Ib qho tshuaj ntsuam tshiab rau kev nthuav qhia kev tswj hwm ntawm Telomeric Protein TRF2 txheeb xyuas cov molecules me me uas cuam tshuam TRF2 nyob ntawm kev tiv thaiv kab mob thiab qog hlav loj hlob
Oct 10, 2022
Thov hu rauoscar.xiao@wecistanche.comyog xav paub ntxiv
Cov ntsiab lus yooj yim:Lub telomeric protein TRF2 (Telomeric rov-binding yam 2) yog upregulated nyob rau hauv tib neeg mob qog noj ntshav thiab txuam nrog tsis zoo prognosis. TRF2 oncogenic zog cia siab rau nws lub luag hauj lwm telomere tiv thaiv, tab sis kuj ntawm cell-extrinsic teebmeem los ntawm immunosuppressive thiab angiogenic kev ua ub no. Yog li ntawd, kev tsom mus rau TRF2 zoo li yog qhov kev cog lus kho mob los tiv thaiv qog noj ntshav. Hauv txoj kev tshawb no, peb tau tsim ib txoj hauv kev ntawm tes los tshuaj xyuas TRF2 inhibitors tso cai rau peb txheeb xyuas ob lub tebchaw uas ua rau TRF2 pro-oncogenic zog hauv vivo.
Paub meej: Telomeric rov-binding yam tseem ceeb 2 (TRIF2) yog ib tug subunit ntawm lub vaj tse protein complex, uas khi rau thiab tiv thaiv telomeres los ntawm pliaj DNA kev puas tsuaj teb (DDR) ua kom. TRF2 qhia ua lub luag haujlwm tseem ceeb hauv kev laus thiab mob qog noj ntshav, raug txo qis thaum lub sijhawm cellular senescence thiab overexpressed thaum lub sij hawm oncogenesis. Cov qog nqaij hlav ntau dhau ntawm TRF2 feem ntau pom qhov tsis zoo. Hauv cov qog nqaij hlav qog noj ntshav, TRF2 ua haujlwm ntau yam, suav nrog kev tiv thaiv telomere thiab tsis yog cell autonomous lub luag haujlwm, txhawb nqa neo-angiogenesis thiab kev tiv thaiv kab mob.puritans vitamin cPeb nthuav tawm ntawm no yog thawj qhov kev tshuaj ntsuam xyuas lub tswv yim, uas ua rau kev txheeb xyuas cov molecules me me uas txo qis lossis nce TRF2 qhia. Los ntawm kev tshuaj xyuas lub tsev qiv ntawv me me ntawm Lub Chaw Haujlwm Saib Xyuas Khoom Noj thiab Tshuaj (FDA)-pom zoo tshuaj, peb tau txheeb xyuas ob lub molecules (AR-A014418and alexidine-2HCl) uas cuam tshuam cov qog loj hlob, neo-angiogenesis thiab kev tiv thaiv kab mob los ntawm kev txo qis TRF2 qhia hauv nas. xenograft qauv. Cov txiaj ntsig no txhawb nqa cov kws kho mob lub tswv yim ntawm kev txo qis TRF2 kev qhia los kho cov qog nqaij hlav hauv tib neeg hnyav thiab ua kom muaj qhov kev ntsuam xyuas ntawm tes no muaj peev xwm kuaj xyuas qhov muaj peev xwm tiv thaiv kab mob qog noj ntshav thiab tiv thaiv kev laus los ntawm kev hloov kho TRF2 qhia qib.
Ntsiab lus:TRF2; mob qog noj ntshav; kev laus; kev tshuaj ntsuam xyuas ntawm tes; neo-angiogenesis; tiv thaiv kab mob
Thov nias ntawm no kom paub ntxiv
1. Taw qhia
Telomeres yog cov qauv tshwj xeeb nucleoprotein pom nyob rau ntawm qhov kawg ntawm cov kab chromo-somes uas tswj hwm los ntawm telomere-koom nrog xws li telomerase, shelterin protein complexes thiab non-coding telomeric rov-muaj RNA [1]. Thaum tswj tau zoo, telomeres tiv thaiv chromosomes tiv thaiv tsis ruaj khov thiab tsis muaj zog. Telomeric DNA luv-ening tshwm sim raws li ib feem ntawm programmed physiological kev loj hlob thiab kev laus [2]. Txawm li cas los xij, ntau dhau telomere DNA shortening ua rau cov kab mob progeroid tsis tshua muaj, xws li dyskeratosis congenita [3]. Ntxiv mus, dysregulated telomere xeev muaj feem cuam tshuam rau ntau yam kab mob uas tshwm sim thoob plaws hauv cov pej xeem, suav nrog yuav luag txhua hom mob qog noj ntshav thiab ntau yam kab mob degenerative [4]. Yog li, kev tsim kho tshuaj kho mob rau lub hom phiaj tshwj xeeb telomere yog cog lus los tiv thaiv thiab kho cov kab mob no.
Raws li deb li telomere thiab mob qog noj ntshav muaj kev txhawj xeeb, qhov tseem ceeb DNA kev puas tsuaj teb (DDR) cov ntsiab lus tshawb xyuas qee zaum ua tsis tiav; Qhov no tuaj yeem ua rau ntau dhau telomeric DNA shortening thiab aberrant chromosome rearrangements, uas nyob rau hauv lem tuaj yeem ua rau oncogenesis. Tsis tas li ntawd, kev txhim kho ntawm telomerase yog qhov tshwm sim tseem ceeb hauv kev txhim kho txog li 90 feem pua ntawm tag nrho cov qog nqaij hlav, vim qhov no tuaj yeem ua rau tsis muaj kev loj hlob rau cov qog nqaij hlav cancer [5]. Vim li no, telomerase inhibition yog lub hom phiaj ntawm ntau qhov kev tshawb fawb nrhiav kev kho mob qog noj ntshav [6]. Txawm hais tias tsis ntev los no, muaj qee qhov kev txwv rau kev siv tshuaj tiv thaiv telomerase. Piv txwv li, txhawm rau tiv thaiv kev loj hlob ntawm cov qog nqaij hlav cancer, qhov tseem ceeb luv luv telomeric DNA ntev yuav tsum tau mus txog, thiab cov teebmeem los tiv thaiv oncogenic ntawm shortened telomeres tau ploj thaum tsis muaj p53 qog sup-pressor noob [7,8]. Lub sijhawm lag luam no txo cov kev kho mob zoo thiab ua rau muaj kev cuam tshuam rau kev laus los ntawm kev txwv kev rov ua dua tshiab ntawm tes thiab txhawb kev ua kom muaj kev sib txuas ntxiv-raws li telomere elongation mechanisms [9,10].sibYog li ntawd, cov tswv yim los tiv thaiv telomerase tej zaum yuav zoo dua rau kev tsom mus rau cov qog nqaij hlav cancer uas twb muaj cov telomeres luv luv [11].
