Simulated Microgravity Influences Immunity-Related Biomarkers hauv ntsws Cancer
Aug 10, 2023
Abstract: Microgravity yog ib lub tswv yim tshiab uas tuaj yeem ua cov cuab yeej ntxiv los tsim kho mob qog noj ntshav yav tom ntej. Hauv kev mob ntsws cancer, kev cuam tshuam ntawm microgravity ntawm cov txheej txheem cellular thiab lub peev xwm tsiv teb tsaws ntawm cov hlwb tau hais zoo. Txawm li cas los xij, nws cov txiaj ntsig ntawm cov txheej txheem uas ua rau kev mob qog nqaij hlav ntsws tseem nyob hauv nws cov menyuam mos. Hauv txoj kev tshawb no, 13 qhov sib txawv ntawm cov noob tau pom tias muaj feem cuam tshuam nrog kev mob ntsws cancer hauv qab simulated microgravity (SMG). Siv cov gene set enrichment tsom xam, cov genes no enriched nyob rau hauv humoral kev tiv thaiv kab mob. Hauv kev hais lus, alveolar basal-epithelial (A549) hlwb tau raug rau SMG ntawm 2D clinostat system hauv vitro. Ntxiv nrog rau kev hloov pauv ntawm morphology thiab txo qis ntawm kev loj hlob, SMG tau thim rov qab qhov kev hloov pauv ntawm epithelial-to-mesenchymal (EMT) phenotype ntawm A549, ib qho tseem ceeb hauv kev mob qog noj ntshav. Qhov no tau ua pov thawj los ntawm kev nthuav qhia epithelial E-cadherin thiab txo mesenchymal N-cadherin qhia, yog li nthuav tawm lub xeev metastatic tsawg dua. Qhov zoo siab, peb tau pom cov lus qhia ntau ntxiv ntawm FCGBP, BPIFB, F5, CST1, thiab CFB thiab lawv qhov kev sib raug zoo rau EMT hauv qab SMG, ua rau lawv muaj peev xwm qog nqaij hlav qog nqaij hlav biomarkers. Ua ke, cov kev tshawb pom no nthuav tawm cov hauv kev tshiab los tsim cov tswv yim kho tshiab rau kev kho mob qog noj ntshav.
Ntsiab lus: simulated microgravity; EMT; mob ntsws cancer; metastasis; mob qog nqaij hlav; biomarker
Cov teebmeem ntawm Cistanche tshuaj ntsuab-Antitumor
1. Taw qhia
Qhov chaw paub tias tsis muaj lub ntiajteb txawj nqus vector, uas cuam tshuam rau lub cev ntawm lub cev, cov ntaub so ntswg, thiab qib cellular. Microgravity, ib qho xwm txheej ntawm qhov tsis muaj qhov hnyav, yog qhov chaw ntxhov siab tseem ceeb uas paub tias muaj kev cuam tshuam rau tib neeg kev noj qab haus huv, xws li pob txha, nqaij atrophy, thiab mob plawv [1,2]. Qhov cuam tshuam ntawm microgravity ntawm cov qog nqaij hlav qog noj ntshav kuj tseem yog qhov tseem ceeb ntawm kev txaus siab hauv qhov chaw thiab kev tshawb fawb txog qog noj ntshav. Microgravity tau pom tias yuav txwv cov kev ua ntawm lub cev tiv thaiv kab mob thiab cuam tshuam ntau lub cev, uas yuav ua rau muaj kev pheej hmoo ntawm kev mob qog noj ntshav [2–4]. Vim cov teeb meem kev noj qab haus huv no, qhov chaw microgravity tau ua rau cov kws tshawb fawb los kawm txog biophysical mechanisms cuam tshuam los ntawm microgravity thiab pab nrhiav kev kho mob rau neurodegenerative disorders, immunotherapies, thiab muaj peev xwm zoo dua thiab ntau hom kev kho mob qog noj ntshav [4-6]. Ntau cov kev tshawb fawb tau sau tseg zoo thiab tshuaj xyuas qhov cuam tshuam loj uas microgravity muaj nyob rau ntawm cellular kev loj hlob, kev loj hlob, thiab apoptosis nyob rau hauv myriad qog cell kab, nrog rau mob ntsws cancer [7–12]. Txawm li cas los xij, ntau lwm yam kev tshawb fawb txog microgravity tau pom tias yuav ua rau muaj kev hloov pauv hauv cov noob qhia uas koom nrog hauv kev mob qog noj ntshav, metastasis, thiab kev ciaj sia, hloov cov hlwb mus rau qhov tsis tshua muaj phenotype [13,14]. Qhov no tuaj yeem ua rau pom kev nkag siab tshiab ntawm qog nqaij hlav biology thiab kev kuaj mob, yog li qhia txog microgravity raws li cov cuab yeej tshiab los nrhiav cov hom phiaj tshiab hauv kev cia siab ntawm kev tsim cov kev tshawb fawb tshiab thiab txhim kho cov tswv yim kho mob. Mob ntsws cancer, nrog rau cov qog nqaij hlav tsis me me (NSCLC) yog hom tseem ceeb tshaj plaws, yog qhov ua rau tuag taus, suav txog 12% ntawm tag nrho cov qog nqaij hlav, thiab feem ntau kuaj mob cancer [15]. Hauv kev tshawb fawb txog microgravity, uas yuav coj peb kom nkag siab zoo dua cov txheej txheem carcinogenic, cov qog nqaij hlav cancer hauv ntsws tau pom tias poob lawv cov qia tom qab raug rau microgravity, yog li cuam tshuam rau kev loj hlob ntawm cov qog nqaij hlav cancer thiab kev ua haujlwm [11]. Kev tshawb fawb los ntawm Ahn et al. qhia tau hais tias nthuav tawm cov qog nqaij hlav cancer hauv lub ntsws (A549) rau microgravity induced sai tsiv teb tsaws thiab kev loj hlob. Qhov no tau piav qhia los ntawm kev tswj hwm qib ntawm matrix metalloproteases (MMP-2 thiab MMP-9) nyob rau hauv microgravity, yog li ua lub luag haujlwm tseem ceeb hauv kev mob qog noj ntshav thiab tsiv teb tsaws [10]. Hauv lwm txoj kev tshawb fawb, Chung et al. pom tias simulated microgravity tsis cuam tshuam rau lub ntsws cancer cell proliferation tab sis ntau zog tsiv teb tsaws piv nrog rau pawg tswj nyob rau hauv ib txwm nqus [16]. Ntawm qhov tsis sib xws, Chang et al. pom qhov txo qis hauv kev tsiv teb tsaws chaw hauv A549 kab mob qog noj ntshav hauv lub ntsws tom qab 24 teev ntawm kev raug mob microgravity [17]. Txawm hais tias muaj qhov cuam tshuam tseem ceeb ntawm microgravity ntawm tus cwj pwm ntawm tes, uas suav nrog apoptosis, kev tsiv teb tsaws, thiab kev tawm tsam, nws cov txiaj ntsig ntawm mob qog noj ntshav tseem tsis tau paub meej. Nws tau tsim tau zoo tias cov hlwb