MiR-199-3p Txhim kho cov nqaij ntshiv thiab ua kom cov nqaij laus laus thiab cov leeg mob plab

Thov hu rau oscar.xiao@wecistanche.com yog xav paub ntxiv

Parabiosis thwmsim qhia tias molecular yam cuam tshuam rau rejuvenation thiab aging circulate nyob rau hauv cov ntshav. Ntawm no, peb qhia tau hais tias miR-199-3p, uas circulates hauv cov ntshav raws li ib tug cell-dawb miRNA, yog ho txo ​​nyob rau hauv cov ntshav ntawm cov nas hnub nyoog piv rau cov hluas nas; thiab miR-199-3p muaj peev xwm txhawm rau txhim kho myogenic sib txawv thiab cov leeg pob txha. Kev tswj hwm ntawm miR-199 mimics, uas muab miR-199-3p, rau cov nas muaj hnub nyoog ua rau cov nqaij fiber ntau hypertrophy thiab qeeb ntawm cov leeg nqaij. Kev tswj hwm ntawm miR-199 ua rau cov nas mdx, tus qauv tsiaj paub zoo ntawm Duchenne leeg nqaij dystrophy (DMD), tau ua kom cov leeg muaj zog ntawm nas. Noj ua ke, cell-free miR-199-3p hauv cov ntshav yuav muaj kev tiv thaiv kev laus xws li cov nyhuv ahypertrophic hauv cov laus cov nqaij fibers thiab tuaj yeem muaj peev xwm ua tau zoo li RNA kho tshiab rau DMD nrog rau cov kab mob muaj hnub nyoog. Cov kev tshawb pom muab peb cov kev nkag siab tshiab rau cov ntshav-cim miRNAs raws li cov hnub nyoog ntsig txog molecules.

Thov nias ntawm no kom paub ntxiv

Lub luag haujlwm tseem ceeb, xws li cov leeg nqaij, kev ua haujlwm ntawm lub paj hlwb, ua haujlwm ua pa, tiv thaiv kab mob, thiab tiv thaiv kab mob, poob qis nrog kev laus. Cov kab mob thiab kev ua haujlwm tsis zoo cuam tshuam nrog kev ua haujlwm poob qis yog nce thiab ua teeb meem loj hauv cov neeg laus. Ntau yam kev hloov pauv, xws li kev hloov pauv hauv metabolic thiab noob caj noob ces, tshwm sim hauv lub cev nrog kev laus -3; thiab kawm cov kev hloov pauv no yuav pab kom nkag siab txog kev laus thiab muab cov tswv yim los daws cov teeb meem ntsib cov neeg laus. Biomolecules (nrog rau cov noob caj noob ces) ntsig txog kev laus tau raug txheeb xyuas thiab tej zaum yuav muaj txiaj ntsig zoo hauv kev tshawb fawb kev laus; Piv txwv li, -galactosidase thiab plolnk4a qhia tau txuam nrog cellular senescence4-6, thiab tus qauv nas uas tsis muaj klotho noob yog lub npe hu ua tus qauv laus nrog ntau cov phenotypes, zoo li tib neeg kev laus ntxov ntxov.

Parabiosis yog kev sib koom ua ke ntawm ob tus neeg uas sib koom ua ke ntawm cov hlab ntsha, thiab tuaj yeem tsim kev sim los ntawm kev phais8. Kev sim nrog parabiosis, uas cov nas me thiab cov laus tau sib koom ua ke los sib koom ua ke ntawm cov hlab ntsha, pom tias cov txheej txheem kev laus hauv cov tsiaj hluas tau nrawm dua, thaum cov tsiaj muaj hnub nyoog tau rov zoo dua 9-12. Cov kev tshawb pom pom tau hais tias qee yam muaj feem cuam tshuam txog kev rov ua dua tshiab thiab kev laus tam sim no, thiab ncig nrog cov ntshav hauv cov hlab ntsha; txawm li cas los xij, cov xwm txheej zoo li no tseem tsis tau nkag siab tag nrhol3.

Cistanche tuaj yeem tiv thaiv kev laus

MicroRNAs (miRNAs) yog 21-23-nucleotide-ntev me me uas tsis yog-coding RNAs, uas tau ua tiav los ntawm cov ntawv sau ntev dua (pri-mary miRNAs) los ntawm kev zom zaub mov, nrog rau microprocessor complex hauv lub nucleus thiab Dicer hauv cytoplasm, thiab muab tso rau hauv lub RNA-induced silencing complex (RISC) 14,15.MiRNA hauv RISC ua haujlwm ua tus neeg nruab nrab hauv gene silencing, qhov twg kev txhais lus ntawm tus xa xov RNAs (mRNAs) muaj ib feem ntawm kev ua tiav rau miRNA yog inhibited lossis mRNAs nqa ze li ntawm cov kab ke sib txuas rau miRNA yog lus 4,15.Cistanche Extract Anti RadiationYog li, miRNAs ua lub luag haujlwm tseem ceeb hauv kev tswj cov noob ntawm sup-nias lawv lub hom phiaj gene qhia. Ntau txhiab tus miRNA noob tau pom nyob rau hauv cov tsiaj thiab nroj tsuag [saib microRNA database (miRBase): http://www.mirbase.org/index.shtml]. Cov ntaub ntawv qhia txog miRNAs tau raug tshuaj xyuas, thiab cov ntaub so ntswg- thiab kev loj hlob theem-kev qhia tshwj xeeb thiab kab mob-kev qhia txog miRNAs tau raug kuaj poml6-21. Cov kev cai ntawm cov noob uas muaj miRNAs muaj feem cuam tshuam nrog ntau yam haujlwm tseem ceeb thiab lub neej tshwm sim, suav nrog cov kab mob14,16,21-24