Ntxiv nrog rau telomerase, kev hloov pauv hauv kev qhia thiab kev ua ub no ntawm cov chaw nyob hauv cov subunits (TRF1, TRF2, RAP1, TIN2, TPP1 thiab POT1) koom nrog cov qog nqaij hlav, thiab qee zaum ntawm nws tus kheej ntawm telomere ntev [12-16]. Yog li, lub hom phiaj ntawm cov tsev nyob rau kev kho mob qog noj ntshav tuaj yeem yog lwm txoj hauv kev zoo rau kev tiv thaiv telomerase. Shel-terin subunit TRF2 sawv cev rau tus neeg sib tw nthuav; TRF2 yog overexpressed nyob rau hauv ntau yam tib neeg malignancies, ob qho tib si nyob rau hauv mob qog noj ntshav thiab vascular hlwb thiab qhov no feem ntau yog txuam nrog ib tug tsis zoo prognosis [17-20]. Tshwj xeeb tshaj yog, TRF2 overexpression tuaj yeem txhawb cov qog nqaij hlav uas tsis yog cell autonomously, ua rau kev tiv thaiv kab mob thiab neo-angiogenesis[13,18-20]Tshwj xeeb, TRF2 upregulation tsim lub zog tiv thaiv kab mob microenvironment. Los ntawm kev hloov pauv cov lus qhia ntawm heparan sulfate proteoglycans, TRF2 ncaj qha nrhiav thiab ua kom cov hlwb los ntawm myeloid-derived suppressive hlwb (MDSCs) los ntawm TLR2 txoj kev [21]. Thaum TRF2 overexpression nyob rau hauv cov qog nqaij hlav cancer muaj zog inhibits NK cell recruitment nyob rau hauv lub qog microenvironment los ntawm cov kev cai ntawm HSPG synthesis [13], TLR2 activation ntawm MDSC induced los ntawm overexpression ntawm TRF2 ua rau muaj zog inhibition ntawm NK cell immunosurveillance nrog ib tug muaj zog txo. ntawm NK hlwb degranulation, IFNgamma ntau lawm thiab tua [21].cistanche yog dab tsiYog li, TRF2 overexpression muaj zog blunts thaum ntxov theem ntawm kev tiv thaiv qog nqaij hlav los ntawm ncaj qha inhibiting NK hlwb recruitment thiab indirectly lub functionality ntawm NK hlwb los ntawm shaping ntawm MDSC-dependent immunosuppressive microenvironment. Yog li ntawd, kev tsom mus rau TRF2 hauv cov qog nqaij hlav tuaj yeem yog lub tswv yim muaj txiaj ntsig ntau ntawm cov txheej txheem ntawm tes-autonomous thiab non-cell-autonomous txheej txheem los ntawm kev txhawb nqa senesce nrog rau kev cuam tshuam neo-angiogenesis thiab kev tiv thaiv kab mob.
cistanche tuaj yeem tiv thaiv kev laus
Txog rau tam sim no, tag nrho cov txheeb xyuas me me uas lub hom phiaj TRF2 cuam tshuam nws lub peev xwm los tiv thaiv chromosome xaus ntawm DDR yog li muaj peev xwm tiv thaiv kev laus [22]. Peb txoj haujlwm yav dhau los tau pom tias qhov txo qis ib feem ntawm TRF2 qhia tuaj yeem thim rov qab mob qog noj ntshav hauv cov qauv nas los ntawm cov teebmeem tsis-cell-autonomous, yam tsis muaj kev cuam tshuam DDR[13]. Yog li ntawd, peb tau txiav txim siab tias kev tsom mus rau kev txo qis TRF2 ntau dhau ntawm cov qog nqaij hlav uas tshwm sim los ntawm nws qhov kev nthuav tawm ntau dhau tuaj yeem yog lub tswv yim nthuav los kho mob qog noj ntshav yam tsis muaj kev cuam tshuam rau kev laus. Hauv txoj kev tshawb no, peb nthuav tawm thawj qhov kev tshuaj ntsuam xyuas los txheeb xyuas cov khoom me me-phaus tsom rau TRF2 kev ruaj ntseg. Peb qhia tau hais tias ob sab saum toj hits inhibited cov qog nqaij hlav ntawm TRF2 overexpression. Txoj kev tshawb no pom tau hais tias TRF2 kev qhia tuaj yeem hloov kho tshuaj kho mob thiab peb qhov kev tshuaj ntsuam xyuas yog ib txoj hauv kev txhim khu kev qha rau kev xaiv cov tshuaj uas muaj peev xwm hloov kho TRF2 qib qhia, uas muaj peev xwm tiv thaiv qog noj ntshav thiab tiv thaiv kev laus.
2. Cov ntaub ntawv thiab cov txheej txheem 2.1.Cells
Tib neeg lub raum embryonic (HEK)293-T hlwb (ATCC CRL-1573) thiab BJ-HELTRAs hlwb[13] tau loj hlob hauv Dulbecco's Modified Eagle's Medium (DMEM) (Lonza, Levallois Perret, Fabkis) ntxiv nrog 10 feem pua fetal calf serum (FCS), 100 IU/mL penicillin thiab 100ug/mL streptomycin (Invitrogen, Cergy Pontoise, Fabkis).