epithelial tau dhau los ua ntau yam kev hloov pauv biochemical los tsim kom muaj mesenchymal phenotype uas txhim kho apoptosis tsis kam, invasiveness, thiab migratory muaj peev xwm. Cov txheej txheem lom neeg zoo li no hu ua epithelial-to-mesenchymal hloov pauv (EMT), ib qho cim ntawm kev mob qog noj ntshav [18]. Kev sib raug zoo ntawm EMT rau kev mob qog noj ntshav tau tham zoo. Cov qog tau paub tias yuav ua kom EMT los ntawm kev tswj hwm cov txheej txheem kev hloov pauv, nthuav tawm cov cell-nto proteins, thiab tsim ECM-degrading enzymes. Thaum EMT tau qhib, cov qog hlwb poob lawv cov cell-cell adhesion thiab tau txais cov khoom tsiv teb tsaws thiab cuam tshuam [19]. Tsuas yog ob txoj kev tshawb fawb tau pom tias simulated microgravity induces transient epithelial-to-mesenchymal hloov (EMT) hauv keratinocytes [9]. Tsis tas li ntawd, cov xwm txheej zoo li no tau hais txog hauv MCF-7 hlwb thiab HUVEC [20]; Txawm li cas los xij, tsis muaj kev tshawb fawb tau tsom mus rau qhov cuam tshuam ntawm microgravity ntawm EMT hauv ntsws cancer. Txawm hais tias muaj ntau qhov kev tshawb pom tseem ceeb tau ua los ntawm lub peev xwm ntawm microgravity los hloov kho cov qog nqaij hlav thiab cov kab mob metastatic ntawm cov qog nqaij hlav, cov txheej txheem tseeb uas tau tsav los ntawm microgravity nroog tseem tsis tau paub, yog li ua rau qhib cov lus nug txog kev hloov pauv uas tshwm sim ntawm qib molecular. Hauv txoj kev tshawb no, peb tau txheeb xyuas qhov kos npe ntawm cov kab mob tiv thaiv kab mob (FCGBP, BPIFB1, F5, CFB, thiab CST1) uas tau nthuav tawm ntau ntxiv mRNA qhia hauv A549 hlwb nyob rau hauv cov nyhuv ntawm simulated microgravity (SMG). Tsis tas li ntawd, peb tau soj ntsuam qhov kev koom tes ntawm SMG hauv A549 kev mob qog noj ntshav ntawm EMT txoj cai. FCGBP, BPIFB1, F5, CFB, thiab CST1 thiab lawv qhov kev sib raug zoo rau EMT cov cim tseem ceeb tau pom tias muaj feem cuam tshuam tsawg dua metastatic phenotype ntawm mob ntsws cancer hauv qab SMG. Qhov kev tshawb pom ntawm txoj kev tshawb no muab lub hauv paus rau kev tshawb nrhiav yav tom ntej uas yuav tsum tau txheeb xyuas cov txheej txheem hauv qab, uas yuav tsim peb txoj kev nkag siab thiab pab txhim kho cov tswv yim kho tshiab rau kev kho mob ntsws cancer.
Cov txiaj ntsig ntawm cistanche tubulosa-Antitumor
2. Cov txiaj ntsig
2.1. Sab saum toj Zoo Tshaj Plaws Tshaj Tawm Ntau Yam Qhia Txog Cov Kab Mob qog noj ntshav hauv lub ntsws nyob rau hauv Simulated Microgravity (SMG) piv nrog av Gravity (GG)
Txhawm rau txheeb xyuas cov noob muaj feem cuam tshuam nrog mob qog noj ntshav nyob rau hauv simulated microgravity (SMG), peb pib tshawb xyuas cov noob uas muaj qhov sib txawv hauv qab SMG piv nrog av nqus (GG) rau tib neeg lub ntsws qog noj ntshav uas siv cov Gene Expression Omnibus (GEO) cov ntaub ntawv tshaj tawm. Ob lub datasets raug xaiv, GSE78210 thiab GSE36931, uas suav nrog cov noob qhia rau ob tus tib neeg lub ntsws qog noj ntshav sib txawv, A549 thiab Colo699, cog rau hauv 2D thiab 3D kab lis kev cai ntawm tes. Qhov sib txawv ntawm cov kev qhia caj ces ntawm cov qauv SMG thiab GG tau nthuav tawm hauv cov phiaj xwm hluav taws xob (Daim duab 1). Cov kev txheeb xyuas qhov sib txawv ntawm cov noob (DEGs; Daim duab 2) rau txhua cov ntaub ntawv tau muab faib ua cov uas tau kho hauv lub ntsws cancer (21 noob hauv A549 thiab 29 noob hauv Colo699 ntawm GSE78210 dataset thiab 47 noob hauv A549 ntawm GSE36931 cov ntaub ntawv). uas yog downregulated I ntsws cancer (40 genes nyob rau hauv A549 thiab 27 noob Colo699 ntawm GSE78210 dataset thiab 87 noob nyob rau hauv A549 ntawm GSE36931 dataset), raws li sawv cev nyob rau hauv Table 1. Kaum peb DEGs tau loj heev nthuav tawm nyob rau hauv A549 cov ntaub ntawv nyob rau hauv GSE1620 thiab GSE37 hlwb. SMG piv nrog GG mob. Cov noob loj tshaj plaws tau txheeb xyuas siv InteractiVienn lub vev xaib (Daim duab 2A). Cov noob neeg sib tw muaj xws li AZGP1, CFB, NOX1, VTCN1, AGR3, GDA, TCN1, CST1, F5, CEACAM6, BPIFB1, FCGBP, thiab BPIFA1. Txhawm rau txheeb xyuas seb DEGs tau txheeb xyuas puas muaj feem cuam tshuam rau txoj hauv kev, cov noob neeg sib tw tau nce mus rau Metascape (http://metascape.org; nkag mus rau 11 Kaum Ib Hlis 2021). Interestingly, cov noob no yog enriched nyob rau hauv txoj kev uas humoral tiv thaiv kab mob thiab tswj exocytosis (Daim duab 2B).
Daim duab 1. Volcano zaj duab xis uas qhia cov noob sib txawv (DEGs) siv GEO Omnibus. (A) Volcano zaj duab xis qhia DEGs hauv qab SMG thiab GG teebmeem hauv A549 hlwb siv (A) GSE78210 thiab (B) GSE36931 datasets. (C) Volcano zaj duab xis qhia DEGs nyob rau hauv SMG thiab GG teebmeem hauv Colo699 cell kab siv GSE78210 dataset. Cov xim liab qhia txog cov noob caj noob ces, thaum lub xim xiav qhia txog cov noob caj noob ces.
Table 1. Tag nrho cov noob uas muaj qhov sib txawv ntawm SMG piv nrog GG hauv cov kab mob ntsws ntsws A549 thiab Colo699. Txhua dataset tau hloov pauv hloov kho kom tshem tawm sab saum toj 5% ntawm DEGs. Rau GSE36931, kev hloov pauv (FC) tau hloov kho kom suav nrog cov noob nkaus xwb nrog FC ntau dua lossis sib npaug rau 4 lossis tsawg dua lossis sib npaug rau −4. Rau GSE78210, FC tau hloov kho kom suav nrog cov noob nkaus xwb nrog FC ntau dua lossis sib npaug rau 3 lossis tsawg dua lossis sib npaug rau −3.