MiRNAs muaj nyob sab nraud, nrog rau hauv lub hlwb25. Piv txwv li, miRNAs muaj nyob rau hauv cov ntshav raws li cell-free nucleotides thiab circulate nyob rau hauv lub vascular system2627. Xws li cell-free miRNAs (cf-miRNAs) tau muab tso rau hauv cov hlwv me me hu ua extra-cellular vesicles los yog txuam nrog cov proteins, yog li tau txais kev tiv thaiv los ntawm extracellular nucleases25,28. Lub xub ntiag ntawm qee qhov cf-miRNAs yuav yog qhov tseem ceeb; Piv txwv li, kev koom tes tseem ceeb ntawm cf-miRNAs thiab qee yam kab mob tau kuaj pom26,27,29-31. Cf-miRNAs tuaj yeem cuam tshuam txog kev hloov pauv ntawm lub cev nqaij daim tawv; Yog li, xws li miRNAs tej zaum yuav muaj peev xwm biomarkers rau kev saib xyuas lub neej, nrog rau cov kab mob.

Txoj kev tshawb fawb tam sim no tau ua los tshuaj xyuas cov koom haum ntawm cf-miRNAs thiab kev laus, thiab cf-miRNAs hauv cov ntshav ntawm cov nas hluas thiab cov laus tau tshawb xyuas. Cov txiaj ntsig tau nthuav tawm cov kev hloov pauv hauv cf-miRNAs ntawm cov menyuam yaus thiab hnub nyoog cov ntshav nas. Ntawm cov miRNAs, miR-199-3p tau poob qis hauv cov ntshav hnub nyoog piv rau cov ntshav hluas. Interestingly, miR-19-3p muaj peev xwm txhim kho cov leeg nqaij sib txawv thiab rov tsim dua tshiab, thiab nws cov kev tswj hwm rau cov nas muaj hnub nyoog ua rau cov nqaij fiber ntau nthuav. Thaum miR-199-3p tau nkag mus rau hauv mdx nas, tus qauv tsiaj ntawm Duchenne muscular dystrophy (DMD) cov leeg nqaij ntawm nas tau zoo dua. Txoj kev tshawb fawb tam sim no muab kev nkag siab tshiab rau cov ntshav-cf-miRNAs hais txog kev laus, muaj peev xwm latent, thiab kev siv kho mob.

Cov txiaj ntsig

Cell-free miRNAs in the blood of young and aged mice. We investigated cf-miRNAs in the blood of young (6-week-old) and aged (>23-hnub nyoog) C57BL/6J nas, siv DNA microarray los txheeb xyuas qhov sib txawv ntawm hnub nyoog. Cov ntaub ntawv ntawm cf-miRNAs tau pom qhov sib txawv ntawm cov nas me thiab cov laus, piv txwv li, cf-miRNAs uas cuam tshuam rau cov menyuam yaus thiab hnub nyoog nas ntshav tau kuaj pom (Table 1). Ntawm cov miRNAs, myogenic miRNAs (piv txwv li, miR-1 thiab miR-133) 1833 muaj nyob rau hauv pawg miRNA uas muaj ntau nyob rau hauv cov ntshav hluas; Hauv lwm lo lus, myogenic miRNAs tau pom tsawg dua hauv cov ntshav hnub nyoog (Table 1).cistanche tshuaj ntsuabQhov sib txawv ntawm cov miRNAs ntawm cov menyuam yaus thiab cov hnub nyoog cov ntshav tau lees paub los ntawm ntau qhov rov qab hloov pauv-polymerase saw cov tshuaj tiv thaiv (qRT-PCR; Fig. la), thiab rov tsim dua tshiab siv RNA qhov chaw muab rho tawm los ntawm cov hlwv me, yog li hu ua exosomes, cais tawm ntawm cov ntshav ( Ntxiv Fig.1).

Kev sim kab lis kev cai ntawm tes, ntawm qhov tod tes, qhia tias cov nas me me muaj peev xwm ua rau muaj qhov sib txawv ntawm myogenic, thiab lub peev xwm muaj zog dua li cov nas muaj hnub nyoog (Fig. 1b). Insulin-zoo li kev loj hlob zoo li I (IGF-I), uas yog ib qho tseem ceeb muaj peev xwm ntawm inducing myogenic sib txawv nyob rau hauv cov ntshav 34,35, raug kuaj; Txawm li cas los xij, tsis muaj qhov sib txawv tseem ceeb ntawm cov ntshav hluas thiab hnub nyoog (Fig. lc). Yog li, lwm yam uas tsis yog IGF-I hauv cov ntshav hluas tuaj yeem cuam tshuam rau kev sib txawv ntawm myogenic.

