Hauv zos Non-pituitary Loj hlob Hormone raug cuam tshuam nrog kev laus thiab pab txhawb kev puas tsuaj ntawm epithelial

Thov hu rauoscar.xiao@wecistanche.comyog xav paub ntxiv

TSEEM CEEB

Microenvironmental factor modulating age-related DINA kev puas tsuaj tsis paub meej. Tsis yog-pituitary loj hlob hormone (np) yog induced nyob rau hauv tib neeg txoj hnyuv, uas tsis yog-transformed tib neeg txoj hnyuv hlwb, thiab fibroblasts, thiab nyob rau hauv 3-dimensional plab hnyuv organoids nrog hnub nyoog-associated DNA puas. Autocrine/paracrine npGH suppresses p53 thiab attenuates DNA puas cov lus teb (DDR) los ntawm inducingTRIM29 thiab txo cov ATM phosphorylation, ua rau txo DNA kho thiab DNA puas tsub zuj zuj. Organoids kab lis kev cai mus txog 4 lub hlis nthuav tawm cov cim kev laus, p16, thiab SA- -galactosidase thiab txo qis telomere ntev, nrog rau DNA puas tsub zuj zuj, nrog nce npGH, suppressed p53, thiab attenuated DDR. Ncua GH hauv cov laus organoids nce p53 thiab txo cov DNA puas. WT nas pom muaj hnub nyoog raws li txoj hnyuv DNA puas tsub zuj zuj, thaum nyob rau hauv cov nas laus uas tsis muaj GH signaling, DNA puas tseem qis, nrog siab p53. Raws li lub hnub nyoog txuam nrog npGH induction enables pro-proliferative microenvironment, abrogating npGH signaling yuav raug tsom raws li kev tiv thaiv kev laus los ntawm impeding DNA puas thiab muaj hnub nyoog pathologies.

Taw qhia

Kev ua haujlwm lom neeg poob qis nrog kev laus ua ke nrog cov kab mob muaj hnub nyoog ntxiv.flavonoidsCov hnub nyoog ntsig txog pathologies yog tsav los ntawm kev puas tsuaj DNA ntau ntxiv thiab qhov tsis zoo hauv DNA kho uas ua rau muaj kev hloov pauv thiab muaj hnub nyoog cuam tshuam nrog chromosomal aberrations. Kev puas tsuaj DNA tsis tu ncua kuj tseem tuaj yeem thaiv cov cell proliferation, ua rau muaj kev loj hlob thiab apoptosis, yog li ua rau kev laus ntxiv (Ou thiab Schumacher, 2018). DNA kev puas tsuaj accumulates nrog lub hnub nyoog nyob rau hauv qus-hom (WT), uas tsis yog-transformed tib neeg thiab murine hlwb, raws li muaj pov thawj los ntawm ntau zog phosphorylated histone 2A variant (yH2AX), ib tug marker ntawm ob-strand DNA so (DSBs) (Sedelnikova li al., 2004) thiab tsis kho DNA puas (Dolle li al., 1997; Hasty li al., 2003; Lombard li al., 2005; Petr et al., 2020). Genomic instability uas tshwm sim los ntawm kev kho DNA puas tuaj yeem ua rau kev hloov pauv ntawm tes (Negrini li al., 2010). DNA puas ua rau phosphorylation ntawm ataxia-telangiectasia-mutated (ATM) noob, lub luag hauj lwm rau kev kho ntawm ib leeg-strand DNA so thiab DSBs. ATM phosphorylates thiab stabilizes qog suppressor p53, yog li ua kom DNA puas tsuaj kho los ntawm kev pab txhawb ntau txoj hauv kev kho DNA. Yog li, ATM ua lub luag haujlwm tseem ceeb hauv kev ua kom muaj kev puas tsuaj DNA (DDR) thiab kho DNA (Ou thiab Schumacher, 2018).

Thov nias ntawm no kom paub ntxiv

ATM kev qhia thiab kev ua haujlwm tsis zoo nrog lub hnub nyoog (Feng li al..2007; Gutierrez-Martinez li al.,2018), tej zaum yuav ua rau muaj hnub nyoog txog kev ua haujlwm p53 poob thiab ua rau cov DNA puas thiab chromosomal instability (Simon et al, 2009) .p53 cov lus teb rau cov kev ntxhov siab xws li hluav taws xob qis hauv cov nas laus (Feng li al.,2007) thiab hauv tib neeg dermal fibroblasts muab los ntawm cov neeg laus (Goukassian li al.,2000). Yog li, cov cim ntawm kev laus muaj xws li kev txo qis DDR kev ua haujlwm thiab p53 kev tawm tsam nrog kev puas tsuaj DNA ntau ntxiv, txhua yam ua rau muaj kev cuam tshuam zoo rau kev loj hlob ntawm epithelial (Lopez-Otin li al., 2013).