Daim duab 2. Kaum peb feem ntau sib koom DEGs ntawm GSE78210 thiab GSE36931 datasets hauv ntsws cancer. (A) Venn daim duab qhia 134 DEGs hauv GSE36931 (A549 cell line), 61 DEGs hauv GSE78210 (A549 cell line), thiab 56 DEGs hauv GSE78210 (Colo699 cell line) nyob rau hauv SMG tej yam kev mob piv nrog GG. Tawm ntawm tag nrho cov txheeb cais 251 noob, 13 DEGs tau pom tias muaj ntau ntawm ob cov ntaub ntawv, GSE78210 thiab GSE36931, hauv A549 cell kab. Venn daim duab tau tsim los siv InteractiVinn.
2.2. Qhov txawv ntawm cov Genes Correlated nrog Lung Adenocarcinoma Cov Neeg Mob 'Clinical Prognosis
Cov Genes txawv txav Correlated nrog Lung Adenocarcinoma Cov Neeg Mob 'Clinical Prognosis Los soj ntsuam cov txiaj ntsig soj ntsuam ntawm tus neeg sib tw cov noob rau cov neeg mob ntsws adenocarcinoma, Kaplan–Meier plotter (http://www.kmplot.com/; nkag rau 11 Kaum Ib Hlis 2{ {44}}21) tau siv los sib piv tag nrho cov ciaj sia taus (OS) ntawm lub ntsws adenocarcinoma (LUAD) cov neeg mob uas muaj siab / nruab nrab piv rau cov neeg sib tw cov noob (Daim duab 3). Siab mRNA qhia theem ntawm NOX1 (kev phom sij piv (HR), 1.45; 95% kev ntseeg siab lub sijhawm (CI), 1.11–1.9; p=0.0058), GDA (HR , 1.74; 95% CI, 1.33–2.26; p=3.2e-05), TCN1 (HR, 1.62; 95% CI, 1.26–2.08; p=0.00014), thiab BPIFA1 (HR, 1.44; 95% CI, 1.12–1.84; p=0.0026) tau pom tias muaj kev cuam tshuam loj heev nrog rau qhov tsis zoo (Daim duab 3A–D). Ntawm qhov tod tes, siab mRNA qhia theem ntawm FCGBP (HR, 0.63; 95% CI, 0.46–0.85; p=0.0026) tau cuam tshuam nrog kev soj ntsuam zoo dua thiab kev muaj sia nyob ntau dua hauv cov neeg mob LUAD (Daim duab 3E). Tsis muaj kev sib raug zoo ntawm mRNA
Daim duab 3. FCGBP, CST1, F5, CFB, thiab BPIFB1 cuam tshuam rau cov neeg mob ntsws adenocarcinoma qhov kev kuaj mob. Kaplan-Meier ciaj sia taus nkhaus ntawm mRNA qhia theem ntawm cov noob neeg sib tw: (A) NOX1; (B) GDA; (C) TCN1; (D) PLUS; (E) FCGBP; (F) AZGP1; (G) H2Bf; (H) B7X; (I) AGR3; (J) CEACAM6; (K) F5; (L) BPIFB1; thiab (M) CST1 hauv cov neeg mob ntsws adenocarcinoma. HR: txaus ntshai piv.
cistanche ntxiv cov txiaj ntsig-Antitumor
Nyem qhov no mus saib Cistanche cov khoom
【Nug ntxiv】 Email: cindy.xue@wecistanche.com / Whats App: 0086 18599088692 / Wechat: 18599088692
2.3. Kev nthuav qhia ntawm FCGBP, BPIFB1, F5, CFB, thiab CST1 hauv A549 Cells Post-Simulated Microgravity
Txhawm rau kom lees paub qhov kev qhia ntawm silico txheeb xyuas cov noob (AZGP1, CFB, NOX1, VTCN1, AGR3, GDA, TCN1, CST1, F5, CEACAM6, BPIFB1, FCGBP, thiab BPIFA1), hauv vitro daim ntawv thov siv qRT-PCR tau ua. Interestingly, siv qRT-PCR, peb cov ntaub ntawv tso tawm ib tug tseem ceeb upregulation ntawm FCGBP mRNA qhia, tshwj xeeb tshaj yog nyob rau hauv 48 thiab 72 h nyob rau hauv A549 hlwb, post-SMG raws li piv nrog GG (Daim duab 4A). Tsis tas li ntawd, mRNA cov lus qhia ntawm CFB, F5, thiab BPIFB1 tau nce siab heev ntawm 72 h tom qab SMG, thiab piv nrog GG, hauv A549 hlwb (Daim duab 4B, C, E). Kev nce me ntsis, txawm hais tias tsis tseem ceeb, hauv mRNA qhia tau kuaj pom rau CTS1 nyob rau hauv SMG tej yam kev mob piv nrog GG (Daim duab 4D). Cov kab lus ntawm AZGP1, AGR3, GDA, VTCN1, BPIFA1, NOX1, CEACAM5, thiab TCN1 tsis tau txiav txim siab rau SMG thiab GG (cov ntaub ntawv tsis qhia).
Daim duab 4. Upregulation ntawm FCGBP, CST1, F5, CFB, thiab BPIFB1 nyob rau hauv SMG tej yam kev mob nyob rau hauv vitro. mRNA qhia theem ntawm (A) FCGBP, (B) CFB, (C) F5, (D) CTS1, thiab (E) BPIFB1, ntsuas los ntawm qRT-PCR thiab normalized rau GAPDH hauv A549 hlwb raug GG thiab SMG tej yam kev mob rau 24 48, 72 h. Bar graphs qhia cov txiaj ntsig ntawm 3 qhov kev sim ywj pheej (n=3). * p < 0.
Zuag qhia tag nrho, cov ntaub ntawv no qhia tau hais tias qhov sib txawv ntawm FCGBP, F5, CFB, thiab BPIFB1 hauv kev mob ntsws cancer tuaj yeem ua lub luag haujlwm tseem ceeb hauv kev mob ntsws cancer.
2.4. Simulated Microgravity Txo Cell Viability thiab Reverses Epithelial-rau-Mesenchymal Kev Hloov Pauv hauv A549 Lung Cancer Cell Kab
Txhawm rau tshawb xyuas seb SMG puas cuam tshuam rau qhov muaj peev xwm loj hlob ntawm A549 hlwb, qhov kev ntsuam xyuas ntawm tes tau ua tiav siv trypan xiav cais tawm. Peb cov ntaub ntawv tau tshaj tawm tias SMG tau ua rau muaj kev cuam tshuam rau lub sijhawm ntawm kev loj hlob ntawm tes (Daim duab 5B). Qhov txo qis hauv cell proliferation yog qhov tseem ceeb tom qab 48 thiab 72 h tom qab SMG piv nrog GG, uas pom tias muaj kev nce hauv cell proliferation ntawm A549 hlwb. Nyob rau tib lub sijhawm, kev hloov pauv hauv cellular morphology tau kuaj pom hauv A549 hlwb hauv qab SMG. Txhawm rau soj ntsuam cov kev hloov pauv no, cov hlwb raug tshem tawm los ntawm SMG mob, cog rau hauv 24- cov phiaj xwm zoo, thiab pom nyob rau hauv lub teeb pom kev zoo tom qab 1 teev ntawm SMG daim ntawv thov. Interestingly, cov hlwb raug rau SMG qhia ib qho granular, clumped morphology post-SMG. (Daim duab 5A).