miR-199-3p muaj peev xwm ua rau muaj kev cuam tshuam myogenic sib txawv-entiation. Peb tau tshawb xyuas qhov cuam tshuam ntawm miRNAs ntau hauv cov ntshav cov tub ntxhais hluas ntawm kev sib txawv ntawm myogenic. Synthetic miRNA mimics tau nkag mus rau hauv C2C12 hlwb, nas myoblast cell kab, thiab kev qhia ntawm myogenic sib txawv cov cim, cov myogenin (Myog) thiab myosin hnyav saw 1 (Myh1) noob, raug tshuaj xyuas. Cov txiaj ntsig tau npaj txhij txog thiab nyiam peb cov kev txaus siab: miR-199-3p, uas tau kwv yees ua qhov tsis zoo miRNA vim yog cov ntaub ntawv me me ntawm kev sib txawv ntawm myogenic, ua rau muaj kev cuam tshuam loj heev rau kev qhia ntawm Myog thiab Myh1 (Fig. 2a, b). Tsis tas li ntawd, qhov induction ntawm miR-199-3p tau muaj zog dua li cov qauv myogenic miRNAs: qhov kev qhia ntawm myosin hnyav saw nyob rau hauv lub xub ntiag ntawm miR-199-3p yog qhov zoo tshaj plaws (Fig. 2b, c).

Txhawm rau kom paub meej txog kev sib raug zoo ntawm miR-199-3p thiab qhov sib txawv ntawm myogenic, peb tau tshawb xyuas cov cellular thiab extracellular (exoso-mal) miR-199-3p thaum lub sij hawm sib txawv ntawm myogenic, thiab tseem cuam tshuam ntawm miR-199-3p inhibitors ntawm qhov sib txawv. Qhov kev qhia ntawm endogenous (cellular)miR-199-3p tau upregulated tom qab pib ntawm kev sib txawv. Exosomal miR-19-3p tau nce ib zaug thiab txo qis tom qab ntawd (Cov duab ntxiv 2a). Inhibitor kev kho mob suppressed cov kev qhia ntawm myogenic sib txawv cov cim, Myog, Myh1, thiab creatine kinase (Ckm) (Suab ntxiv Fig. 2b).cistanche noov loj hlobYog li, qhov kev tshawb pom qhia tias miR-199-3p koom nrog kev sib txawv ntawm myogenic.

Tsim miR-199 mimics. Peb tsim miR-199 mimics xws li cov mimics muaj peev xwm ua tau zoo miR-199-3p raws li ib tug func-tional mediator (cov lus qhia strand) nyob rau hauv gene silencing. Cov qauv miRNA mimics tau raug tshuaj xyuas thiab miR199#4 raug xaiv los ua tus muaj peev xwm miR-199 mimic (Cov duab ntxiv 3). Tom qab ntawd, miR199 # 4 tau siv ob qho tib si hauv vivo thiab hauv vitro kev sim.

Target noob ntawm miR-199-3p hauv myogenic txawv. Txhawm rau txheeb xyuas cov noob hom phiaj rau miR-199-3p nyob rau hauv myogenic sib txawv-entiation, peb tau tshawb nrhiav cov noob neeg sib tw los ntawm TargetScan database thiab tsom mus rau Lin28b thiab Suz12 cov noob, ob qho tib si tau qhia hauv C2C12 myoblasts. Lin28b thiab Suz12 muaj putative miR-199-3p kev sib txuas ua ke hauv lawv cheeb tsam 3'untranslated (UTR; Fig. 3a). Peb pom tau hais tias cov kev sib txuas ua ke yog qhov tseeb khi qhov chaw rau miR-19-3p, siv cov noob caj noob ces Luciferase nqa cov kev sib txuas thiab cov sib txuas tsis sib xws (Daim duab 3a, b). Cov noob Luciferase uas nqa cov kev sib txuas ua ke tau raug tshem tawm los ntawm miR-199-3p [pLin28b-(81), pLin{18}}(983), thiab pSuz12-(973)], whereas the mismatched sequences, uas muaj lub hauv paus hloov pauv nyob rau hauv cov noob [pLin28b-(81) mut, cheeb tsam, abrogated nws suppression nyhuv [pSuz12-(973)mut]. pLin28-(983)mut, thiab Thaum miR{29}} mimics tau qhia rau C2Cl2 hlwb, qhov kev qhia ntawm Lin28b thiab Suz12 tau raug txwv tsis tu ncua (Daim duab 3c).Kom ntsuas seb qhov kev tawm tsam ntawm Lin28b thiab/ los yog Suz12 ncaj qha con-tributes rau myogenic sib txawv, tshwj xeeb inhibition ntawm Lin28b thiab Suz12 tau ua los ntawm gene silencing, siv RNA inter-ference (RNAi; Fig. 3d, e). Raws li qhov xav tau, qhov kev qhia ntawm myo-genic sib txawv cov cim, xws li Ckm, Myh1, thiab Myog raug kho raws li Lin28b thiab Suz12 gene silencing (Fig. 3f, g).

Txij li Lin28b yog RNA-binding protein koom nrog hauv kev ua cov miRNAs3637, peb tau tshawb xyuas cov teebmeem ntawm miR-19 mimics ntawm Lin28b ntawm myogenic miRNA ua. Qib ntawm miR-1, ib qho tseem ceeb myogenic miRNA, tau raug tshuaj xyuas vim tias kev ua haujlwm ntawm nws qhov ua ntej yog tswj hwm los ntawm Lin28b37. Matured miR-1 tau nce ntxiv los ntawm miR-199 kev kho mob qog noj ntshav (Daim duab 3h), qhia tias miR-199-3p tuaj yeem tswj hwm kev ua haujlwm ntawm miR-1 los ntawm kev tawm tsam ntawm nws lub hom phiaj Lin28b. Ua ke, qhov kev tshawb pom qhia tias miR-199-3p koom nrog hauv kev sib txawv ntawm myogenic los ntawm kev tawm tsam ntawm nws cov hom phiaj, Lin28b thiab Suz12.