Raws li pituitary kev loj hlob hormone (GH) secretion poob qis nrog lub hnub nyoog, hloov GH signaling tau cuam tshuam rau hauv cov txheej txheem kev laus (Ho thiab Hoffman, 1993) Txawm li cas los xij, lub cev ntawm cov pov thawj txhawb cov txiaj ntsig ntawm GH attenuation ntawm kev laus. , thiab tib neeg kab mob qauv (Brown-Borg li al., 1996; Junnila et al., 2013), cov txiaj ntsig ntawm GH deficiency feem ntau yog los ntawm kev soj ntsuam cov yam ntxwv thib ob uas ua rau lub neej ntev, suav nrog kev txhim kho insulin rhiab heev, txo cov kab mob siab oxidation ntawm cov protein. , txo qis kev mob qog noj ntshav, thiab txo qis hnub nyoog sib xws (Aguiar-Oliveira thiab Bartke, 2019; Bartke, 2016; Junnila et al., 2013; Spadaro et al., 2016). Cov xeeb ntxwv ntawm Leiden kawm cov tsev neeg uas muaj tsev neeg nyob ntev thiab qis dua kev tuag. tshaj li kev tswj hwm ib puag ncig tau qis dua kev sib koom ua ke ntawm GH theem (van der Spoeletal., 2016), thaum GHexcess txo qis lub neej raws li pom hauv cov neeg mob acromegaly (Chesno-kova li al., 2019a; Melmed, 2020; Dej thiab Barclay, 200 7) thiab hauv transgenic murine qauv overexpressing GH (Bartke, 2003).hesperidin sivQee tus tau hais txog qhov tsis zoo ntawm qhov siab thiab kev ua neej ntev hauv tib neeg (Nws li al., 2014; Samaras thiab Storms, 1992) thiab hauv dev (Greer li al., 2007), Peb xav tias qhov tsis zoo GH cuam tshuam rau kev laus, yam tsawg kawg. ib feem, raug ntaus nqi los ntawm kev puas tsuaj DNA ntau ntxiv (Ches-nokova thiab Melmed, 2020). Hauv kev txhawb nqa ntawm qhov chaw no, lymphocytes muab tau los ntawm cov neeg mob acromegaly pom DNA puas nrog chromosomal aberrations (Bayram li al, 2014), thaum unrepaired DNA puas accumulates nyob rau hauv daim siab ntawm ib tug zebrafish acromegaly qauv (Elbialy li al.,2018). High circulating GH theem attenuate murine nyuv DNA kho, ua rau DNA puas tsub zuj zuj, thaum DNA kev puas tsuaj yog attenuated tom qab thaiv GH receptor (GHR) nyob rau hauv tib neeg txoj hnyuv hlwb thiab cov nas nrog abrogated GHR signaling (Chesnokova li al., 2019a).

Non-pituitary GH (np) synthesized hauv zos nyob rau hauv peripheral cov ntaub so ntswg yog zoo tib yam rau endocrine GH1 tsim los ntawm pituitary thiab ua los ntawm autocrine / paracrine mechanisms los ntawm kev nthuav dav GHR (Ballesteros li al., 2000) uas lees paub ob qho tib si pituitary GH thiab npGH ligand. Dej thiab Barclay, 2007; Waters et al, 2006).poob teb chaws Ottoman cistancheRaws li DDR activation induces npGH(Chesnokova et al.,2013), peb xav tias muaj hnub nyoog txuam nrog cov nyuv DNA puas induces npGH qhia, uas, nyob rau hauv lem, yuav ntxiv constrain DNA puas kho.

Cistanche tuaj yeem tiv thaiv kev laus

Siv ob qho tib si hauv vitro thiab ex vivo tib neeg cov qauv qhia txog ag-ing, peb qhia ntawm no tias npGH raug ntxias thiab DNA puas tsuaj hauv cov neeg laus cov ntaub so ntswg, raws li pov thawj los ntawm H2AX phosphorylation (yH2AX). Tsis tas li ntawd, txoj hnyuv DNA puas tau nce ntxiv rau cov nas muaj hnub nyoog WT, thaum qhov kev puas tsuaj ntawm epithelial DNA tseem qis hauv cov hnub nyoog sib xws GHR--cov nas uas tsis muaj GH signaling thiab hauv cov nas tshwj xeeb GHR knockout nas (GHRcolKO).

Lub zos npGH kuj raug ntxias nyob rau hauv hnub nyoog 3-dimensional (3D) nyob rau hauv-testinal organoids generated los ntawm induced human pluripotent qia hlwb (iPSCs)(Barrett li al,2014; Chesnokova li al, 2019a). Organoid npGH, nyob rau hauv lem, suppressed p53 thiab attenuated DNA puas kho.micronized purified flavonoid feem 1000 mg sivCov teebmeem ntawmnpGH yog qhov zoo li paracrine, raws li kev sib koom ua ke ntawm GH-kev nthuav qhia fibroblasts nrog Colon Intestine-Chip microfluidic cov cuab yeej kos nrog tib neeg colonoidsled rau sup-pressed p53 / p21 thiab txhim kho DNA puas. Los ntawm qhov sib txawv, suppressing GH hauv cov laus organoids tiv thaiv DNA puas.

Yog li, peb qhia tau hais tias epithelial npGH muaj zog induced thaum lub sij hawm aging thiab hais tias npGH txo DNA kho thiab pab txhawb lub hnub nyoog-txog DNA puas tsub zuj zuj. Zuag qhia tag nrho, cov txiaj ntsig tau qhia tias cov hnyuv hauv zos epithelial npGH ua rau muaj kev hloov pauv hauv mucosal, tsim kom muaj ib qho milieu ntawm chromosomal instability uas yog qhov zoo-muaj peev xwm rau kev loj hlob neoplastic hauv cov ntaub so ntswg laus.

Qhov tseem ceeb, txawm hais tias muaj ntau qhov tsis tsim nyog siv GH ua "tiv thaiv kev laus" cov tshuaj hormones txawm tias tsis muaj qhov cuam tshuam rau kev laus (Blackman li al.,2002; Clemmons li al..2014; Lieut al.,2007; Melmed, 2019), peb cov txiaj ntsig tau pom tias cov teeb liab GH hauv zos pab txhawb rau cov ntaub so ntswg microenvironment, nyiam kev loj hlob ntawm cov hnub nyoog ntsig txog pathologies. Nthuav npGH hauv zos qhov taw qhia kom txwv tsis pub muaj kev puas tsuaj rau epithelial DNA tuaj yeem ua rau muaj lub hom phiaj kho anovulant-aging. Cov txiaj ntsig ntawm cov kev tshawb fawb no qhia txog txoj hauv kev tshiab rau kev ncua sij hawm (piv txwv li, kev tawm tsam ntau dua li induction ntawm lub zos GH signaling). TSEEM CEEB