Daim duab 5. Simulated microgravity txo cov cell viability thiab induces morphological hloov nyob rau hauv A549 hlwb nyob rau hauv ib lub sij hawm-nyob ntawm seb. (A) Cov neeg sawv cev micrographs ntawm niam txiv A549 hlwb (GG) thiab A549 hlwb raug rau SMG rau 24, 48, thiab 72 teev ua ntej lawv rov qab rau 1hr hauv GG. Cov duab raug coj los ntawm 10X magnification. (B) Cell viability ntawm A549 hlwb raug soj ntsuam siv trypan xiav dye exclusion assay. Qhov nruab nrab ntawm tes muaj peev xwm ntawm 3 qhov kev sim ywj pheej tau tshwm sim raws li kev tswj feem pua. **** p < 0.0001. Daim duab 5. Simulated microgravity txo cov cell viability thiab induces morphological hloov nyob rau hauv A549 hlwb nyob rau hauv ib lub sij hawm-nyob ntawm seb. (A) Cov neeg sawv cev micrographs ntawm niam txiv A549 hlwb (GG) thiab A549 hlwb raug rau SMG rau 24, 48, thiab 72 teev ua ntej lawv rov qab rau 1hr hauv GG. Cov duab raug coj los ntawm 10X magnification. (B) Cell viability ntawm A549 hlwb raug soj ntsuam siv trypan xiav dye exclusion assay. Qhov nruab nrab ntawm tes muaj peev xwm ntawm 3 qhov kev sim ywj pheej tau tshwm sim raws li kev tswj feem pua. **** p <0.0001.
Suav tshuaj ntsuab cistanche cog-Antitumor
Txhawm rau nkag siab txog cov ntaub ntawv tau txais ntawm kev txo qis ntawm cov cellular proliferation hauv A549, peb tau soj ntsuam qhov kev koom tes ntawm SMG rau cov txheej txheem epithelial-to-mesenchymal hloov pauv (EMT). EMT yog lub cim tseem ceeb thiab lub luag haujlwm tseem ceeb uas ua rau kev mob qog noj ntshav thiab metastasis, yog li txhim kho nws cov cell motility thiab invasive zog [19]. Cov cim tseem ceeb EMT (E-cadherin, N-cadherin, ZO-1, thiab Snail) thiab metalloproteinases MMP-2 thiab MMP-9 mRNA qhia theem tau ntsuas los ntawm qRT-PCR hauv A549, hauv qab GG thiab SMG (Daim duab 6). Raws li SMG daim ntawv thov, ib qho kev nce ntxiv hauv E-cadherin mRNA qhia tau pom, piv nrog qhov txo qis ntawm N-cadherin mRNA qhia, ntawm 48 thiab 72 h tom qab SMG (Daim duab 6A, B). Kev nce tsis tseem ceeb hauv ZO-1 thiab MMP-9 thiab qhov txo qis ntawm Snail transcription factor tau kuaj pom raws li SMG (Daim duab 6C–E). MMP-2 tau txo qis hauv A549 ntawm 48 teev tom qab SMG (Daim duab 6F), qhia tias muaj kev cuam tshuam tsawg dua. Ua ke, cov ntaub ntawv no txhawb kev hloov pauv mesenchymal-epithelial (MET) phenotype raug ntxias los ntawm SMG hauv A549.
Daim duab 6
2.5. Epithelial-rau-Mesenchymal Kev Hloov Pauv Hloov Pathway cuam tshuam nrog kev nthuav qhia ntawm FCGBP, BPIFB1, F5, CFB, thiab CST1
Txhawm rau kom tau txais kev pom zoo ntxiv rau hauv lub luag haujlwm ntawm cov noob caj noob ces hauv A549 thiab lawv qhov kev sib raug zoo rau EMT nyob rau hauv SMG cov xwm txheej, kev tshuaj ntsuam gene-kev sib cuam tshuam tau ua los ntawm GeneMANIA tawm tsam EMT gene markers. Daim duab 7 qhia txog cov noob kev sib cuam tshuam ntawm EMT genes E-cadherin (CDH1), N-cadherin (CDH2), TJP1, CTNNB1, SNAI1 (Snail), ZO-1, thiab -catenin, feem. Paub txog lawv lub luag haujlwm tseem ceeb hauv kev mob qog noj ntshav thiab kev tsiv teb tsaws, cov metalloproteinases MMP-2 thiab MMP-9 kuj tau suav nrog hauv kev tshuaj xyuas. Cov nodes txheeb xyuas cov qauv kev sib koom ua ke ntawm cov noob ntawm qhov sib txawv zog raws li qhov tuab ntawm cov node. Tus neeg sib tw genes qhia ib qib ntawm kev sib tham ntawm ib leeg; BPIFB1 yog sib koom nrog CFB thiab CST1; FCGBP tau qhia nrog F5, CST1, thiab CFB; CST1 tau qhia nrog CFB, thiab F5 yog qhia nrog CFB, CST1, thiab BPIFB1. Co-expression nodes kuj tau txheeb xyuas ntawm EMT cov noob thiab cov noob neeg sib tw; CDH1 tau qhia nrog FCGBP thiab F5; CDH2 tau qhia nrog F5, CST1, BPIFB1, thiab FCGBP; CTNNB1 tau qhia nrog CST1 thiab FCGBP; SNAI1 tau qhia nrog CST1, F5, thiab BPIFB1; TPJ1 tau qhia nrog CST1, FCGBP, thiab F5; CTNNB1 tau qhia nrog CST1 thiab FCGBP; MMP2 tau qhia nrog BPIFB1 thiab CST1; thiab MMP9 tau qhia nrog F5, CST1, FCGBP, thiab CFB. Cov kev tshawb pom no qhia txog kev sib raug zoo ntawm cov noob caj noob ces tshiab thiab EMT cov cim nyob rau hauv SMG cov xwm txheej, tejzaum nws muaj feem xyuam nrog EMT txoj hauv kev.
