on/off, on ice) na centrifuge (13,000). rpm)., 30 min, 4°C).A na-ebunye ihe dị elu na Ni-NTA resin (QIAGEN) ugboro 3 na 4 Celsius C, jiri ihe nchekwa sachaa ugboro anọ (50 mM Tris, pH 8.0, 40 mM imidazole, 250 mM NaCl) wee elu ugboro 3, yana ngụkọta. nke 10 ml eluent buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 300 mM imidazole).Ekpebiri protein dị ọcha site na iji WB wee kpebisie ike itinye uche site na iji ngwa nyocha protein Qubit ™ (Thermo Fisher Scientific; Q 33212).
Emere nyocha RIP dị ka akọwara na mbụ, yana mgbanwe.Na nkenke, 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 mmanya. (Roche, 1 mM DTT) na centrifuge na 13,000 rpm maka 15 min na 4 Celsius C.Ejiri protein A+G magnetik (Millipore) jikọtara ya na 5 μg nke mgbochi mgbochi PACT (Abeam) ma ọ bụ IgG (CST).Ejiri 5x RIP buffer sachaa beads ahụ ugboro ise, wee gbarie ya na proteinase K (NEB).Ejiri Trizol weputara RNA wee kpebie site na RT-qPCR.A na-egosi ọkwa ọkwa na tebụl mgbakwunye 5.
Emere nyocha in vitro RIP dị ka ọkọlọtọ RIP assay protocol gbanwetụrụ.Ngụkọta 5 pmol nke in vitro edegharịrị RNA gbajiri 1x na ihe nchekwa RIP wee kpochie ya site na incubation na 65°C maka nkeji 5 na-esochi nwayọ nwayọ ruo ụlọ okpomọkụ.Ngụkọta 5 pmol nke protein na-adịghị mma ma ọ bụ mutated nke akpọrọ ọkọlọtọ PACT dị ọcha site na E. coli.Tinye RNA renatured maka awa 2 na 4 Celsius ma soro usoro dị n'elu maka nyocha RIP maka IP mgbochi ọkọlọtọ.
Maka nyocha ndọtị RNA, sel 1 × 107 ejiri 1xRIP kpuchie ya.Mgbe centrifugation na 13,000 rpm maka 15 min na 4 Celsius C, a na-eji 30 μl nke streptavidin magnetik beads (Beckman) mee ihe dị elu maka 2 h na 4 ° C.E wetara lysate a dị ọcha na tRNA yist wee tinye 40 pmol nke RNA renatured n'abali na 4 Celsius, wee tinye maka awa 2 ọzọ na 20 μl nke ọhụrụ streptavidin magnetik beads nke ejiri BSA kpuchiri.Nzọụkwụ ịsa ahụ nwere ugboro anọ nwere ihe nchekwa RIP 5x yana ugboro anọ nwere ihe nchekwa 1x RIP.E jikwa biotin elution buffer (25 mM Tris-HCl, pH 7.5, 12.5 mM D-biotin, PMSF) wee kewaa na NuPAGE 4-12% Bis-Tris Gel (Invitrogen).Mgbe ntụpọ ọla ọcha gachara (Beyotime Biotechnology), MS wepụrụ wee nyochaa ụfọdụ ndị egwu.
Emere nyocha ngalaba-IP iji nwalee mmekọrịta dị n'etiti PACT na PKR.Na nkenke, a kwadebere lysates dị elu site n'itinye sel 1 x 107 lysed na 1 x RIP buffer sochiri centrifugation na 13,000 rpm maka nkeji 15 na 4 Celsius.Ebujuru lysates na protein A + G magnetik beads, jikọtara ya na 5 μg nke mgbochi PACT, ma jiri nwayọ tụgharịa n'otu abalị na 4 Celsius.Ejiri ihe nchekwa 5 × RIP sachara chaịlị ahụ ugboro 3, jiri ihe nchekwa 1 × RIP mee ya ugboro abụọ wee jiri ihe nchekwa 1 × SDS kpuchie ya.SDS-PAGE gel nyochara protein ahụ enwetara wee chọpụta ya site na WB.
A sachara pmol abụọ nke PACT ọkọlọtọ na 1 pmol nke PKR site na E.coli.Tinye n'ime ihe nchekwa 1 × RIP ma tinye 10 pmol nke RNA renatured maka awa 2 na 4 Celsius.Mgbe nke ahụ gasịrị, etinyere ha na protein A+G magnetik bead conjugated anti-labeled antibody maka awa abụọ ọzọ.Ejiri 1x RIP buffer sachaa chaịlị ndị ahụ ugboro anọ wee jiri 1x SDS buffer kpuchie ya.WB chọtara PACT na PKR nke pụtara.
Oge nzipu: Sep-23-2022