Tib neeg txoj hnyuv DNA puas thiab npGH sib sau nrog kev laus

Kev puas tsuaj DNA hnub nyoog muaj hnub nyoog raug soj ntsuam hauv cov qog nqaij hlav hauv tib neeg cov plab hnyuv paraffin cov qauv uas tau muab los ntawm cov neeg mob cohorts spanning 3 pawg hnub nyoog: hluas (18-39 xyoo), hnub nyoog nruab nrab (40-60 xyoo), thiab hnub nyoog ( 61-88 xyoo). Thaum tau qhab nia rau yH2AX, 3 ntawm 11 tus qauv (27 feem pua) hauv pawg tub ntxhais hluas tau nthuav tawm DNA kev puas tsuaj, 8 ntawm 16 (50 feem pua) tau nthuav tawm DNA kev puas tsuaj rau hnub nyoog nruab nrab, thiab 8 ntawm 18 (45 feem pua) cov qauv hnub nyoog tau nthuav tawm DNA puas (Figures 1A-1C thiab 1J).

Raws li peb yav dhau los tau pom tias DNA kev puas tsuaj induces npGH (Chesnokova li al, 2013), peb tau nrhiav los txiav txim seb puas muaj hnub nyoog raws li kev puas tsuaj ntawm DNA puas cuam tshuam nrog GH qhia hauv cov nqaij mos uas tsis yog qog nqaij hlav. Raws li tsis muaj qhia nyob rau hauv neuroendocrine nyuv hlwb (Chesnokova li al, 2016), peb cais cov hlwb los ntawm kev tsom xam.

Epithelial GH qhia tau pom nyob rau hauv 16 feem pua ​​-17 feem pua ​​​​ntawm cov qauv hnyuv ib txwm muab los ntawm cov hnub nyoog nruab nrab thiab cov laus (38 thiab 42 cov qauv, raws li) tab sis tsuas yog 2 feem pua ​​​​ntawm cov tub ntxhais hluas pawg (37 cov qauv), thaum GH qhia tau qhab nia tau nce hauv cov neeg laus hnub nyoog nruab nrab thiab tau nce ntxiv hauv cov neeg laus cov qauv (p<0.01) (figures="" 1d-1f="" and="" 1k).="" furthermore,="" colon="" mrna="" gh="" expression="" increased="" in="" both="" middle-aged="" (30%="" of="" 10="" samples)="" and="" aged="" (26%="" of="" 14="" samples)="" versus="" young="" cohort="" samples(17%="" of9=""><0.01 and=""><0.05, respectively)(figures="" 1g-1).="">oteflavonoidHauv cov ntsiab lus, cov txiaj ntsig no taw qhia txog DNA puas tsub zuj zuj uas tsis muaj kev cuam tshuam hauv txoj hnyuv laus.

DDR activation induces zos npGHin murine thiab tib neeg txoj hnyuv

Peb tom ntej no tau txiav txim siab seb puas tau qhib DDR ua rau cov kab mob hauv zos npGH qhia thiab kho cov nas nrog nutlin3 kom stabilize p53 (Vassilev et al, 2004) thiab pom induced nyuv p53 thiab nce (Daim duab 2A). Tsis tas li ntawd, nyob rau hauv txoj hnyuv adenocarcinoma specimens muab los ntawm tib cov neeg mob. ua ntej thiab tom qab DNA kev kho puas tsuaj (Table S1), peb tau pom me ntsis kev kho mob ua ntej lub hauv paus epithelial npGH qhia thiab nce npGH qhia tom qab kho hauv tag nrho 6 tus neeg mob kuaj tau kawm (p<0.01) (figure="" 2b).="" enhanced="" gh="" expression="" was="" also="" observed="" in="" tumor-associated="" fibroblasts="" after="" dna="" damage(figure="" 2c),="" indicating="" that="" these="" findings="" are="" likely="" not="">

DDR qhib GH qhia hauv vitro

Peb tau siv cov topoisomerase Il inhibitor etoposide los tsim ob leeg-strand DNA so thiab DSBs (Walles li al, 1996) thiab los txiav txim seb DDR activates GH hauv vitro. Lub DNA puas-induced npGH hauv NCC, hNCF, thiab HCT116 human colon adenocarcinoma cells (Figures 3A thiab S1A-S1C); hauv organo-ids, etoposide-induced GH thiab GH mRNA (Figures 3B thiab S1D), thaum IGF1 thiab prolactin mRNA qib tsis hloov pauv (Daim duab S1E). STAT5 phosphorylation, ib tug downstream GH teeb liab (Waters, 2016), tau induced nyob rau hauv tag nrho cov hom ntawm tes (Figures 3A-3C thiab S1A-S1C). Los ntawm qhov sib piv, tshem tawm npGH nrog GH me me cuam tshuam RNA (siRNA) tiv thaiv etoposide los ntawm inducing pSTAT5 (Fig-ures 3C thiab S1F), muab pov thawj tias DNA puas-induced nyuv npGH ua haujlwm. Kev yees duab ntawm NCC kho nrog etoposide rau 24 h pom GH thiab yH2AX co-expression hauv ntau lub hlwb (Daim duab 3D).

Epithelial hlwb raug kev puas tsuaj DNA secrete GH Los txiav txim seb puas intracellular npGH exerts autocrine/paracrine teebmeem, peb soj ntsuam GH qib secreted rau hauv NCC thiab organoid kab lis kev cai nruab nrab tom qab kho etoposide. Nruab nrab sau los ntawm ANCC ntawm 8 thiab 24 teev tom qab kev kho mob thiab los ntawm organoids ntawm 4-48 h tom qab kev kho mob pom lub sij hawm-dependent GH nce tom qab DDR activation (Daim duab 3E). Vim tias NCC thiab organoids nthuav qhia GH receptors raws li tib neeg cov kab mob plab (Chesnokova li al., 2016), cov txiaj ntsig no qhia tias GH hauv zos raug ntxias los teb rau DNA puas tuaj yeem ua rau cov ntaub so ntswg laus rau autocrine / paracrine ua.