Txhim kho cov nqaij ntshiv los ntawm miR199 # 4 hauv cov leeg nqaij nas.cistanche salsa cov txiaj ntsigPeb tau tshuaj xyuas cov teebmeem ntawm miR-199 mimic (miR199#4) hauv vivo siv cov qauv raug mob (Fig. 4a, b). Yim-lub lim tiam-laus C57BL / 6J nas tau txhaj nrog BaClz mus rau tibialis anterior (TA) txhawm rau ua rau cov leeg raug mob, thiab miR19 # 4 tau muab rau cov chaw puas 24 teev tom qab. Ob hnub tom qab kev tswj hwm miR199 # 4, qhov kev qhia ntawm myogenic marker noob tau tshuaj xyuas. Myog qhia tau nce zuj zus thiab qhov sib txawv ntawm myogenic 1 (Myod1) thiab myogenic factor 5 (Myf5) kuj tau tswj hwm los ntawm miR199#4 kev tswj hwm (Fig. 4c). Cov ntaub ntawv zoo ib yam nrog cov txiaj ntsig hauv vitro tau piav qhia saum toj no (Fig. 2).

Immunohistochemical tsom xam ntawm regenerating cov leeg tau ua 9 hnub tom qab kev tswj hwm miR199 # 4. Cov chaw hla ntu ntawm cov leeg nqaij (myofibers), uas yog lub hauv paus nuclei, raug kuaj xyuas (Daim duab 4b). Qhov nruab nrab ntu ntu ntawm regenerating myofibers kho nrog miR199 # 4 yog qhov loj dua li ntawm kev tswj hwm myofibers kho nrog kev tswj tsis tu ncua RNA duplex (nsCont; Fig. 4d), nrog qhov sib txawv mus txog qhov tseem ceeb ntawm cov ntaub ntawv. Ntxiv rau qhov nruab nrab ntawm qhov chaw hla ntu, qhov loj-sib faib histogram ntawm thaj chaw hla ntu tau pom qhov nce ntawm myofibers loj nyob rau hauv lub xub ntiag ntawm miR199#4(Fig.4e).

Hypertrophic nyhuv ntawm miR199 # 4 ntawm cov laus cov leeg nqaij. Peb tau tshuaj xyuas cov teebmeem ntawm miR199#4 ntawm cov nas muaj hnub nyoog (~ 24 hli). Cov nas muaj hnub nyoog pom qhov txo qis hauv cov leeg miR-199-3p (Fig. 5a), xws li hauv cov ntshav ncig cf-miR-19-3p, com-pared rau cov nas me. Peb muab miR199#4 rau TA cov leeg ntawm cov nas muaj hnub nyoog, thiab 2 hnub tom qab ntawd cov leeg TA tau kuaj xyuas los ntawm kev tshuaj ntsuam xyuas immunohistochemical (Fig. 5b). Qhov nruab nrab ntu ntu ntawm myofibers kho nrog miR199#4 yog qhov loj dua li kev tswj hwm myofibers kho nrog nsCont (Fig. 5c). Raws li qhov no, qhov loj-sib faib histogram tsom xam pom qhov nce hauv myofibers loj nyob rau hauv lub xub ntiag ntawm miR199#4(Fig.5d).

Txij li cov ntshav ncig cf-miR-199-3p txo qis hauv cov nas muaj hnub nyoog, peb tau qhia miR199#4 rau hauv cov ntshav ncig ntawm cov nas hnub nyoog dhau los ntawm tus Tsov tus tw vain. Tom qab txhaj tshuaj ntawm miR19 # 4, qhov chaw hla ntu ntawm myofibers tau kuaj xyuas thiab tshuaj xyuas, raws li tau piav qhia saum toj no. Cov txiaj ntsig, raws li tau pom hauv daim duab 5e, zoo ib yam nrog cov txiaj ntsig ntawm kev tswj hwm hauv cheeb tsam ntawm miR199#4 rau cov leeg laus (Fig. 5c, d).

Nws yuav tsum raug sau tseg tias muaj qhov sib txawv ntawm cov leeg nqaij loj ntawm cov leeg nqaij mob thiab cov nas muaj hnub nyoog. Central nuclei yog pom nyob rau hauv regenerating nqaij fibers (Fig. 4b), tab sis xws li myofibers tsis tau pom nyob rau hauv cov laus nas kho nrog miR199#4 (Fig. 5b). Qhov no qhia tau hais tias cov leeg nqaij ua ntej tuaj yeem ua rau cov nas muaj hnub nyoog tom qab kho miR199 # 4. Peb xav tias lwm cov txheej txheem sib txawv ntawm cov nqaij pob txha tuaj yeem koom nrog hauv myofiber o hauv cov nas hnub nyoog, thiab tau hais txog kev koom tes ntawm cov leeg nqaij atrophy.cistanche tubulosa noj redditAtroginl thiab MuRFI cov noob muaj feem cuam tshuam rau cov leeg nqaij atrophy38, thiab lawv cov qib qhia tau nce siab hauv cov leeg nqaij laus piv rau cov leeg hluas (Fig. 6a). Peb tau tshuaj xyuas qib ntawm Atroginl thiab MuRFI hauv cov leeg ntawm cov nas hnub nyoog tom qab kev tswj hwm ntawm miR199 # 4. Atroginl thiab MuRFI tau txo qis hauv ntau cov leeg los ntawm miR19#4 kev tswj hwm (Daim duab 6b), qhia tias kev tawm tsam ntawm cov leeg nqaij atrophy los ntawm miR199#4 tuaj yeem ua rau myofiber nthuav dav hauv cov nas muaj hnub nyoog.