2.2.SDS-PAGE thiab Western Blotting
Tag nrho cov cell lysates tau npaj, sib cais los ntawm electrophoresis thiab blotted raws li tau piav qhia yav dhau los [14]. Luv luv, cov hlwb raug sau thiab lysed hauv lysis buffer (8.76 g/L NaCl 10 mM Tris-HCL pH7.2,0.1 feem pua SDS,0.1 feem pua Triton X -100,10 g/L sodium deoxycholate, 5 mM ethylenediaminetetraacetic acid (EDTA), 1{{50}} ug/mL leupeptine, 1 mM AEBSF thiab 19 ug/mL aprotinin). Cov qauv tom qab ntawd tau titrated siv BCA protein assay kit (Interchim, Monlucon, Fabkis). Cov qauv (60 ug / kab) tau ua kom sov ntawm 95 degree rau 5 min hauv kev thauj khoom tsis sib xws (500 mM Tris-HCl, 100 mM DTI, 2% SDS, 0.1% bromophenol xiav, 10 feem pua glycerol pH 6.8). Tom qab ntawd cov qauv raug thauj khoom ntawm 10 feem pua polyacrylamide gels thiab khiav rau 45 feeb ntawm 160 V. Cov Proteins raug xa mus rau Immobilon-FL daim nyias nyias (Millipore, Upstate New York, USA) siv Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio- Rad. 250; thiab luav IgGanti-Beta-actin diluted ntawm 1:10,000) hauv Intercept Blocking tsis muaj 0.5 feem pua Tween20. Tom qab tau ntxuav hauv PBS 0.1 feem pua Tween20, cov membranes tau incubated rau 1 h nyob rau hauv chav tsev kub hauv Intercept blocking tsis muaj 0.25 feem pua Tween20 nrog ib tug sib tov ntawm lwm cov tshuaj tiv thaiv: tshis-anti-nas IRDye 680 rau anti-TRF2 antibody thiab tshis los tiv thaiv luav IRDye 800CW rau anti-Beta-actin antibody (1:15,{68}} dilution).Anti aging cistancheThawj thiab IRDye cov tshuaj tiv thaiv kab mob thib ob uas tau siv tau teev tseg hauv qab no hauv daim ntawv tshuaj tiv thaiv kab mob (Table 1). Thaum kawg, cov kab protein tau pom pom siv LiCor Odyssey 9120 imaging system (LI-COR).TRF2 qhia tau ntsuas los ntawm normalizing TRF2 band siv rau qhov siv ntawm actin band thiab keeb kwm yav dhau.
2.3.Cloning Strategy
Tib neeg TRF2 cDNA ib ntus tau cloned ntawm BamHI / BclI txwv qhov chaw ntawm lentiviral SFFV-GPR plasmid uas yog HIV-SFFV-GFP-WPRE derivative [23]. SFFV-GPR plasmid muaj cov kab mob spleen focusing forming virus (SFV) txhawb nqa tswj GPR polycistronic gene uas muaj cDNA sequences rau ntsuab fluorescent protein (GFP), ib tug puromycin tsis kam protein thiab Tag-red fluorescent protein (RFP)-T (S158T mutated Tag -RFP), txhua qhov sib cais los ntawm E2 thiab T2 Picornaviridae sequences. hTRF2 cDNA tau muab tso rau ntawm thaj tsam C-terminal ntawm RFP-T, txhawm rau ua kom muaj kev qhia ntawm RFP-TRF2 fusion protein.
2.4.Lentivirus Production
Rau lentivirus ntau lawm, 5 × 10* HEK 293-T hlwb tau kis nrog 8.6 ug ntawm khoob SFFV-GPR lossis RFP-TRF2- nthuav qhia SFFV-GPR vector, 8.6 ug ntawm Lenti -Delta 8.91 thiab 2.8 ug ntawm VSV-g ntawm calcium phosphate-mediated transfection. Cov hlwb hloov pauv tau coj mus rau hauv DMEM ntxiv nrog 10 feem pua FCS ntawm 37 degree nrog 5 feem pua CO2 hauv 10-cm cov tais diav. Supernatants uas muaj cov kab mob tsim tshiab tau sau tom qab 48 teev, tom qab ntawd dhau los ntawm 0.45-μm Millipore lim. Tom qab tus kab mob titration, 1∶1 (virus∶cell) piv tau siv los kis rau BJ-HELTRAs cell kab, xaiv rau nws cov kev tiv thaiv rau TRF2 tsis xws luag[13].cistanche cov txiaj ntsigIb qho clone uas qhia GFP thiab cov fused RFP-TRF2 protein mus rau theem nrab tau raug cais tawm los ntawm fluorescence-activated cell sorting (FACS).
2.5.Flow Cytometry Kev Ntsuam Xyuas
BJ-HELTRAs clonal kab hlwb uas muaj SFFV-GPR vector thiab qhia txog RFP-TRF2 fusion protein tau coj los ua kab lis kev cai hauv 96-cov phiaj zoo hauv DMEM nruab nrab ntxiv nrog 10 feem pua FCS thiab 1 feem pua penicillin/streptomycin. Tom qab 24 teev ntawm kev kho tshuaj, cov hlwb tau sim sim thiab ntxuav hauv phosphate-buffered saline (PBS) uas muaj 0.5 mM EDTA thiab 2 feem pua FCS ua ntej kho nrog 0.5 feem pua formaldehyde (FA). Cov hlwb raug tom qab ntawd. kom ntws cytometry siv FacsCalibur high-throughput sampler (BD Biosciences, Franklin Lakes, NJ, USA).