Autocrine/paracrine GH suppresses tib neeg txoj hnyuv cell p53

Raws li endocrine GH suppresses murine nyuv p53 (Chesnokova li al, 2016), peb tau txiav txim siab seb GH hauv zos ua rau cov teebmeem au-autocrine / paracrine zoo li hauv tib neeg lub plab hnyuv loj. Kev tshuaj xyuas ntawm ob kab NCC cais tau los ntawm ob tus neeg mob sib txawv tom qab nucleofection nrog hGH lossis kab mob nrog lentivirus express-ing GH (lenti-GH) (Figures 4A, 4B, thiab S2) nrog rau cov organoids stably qhia hGH (Daim duab 4C) pom tau hais tias suppressed. p53 thiab concomitant overexpression. Cells muab tau los ntawm organo-ids kis nrog lenti-GH los yog khoob vector (lenti-V) pulsed nrog bromodeoxyuridine (BrdU) pom p53 suppression ua raws li kev coj tus kheej tab sis muaj zog BrdU incorporation raws li-sessed los ntawm fluorescence-activated cell sorting (FACS)(Daim duab S3A). Los ntawm qhov sib txawv, GH kev tawm tsam nrog lentivirus nthuav tawm cov plaub hau me me GH (GH) ua rau txo qis kev tsim thiab cov cheeb tsam loj hauv NCC (Figures S3B thiab S3C), qhia txog kev ua haujlwm zoo rau cov ntawv npGH.

Paracrine GH cov teebmeem hauv tib neeg Colon Intestine-Chip Los soj ntsuam cov teebmeem paracrine ntawm npGH ntawm cov hnyuv mucosal epithelial hlwb, peb tau sib koom ua ke UNCF kis kab mob lenti-GH thiab Colon Intestine-Chip microfluidic devices (Kasendra li al, 2020) noob nrog kev noj qab haus huv colonic organoid-derived epithelial hlwb rau 8 hnub. Hauv zos GH tso tawm los ntawm hNCF mus rau hauv nruab nrab tau lees paub los ntawm ELISA(338ng/mL/1 × 10 degree hlwb).5-Ethynyl-2'-deoxyuridine (EdU) (20 μM) tau ntxiv rau hnub 7 , chips raug kho tom qab 24 teev, thiab stained rau EdU, thiab H2AX, thiab EdU ntxiv nuclei raug suav rau hauv cov hlwb epithelial. Ib txheej ntawm chips seeded

nrog cov hlwb epithelial tau siv los ntsuas p53 / p21 los ntawm western blot. Kev loj hlob ntawm nti epithelial hlwb nce (p<0.05), likely="" due="" to="" decreased="" p53="" and="" p21="" (figures="" 4d="" and="" 4e).="" moreover,="" dna="" damage="" accumulation="" was="" evident,="" with="" increased="" numbers="" of="" cells="" expressing="" γh2ax="" （p=""><0.01） （figure="">

Autocrine/paracrine GH suppresses DDR

Ua kom lub ATM thaum DNA puas phosphorylates H2AX, uas cim qhov chaw DNA puas los qhib DDR thiab txhawb kev kho (Turinetto thiab Giachino, 2015).ATM kuj phosphorylates p53, ntxiv txhim kho DNA (Blackford thiab Jackson 2017). Hauv hNCC thiab hauv organoids kis nrog pIRES2-ZsGreen1hGH lossis nrog lentil-GH, GH overexpression suppressed lub phosphorylation ntawm ATM thiab DNA-protein kinase (PK)cs, ob qho tib si koom nrog hauv DNA kho (Blackford thiab Jackson. 2017), thiab txo phosphorylation ntawm lub hom phiaj protein p53 (Figures 5A-5C).

Tip60 histone acetylase activates ATM (Jackson thiab Bartek, 2009) thiab raug tswj tsis zoo los ntawm TRIM29 (Sho et al., 2011; Sun et al, 2005). Peb pom tias GH overexpression hauv NCC thiab hauv organoids kuj ua rau TRIM29 induction, uas nyob rau hauv lem suppressed Tip60 (Figures 5A-5C). Cov txiaj ntsig zoo sib xws tau pom hauv kab thib ob NCC 48 teev tom qab nucleofection nrog plasmid qhia GH thiab ntawm 6 hnub tom qab kis kab mob nrog lenti-GH (Figures S4A thiab S4B).

Autocrine / paracrine GH txo qis kev kho DNA

Peb tom ntej ntsuas qhov cuam tshuam ntawm DSB kho cov txheej txheem, tshwj xeeb yog kev ua yuam kev tsis sib xws tsis yog homologous kawg koom nrog (NHEJ)(Jackson thiab Bartek, 2009) thiab yuam kev-pov thawj homologous recombination (HR)(Beucher li al.,2009 ; Blackford thiab Jackson, 2017; Chapman et al., 2012). Peb nucleofected NCC ruaj khov qhia NHEJ thiab HR cov neeg sau xov xwm cov ntawv xov xwm nrog pcDNA3.1- tib neeg GH1 (hGH1) plasmids, ntxias DSBs los ntawm kev sib koom ua ke nrog plasmid encoding I-Excel endonuclease, thiab ntsuas qhov rov ua haujlwm ntawm DSB kho cov neeg sau xov xwm. Transfection efficiency yog tswj los ntawm co-transfecting nrog plasmid encoding DsRed (Seluanov li al, 2010). NHEJ efficiency tom qab pcDNA3.1-hGH1 nucleofection yog 0.<0.05)(figure5d), and="" hr,while="" low(0.05),="" consistent="" with="" other="" reports="" (chitnis="" et="" al,2014;gatei="" et="" al.,2011),showed="" consistently="" decreased="" efficiency="" by="" up="" to="" 38.7%±3.5%="" (n="4" independent=""><0.05) at="" 72="">