Daim duab 7. FCGBP, CST1, F5, CFB, thiab BPIFB1 cuam tshuam nrog EMT txoj kev. Cov noob caj noob ces sib cuam tshuam network ntawm cov noob xaiv xaiv; FCGBP, CST1, F5, CFB, thiab BPIFB1 nrog EMT noob; CDH1, CDH2, TJP1, CTNNB1, thiab SNAI1, as Well as cell-migration-related genes MMP2 thiab MMP9 generated by GeneMANIA (http://genemania.org/; nkag tau rau 11 Kaum Ib Hlis 2021) txhawm rau txheeb xyuas kev cuam tshuam ntawm cov noob neeg sib tw nrog EMT thiab cell migration markers. Cov xim sib txawv ntawm lub network ntug qhia txog bioinformatics txoj kev siv: lub vev xaib twv txiaj (txiv kab ntxwv), kev sib cuam tshuam ntawm lub cev (liab), co-expression (liab), sib koom cov protein ntau (xim av), txoj hauv kev (lub teeb xiav), co-localization (dub. xiav), thiab kev sib txuas ntawm caj ces (ntsuab)
3. Kev sib tham
Cistanche tshuaj yej
Kev Sib Tham Mob ntsws cancer, ib qho ntawm cov kab mob tseem ceeb thiab thoob ntiaj teb kev hem thawj, tsis ntev los no tau txais kev saib xyuas ntau heev hauv "Space research". Txawm li cas los xij, txawm tias muaj kev vam meej hauv daim teb no, kev mob qog noj ntshav hauv lub ntsws thiab nws cov lus teb rau kev kho mob tseem muaj teeb meem vim tsis muaj cov tswv yim meej ntawm kev ntsuam xyuas uas pab tiv thaiv lossis kho mob qog noj ntshav [21]. Rau peb txoj kev paub, qhov no yog thawj daim ntawv tshaj tawm los txheeb xyuas cov noob caj noob ces yog kos npe rau kev mob qog noj ntshav hauv lub ntsws nyob rau hauv ib puag ncig kev tsim kho los ntawm simulated microgravity (SMG). Peb qhia txog kev nthuav dav ntxiv ntawm cov tshuaj tiv thaiv kab mob tiv thaiv kab mob FCGBP, BPIFB, F5, CST1, thiab CFB, thiab lawv qhov kev sib raug zoo rau kev hloov pauv ntawm epithelial-to-mesenchymal (EMT) raws li SMG, ua rau lawv muaj peev xwm biomarkers ntawm kev mob ntsws cancer. Ntawm no, peb kuj tseem hais txog cov txiaj ntsig ntawm kev ntxias SMG ntawm EMT kev tswj hwm, lub cim ntawm kev mob qog noj ntshav [18,22], hauv kev txhawb nqa kev hloov pauv mesenchymal mus rau epithelial (MET) phenotype. Zuag qhia tag nrho, txoj kev tshawb no muab lub hauv paus rau kev koom tes ntawm cov noob caj noob ces nrog EMT; Txawm li cas los xij, kev tshawb nrhiav ntxiv yuav tsum tau nthuav tawm cov txheej txheem tseeb uas pab tsim kho tshiab hom phiaj kho mob ntsws cancer. Hauv txoj kev tshawb no, peb tau nthuav tawm kaum peb lub noob (AZGP1, CFB, NOX1, VTCN1, AGR3, GDA, TCN1, CST1, F5, CEACAM6, BPIFB1, FCGBP, thiab BPIFA1) kom tau txais kev pom zoo hauv ntsws cancer hauv qab simulated microgravity (SMG) raws li piv nrog av gravity hauv silico. Peb cov ntaub ntawv qhia tau hais tias cov noob caj noob ces sib txawv no muaj ntau txoj hauv kev cuam tshuam feem ntau rau kev tiv thaiv kab mob humoral thiab tswj exocytosis. Kev txaus siab nyob rau tib lub sijhawm nrog peb qhov kev tshawb pom, txawm hais tias lub luag haujlwm tseem ceeb ntawm FCGBP tseem tsis meej [23]. Qhov no tej zaum yuav yog ib feem vim yog lub luag haujlwm tsis sib xws nws nthuav tawm hauv cov qog sib txawv [25]. Piv txwv li, nws tau pom tias FCGBP tseem cuam tshuam nrog kev pom zoo tag nrho thiab cov kab mob tshwj xeeb muaj sia nyob hauv lub taub hau thiab caj dab mob qog noj ntshav, mob qog noj ntshav, thiab cov neeg mob osteosarcoma [25-27], thaum nyob hauv zes qe menyuam thiab qog nqaij hlav prostate, kev qhia siab ntawm FCGBP tau cuam tshuam nrog kev muaj sia nyob tsis zoo [28]. Ntawm qhov kev ceeb toom, lub luag haujlwm ntawm FCGBP lub luag haujlwm tau raug ntaus nqi rau lub cev tiv thaiv kab mob, cov lus teb tiv thaiv kab mob, nrog rau kev tiv thaiv ntawm tes [29,30], yog li ua rau nws yog ib qho tseem ceeb prognostic marker [31]. Siv hauv vitro daim ntaub ntawv, peb tau soj ntsuam qhov qhia txog cov noob caj noob ces nyob rau hauv lub ntsws cancer thiab nyob rau hauv SMG tej yam kev mob ntawm ib tug 2D clinostat. Muaj kev xaiv dav ntawm clinostat hom thiab lwm yam microgravity platforms uas tau tsim. Cov no suav nrog 1/2/3 D clinostat systems, random positioning machines nruab nrog slide flasks (tsuas yog rau cov thyroid cancer hlwb), thiab NASA-tsim rotating phab ntsa hlab ntsha. Txhua qhov yog tsim los ua haujlwm rau qee lub hom phiaj kev tshawb fawb, qhov twg qee qhov yog tsim los cog qoob loo los yog raug tshem tawm cov hlwb, thiab lwm tus yog siv rau kev ntsuas online ntawm cov lus teb kinetic. Nco ntsoov, clinostat yog ib qho yooj yim tshaj plaws thiab hloov tau yooj yim tshaj plaws platforms siv los ntawm kev sim sib txawv. Raws li txoj cai, 2D clinostat yog tus cwj pwm los ntawm kev sib hloov axis uas tig tsis tu ncua ntawm qhov hloov kho qhov ceev thiab nyob rau hauv ib qho kev taw qhia uas yog perpendicular mus rau cov kev taw qhia ntawm lub ntiaj teb lub ntiajteb txawj nqus vector, li no tsim centrifugal rog uas mimic tiag microgravity [14,32,33. ]. Qhov zoo siab, ntawm kaum peb cov noob, FCGBP, BPIFB, F5, CST1, thiab CFB cov lus qhia tau nce ntxiv hauv cov lus teb rau 2D clinostat-induced SMG hauv A549 hlwb. Cov lus qhia ntawm FCGBP thiab BPIFB yog qhov cuam tshuam tshaj plaws, uas qhia tau tias muaj kev nce ntxiv ntawm txhua lub sijhawm ntsuas: 24, 48, thiab 72 h tom qab SMG. Zoo ib yam li FCGBP, BPIFB1 paub tias yuav pab txhawb kev tiv thaiv kab mob hauv lub cev [34]. BPI quav uas muaj tsev neeg B tus tswv cuab 1 cov protein (BPIFB1), uas yog tsim los ntawm cov hlab ntsws epithelia, tau ua pov thawj los koom nrog hauv kev tiv thaiv tus tswv tsev, nrog rau cov kab mob bactericidal thiab tiv thaiv kab mob [35]. Hauv cov kab mob ua pa, BPIFB1 tau pom tias muaj tshuaj tiv thaiv qog thiab tiv thaiv kab mob metastatic; Txawm li cas los xij, cov txheej txheem tseeb tseem tsis meej, lav kev tshawb nrhiav ntxiv [34,36]. Ib txoj kev tshawb fawb los ntawm Wei et al. pom tias BPIFB1 inhibits migration thiab ntxeem tau ntawm nasopharyngeal carcinoma [37]. Muab