2.6.Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)
Tag nrho RNA raug cais siv RNeasy Mini Kit (Qiagen, Venlo, Netherlands). Kev hloov pauv rov qab tau ua tiav siv Superscript II thim rov qab transcriptase (Invitrogen) nrog 1 ug ntawm tag nrho RNA. Qhov kev qhia ntawm txhua tus noob tau zoo li qub rau ntawm GAPDH. Cov primers hauv qab no tau siv: hTRF2 Fw 5'-GCTGCCTGACTIGAACAGT-3';hTRF2 Rv 5'-CCGTTCTCAACCAACCCCTC-3';hGAPDHFw5'-AGCCACATCGCTCAGACAC-3'hGACACTAG RCCCACCCC 5' 14}} ib. 2.7.AlamarBlue
Nyob rau hauv ib tug 96-zoo tiaj tiaj-hauv qab phaj, 5 × 103 hlwb ntawm ib qhov dej tau noob nyob rau hauv 200 uL DMEMsup-suppleted nrog 10 feem pua FCS. Thaum 24 teev tom qab kev kho tshuaj, 10 l ntawm AlamarBlue (Bio-Rad) tau ntxiv thiab nqus tau ntsuas ntawm 570 nm thiab 600 nm rau 36 h hauv Spectrostar Nano phaj nyeem (BMGLabtech, Ortenberg, Lub teb chaws Yelemees). Qhov feem pua ntawm oxidation-txo ntawm AlamarBlue tau txiav txim siab raws li tau piav qhia los ntawm cov chaw tsim khoom.
2.8. Tsiaj
Cov kev sim tau ua nyob rau ntawm 8- rau 12-lub lim tiam-laus NMRI poj niam liab qab nas los ntawm Janvier Labs (Fabkis). Txhua qhov kev sim nas tau ua raws li cov txheej txheem hauv zos thiab thoob ntiaj teb thiab tau txais kev pom zoo los ntawm Pawg Saib Xyuas Tsiaj txhu ntawm IRCAN thiab cheeb tsam (CIEPAL Cote d'Azur #187 thiab #188) thiab lub tebchaws (Fabkis Ministry of Research #03482.01/02482.2 thiab #02973.01/02973.2) cov cai.
2.9.Tumor Growth Experiments
Txhua NMRI liab qab nas tau txhaj tshuaj subcutaneously nyob rau tom qab nrog 1 x 106 BJ-HELTRAs hlwb raug tshem tawm hauv 100 μL ntawm PBS (n=8 nas rau ib pawg). Cov nas raug kho rau hnub 16, 18, 20 thiab 22 nrog kev txhaj tshuaj intraperitoneal ntawm 100 ul ntawm DMSO (45 feem pua), alexidine-2HCl (1 mg / kg) lossis AR-A014418 (5 mg / kg), tom qab ntawd ua raws li hnub tim 26. Cov qog nqaij hlav tau soj ntsuam los ntawm palpation txhua hnub. Cov qog loj tau ntsuas txhua txhua 2-3 hnub siv lub caliper. Cov qog ntim tau txiav txim siab siv cov qauv hemi-ellipsoid: π × (L × 1 × h) / 6, qhov twg L sib raug rau qhov ntev, Kuv mus rau qhov dav thiab h mus rau qhov siab ntawm cov qog, raws li.
2.10.Matrigel Plug Assay
BJ-HELTRAs hlwb tau kho nrog DMSO (1 feem pua), alexidine-2HCl (1 uM) lossis AR-A014418 (10 μM) rau 2 hnub. Tom qab ntawd, 100 μL ntawm 1 × 10 degree kho cov hlwb raug tshem tawm hauv PBS ua ke nrog 400 uL ntawm kev loj hlob-yam-txo Matrigel (Corning, New York, USA) tau inoculated subcutaneously rau sab nraum qab ntawm NMRI liab qab nas nyob rau hauv isoflurane tshuaj loog. Hnub 5 tom qab inoculation, Matrigel plugs tau sau, thiab infiltrating hlwb tau sau los ntawm enzymatic dissociation ntawm dispase (Corning), collagenase A (Roche, Bale, Switzerland) thiab DNAseI (Roche) digestion rau 30 min ntawm 37 degree [13. ]. Cells tau saturated rau 15 feeb ntawm dej khov nrog Fe-Block anti-CD16 / CD32 cov tshuaj tiv thaiv (clone 2.4G2) ua ntej staining nrog cov tshuaj tiv thaiv sib txuas rau 30 min ntawm 4 degree. Cov tshuaj tiv thaiv conjugated siv tau teev nyob rau hauv daim ntawv tshuaj tiv thaiv. Cells raug ntxuav hauv PBS nrog 0.5 mM EDTA, 2% FCS thiab kho nrog 0.5% FA. Stained cells tau txheeb xyuas siv ARIA III cytometer nrog DIVA6 software (BD Biosciences) thiab FlowJo 10 (LLC).
2.11.Statistics
Tag nrho cov duab thiab cov kev txheeb xyuas txheeb cais tau tsim los siv GraphPad Prism software (San Diego, CA, USA).Tag nrho cov txiaj ntsig tau sawv cev raws li qhov nruab nrab ± tus qauv sib txawv (sd) lossis txhais tau tias ± tus qauv yuam kev ntawm qhov nruab nrab (SEM). Qhov sib txawv tseem ceeb ntawm txoj kev tau txiav txim siab siv Mann-Whitney ob-tailed test. Qhov kev ntsuas ntsuas (Mantel-Cox) tau siv los txiav txim siab qog noj ntshav. Rau txhua qhov kev xeem, p<0.05 was="" considered="" statistically="">
3. Kev tshwm sim
3.1.Identification ntawm TRF2 Inhibitory Molecules
Txhawm rau tshuaj xyuas cov tshuaj uas muaj peev xwm hloov kho TRF2 cov protein ntau, peb tau siv cov kab mob lentiviral los koom ua ke ntawm GFP noob thiab RFP fused rau N-terminus ntawm TRF2. Ua raws li cov tswv yim dav dav piav qhia lwm qhov [23], peb tsim GRT lentivirus, uas SFFV tus txhawb nqa tau tswj cov kev qhia ntawm poly-cistronic gene encoding GFP, puromycin tsis kam protein thiab RFP-TRF2 lossis RFP nkaus xwb raws li kev tswj hwm (Daim duab 1A). Yog li ntawd, tag nrho cov hlwb transduced qhia GFP thiab RFP-TRF2. Cov txheej txheem no tau tsim los txheeb xyuas cov tshuaj uas hloov kho TRF2 kev qhia, los ntawm kev ntsuas RFP kev siv cov cytometry ntws, thaum GFP siv los ua kev tswj xyuas sab hauv rau kev sau ntawv ntawm tus neeg sau xov xwm tsim.