Autocrine / paracrine GH ua rau kom tsis muaj DNA puas tsuaj

Kev tshuaj xyuas seb npGH nce DNA puas tsuaj, peb pom tias NCC thiab organoids ruaj khov hloov nrog lenti-GH pom tias muaj kev puas tsuaj rau cov DNA uas tsis tau kho dua tshiab raws li ntsuas los ntawm Comet assay (p< 0.05)="" (lee="" and="" paull,="" 2007)(figures="" 5e="" and="" 5f).="" levels="" of="" unrepaired="" dna="" were="" also="" markedly="" increased="" in="" a="" second="" ncc="" line="" in="" cells="" nucleofected="" with="" a="" plasmid="" expressing="" gh="" or="" infected="" with="" lenti-gh="" (figures="" s4c="" and="" s4d).="" overall,="" these="" results="" show="" that="" npgh="" is="" induced="" in="" response="" to="" dna="" damage="" and="" enhances="" dna="" damage="" accumulation="" by="" interfering="" with="" both="" ddr="" and="" dna="" repair="">

GH yog induced nyob rau hauv lub hnub nyoog 3D tib neeg plab hnyuv organoids thiab attenuates DDR

Ua raws li pom tau nce GH qhia thiab DNA kev puas tsuaj hauv cov neeg laus cov kab mob qog noj ntshav, peb "hnub nyoog" organo-ids tsim los ntawm peb tus kab mob ywj pheej iPSC los rov sau cov qauv ntawm cov laus tib neeg cov ntaub so ntswg. Cov organoids no muaj xws li enterocytes, goblet hlwb, Paneth hlwb, thiab enteroendocrine hlwb thiab qhia caudal-hom homeobox 2 (CDX2)(Barrett li al..2014; Gao li al.,2009; Workman li al.,2017). Ob ntawm peb iPSC kab (kab 1 thiab 3) tau txais los ntawm cov neeg laus dua 60 xyoo, thiab thib peb iPSC kab (kab 2) tau muab los ntawm fibroblasts ntawm ib tus neeg muaj hnub nyoog 30- xyoo. Organoids tsim los ntawm kab 1 thiab 3 tau coj mus rau 2 lub hlis, thaum lub sijhawm cell proliferation qeeb heev. Los ntawm qhov sib txawv, organoids tsim los ntawm kab 2 proliferated robustly nyob rau hauv kab lis kev cai mus txog rau 4 lub hlis. Cellular aging tau lees paub nyob rau hauv tag nrho 3 kab los ntawm kev nce p16, nrog rau kev txo qis telomere ntev (p<0.05)(figures 6a="" and="" 6b).="" in="" aged="" organoids,="" we="" also="" found="" increased="" senescence-associated="" β-galactosidase="" (sa-β-galactosidase)="" activity,="" which="" has="" been="" shown="" to="" increase="" with="" age="" (kurz="" et="" al.,="" 2000;="" lee="" et="" al.,="" 2006),="" as="" well="" as="" increased="" β-galactosidase="" protein="" expression,="" which="" correlates="" with="" sa-β-galactosidase="" enzymatic="" activity(figures="" 6a="" and="">

Raws li cov yam ntxwv hauv vitro organoid tshwm sim rau kev ncaj ncees rov hais dua cov kev hloov pauv ntawm tes raws li kev laus ntawm tib neeg cov ntaub so ntswg (Baker li al.,2011; Lieut al.,2019; Lopez-Otin li al.,2013), peb siv qhov no qauv los kawm ntxiv txog kev taw qhia mechanisms hauv qab tib neeg txoj hnyuv epithelial aging. Hauv kab 1, GH mRNA qib tau raug ntxias tom qab 1 lub hlis hauv kab lis kev cai thiab nce ntxiv tom qab 2 lub hlis (p <0.05)(daim duab="" 6c),="" uas="" zoo="" ib="" yam="" nrog="" nce="" npgh="" pom="" hauv="" cov="" laus="" organoids="" ntawm="" ob="" sab="" hnub="" poob="" blot="" thiab="" immunohistochemistry="" (ihc)="" (daim="" duab="" 6d,="" 6e,="" thiab="" s5b).="" yh2ax="" kuj="" tau="" ua="" rau="" muaj="" kev="" cuam="" tshuam="" nrog="" kev="" coj="" noj="" coj="" ua="" ntev,="" qhia="" txog="" kev="" puas="" tsuaj="" dna="" endogenous="" nrog="" hauv="" vitro="" ag-ing.="" raws="" li="" cov="" txiaj="" ntsig="" uas="" qhia="" pom="" p53="" hauv="" ncc="" thiab="" organoids="" transfected="" nrog="" gh="" (figures="" 4a-4c),="" peb="" pom="" suppressed="" p53="" ntawm="" 1="" thiab="" 2="" lub="" hlis="" ntawm="" kab="" lis="" kev="" cai,="" concordant="" nrog="" nce="" npgh="" (figures="" 6e="" thiab="" s5b).="" cov="" txiaj="" ntsig="" zoo="" sib="" xws="" (="" a="" thiab="" b)="" western="" blot="" ntawm="" (a)="" ancc,="" hncf,="" thiab="" hct116="" hlwb="" thiab="" (b)="" organoids.="" cov="" hlwb="" raug="" kho="" nrog="" cov="" tshuaj="" etoposide="" qhia="" thiab="" tshuaj="" xyuas="" 24="" teev="" tom="" qab.="" organoids="" tau="" kho="" nrog="" 3="" um="" etoposide="">

(C) NCC nucleofected nrog GH siRNA (siGH RNA) lossis scramble RNA (Scr RNA) raws li kev tswj hwm rau 24 teev tau kho nrog cov tshuaj qhia ntawm etoposide (Etop) thiab tshuaj xyuas 24 teev tom qab. ImageJ kom muaj nuj nqis ntawm sab hnub poob blots yog piav qhia hauv daim duab S1.