MiR199 # 4 ncua kev poob ntawm cov leeg nqaij hauv cov nas hnub nyoog. Cov nyhuv ntawm miR199 # 4 ntawm cov leeg ua haujlwm hauv cov nas muaj hnub nyoog yog qhov txaus siab. Kev ntsuas lub zog tuav tau ua rau cov nas muaj hnub nyoog ua ntej thiab tom qab kev tswj hwm ntawm miR199 # 4. Qhov kev sim tau ua nyob rau hauv ob qhov muag tsis pom kev; kev txhaj tshuaj RNA thiab 100 feem pua ​​​​tuaj yeem ntsuas tau ua los ntawm cov neeg sim sib txawv yam tsis tau qhia kuv cov ntaub ntawv. C57BL/6 nas (80-lub lim tiam) txiv neej tau tuav-kuaj, tom qab ntawd 1 lub lis piam tom qab, lawv tau txhaj tshuaj intrave-nously nrog miR199#4 thiab nsCont, thiab tuav-kuaj dua. Kev hloov pauv ntawm cov leeg nqaij tom qab miR199#4 txhaj tshuaj yog MiR199#4 txhim kho cov leeg nqaij ntawm mdx nas. Txij li thaum miR199#4 muaj kev cuam tshuam zoo rau cov leeg, peb tau ua ib qho kev sim los soj ntsuam cov txiaj ntsig ntawm miR199#4 kev tswj hwm hauv mdx nas, paub zoo DMD tsiaj model32. Yim-lub lim tiam-hnub mdx nas tau txhaj tshuaj rau hauv lub cev nrog miR199#4 (1.6 mg / kg) ob zaug (1 lub lis piam ib ntus), thiab kev ntsuas tuav tau ua 1 lub lis piam tom qab kev tswj hwm zaum kawg. Qhov kev sim tau ua nyob rau hauv ob qhov muag tsis pom kev raws li qhia hauv Fig.7. Cov txiaj ntsig (Fig. 8a) qhia txog kev txhim kho cov leeg nqaij muaj zog hauv mdx nas kho nrog miR199#4 piv rau mdx nas kho nrog nsCont. Tsis tas li ntawd, kev txo qis hauv creatine kinase (CK) kev ua haujlwm thiab cell-free miR-1 tau pom nyob rau hauv cov ntshav ntawm mdx nas kho nrog miR199#4 (Fig. 8b, c), uas yog cov cim qhia ntawm tus kab mob. kev txhim kho.

Raws li kev xa tshuaj reagent, atelocollagen yog nkig thiab nyuaj rau kev daws, thiab cov nas uas tau kho nrog atelocollagen tshwm sim los cuam tshuam tsis zoo vim lub tsheb. Yog li, peb tau nrhiav lwm txoj hauv kev rau atelocollagen los txhim kho peb txoj kev xa tshuaj (DDS) thiab pom DC-CHOL / DOPE cationic liposomes raws li DDS reagent tshiab uas sib npaug lossis zoo dua rau atelocollagen (Supplementary Fig. 4). Kev faib tshuaj ntawm miR199#4 nrog cov cationic liposomes qhia tau tias cov tshuaj miR19#4 tau xa mus rau cov leeg thiab coj mus tas li nyob rau hauv ntau ntau hauv daim siab (Ntxiv daim duab 5). Extracellular miR199#4 xa mus rau cov leeg nqaij tuaj yeem ua haujlwm ua tus neeg nruab nrab hauv cov gene silencing thiab pab txhawb kev tswj cov noob koom nrog kev txhim kho cov leeg nqaij.

Peb tau sim txheeb xyuas cov biomolecules ntsig txog kev txhim kho tom qab miR199#4 kev tswj hwm. Ntau yam poly-peptides ntsig txog DMD tau tshuaj xyuas los ntawm western blotting; txawm li cas los xij, cov cim qhia txawv ntsig txog kev txhim kho tsis raug kuaj pom hauv qhov kev tshawb fawb no (Cov duab ntxiv 6). Peb yuav tham txog qhov no ntxiv nyob rau hauv nqe lus hauv qab no.

Kev sib tham

Biomolecules hloov zoo thiab ntau nrog kev laus --3. Kev ntes cov kev hloov pauv no thiab kawm lawv yog qhov tseem ceeb rau kev nkag siab txog kev laus, thiab tuaj yeem nthuav tawm cov tswv yim los daws cov teeb meem hauv cov neeg laus. Cov kev tshawb fawb Parabiosis uas cov tsiaj hluas thiab cov laus tau koom ua ke los qhia txog cov kab mob vascular tau muab kev nkag siab tseem ceeb rau kev laus thiab rov ua dua tshiab 9-l2. Cov kev tshawb pom pom tau hais tias muaj cov rejuvenating thiab aging yam tseem ceeb nyob rau hauv cov hluas thiab cov laus circulating ntshav, feem. Cov kev tshawb fawb yav dhau los tau tshaj tawm ntau yam molecules raws li cov neeg sib tw cuam tshuam txog kev rov ua dua tshiab thiab kev laus, tab sis cov txiaj ntsig tau sib cav sib ceg3.