qhov ntawd, nws tau pom tias kev hloov pauv tshwm sim hauv BPIFB1 txhawb txoj kev pheej hmoo ntawm mob qog noj ntshav hauv lub ntsws, thiab nws qhov kev txo qis ua rau cov neeg mob qog noj ntshav tsis zoo [38,39]. Ntxiv nrog rau lub luag haujlwm ntawm FCGBP thiab BPIFB, qhov sib ntxiv ntawm B (CFB) thiab chimeric qog suppressor 1 (CST1) tau pom tias muaj lub luag haujlwm tiv thaiv kab mob qog noj ntshav. Piv txwv li, nws tau tshaj tawm tias qhov kev qhia siab ntawm CFB tau cuam tshuam nrog cov neeg mob ntau ntxiv thiab tsis muaj kab mob nyob hauv cov neeg mob qog noj ntshav [40]. Tsis tas li ntawd, cov txheej txheem ntxiv ua haujlwm ua thawj kab ntawm kev tiv thaiv kab mob, ua haujlwm tseem ceeb ntawm ob qho tib si hauv nruab nrog cev thiab tau txais kev tiv thaiv kab mob [41]. Raws li rau CST1, cov kev tshawb fawb sib txawv tau hais txog nws cov khoom nthuav dav, xws li nws lub peev xwm los txo qis kev loj hlob ntawm tes, induce apoptosis, thiab nws tsis kam mus rau inactivation los ntawm oncogenic cov ntaub ntawv ntawm p53, rendering nws ib tug txaus nyiam, tsis tau lwm txoj kev kho lub hom phiaj ntawm qus-hom p{ {74}}cov qog tib neeg tiv taus [42]. Ntxiv rau qhov kev nthuav qhia ntau ntxiv ntawm FCGBP, BPIFB1, F5, CFB, thiab CST1, peb cov ntaub ntawv nthuav tawm ob lub xov tooj ntawm tes-txog phenotypes ntawm A549 hlwb nyob rau hauv SMG-kev hloov pauv morphology rau hauv aggregates thiab clump-zoo li lub hlwb thiab txo qis hauv kev loj hlob. tus nqi tom qab SMG. Qhov no yog nyob rau tib lub sijhawm nrog qee qhov kev tshawb fawb, qhov twg cov hlwb uas raug SMG nthuav tawm cov pob me me lossis ntau txheej cell aggregates [43,44]. Cov kev sib txawv morphological no yog tsom los ntawm kev hloov pauv hloov pauv hauv cov txheej txheem ntawm tes, ib feem ntawm EMT. EMT yog lub cim tseem ceeb ntawm cov txheej txheem metastatic, cuam tshuam nrog kev khiav tawm ntawm kev tiv thaiv kev tiv thaiv kab mob thiab kev cuam tshuam ntawm vasculature, yog li tso cai rau cov hlwb kom nthuav tawm mus rau lwm yam kabmob [19,22]. Nyob rau hauv txoj cai, malignant epithelial hlwb tau txais ib tug EMT mechanism nyob rau hauv lub thawj theem ntawm lub qog txoj kev loj hlob, qhov twg cov hlwb yuav qhia mesenchymal zog, ua kom muaj zog motility uas pab txhawb lawv txoj kev khiav tawm ntawm cov thawj niche, whereas lub metastatic hlwb tsim nyob rau hauv lub thib ob sab zaub. tsawg dedifferentiated zog piv nrog rau lawv cov thawj qog nqaij hlav. Ntawm no, MET (mesenchymal-rau epithelial) txheej txheem yog ib feem ntawm xws li kev loj hlob ntawm metastatic qog tsim. Txawm li cas los xij, qhov ntawd tsis tso tseg qhov kev koom tes ntawm EMTs ntawm ntau theem ntawm kev mob qog noj ntshav vim tias cov qog nqaij hlav qog nqaij hlav ntawm cov chaw thib ob yuav loj hlob thiab loj hlob los yog nyob rau hauv dormancy [45,46]. Xws li ntau yam kev sib koom tes ntawm cov noob thiab cov tsos ntawm cov cim migratory uas tswj cov metastatic kev loj hlob ntawm cov qog nqaij hlav cancer yuav ua rau muaj qhov sib txawv ntawm qhov raug SMG [47]. Piv txwv li, nyob rau hauv microgravity tej yam kev mob, txawv qog nqaij hlav cancer ntawm lub mis nthuav tawm txawv morphologies, cell adhesion, thiab migratory zog thaum raug microgravity [5]. Ntawm qhov kev ceeb toom, tus yam ntxwv tseem ceeb ntawm EMT yog qhov poob ntawm cov cell adhesion molecule, E-cadherin, yog li cuam tshuam rau lub quiescence ntawm lub hlwb 'kev ncaj ncees. Ntawm qhov tod tes, qhov nce ntawm neural cadherin, N-cadherin, ua rau kev hloov pauv ntawm cov cell adhesion. Qhov no yog induced tsuas yog los ntawm kev hloov pauv kev loj hlob (TGF- ), uas ua rau pleiotropically qhia transcription yam, xws li Snail, Twist, thiab Zeb proteins thiab lwm yam kev taw qhia txoj kev siv nyob rau hauv metastatic txoj kev [18,48,49]. Ntawm no, peb qhia txog kev txhim kho ntawm epithelial marker E-cadherin (CDH1) thiab downregulation ntawm mesenchymal N-cadherin (CDH2) mRNA qib, cov cim tseem ceeb ntawm EMT [49]. Qhov no txhais tau hais tias ua ke nrog peb cov ntaub ntawv ntawm kev txo qis ntawm A549, cov hlwb tau nthuav tawm lub xeev metastatic tsawg dua. Interestingly, qhov kev hloov pauv ntawm cov cim EMT tseem ceeb kuj tau nrog los ntawm kev txo qis ntawm matrix metalloproteinase MMP{105}}, uas yog ib qho tseem ceeb ntawm lub hauv paus membrane uas feem ntau cais cov txheej epithelial los ntawm ib puag ncig mesenchyme, yog li ua rau Yuav ua li cas ua kom lub qhov cub hauv qab [50] Qhov no yog nyob rau tib lub sijhawm nrog txoj kev tshawb fawb los ntawm Chang li al., qhov uas lawv tau tshaj tawm lawv qhov kev tshawb pom ntawm kev txo qis metastatic peev xwm ntawm tib neeg lub ntsws adenocarcinoma hlwb los ntawm kev hloov pauv ntawm MMP2 qhia [17]. Feem ntau, lub luag haujlwm ntawm MMP tau cuam tshuam zoo rau kev mob qog noj ntshav, suav nrog cov kab mob metastasis, qhov twg qhov txo qis MMP (MMP-2 thiab MMP-9) kev qhia thiab kev ua haujlwm enzymatic yog tus yam ntxwv ntawm tsawg dua metastatic phenotype [ 50–52] ib. Muaj lub luag haujlwm tseem ceeb ntawm EMT hauv metastasis, peb cov ntaub ntawv qhia txog qhov tseem ceeb ntawm kev sib raug zoo ntawm FCGBP, BPIFB, F5, CST1, thiab CFB nrog EMT gene markers nrog rau cov cell-migration-related genes MMP2 thiab MMP9. Interestingly, Xiong et al. tau qhia FCGBP los ua tus tswj hwm tseem ceeb ntawm EMT hauv lub zais zis [53]. Qhov no qhia tau hais tias cov noob caj noob ces tuaj yeem ua lub peev xwm biomarkers rau cov kev tshawb fawb yav tom ntej uas tuaj yeem kwv yees thiab nkag siab ntxiv txog kev mob ntsws cancer. Sib sau ua ke, peb qhov kev tshawb pom tau faib cov noob uas tau txheeb xyuas raws li tus tswj hwm tseem ceeb ntawm EMT, txhawb kev txhim kho ntawm qhov tsis tshua muaj metastatic phenotype ntawm kev hloov pauv mesenchymal mus rau epithelial (MET) ntawm lub ntsws cancer hlwb.