(D) Cov duab sawv cev ntawm NCC kho nrog 20 μM etoposide rau 24 teev. GH, liab; yH2AX, ntsuab; phalloidin, grey; DAPI, xiav. Lub ci ntsuab nucleus qhia tias apoptosis. Scale bar, 20 um. Tswj, tsis kho hlwb.

(E) Sab hnub poob blot ntawm GH nyob rau hauv kab lis kev cai nruab nrab ntawm ANCC thiab organoids kho nrog 20 los yog 5 uM etoposide, feem. Qhov nruab nrab tau sau los ntawm tshav kub 8 thiab 24 teev thiab los ntawm organoids ntawm 4-48 h tom qab kho. Ponceau tau siv los tswj kev thauj khoom. Cov neeg sawv cev blots los ntawm tsawg kawg 3 qhov kev sim ywj pheej tau pom.

tau txais los ntawm culturing organoids generated los ntawm iPSC kab 2 thiab 3 rau 4 thiab 2 lub hlis, raws li (Figures S6A-S6D).

Tsis tas li ntawd, nce npGH nyob rau hauv cov laus organoids yog txuam nrog attenuated DDR, nrog suppressed phosphorylation ntawm ob lub ATM thiab DNA-PKcs thiab qis dua theem ntawm phosphorylated p53 (Figures 6E thiab S5B). Cov txiaj ntsig no qhia tias kev ua haujlwm tsis zoo ntawm DDR ua rau muaj kev puas tsuaj DNA ntau ntxiv, raws li tau pom los ntawm yH2AX induction (Figures 6E thiab S5B). Tom qab 2 lub hlis nyob rau hauv kab lis kev cai, cov laus organoids kis GH shRNA pom muaj zog p53 (Figures 6F thiab S7A) thiab txo DNA puas tsuaj ntsuas los ntawm Comet assay (p<0.01)(figure 6g),="" indicating="" a="" requirement="" for="" npgh="" in="" age-related="" dna="" damage="" accumulation="" in="" this="">

GH signaling deficiency attenuates nyob rau hauv vivo nyuv DNA puas nyob rau hauv cov laus nas

Txhawm rau kom paub meej tias peb cov txiaj ntsig tau txais hauv cov hnub nyoog organoids, peb tau kawm hnub nyoog WT thiab GHR-7 nas hauv vivo. Colon DNA puas tau nce ntxiv hauv cov laus (24-hli) piv rau cov hluas (3-hli) WT nas(p<0.01), but,="" strikingly,="" was="" not="" increased="" in="" aged="" ghr-mice="" (figures="" 7a="" and="" s7b),="" likely="" due="" to="" p53="" in-duction.="" we="" also="" generated="" transgenic="" mice="" with="" colon-specific="" ghr="" excision="" (car1-cre/ghr"loxmox,="" ghrcolko)(figure="" s7d).="" control="" mice="" were="" derived="" from="" the="" same="" breeding,="" but="" they="" did="" not="" harbor="" the="" cre="" enzyme.="" colon="" dna="" damage="" was="" much="" reduced="" in="" 20-month-old="" male="" ghrcolko="" mice="" with="" ghr="" excision="" compared="" to="" control="" mice,="" but="" not="" in="" the="" small="" intestine="" with="" intact="" ghr(figure="" 7d).="" thus,="" it="" appears="" that="" dna="" is="" repaired="" more="" efficiently="" in="" mice="" devoid="" of="" gh="" signaling,="" likely="" due="" to="" the="" higher="" colon="" p53="" expression="" we="" observed="" in="" both="" ghr-/-and="" in="" ghrcolko="" animals(figures="" 7b,7c,="" 7e,="" s7c,="" and="" s7e).="" these="" in="" vivo="" results="" buttress="" our="" hypothesis="" that="" npgh="" en-hances="" age-associated="" colon="" dna="" damage="">

Kev sib tham

Peb qhia ntawm no tias npGH nce nrog lub hnub nyoog hauv tib neeg cov hnyuv thiab hauv 3D tib neeg txoj hnyuv organoids. Peb pom ntau cov ntaub ntawv pov thawj qhia tias nyob rau hauv cov laus tib neeg txoj hnyuv cov ntaub so ntswg, npGH yog induced los ntawm DNA puas, raws li tau pom nyob rau hauv tib neeg qauv, xws li epithelial non-tumorous cells, ib txwm nyuv fibroblasts, organoids, thiab cov nqaij mos mob cancer ntawm cov neeg mob nyob rau hauv lub DNA puas kho. ; peb kuj tseem qhia cov kev tshawb pom zoo sib xws hauv vivo hauv cov nyuv ntawm nas nrog qhib DDR.

Siv 3D tib neeg txoj hnyuv organoids, peb pom muaj zog p16, senescence, thiab telomere shortening, tag nrho raws li cov hnub nyoog-kev hloov pauv ntawm tib neeg cov ntaub so ntswg (Baker et al., 2011; Liu et al., 2019; Lopez-Otin et al., 2013 ). Txawm hais tias muaj kev txwv ntawm tus qauv organoid, peb cov txiaj ntsig tau txhawb cov plab hnyuv organoids raws li kev ncaj ncees rau tib neeg rov qab los kawm txog cov teeb meem ntawm cov kev hloov hauv cov hnub nyoog uas muaj feem cuam tshuam rau txoj hnyuv (Hu et al., 2018).

Peb pom tias npGH hauv zos, ua yeeb yam hauv autocrine / paracrine zam, suppresses p53 thiab ua rau cov neeg nyob sib ze zuj zus. Induced GH kuj suppresses DDR thiab DNA kho, ntxiv txhim kho DNA puas tsub zuj zuj. Cov txiaj ntsig kuj tseem muaj txiaj ntsig nrog cov txiaj ntsig tau pom ntawm DNA puas tsub zuj zuj hauv GH-secreting tib neeg cov qog pituitary (Ben-Shlomo li al., 2020).