Peb cov kev sim hauv vitro cell kab lis kev cai siv cov tub ntxhais hluas thiab cov hnub nyoog sera zoo ib yam li kev sim parabiosis (Daim duab lb), thiab peb pom miR-199-3p, uas hloov nrog hnub nyoog, hauv cov ntshav ncig; cov miRNA raug txheeb xyuas kom txo qis hauv cov ntshav hnub nyoog (Table 1 thiab Fig. la). Tsis tas li ntawd, kev qhia ntawm cov leeg nqaij (cellular)miR-199-3p tau txo qis hauv cov nqaij leeg hnub nyoog com-pared rau cov leeg hluas (Fig. 5 a); yog li, cf-miR-199-3p hauv cov ntshav tuaj yeem cuam tshuam nrog cov leeg miR-199-3p. Qhov txo qis hauv kev qhia ntawm miR-199-3p nrog kev laus kuj tseem pom nyob rau hauv mesenchymal qia hlwb muab los ntawm rhesus macaque pob txha marrow39. Yog li ntawd, kev qhia ntawm cellular miR-19-3p tej zaum yuav nyob rau hauv kev tswj hnub nyoog. Nyob rau hauv sib piv rau miR-19-3p, kev qhia ntawm cellular myogenic miRNAs, xws li miR-1 thiab miR{14}}, nyob twj ywm unchanged nyob rau hauv cov tub ntxhais hluas thiab cov laus cov leeg (Fig. 5a), tab sis theem ntawm lawv cov extracellular miRNAs hauv cov ntshav tau txo qis hauv cov nas muaj hnub nyoog (Fig. la). Qhov txawv ntawm miR-199-3p thiab myogenic miRNAs(miR{17}} thiab-133) tuaj yeem cuam tshuam qhov sib txawv ntawm kev ua cf-miRNA thiab/lossis hauv kev tswj hnub nyoog ntsig txog cf-miRNAs ntawm lawv . Txhawm rau txheeb xyuas qhov sib txawv no, kev tshawb fawb ntxiv yuav tsum tau ua yav tom ntej.

Cov kev tshawb fawb yav dhau los tau qhia tias miR-199-3p koom nrog ntau lub zog ntawm tes thiab cov xwm txheej tseem ceeb los ntawm kev tawm tsam ntawm nws cov hom phiaj noob; Piv txwv li, miRNA tuaj yeem txhawb nqa kev loj hlob thiab kev tsiv teb tsaws ntawm endothelial tip cells thiab modulate qhov pib ntawm kev puberty, ntsig txog, los ntawm kev txo qis ntawm lub hom phiaj semaphorin -3A gene40 thiab los ntawm kev tawm tsam ntawm cov hom phiaj (Cdc42, Map3k2, Map3k4, thiab Taok1) koom nrog hauv p38MAPK txoj kev41. Txoj kev tshawb fawb tam sim no tau pom tias miR-19-3p tau koom nrog hauv kev sib txawv ntawm myogenic los ntawm kev tawm tsam ntawm nws cov hom phiaj, Lin28b thiab Suz12 (Fig. 3). Lin28b yog ib qho RNA-binding protein thiab inhibits kev loj hlob ntawm miRNAs, suav nrog miR{17}}, uas yog cov nqaij leeg tshwj xeeb miRNA thiab pro-motes myogenesis37. Kev tawm tsam ntawm Lin28b los ntawm miR-19-3p ua rau muaj kev loj hlob ntawm miR-1 (Daim duab 3), uas tuaj yeem tso cai rau kev sib txawv ntawm myogenic mus ntxiv.

Suzl2 yog ib qho tseem ceeb subunit ntawm polycomb repressive complex 2 (PRC2), uas koom nyob rau hauv repressing chromatin42. Gene regulation ntawm PRC2 muaj kev koom tes hauv kev tswj cov qia cell, embryonic txoj kev loj hlob, cell proliferation thiab sib txawv, nrog rau myogenic differentiation42.PRC2 yog tam sim no nyob rau hauv ntsiag to leeg nqaij tshwj xeeb loci, xws li Myog thiab nqaij creatine kinase (MCK) nyob rau hauv undifferentiated myoblasts, tab sis yog dissociated. los ntawm tej loci nyob rau hauv txawv myotubes43. Gene silencing tawm tsam Suz12 los ntawm RNAi ua rau kev txhim kho ntawm myogenic noob qhia (Fig. 3). Kev tshem tawm ntawm qhov kev txhim kho ntawm zeste homolog 2 (Ezh2) protein, lwm qhov tseem ceeb ntawm PRC2, kuj ua rau myogenic sib txawv44. Cov kev tshawb pom no qhia tias inhibition ntawm PRC2 tsim vim kev hem thawj ntawm nws cov core subunits tuaj yeem ua rau tsim lub xeev genomic rau cov leeg nqaij sib txawv. Yog li, kev tawm tsam ntawm Suz12 los ntawm miR-199-3p tuaj yeem ua rau muaj qhov sib txawv ntawm myogenic. Ua ke, miR-19-3p tuaj yeem ua lub luag haujlwm tseem ceeb hauv kev sib txawv ntawm myogenic los ntawm inhibition ntawm ob txoj kev ywj pheej uas muaj feem xyuam nrog Lin28b thiab Suzl2, uas yog txhua tus koom tes hauv kev saib xyuas ntawm lub xeev tsis txawv ntawm myoblasts.