Peb tau hloov GRT lentiviruses rau hauv tib neeg BJ-HELTRAs fibroblasts uas tsis txawj tuag los ntawm SV40 thiab hTERT thiab ua rau oncogenic los ntawm Ras v12[13]. Txhawm rau ua kom yooj yim rau kev ntsuas ntawm ob qho tib si nce- thiab qis-txoj cai ntawm TRF2, ib qho puromycin-resistant clone uas qhia me ntsis GFP thiab RFP-TRF2 cov proteins raug cais tawm siv FACS sorting thiab hu ua GRI-BJ-HELTRAs (Daim duab 1A). Raws li kev tswj hwm zoo, peb tau kho GRT-BJ-HELTRAs hlwb nrog 10 uM gemcitabine, ib qho kev piav qhia yav dhau los ntawm TRF2 stabil-ity [24], rau 24h. Thaum tsis muaj RFP kev hloov kho tau kuaj pom hauv cov hlwb hloov nrog cov vector khoob, qhov txo qis hauv RFP / GFP txhais tau tias fluorescence siv (MFI) piv tau pom hauv gemcitabine-kho GRT-BJ-HELTRAs hlwb piv rau DMSO-kho tswj (82 feem pua. tswj; p<0.0001)(figure 1b).moreover,="" gemcitabine="" specifically="" diminished="" rfp-trf2="" protein="" levels="" without="" affecting="" gfp="" levels="" (figure="" s1a).="" of="" note,="" we="" observed="" here="" that="" gemcitabine="" is="" reducing="" trf2="" level="" in="" contrast="" to="" the="" published="" work[24],="" a="" difference="" that="" may="" be="" explained="" by="" cell="" type="" differences="" in="" the="" dna="" damage="" response="" induced="" by="" gemcitabine="" since="" trf2="" stability="" can="" be="" altered="" in="" a="" p53-dependent="" manner="" [25].="" this="" effect="" was="" further="" confirmed="" by="" western="" blotting="" analyses="" where="" we="" observed="" that="" endogenous="" trf2="" levels="" were="" affected="" by="" gemcitabine="" treatment="" on="" untrans-duced="" bj-heltras="" cells="" (figure="" s1b).therefore,="" grt-bj-heltras="" cells="" enabled="" detection="" of="" specific="" variations="" in="" trf2="" protein="" levels="" induced="" by="" drug="" treatment.="" or="" gemcitabine="" (right="" panel)(n=""><0.001;two-tailed student'st="" test).="" (c)representative="" rfp/gfp="" flow="" cytometry="" plots="" of="" trf2="" vector-transduced="" bj-heltras="" cells="" treated="" with="" 10="" um="" of="" alexidine-2hcl,="" ar-a014418="" or="" dmso="" (left="" panel).="" box-plot="" quantification="" of="" rfp-trf2="" fusion="" (trf2="" vector)="" proftein="" levels="" following="" treatment="" with="" dmso,="" alexidine-2hcl="" or="" ar-a014418(right="" panel)(n=""><0.05;mann-whitney test).(d)representative="" western="" blotting="" showing="" reduced="" trf2="" protein="" levels="" following="" treatment="" with="" 10="" um="" alexidine2hcl="" or="" ar-a014418="" compared="" to="" dmso="" control="" treatment="" in="" untransduced="" bj-heltras="" cells="" (left="" panel;="" full="" western="" lolotting="" image="" is="" presented="" in="" figure="" s2a).="" quantification="" of="" trf2="" protein="" levels="" after="" treatment="" with="" 10="" μm="" alexidine-2hccl="" or="" ar-a014418="" compared="" to="" dmso="" control="" treatment="" （right="" panel)(n="2;mean" +="" standard="" error="" of="" the="" mean).(e)quarntification="" of="" trf2="" mrna="" levels="" analyzed="" by="" rt-qpcr="" after="" treatment="" with="" 10="" um="" alexidine-2hcl="" or="" ar-a014418="" compared="" to="" dmso="" control="">
Siv cov cytometry ndlwg, peb mam li tshuaj xyuas 396 Food and Drug Administration (FDA) pom zoo cov tshuaj pharmacological muaj peev xwm tsom rau 6 pawg tseem ceeb ntawm cov txheej txheem biologic: ion channels, phosphatase, kinases, epigenetic yam, nuclear receptor ligands thiab Wnt txoj kev (Daim duab S1C. ).GRT-BJ-HELTRAs hlwb raug kho thawj zaug nrog 10 uM ntawm txhua qhov ntawm 396 lub tebchaw lossis DMSO rau 24h ua ntej kev ntsuas cytometry (Daim duab S1D). Hauv qhov no, 84 lub tebchaw tau pom los hloov kho TRF2 cov protein ntau (Daim duab S1D, txoj cai vaj huam sib luag; Table S1), thiab tau siv rau lub vijtsam thib ob (Daim duab S1E; Table S1). Hauv qhov kev tshuaj ntsuam thib ob no, ntxiv rau kev kho GRT-BJ-HELTRAs hlwb nrog 10 uM ntawm cov khoom sib txuas, uas tsis yog-transduced BJ-HELTRAs hlwb los yog BJ-HELTRAs hlwb transduced nrog ib tug khoob vector raug kho zoo ib yam li muab pov tseg tsis tseeb zoo emit liab. lossis ntsuab autofluorescence lossis cuam tshuam rau RFP lossis GFP cov proteins. 