GH kev ua hauv cov plab hnyuv ib txwm nyiam kev loj hlob ntawm pro-proliferative milieu uas tuaj yeem ua rau muaj hnub nyoog txog kev nce qib ntawm cov nyuv polyps, piv txwv los ntawm cov hnub nyoog sib txuas nrog DNA dam-hnub nyoog thiab chromosomal instability raws li tus neeg tsav tsheb ntawm kev mob qog noj ntshav hauv cov neeg mob laus dua. 50 xyoo (Aunan et al., 2017). Lub hnub nyoog cuam tshuam txog DNA kev puas tsuaj uas peb qhia ntawm no kuj tseem ua raws li cov lus ceeb toom tias yH2AX nce linearly nrog lub hnub nyoog hauv cov kab mob hnyuv ib txwm (Risques et al., 2008). Peb tau pom tias muaj kev cai lij choj ntawm yH2AX nyob rau hauv cov laus organoids yog qhia txog DNA puas tsub zuj zuj, raws li yH2AX cim DNA puas qhov chaw kom nyiam DNA kho proteins (Turinetto thiab Giachino, 2015). Cov txiaj ntsig no yuav zoo li tsis sib haum nrog qhov tseeb tias GH induced hauv cov hlwb laus suppresses H2AX phosphorylation. Txawm li cas los xij, peb txhais cov kev soj ntsuam no los qhia tias GH de-creases DDR kev ua haujlwm los ntawm kev txo qis phosphorylation ntawm ATM, uas, nyob rau hauv lem, txo (tab sis tsis tag tshem tawm) phosphorylation ntawm downstream proteins nrog rau H2AX. Thaum DDR tsis tsim nyog qhib, kho DNA hloov pauv, tab sis yH2AX tseem khaws cia raws li nws cim qhov chaw puas DNA tsis kho. Yog li, yH2AX qib tau pom nyob rau hauv cov laus tib neeg cov ntaub so ntswg thiab organoids muaj kev cuam tshuam cov txiaj ntsig ntawm DDR kev ua haujlwm thiab tus nqi ntawm DNA tsis kho.

DNA kev puas tsuaj tuaj yeem sau rau hauv cov ntaub so ntswg laus vim qhov poob ntawm lub hauv paus excision kho, tej zaum los ntawm qhov poob ntawm p53 teb rau DNA puas (Cabelof li al., 2006; Simon et al., 2009, 2012). Hauv peb qhov kev sim, p53 tsis kam nyob rau hauv cov hnyuv thiab cov organoids ex-nias npGH, raws li kev tshawb pom ua ntej ntawm GH suppressing p53 (Chesnokova li al., 2016). Los ntawm qhov sib piv, cov laus GHR-/- nas, uas tiv taus mob qog noj ntshav thiab muaj lub neej ntev (Basu li al., 2018), nrog rau cov kab mob tshwj xeeb GHRcolKO cov nas pom muaj kev nthuav qhia ntawm cov nyuv p53, yuav txhim kho DNA kho. Raws li GH suppresses p53 (Chesnokova li al., 2013), npGH induction tej zaum yuav ua rau muaj kev hloov hauv lub zos microenvironment raws li hnub nyoog-txo qis p53 (Feng li al., 2007; Goukassian li al., 2000). GH txhawb nqa muaj p53 khi qhov chaw (Chesnokova li al., 2013), thiab DNA puas-induced p53 activates GH qhia (Chesnokova li al. 2013,2019a). Yog li, peb cov txiaj ntsig tau pom tias nyob rau hauv cov laus organoids, DNA puas tsub zuj zuj induces npGH, uas, nyob rau hauv lem, down-regulates p53.

Qhov tseem ceeb, raws li npGH yog zais hauv zos los ntawm DNA- puas hlwb, ib qho kev txiav txim paracrine rau zais GH ntawm cov hlwb nyob sib ze tuaj yeem ua haujlwm. Thaum co-culturing GH-expressing hNCF nrog Colon Intestine-Chip, peb tau pom ntau DNA puas thiab proliferation thiab downregulated p53/p21 nyob rau hauv nti-embedded epithelial hlwb raug rau paracrine teebmeem ntawm fibroblast-derived. Cov txiaj ntsig no qhia tias kev laus hauv zej zog tsis muaj kev cuam tshuam hauv tib neeg cov ntaub so ntswg hloov cov ntaub so ntswg micro-ib puag ncig los ntawm kev txo cov qog nqaij hlav proteins thiab pab txhawb kev loj hlob ntawm epithelial thiab DNA puas.

Cov txiaj ntsig tau zoo ib yam nrog cov kev soj ntsuam uas exogenous GH kev kho mob ua rau muaj peev xwm loj hlob, tsim cov kab mob, thiab metastases hauv DJ-1 KO nas (Chien et al., 2016), thiab nrog cov pov thawj tias GH induction hauv tib neeg cov qog hlwb cuam tshuam nrog kev loj hlob ntawm ntau cov qog nqaij hlav (Perry li al., 2017); cov kev sib tham no nrog peb cov txiaj ntsig uas qhia pom tsawg dua colony formations thiab me me colony loj nrog GH suppression.