4. Cov ntaub ntawv thiab cov txheej txheem
4.1. Cov ntaub ntawv los txheeb xyuas cov noob sib txawv (DEGs) hauv cov kab mob qog nqaij hlav qog nqaij hlav raug rau Simulated Microgravity
Cov ntaub ntawv cuam tshuam los ntawm cov ntaub ntawv muaj nyob hauv Gene Expression Omnibus (GEO) tau muab rho tawm rau thawj zaug kev tshuaj xyuas. Cov ntaub ntawv no yog siv los ua kev tshawb fawb txog kev ua haujlwm genomics ntawm cov ntaub ntawv qhia txog noob caj noob ces thiab microarrays. Cov ntaub ntawv xaiv tau ua tiav cov txheej txheem hauv qab no: cov ntaub ntawv siv tib neeg lub ntsws qog nqaij hlav hlwb nkaus xwb, cov kev tshawb fawb uas suav nrog kev tswj hwm hauv av hauv av (GG) cov xwm txheej, cov ntaub ntawv nrog cov kev faib tawm ntawm cov kab mob qog nqaij hlav hauv lub ntsws, thiab cov ntaub ntawv nrog tib neeg lub ntsws qog noj ntshav cell gene qhia. siv microarray. Ob cov ntaub ntawv haum rau cov qauv no, GSE78210 thiab GSE36931. Nyob rau hauv tag nrho, 25 tus qauv tau suav nrog hauv cov kev tshawb fawb, thiab 14 kab mob qog noj ntshav hauv cov kab mob qog noj ntshav hauv simulated microgravity (SMG) tau muab piv nrog 11 GG tswj, raws li qhia hauv Table 2 (https://www.ncbi.nlm.nih.gov /geo/; nkag rau 11 Kaum Ib Hlis 2021).
Table 2. Cov ntsiab lus ntawm cov ntaub ntawv tau muab rho tawm los ntawm Gene Expression Omnibus (GEO) siv rau Kev Taw Qhia Thawj Zaug ntawm DEG Ntawm Simulated Microgravity (SMG) thiab Av Gravity (GG) Cov Kab Mob Mob ntsws.
4.2. GEO2R Gene teeb Enrichment Analysis los tsim DEGs hauv Txhua Datase
GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r; nkag rau 11 Kaum Ib Hlis 2021), ib qho kev sib tham hauv online uas siv los sib piv GEO series, tau siv nyob rau hauv txhua cov ntaub ntawv rau pawg thiab txheeb xyuas cov noob caj noob ces hauv cov xwm txheej SMG piv nrog GG cov xwm txheej. Cov noob nrog p-tus nqi tsawg dua 0.05 thiab hloov pauv ntau dua 2 tau raug xaiv raws li qhov sib txawv ntawm cov noob DEGs hauv SMG cov xwm txheej. Yog li ntawd, DEGs ntawm txhua cov ntaub ntawv sib cuam tshuam nrog ib leeg, thiab cov noob caj noob ces raug txheeb xyuas.
4.3. Quantification ntawm Clinicopathological kev koom tes ntawm cov noob caj noob ces
Tom ntej no, cov txiaj ntsig ntawm cov noob luv luv tau raug lees paub siv Kaplan-Meier Plotter (http://www.kmplot.com/; nkag mus rau 11 Kaum Ib Hlis 2021), cov ntaub ntawv pej xeem hauv online uas ntsuas cov txiaj ntsig ntawm cov noob xaiv ntawm cov neeg mob tau txais kev kho mob. Zuag qhia tag nrho kev ciaj sia (OS) tau txhais tias yog lub sijhawm txij li lub sijhawm kuaj mob (hauv lub hlis) txog thaum tuag. Gene qhia cov ntaub ntawv thiab cov ntaub ntawv muaj sia nyob yog muab los ntawm Gene Expression Omnibus (GEO), Cancer Genome Atlas (TCGA), thiab European Genome-phenome Atlas (EGA). Cov neeg mob tau muab faib ua ob pawg: (1) kev qhia siab (nrog TPM qhov tseem ceeb saum toj kawg nkaus quartile) thiab (2) qhia qis / nruab nrab (nrog TPM qhov tseem ceeb hauv qab quartile sab sauv). Kaplan-Meier cov phiaj xwm tau siv los sib piv OS ntawm lub ntsws adenocarcinoma (LUAD) cov neeg mob uas muaj siab / nruab nrab piv rau cov neeg sib tw cov noob qis.
4.4. Enriched Ontology Clustering rau Cov Cim Cim
Txhawm rau tshawb xyuas seb cov noob caj noob ces sib faib txoj hauv kev, Gene Ontology (GO) txoj hauv kev txhim kho txoj hauv kev rau cov noob caj noob ces tau ua tiav siv Metascape lub vev xaib (https://metascape.org/; nkag mus rau 11 Kaum Ib Hlis 2021) rau cov lus qhia dav dav ntawm cov noob caj noob ces thiab cov ntaub ntawv txheeb xyuas nrog rau GEO2R
4.5. Cell Kab thiab Cell Culture
Tib neeg adenocarcinoma alveolar basal epithelial (A549) cell kab, dav siv los ua qauv rau lub ntsws adenocarcinoma, tau loj hlob hauv Roswell Park Memorial Institute Medium (RPMI)-1640 nruab nrab ntxiv nrog 10% fetal bovine serum (FBS; Sigma, St. Louis, MO, USA) thiab 100 units/mL ntawm penicillin/streptomycin (P/S; Sigma, St. Louis, MO, USA). Cov kab hluav taws xob tau coj mus rau hauv 37 ◦C humidified incubator nyob rau hauv ib qho chaw ntawm 5% CO2. Thaum cov hlwb mus txog qhov sib txuam, lawv tau sau nrog 0.5% trypsin, centrifuged ntawm 100 × g rau 5 min, thiab siv thaum xav tau.