18 qhov zoo tshaj plaws com-pounds tau raug xaiv los txiav txim siab lawv lub peev xwm los hloov kho qhov kev qhia ntawm endoge nous TRF2 hauv cov hlwb uas tsis yog BJ-HELTRAs, raws li Western blotting (Daim duab S2A, B Table S1). Peb txhais hits raws li cov tebchaw modulating los ntawm tsawg kawg yog 20 feem pua ntawm TRF2 ntau npaum li cas (txawm li los yog down). Siv cov qauv no, los ntawm 18 cov tshuaj ntsuam xyuas los ntawm Western blotting, 9 ntawm lawv txo qis thiab ib qho nce ntxiv ntawm cov protein ntau TRF2 (Daim duab S2A, Table S1) Peb mam li txiav txim siab xaiv ob lub tebchaw ntawm cov tshuaj no rau hauv vivo thwmsim los txiav txim seb lawv puas tuaj yeem ua tau. tiv thaiv cov teebmeem pro-oncogenic ntawm TRF2 overexpression. Txhawm rau zam kev ua kom DDR thiab cov kev mob tshwm sim hauv cov hlwb uas tsis yog tumorigenic, peb xaiv cov tshuaj uas tsis txo qis rau qhov siab tshaj plaws los yog qis tshaj TRF2 ntau npaum. Ntawm lawv, AR thiab AD tau ua raws li cov qauv no. Ob lub tebchaw downregulated RFP-TRF2 nyob rau hauv GRT-BJ-HELTRAs hlwb raws li soj ntsuam los ntawm flow cytometry (Daim duab 1C), endogenous TRF2 protein theem raws li qhia los ntawm Western blotting tsom xam (Daim duab 1D; daim duab S2A, B) thiab TERF2 mRNA qib raws li tau txiav txim los ntawm RT -qPCR (Daim duab 1E). Tsis tas li ntawd, kev kho mob nrog LD50 qhov ntau ntawm AR thiab AD txo qis TERF2 mRNA qhia hauv BJ-HELTRAs hlwb thiab hauv TRF2-overexpressing BJ-HELTRAs hlwb (Daim duab S3A-C).
3.2.AR-A014418 thiab Alexidine-2HCl thim rov qab Tumorigenicity Conferred los ntawm High TRF2 Qhia Qib
Peb tom ntej no tau soj ntsuam qhov cuam tshuam ntawm AR thiab AD ntawm cov qog loj hlob. Txhawm rau tswj lawv lub peev xwm los tsom TRF2- mob qog noj ntshav ntau dhau, peb tau txheeb xyuas qhov muaj peev xwm tiv thaiv qog nqaij hlav ntawm AR thiab AD ntawm ob qho tib si tus qauv BJ-HELTRAs hlwb thiab TRF2-overexpressing BJ-HELTRAs hlwb. BJ-HELTRAs hlwb tau hloov nrog TRF2lentiviral vector lossis khoob vector, ces txhaj subcutaneously rau hauv cov nas liab qab. Cov nas tau raug kho nrog DMSO, 1 mg / kg AD lossis 5 mg / kg AR ntawm hnub 16,18,20 thiab 22 tom qab txhaj tshuaj [26,27](Daim duab 2A). Raws li yav dhau los qhia [13,21], TRF2 overexpression txhawb qog ini. tiation thiab kev loj hlob hauv vivo (Daim duab 2B-D; p<0.05). treatment="" with="" neither="" ar="" nor="" ad="" impacted="" tumor="" volume="" in="" bj-heltras="" cells="" transduced="" with="" the="" empty="" vector="" (figure="" 2e,="" upper="" panels)="" neither="" tumor="" growth="" rate(figure="" 2f-h).="" by="" contrast,="" both="" drugs="" induced="" a="" significant="" decrease="" in="" the="" tumor="" volume="" of="" trf2-overexpressing="" xenografted="" tumors,="" leading="" to="" significantly="" smaller="" tumor="" sizes="" at="" day="" 26="" (figure="" 2e,middle=""><001)but also="" of="" the="" tumor="" growth="" rate="" (figure="" 2f-h)="" at="" each="" time-point.="" furthermore,="" the="" vol-ume="" and="" growth="" rates="" of="" trf2-overexpressing="" tumors="" treated="" by="" the="" two="" drugs="" returned="" to="" levels="" similar="" to="" those="" of="" the="" control="" tumors,="" thus="" demonstrating="" the="" trf2-specific="" selectivity="" of="" ar="" and="" ad="" (figure="" 2e,lower="" panels).these="" results="" show="" that="" ar="" and="" ad="" treatment="" reversed="" specifically="" the="" tumorigenicity="" conferred="" by="" trf2="" overexpression.="" or）="" were="" injected="" subcutaneously（1×10°cells/100μl）into="" nmri="" nude="" mice.="" the="" mice="" were="" then="" treated="" with="" dmso,alexidine-2hcl="" (1="" mg/kg)="" or="" ar-a014418(5="" mg/kg)="" at="" days16,18,20="" and="" 22="" post-injection,="" then="" followed="" up="" until="" day="" 26="" (n="8" mice="" per="" group).(b-d)="" the="" percentage="" of="" tumor-free="" mice="" (b)="" and="" tumor="" volumes(c)="" were="" determined="" at="" the="" indicated="" time="" points="" in="" mice="" injected="" with="" bj-heltras="" cells="" overexpressing="" trf2(trf2="" vector)="" or="" empty="" vector.