DNA puas kho txoj hauv kev tiv thaiv chromosomal instability uas tsav cellular transformation (Negrini li al. 2010). Piv txwv li, Tip60 qhib ATM (Jackson thiab Bartek, 2009), thiab ATM, lub hauv paus tswj hwm ntawm DDR, phosphorylates thiab stabilizes p53. Cov hlwb laus ua rau txo qis ATM thiab p53 kev tawm tsam, nrog rau DNA kho dysregulation nrog kev puas tsuaj DNA uas tsis tau kho (Lan li al.,2019). Yog li, nyob rau hauv cov hnyuv, DNA kev puas tsuaj accumulates nrog lub hnub nyoog (Risques li al., 2008; Schu-macher li al., 2008), tsawg kawg yog ib feem vim poob DDR efficiency (Jackson thiab Bartek, 2009), thiab suppressed p53 hauv teb rau y-irradiation nyob rau hauv cov laus nas yog vim muaj hnub nyoog-related poob nyob rau hauv p53-stabilizing ATM(Feng li al,2007).Hauv peb cov kev sim, tsis induced nyob rau hauv teb rau DNA puas tsuaj kuj suppressed Tip60, uas tej zaum yuav coj. kom txo DNA kho raws li pov thawj los ntawm attenuated ATM thiab p53 phosphorylation, feem. Cov txiaj ntsig no qhia tias npGHoverexpression hauv zos tuaj yeem ua rau muaj hnub nyoog ntsig txog dysregulated DDR thiab DNA kho. Hauv cov hnub nyoog organoids, GH induction attenuated DDR, thaum GH activated DDR, raws li pov thawj los ntawm txo DNA puas. Cov txiaj ntsig no tau txais kev txhawb nqa los ntawm peb cov kev soj ntsuam yav dhau los uas thaiv GH teeb liab hauv ANCC ua rau txo qis DNA puas (Chesnokova li al.,2019a). Tsis tas li ntawd, peb qhia tau hais tias txoj hnyuv DNA puas tsis muaj nyob rau hauv cov nyuv-targeted GHRcolKO nas nrog excised GHR, tab sis nws tau sau nyob rau hauv cov hnyuv me, qhov twg GHRwas zoo lawm. Cov txiaj ntsig no, nrog rau peb qhov kev ua qauv qhia tias muaj hnub nyoog GHR-7-cov nas tsis tuaj yeem ua rau cov hnyuv DNA puas, muab kev txhawb nqa vivo rau qhov kev xav tias npGH hauv zos induction nrog cov hnub nyoog mayrender hlwb raug rau kev hloov pauv thiab tej zaum kuj ua rau kev kho mob qog noj ntshav, xws li tau tawm tswv yim (Basu thiab Kopchick, 2019).

Cov txiaj ntsig tau pom ntawm no kuj nce qhov ua tau tias GH cuam tshuam tuaj yeem kho los ntawm insulin-zoo li kev loj hlob yam 1 (IGF1). Tsis zoo li GH, IGF1 signaling activates DNA kho nyob rau hauv tsis-hlwb thiab qog hlwb (Chesnokova thiab Melmed, 2020; Chitnis li al., 2014; Turney li al., 2012), thiab peb tau qhia tias GH suppression ntawm p53 thiab induction ntawm DNA. dam-hnub nyoog tshwm sim ntawm nws tus kheej ntawm IGF1 nyob rau hauv cov hnyuv ib txwm (Ches-nokova li al.,2019a, 2019b). Txawm li cas los xij, hauv cov hlwb thiab cov ntaub so ntswg uas GH activates IGF1, qhov kev loj hlob tom kawg kuj tseem tuaj yeem ua teeb meem hauv cov xov tooj ntawm tes los tiv thaiv GH kev ua ntawm genome stability.

Cov kev tshawb pom pom tau hais tias npGH ua rau muaj kev laus ntawm txoj hnyuv hauv cov lus teb rau kev puas tsuaj DNA ua rau muaj kev puas tsuaj ntxiv rau DNA thiab zoo li yog qhov tsis zoo ntawm kev kho DNA puas. Extrapolation ntawm cov kev tshawb pom no muab lub platform rau ntau yam kev siv tshuaj kho mob. Raws li DNA puas tsub zuj zuj yog lub cim ntawm cov kab mob uas muaj hnub nyoog, tsom GH teeb liab los thaiv autocrine / paracrine tsis muaj kev cuam tshuam yuav ua pov thawj muaj txiaj ntsig zoo rau kev cuam tshuam hnub nyoog cuam tshuam txog kev puas tsuaj ntawm epithelial DNA thiab thaum kawg ameliorating qhov kev loj hlob ntawm cov hnub nyoog ntsig txog epithelial pathologies (Miman li al. , 2016) Ib txoj hauv kev zoo li no kuj tseem yuav muaj txiaj ntsig zoo rau cov neeg mob qog noj ntshav uas tau txais kev kho mob DNA-kev puas tsuaj, nyob rau hauv uas cov chaw epithelial hloov pauv los ntawm autocrine / paracrine tsis muaj kev cuam tshuam yuav ua rau cov ntaub so ntswg nyob sib ze. Nyob rau hauv no hais txog, cuam tshuam GH signaling nrog ib tug pom zoo GHR antagonist (List li al.,2011; Trainer et al, 2000) tej zaum yuav muab kev pab kho mob. Qhov tseem ceeb, tib neeg GH tau txais kev txhawb nqa tsis tsim nyog raws li qhov tsis pom zoo los tiv thaiv ag-ing thiab kev kho kom muaj zog (Giordano li al., 2008; Holt thiab Ho, 2019; Medeiros thiab Siegel Watkins, 2018; Melmed, 2019). Muab cov teebmeem paracrine deleterious ntawm npGH uas peb tau tshaj tawm ntawm no, kev tswj hwm GH mus ntev tsis tsim nyog raws li kev tiv thaiv kev laus elixir tuaj yeem ua rau GHR teeb liab thiab muaj kev pheej hmoo rau kev loj hlob ntawm epithelial cell DNA kev puas tsuaj, nrog rau qhov muaj peev xwm ua kom cov kab mob occult proliferative.

Kab lus no yog muab rho tawm los ntawm Cell Reports 37, 110068, Kaum Ob Hlis 14, 2021, ª 2021 Tus Sau. 1 Qhov no yog ib tsab xov xwm qhib rau hauv CC BY-NC-ND daim ntawv tso cai (http://creativecommons.org/licenses/by-nc-nd/4.0/)