4.6. Microgravity
Cov qog nqaij hlav qog nqaij hlav (A549) tau coj mus rau hauv 2D clinostat system ua raws li qhov zero-zero gravity ib puag ncig (microgravity). Qhov no Rotary Culture Max (RCMW ™; Synthecon® Inc—Houston, TX, USA) yog bioreactor nruab nrog lub cell kab lis kev cai nkoj sib txuas nrog microporous perfusion core thiab ib qho hauv-kab oxygenator system, uas muab cov khoom noj khoom haus thiab sab nraud gassing ntawm cov xov xwm, li no insuring kom raug pa pauv thiab ib tug tsawg-shear cell kab lis kev cai ib puag ncig. Lub chamber tau ntim nrog cov kab lis kev cai xov xwm uas muaj cov hlwb. Lub chamber no horizontally tig ib ncig ntawm ib axis perpendicular rau lub zog ntawm lub ntiajteb txawj nqus ntawm ib tug ceev ntawm 10 rpm. Lub 2D clinostat tau muab tso rau hauv 37 ◦C humidified incubator nyob rau hauv ib qho chaw ntawm 5% CO2. A549 hlwb tau sau tom qab simulated microgravity (SMG) ntawm 24, 48, thiab 72 h. Txhawm rau tsim kom muaj qhov sib piv ntawm lub ntsws cancer ib puag ncig, A549 hlwb tau muab coj los ua kab lis kev cai ntawm GG, nyob rau hauv qhov tsis sib hloov lossis zoo li qub, thiab tau khaws cia rau hauv qhov sib thooj ntawm cov cuab yeej hauv lub tshuab nqus tsev vaum (37 ◦C). Cov hlwb no raug xa mus rau pawg tswj hwm thiab tseem raug txiav tawm ntawm 24, 48, thiab 72 teev.
4.7. Cell Viability Assay
Rau cov xwm txheej GG, A549 hlwb tau muab noob rau hauv 24-cov kab lis kev cai zoo ntawm tes ntawm qhov ntom ntawm 3.0 × 104 hlwb ib 500 µL. Cells tau sau los ntawm trypsinization ntawm 24, 48, thiab 72 h tom qab cog, ces centrifuged ntawm 200 × g rau 5 min. Tau txais cell pellets tau rov tsim dua hauv kab lis kev cai xov xwm. Rau cov xwm txheej SMG, A549 hlwb tau cog rau hauv 2D clinostat rotating system ntawm qhov ceev ntawm 6.0 × 104 hlwb ib 1 mL. Cells tau sau ntawm 24, 48, thiab 72 h tom qab SMG raug, ces centrifuged ntawm 200 × g rau 5 min. Tau txais cell pellets tau rov tsim dua hauv kab lis kev cai xov xwm. Trypan xiav (Sigma, USA) dye exclusion assay tau ua. Cell suav tau txiav txim siv CellDrop™ Automated Cell Counter.
4.8. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Gene qhia theem (mRNA qib) ntawm cov noob hauv silico txheeb xyuas cov noob (AZGP1, CFB, NOX1, VTCN1, AGR3, GDA, TCN1, CST1, F5, CEACAM6, BPIFB1, FCGBP, thiab BPIFA1) nrog rau EMT gene markers (Table 3) kuj tau txiav txim siab hauv A549 hlwb los ntawm qRT-PCR. Luv luv, kev rho tawm tag nrho RNA los ntawm cov hlwb tau ua tiav siv RNEasy Mini Cov Khoom Siv (QIAGEN, Hilden, Lub Tebchaws Yelemees) ntawm 24, 48, thiab 72 h ntawm GG thiab tom qab raug rau SMG los ntawm kev ua raws li cov chaw tsim khoom raws tu qauv. Ib µg ntawm tag nrho RNA tau thim rov qab mus rau ib leeg-stranded complementary DNA (cDNA) hauv 20 µL cov tshuaj tiv thaiv ntim siv cov khoom siv iScript™ cDNA synthesis (Thermo, Waltham, MA, USA). qRT-PCR tau ua tiav siv PowerUp™ Sybr™ Green master mix (Thermo, USA) hauv Quant Studio 5 pcr tshuab (Thermo, USA). PCR amplification cov kauj ruam yog raws li nram no: thawj kauj ruam denaturation ntawm 95 ◦C rau 3 min, annealing kub ntawm lub hom phiaj noob rau 30 s, thiab ces 72 ◦C rau 30 s. Qhov fluorescence pib lub voj voog tus nqi tau txais rau txhua tus noob. ∆∆ Txoj kev Cq tau siv los xam cov txheeb ze hloov pauv hauv cov noob qhia tom qab normalization rau cov neeg tu tsev, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Table 3. Cov npe ntawm tib neeg primers. NOX1, NADPH Oxidase 1; GDA, Guanine Deaminase; TCN1, Transcobalamin 1; FCGBP, Fc Gamma Binding Protein; BPIFA1, BPI Fold Containing Family A Member 1; AZGP1, Alpha-2-Glycoprotein 1; CFB, Complement Factor B; VTCN1, V-Set Domain Muaj T Cell Activation Inhibitor 1; AGR3, Anterior Gradient 3; CTS1, chimeric qog suppressor 1; F5, Coagulation Factor V; CEACAM6, CEA Cell Adhesion Molecule 6; BPIFB1, BPI Fold Containing Family B Member 1; ZO-1, Zonula occludens protein 1; MMP-9, Matrix metalloproteinase 9; MMP-2, Matrix metalloproteinase 2; thiab GAPDH, Glyceraldehyde 3-phosphate dehydrogenase
4.9. Gene-Gene Interaction Analysis
Cov ntaub ntawv los ntawm hauv vitro qRT-PCR tau siv hauv kev tshuaj ntsuam gene-gene cuam tshuam siv hauv silico lub vev xaib GeneMANIA (http://genemania.org/; nkag mus rau 11 Kaum Ib Hlis 2021), lub network algorithm rau kev kwv yees cov noob ua haujlwm siv cov txheej txheem loj. cov ntaub ntawv sib koom ua haujlwm. Qhov no tau ua los soj ntsuam ntxiv kev sib cuam tshuam ntawm / ntawm cov noob neeg sib tw xaiv; FCGBP, CST1, F5, CFB, thiab BPIFB1 nrog EMT noob; CDH1, CDH2, TJP1, CTNNB1, thiab SNAI1, as well as cell-migration-related genes, MMP2 and MMP9.
4.10. Kev txheeb cais
GraphPad Prism software tau siv los ua qhov kev txheeb xyuas txheeb cais. Cov txiaj ntsig yog ex-pressed raws li tus kheej cov ntaub ntawv lossis txhais tau tias ± tus qauv sib txawv (SD). Cov tub ntxhais kawm qhov t-test tau siv los sib piv ntau pawg. Qhov sib txawv ntawm cov pab pawg tau soj ntsuam los ntawm ib txoj kev ANOVA ntawm kev sib txawv (ANOVA). p-tus nqi tau txiav txim siab thiab qhov tseem ceeb ntawm p < 0.05, p < 0.01, p < 0.001 (*, **, thiab ***, raws li) tau suav tias yog qhov tseem ceeb. Txhua qhov kev sim tau ua hauv triplicate (n=3).
Cov ntaub ntawv
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