="" tumor="" take="" based="" on="" palpability="" was="" determined="" at="" the="" indicated="" time="" points="" and="" is="" represented="" as="" a="" percentage="" of="" tumor-free="" mice.p="" values="" were="" determined="" using="" the="" log-rank="" mantel-cox="" test(*=""><0.05). tumor="" volumes="" were="" assessed="" at="" different="" time-points="" (mean="" ±="" standard="" deviation);="" tumor="" volume="" at="" d.ay="" 15="" is="" represented="" in="" (d).p="" values="" were="" determined="" using="" the="" mann-whitney=""><><><0.001). (e)="" tumor="" volumes="" were="" followed="" as="" in="" (c)="" after="" repetitive="" treatments="" with="" dmso,="" alexidine-2hcl="" (1="" mg/kg),="" or="" ar-a014418(5mg/kg).p="" values="" were="" determined="" using="" the="" mann-whitney=""><0.0001;ns: not="" significant).(f-h)="" tumor="" growth="" rate="" of="" ar-a014418(5mg/kg)treated="" mice(f,h)="" or="" alexidine-2hcl="" (1="" mg/kg)(g,h)="" treated="" mice="" were="" determined="" by="" considering="" the="" last="" day="" before="" treatment="" for="" empty="" vector="" or="" trf2="" vector="" as="" 100%.="" variations="" of="" the="" growth="" rate="" in="" both="" treatments="" over="" the="" time="" are="" represented="" in="" (f,g)="" and="" for="" each="" time-point="" (h).p="" values="" were="" determined="" using="" the="" mann-whitney=""><><><>
3.3.AR-A014418 thiab Alexidine-2HCI Counteracted TRF2-Dependent Immunosuppression thiab Neo-Angiogenesis
Txhawm rau txiav txim siab seb qhov cuam tshuam los ntawm AR thiab AD tuaj yeem tsom mus rau qhov tsis yog-cell autonomous oncogenic thaj chaw ntawm TRF2, peb tau tshuaj xyuas lub cev tiv thaiv kab mob hauv lub cev thiab ua kom muaj zog ntxiv nrog rau angiogenesis hauv cov qog kho mob. Peb tau kho tus qauv thiab TRF2-overexpressing BJ-HELTRAs hlwb (Daim duab S3A) nrog 1 uM ntawm AD, 10 uM ntawm AR lossis DMSO rau 48 teev ua ntej lawv txhaj tshuaj subcutaneous nrog Matrigel rau cov nas liab qab. Matrigel plugs tau raug sau tom qab 5 hnub tom qab txhaj tshuaj los soj ntsuam cov qog microenviron-ment los ntawm kev ntws cytometry (Daim duab 3A). Raws li xav tau [13,21], TRF2 overexpression tsis hloov lub ntiaj teb lub cev tiv thaiv kab mob (CD45 ntxiv rau cell) infiltration (Daim duab 3B; Daim duab S4A), tab sis tau inhibit ntuj killer (NK) cell recruitment (Daim duab 3C; Daim duab S4B) thiab NK muaj nuj nqi -ality (Daim duab 3C-E; Daim duab S4C), thiab nce MDSC infiltration (Daim duab 3F). Hauv TRF2-overexpressing BJ-HELTRAs hlwb tshwj xeeb, kev kho mob nrog cov tshuaj muaj zog thoob ntiaj teb kev tiv thaiv kab mob (Daim duab 3B; Daim duab S4A), nrog rau qhov kom muaj nuj nqis thiab kev ua haujlwm ntawm intra-tumoral NK hlwb (Daim duab 3C-E; daim duab S4B, C). Qhov tseem ceeb, ob qho tshuaj tau txais kev cawmdim ntawm NK cell-mediated immune surveillance (CD107a ntxiv thiab CD69 ntxiv rau NK cell) tshwm sim los ntawm TRF2 overexpression (Daim duab 3C). Qhov no tau cuam tshuam nrog kev txo qis hauv MDSC kev nrhiav neeg ua haujlwm (Daim duab 3F; Daim duab S4D) thiab nrog kev nce ntxiv ntawm monocytes thiab macrophages (Daim duab 3G).
Raws li yav dhau los tau tshaj tawm [14,20], qog angiogenesis tau siab dua hauv TRF2-cov qog nqaij hlav overexpressing piv rau kev tswj cov qog. Thaum TRF2 overexpression nce qhov ntau ntawm CD31 ntxiv rau CD45-cov hlwb endothelial nyob rau hauv cov qog nqaij hlav (Daim duab 3H; Daim duab S4E), tsis muaj qhov sib txawv ntawm qhov khoob vector lossis TRF2- overexpressing qog tom qab kho nrog ob qho tshuaj. ing TRF2 (TRF2 vector) lossis khoob vector raug kho nrog 1uM ntawm alexidine-2HCl lossis 10 μM ntawm AR-A014418 lossis DMSO ntawm 2 hnub ua ntej txhaj tshuaj subcutaneous nrog Matrigel (1 × 10 degree hlwb) rau hauv NMRI cov nas liab liab ( n=8). Kev tiv thaiv kab mob thiab endothelial cell infiltration tau soj ntsuam tom qab 5 hnub tom qab txhaj tshuaj los ntawm kev ntws cytometry. (BH). Flow cytometry tsom xam ntawm lub cev tiv thaiv kab mob ntawm Matrigel plug. Cov xov tooj ntawm lub cev tiv thaiv kab mob infiltrating Matrigel plugs ntawm cov hlwb nyob tau qhia. Tag nrho cov tshuaj tiv thaiv kab mob hauv lub cev (CD45 ntxiv rau cov hlwb) tau pom nyob rau hauv (B); natural killer(NK) cell(NKp46 plus cells) infiltration yog qhia hauv (C); activated NK cell (CD107a ntxiv thiab CD69 ntxiv rau NK hlwb) inffiltration yog qhia hauv (D,E); myeloid-derived suppressor cell (MDSC; CD11b plus GR1 plus )) infiltration yog qhia nyob rau hauv (F); monocyte-macrophage infiltration yog qhia nyob rau hauv (G) thiab endothelial cell infiltration yog qhia nyob rau hauv (H).p Qhov tseem ceeb tau txiav txim siv cov Mann- Whitney test(*p<><><0.001;n =="" 8="" mice="" per="">