Cov nyhuv ntawm miR-199-3p ntawm myogenic sib txawv kuj tau pom nyob rau hauv vivo (Fig. 4). Kev puas tsuaj rau cov leeg ua rau cov cell pro-liferation thiab sib txawv vim cov leeg nqaij. Intro-duction ntawm miR199#4, uas muab miR-199-3p, mus rau qhov chaw puas, txhim kho cov nqaij ntshiv thiab nthuav cov myofibers (Fig. 4). Myogenic gene qhia tau raug kho raws li kev kho mob miR199#4 (Fig. 4). Los ntawm qhov kev pom zoo, miR199 # 4 yuav ua tau zoo thiab muaj txiaj ntsig zoo rau kev kho mob phais.

Thaum miR199#4 tau muab rau cov nas muaj hnub nyoog uas tsis muaj ntshav ncig miR-199-3p, cov nas tau nthuav tawm cov myofibers loj heev (Fig. 5) thiab ncua kev poob ntawm cov leeg nqaij nrog kev laus (Fig. 7). Qhov nthuav dav myofibers hauv cov nas muaj hnub nyoog sib txawv los ntawm kev tsim cov myofibers, uas yog loj nyob rau hauv lub xub ntiag ntawm miR199#4; Lub qub yog muab los ntawm preexisting myofibers, thiab tom kawg yog los ntawm neo-myofibers (me nyuam mos myofibers). Yog li, muaj qhov sib txawv, ywj siab mechanisms hauv cov leeg nqaij fiber ntau. Peb txoj kev tshawb fawb qhia tias (i) nyob rau hauv cov ntaub ntawv qub, inhibition ntawm cov leeg nqaij atrophy vim kev tawm tsam ntawm Atroginl thiab MuRFI los ntawm miR199 # 4 kev kho mob coj txog kev loj hlob ntawm cov hnub nyoog myofibers, thiab (ii) nyob rau hauv qhov kawg, kev tawm tsam ntawm lub hom phiaj noob, Lin28b. thiab Suz12, ua rau kom loj hlob ntawm myofibers. Ua ke, sib txawv (ntau) cov noob tswj los ntawm miR-19-3p tuaj yeem koom nrog hauv cov leeg nqaij hypertrophy hauv ntau lub sijhawm. Cov kev tshawb pom kuj qhia tias miR-199-3p tej zaum yuav muaj tsawg kawg yog ib qho kev tiv thaiv ntawm cov leeg nqaij, thiab tias miR199#4 muab miR-199-3p tej zaum yuav muaj peev xwm ua tau ib yam tshuaj tshiab RNA rau cov kab mob uas muaj hnub nyoog, xws li sarcopenia.

Raws li qhov ua tau zoo ntawm miR199 # 4 ntawm cov leeg, peb tau tswj hwm miR199 # 4 rau mdx nas, tus qauv DMD paub zoo, thiab tau txais txiaj ntsig zoo: kev tswj hwm ntawm miR199 # 4 txhim kho cov leeg nqaij hauv cov nas los ntawm ~ 20 feem pua ​​( Fig.8 a). Txhawm rau nkag siab txog cov txheej txheem ntawm cov leeg lub zog rov qab los, peb tau sim txheeb xyuas cov biomolecules uas tau txhim kho (los yog cuam tshuam) los ntawm miR-199-3p. Txawm li cas los xij, tshwj tsis yog kev txhim kho ntawm cov ntshav CK thiab cf-miR-1 (Fig. 8b, c), peb tsis tuaj yeem kuaj pom qhov kev txhim kho ntawm cov qib molecular. Qhov teeb meem no nyob rau hauv kev tshawb nrhiav tej zaum yuav yog vim cov complex cell pejxeem, uas muaj ib tug sib tov ntawm regenerating thiab dis-rupting cov leeg hlwb nyob rau hauv mdx nas. Txawm hais tias miR199 # 4 ua rau muaj kev txhim kho, lub suab nrov tom qab los ntawm cov neeg hauv cell complex yuav npog pov thawj ntawm kev txhim kho. Yuav kom daws tau qhov teeb meem no, kev tshawb fawb ntau dua uas siv cov leeg nqaij sib luag ntawm cov nqaij pob txha los ntawm mdx nas lossis tsom mus rau ntau yam ntaub so ntswg, suav nrog cov leeg nqaij yuav tsum tau ua.

Txawm hais tias cov txheej txheem molecular ntawm cov leeg lub zog rov qab los ntawm miR199 # 4 tseem tsis paub, qhov koob tshuaj ntawm miR19 # 4 rau kev txhim kho cov leeg nqaij yog qhov tsim nyog. Ob zaug admin-istration ntawm ~ 1.6 mg / kg miR199 # 4 txhim kho cov leeg nqaij hauv mdx nas. Cov koob tshuaj zoo zoo li qis dua li ntawm antisense oligonucleotides rau exon-khiav tawm tsam mutant dystrophin noob hauv vivo45-47. Qhov laj thawj yog vim li cas miR199 # 4 muaj peev xwm txhim kho cov leeg nqaij ntawm cov koob tshuaj qis dua tuaj yeem yog vim nws cov txheej txheem sib txawv ntawm kev ua piv nrog antisense oligonucleotides. Lub mechanism ntawm kev txiav txim ntawm miR19 # 4 yog cata-lytic gene silencing. Nyob rau hauv sib piv, lub mechanism ntawm antisense oli-gonucleotides yog raws li lub cev hybridization. Yog li ntawd, qhov sib txawv ntawm cov koob tshuaj uas tsim nyog los qhia txog kev ua tau zoo ntawm cov tshuaj yuav yog vim muaj qhov sib txawv ntawm kev ua haujlwm.