4. Kev sib tham
Ntawm no, peb tshaj tawm txoj kev txhim kho ntawm kev soj ntsuam ntawm tes los tshuaj xyuas cov tebchaw uas hloov pauv kev qhia ntawm telomeric protein TRF2. Los ntawm kev tshuaj xyuas lub tsev qiv ntawv me me ntawm FDA-pom zoo molecules, peb txheeb xyuas cov tebchaw uas tuaj yeem nce lossis txo TRF2 qib qhia. Peb tau tshawb pom tias AD thiab AR txo qis kev tswj hwm TRF2 thiab nthuav tawm cov haujlwm tiv thaiv qog nqaij hlav tshwj xeeb rau cov qog hlwb overexpressing TRF2. Ntxiv ua qauv qhia-ing lawv TRF2-cov haujlwm tshwj xeeb, AR thiab AD kev kho mob cawm tau cov tshuaj tiv thaiv kab mob thiab neo-angiogenesis tau muab los ntawm TRF2 overexpression. Strikingly, qhov tseeb hais tias lub ntiaj teb no tiv thaiv kab mob infiltration yog nce rau TRF2 overexpressing qog tom qab AR los yog AD kev kho mob qhia tias cov tshuaj no muaj zog ntau zog los txhim kho lub cev tiv thaiv kab mob thaum TRF2 yog overexpressed. Cov tshuaj no inhibit qhov kev tiv thaiv kab mob ntawm TRF2 overexpression los ntawm kev rov ua haujlwm ntawm NK cell functionality (CD107a thiab CD69 ntxiv rau NK hlwb) thiab txo qis MDSC infiltration. Qhov no qhia tau hais tias cov tshuaj no ua rau TRF2- nyob ntawm ib qho kev pab cuam tshwj xeeb uas ua rau lub cev tsis muaj zog thiab tiv thaiv kab mob thiab tuaj yeem txhim kho kev tso tawm ntawm Danger Associated Molecules (DAMP) uas txhim kho lub cev tiv thaiv kab mob tshwj xeeb thaum TRF2 yog overexpressed. Ntawm qhov kev ceeb toom, peb pom tias AR thiab AD tau cawm cov tshuaj tiv thaiv kab mob thiab kev tiv thaiv angiogenic ntawm TRF2 overexpression, ob yam ntxwv ntawm TRF2 overexpres-sion uas peb yav dhau los tau pom tias yog DDR ywj pheej. Yog li, peb xav tias AR thiab AD cuam tshuam rau cov qog loj hlob yog DDR-ywj siab, ib lub tswv yim uas tseem yuav tau piav qhia hauv cov kev tshawb fawb ntxiv. Txoj kev tshawb fawb pov thawj tam sim no muab pov thawj tias kev txo cov tshuaj kho mob ntawm TRF2 qhia tuaj yeem yog qhov muaj txiaj ntsig zoo los tiv thaiv qog noj ntshav.
Txawm hais tias txoj kev tshuaj ntsuam xyuas tau suav tias yog TRF2 cov protein ntau ntawm nws tus kheej ntawm cov ntawv sau tseg, ob qho tshuaj txo qis endogenous TERF2 mRNA qib, txhais tau hais tias AR thiab AD tsom mus rau ntau theem ntawm TRF2 txoj cai. Txhawb nqa qhov no, AR yog ib qho inhibitor ntawm Wnt signaling [28], uas yog tus activator ntawm TERF2 transcription [29]. Feem ntau, cov tshuaj tsom mus rau Wnt signaling txoj hauv kev tau ua kom muaj txiaj ntsig zoo thaum kuaj cov kauj ruam (Daim duab S1B-D). Yuav ua li cas AD, tus neeg sawv cev mitochondria-targeting, cuam tshuam rau TRF2 kev qhia tseem yuav txiav txim siab. Txij li cov qog nqaij hlav uas muaj qib siab TRF2 muaj qhov pom tsis zoo thiab pom tias muaj kev tiv thaiv ntau ntxiv rau cov tshuaj kho mob [21], AR thiab AD yog cov tshuaj kho mob uas txaus siab rau cov qog no. Lub peev xwm los uncouple lub telomeric thiab pro-oncogenic kev ua ub no ntawm TRF2 [13] nce qhov muaj peev xwm ntawm pharmacologically downregulating TRF2 yog li conferring multi-ntaus kev pab cuam los tiv thaiv mob qog noj ntshav yam tsis muaj deleterious pro-aging phiv. Yog li, cov kev tshawb fawb yav tom ntej tau lees paub los txiav txim siab cov txiaj ntsig ntawm cov tshuaj no ntawm TRF2 qhia qib hauv kev tshawb fawb soj ntsuam.
Txawm hais tias TRF2 yog upregulated nyob rau hauv ntau yam tib neeg mob qog noj ntshav [13,21], nws cov lus qhia yog downregulated thaum lub sij hawm ob qho tib si ib txwm thiab pathological laus ntawm ntau cov ntaub so ntswg [30,31] Tsis tas li ntawd, ntau cov ntaub ntawv hais txog nas qauv ntawm TRF2 dysregulation hais txog qhov tseem ceeb ntawm TRF2 ntawm lub kev sib tshuam ntawm kev laus thiab mob qog noj ntshav [31-35]. Yog li, cov molecules uas tau txheeb xyuas los ntawm cov txheej txheem kev tshuaj ntsuam tau piav qhia ntawm no yog cov neeg sib tw tshuaj lom neeg, ob qho tib si ua cov tshuaj tiv thaiv kab mob rau TRF2 downregulation raws li tau lees paub hauv txoj kev tshawb no, thiab muaj peev xwm ua tau raws li kev tiv thaiv kev laus los ntawm kev tswj hwm TRF2.
Kab lus no yog muab rho tawm los ntawm Cancers 2021, 13, 2998. https://doi.org/10.3390/cancers13122998 https://www.mdpi.com/journal/cancers