Lub tshuab uas lub cev, cov ntaub so ntswg, lossis cov hlwb tso tawm miRNAs rau hauv cov ntshav ncig tseem tsis tau nkag siab tag nrho, thiab cov kev cai muaj hnub nyoog txog kev tso tawm ntawm miRNAs kuj tseem tsis meej. Txoj kev tshawb no tau pom tias myogenic cf-miR-NAs, suav nrog cf-miR-199-3p ncig nrog cov ntshav hauv cov hlab ntsha thiab txo qis nrog kev laus. Xws li cf-miRNAs tej zaum yuav muaj nyob rau hauv exosomes, piv txwv li, lawv yog exosomal miRNAs.

Myogenic miRNAs koom nrog hauv kev sib txawv ntawm myogenic, kev tsim cov leeg nqaij, thiab kev saib xyuas cov leeg nqaij homeostasis33. Cov kev tshawb fawb yav dhau los tau tshaj tawm tias myogenic miRNAs, xws li miR-1 thiab miR-133, tau pom tias nce ntshav hauv cov lus teb rau kev tawm dag zog lub cev4849. Raws li cov ntawv ceeb toom no, cov nas me nyuam yaus nquag tuaj yeem tso tawm ntau myogenic miRNAs rau hauv cov ntshav ntau dua cov nas uas muaj hnub nyoog qeeb, thiab cov miRNA extracellular, suav nrog cf-miR-199-3p tuaj yeem pab tswj cov leeg nqaij homeostasis hauv cov nas me los ntawm kev ua autocrine. . Txawm li cas los xij, theem ntawm extracellular (exosomal) miRNA tsis yog ib txwm cuam tshuam nrog qib cellular miRNA, thiab qhov sib txawv tau pom nyob rau hauv myogenic miRNAs thiab miR-199-3p ntawm cov nas hluas thiab hnub nyoog (Fig. la thiab 5a) thiab thaum lub sij hawm myogenic sib txawv (Ntxiv daim duab 2a). Yog li, raws li lwm qhov ua tau, nws yog qhov pom tau tias muaj hnub nyoog ntsig txog thiab kev sib txawv ntawm kev tswj hwm ntawm exosome tsim yuav cuam tshuam rau cf-miRNA ntau lawm; tshwj xeeb, tej zaum yuav muaj qhov sib txawv ntawm exosome tsim ntawm cov nas hluas thiab cov laus. Hauv cov ntsiab lus ntawm cov kev xav no, kev tshawb fawb ntxiv yog xav tau los piav qhia txog hnub nyoog txog kev tso tawm myogenic miRNAs rau hauv cov ntshav.

Ib cag ntawm cov txheej txheem tso tawm, thaum cov tub ntxhais hluas myogenic cf-miRNAs uas muaj cf-miR-199-3p ntws mus rau hauv cov laus cov ntshav ncig los ntawm cov tub ntxhais hluas vascular system los ntawm parabiosis, cf-miRNAs tuaj yeem ua rau cov leeg ua kom thiab inhibition ntawm cov leeg. atrophy ntawm cov laus; Lub tswv yim no tau txais kev txhawb nqa los ntawm cov pov thawj los ntawm cov nas muaj hnub nyoog txhaj tshuaj miR19#4 hauv txoj kev tshawb no (Figs. 5-7). Cf-miRNAs hauv cov ntshav tuaj yeem cuam tshuam txog hnub nyoog txog kev ua haujlwm homeostatic thiab tuaj yeem sawv cev hloov pauv homeostasis. Thaum xws li cf-miRNAs muaj ectopically tam sim no, piv txwv li, thaum cf-miRNAs nyob rau hauv cov tub ntxhais hluas cov ntshav raug xa mus rau cov laus cov ntshav-kwv system, qee cov cf-miRNAs yuav qhia rejuvenating los ntawm cov laus, piv txwv li, lawv yuav tsim antiaging los. Hauv qhov sib piv, cf-miRNAs hauv cov ntshav muaj hnub nyoog tuaj yeem sawv cev hloov pauv homeostasis nrog kev laus. Thaum xws li cf-miRNAs raug xa mus rau cov tub ntxhais hluas cov ntshav ncig, qee qhov cf-miRNAs tuaj yeem ua rau muaj kev cuam tshuam txog kev laus thiab yuav ua rau muaj kev laus ntxov ntxov ntawm cov hluas. Cf-miRNAs uas tau nce ntxiv hauv cov ntshav muaj hnub nyoog sawv cev rau cov neeg sib tw uas yuav xav tau cov txheej txheem ntxaws ntxaws kom nkag siab thiab tiv thaiv kev laus.

Hauv kev xaus, peb txoj kev tshawb fawb tam sim no tau muab cov kev nkag siab tshiab rau cf-miRNAs raws li cov hnub nyoog ntsig txog molecules, thiab nthuav tawm cov kev tshawb fawb tshiab los qhia meej txog kev sib raug zoo ntawm kev laus thiab cell-free functional RNAs, suav nrog cf-miRNAs.

Kab lus no yog muab rho tawm los ntawm Kev Sib Txuas Lus BIOLOGY|(2021) 4:427|https://doi.org/10.1038/s42003-021-01952-2|www.nature.com/commsbio