Echinacoside Exerts Anti-tumor Activity nyob rau hauv daim siab mob cancer
Mar 31, 2022
Hu rau:joanna.jia@wecistanche.com/ WhatsApp: 008618081934791
Wen Li*, Jing Zhou, Yajie Zhang, Jing Zhang, Xue Li, Qiao Yan, Jiabing Han, thiab Fangdi Hu*
Abstract
Keeb kwm:Echinacoside (ECH) yog cov khoom xyaw tseem ceeb ntawm Cistanches Herba, uas paub tias muaj kev kho mob ntawm cov qog nqaij hlav metastatic. Txawm li cas los xij, qhov cuam tshuam ntawm ECH ntawm daim siab mob cancer tseem tsis paub meej. Txoj kev tshawb no yog los tshawb xyuas qhov cuam tshuam ntawm ECH ntawm kev ua phem ntawm daim siab mob hlwb.
Txoj kev:Ob hom kab mob qog nqaij hlav hauv siab Huh7 thiab HepG2 tau kho nrog cov koob tshuaj sib txawv ntawm ECH ntawm lub sijhawm sib txawv thiab gradients. MTT thiab colony tsim kev soj ntsuam tau siv los txiav txim qhov cuam tshuam ntawm ECH ntawm kev muaj peev xwm ntawm Huh7 thiab HepG2 hlwb. Transwell assays thiab flow cytometry assays tau siv los txheeb xyuas qhov cuam tshuam ntawm ECH kev kho mob ntawm kev ntxeem tau, kev tsiv teb tsaws, apoptosis, thiab lub voj voog ntawm tes ntawm Huh7 thiab HepG2 hlwb. Western blot tsom xam tau siv los txheeb xyuas qhov cuam tshuam ntawm ECH ntawm kev qhia theem ntawm TGF- 1, smad3, smad7, apoptosis-related proteins (Caspase-3, Caspase-8), thiab Cyto C nyob rau hauv daim siab mob hlwb. Kev sib raug zoo ntawm miR-503-3p thiab TGF- 1 tau kuaj pom siv bioinformatics tsom xam thiab Luciferase tus kws tshuaj ntsuam xyuas.
Cov txiaj ntsig:Cov txiaj ntsig tau pom tias ECH inhibited qhov kev loj hlob, kev ntxeem tau, thiab kev tsiv teb tsaws ntawm Huh7 thiab HepG2 hlwb nyob rau hauv ib koob- thiab lub sij hawm-nyob. Ntxiv mus, peb pom tias ECH ua rau Huh7 thiab HepG2 cell apoptosis los ntawm thaiv cov hlwb hauv S theem. Tsis tas li ntawd, kev qhia ntawm miR-503-3p tau pom tias txo qis hauv daim siab qog nqaij hlav, tab sis kev kho ECH tau nce qhov kev qhia ntawm miR-503-3p hauv Huh7 thiab HepG2 hlwb. Ntxiv rau, peb pom tias TGF- 1 raug txheeb xyuas tias yog lub hom phiaj muaj peev xwm ntawm miR-503-3p. ECH txhawb kev ua kom TGF- 1/Smad signaling pathway thiab nce qib kev qhia ntawm Bax/Bcl-2. Ntxiv mus, ECH tuaj yeem ua rau kev tso tawm ntawm mitochondrial Cyto C, thiab ua rau cov tshuaj tiv thaiv Caspases qib.
Cov lus xaus:Txoj kev tshawb no qhia tau hais tias ECH siv cov tshuaj tiv thaiv kab mob los ntawm miR-503-3p/TGF- 1/Smad axis hauv daim siab mob qog noj ntshav, thiab muab cov tshuaj tiv thaiv qog nqaij hlav zoo thiab muaj txiaj ntsig zoo rau mob qog noj ntshav.
Ntsiab lus: Echinacoside, mob qog noj ntshav, Apoptosis, MiR-503-3p, TGF- 1/smad
Cistanche yog ib qho kev nyab xeeb thiab siv tau los tiv thaiv qog nqaij hlav rau lub siab mob cancer.
Taw qhia
Mob qog noj ntshav siab yog ib qho ntawm cov qog qog nqaij hlav uas tshwm sim ntau tshaj plaws, thiab qhov tshwm sim ntawm daim siab mob cancer tau loj hlob sai [1]. Hepatectomy yog txoj hauv kev zoo tshaj plaws los kho mob qog noj ntshav rau cov neeg mob thaum ntxov [2]. Txawm li cas los xij, feem ntau cov neeg mob tau rov ua dua thiab metastasis tom qab phais. Tus 5-xyoo ciaj sia taus yog li ntawm 3–5 feem pua [2]. Yog li ntawd, cov txheej txheem kho tshiab thiab cov neeg sawv cev yog qhov xav tau sai.
Hauv ob peb xyoos dhau los, kev txhim kho tshuaj kho mob qog noj ntshav thiab kev tshawb fawb ntawm phytochemicals tau pib ua tib zoo mloog rau ethnopharmacology [3, 4]. Ntau cov kev tshawb fawb soj ntsuam thiab kev sim tau pom tias cov tshuaj monomers tuaj yeem cuam tshuam kev loj hlob, metastasis ntawm hepatocellular carcinoma cells, induce apoptosis thiab sib txawv ntawm cov qog nqaij hlav cancer [5]. Cistanches Herba yog cov nroj tsuag ntawm tsev neeg ntawm cov nroj tsuag, uas yog cov tshuaj ib txwm nyob hauv Suav teb thiab hu ua "desert ginseng" [6]. Cistanches Herba yog nplua nuj nyob rau hauv hom tshuaj lom neeg, thiab cov kws tshawb fawb hauv tsev thiab txawv teb chaws tau cais ntau tshaj li ib puas tebchaw los ntawm nws, xws li phenylethanol glycosides, lignans, cycloaliphatic terpenes, qab zib, amino acids, thiab lwm yam. [7]. Echinacoside (ECH) yog qhov tseem ceeb ntawm Cistanches Herba thiab feem ntau suav hais tias yog nws cov khoom xyaw tseem ceeb [6]. Nyob rau hauv xyoo tas los no, cov kev tshawb fawb tau pom tias ECH tej zaum yuav yog ib qho kev kho mob tshiab rau cov qog nqaij hlav loj heev los yog dav metastatic [8]. Cov kev tshawb fawb tau pom tias ECH tuaj yeem tiv thaiv qog nqaij hlav pancreatic, leukemia, mob qog noj ntshav, mob qog noj ntshav, thiab ntau lwm cov qog nqaij hlav [9]. Txawm li cas los xij, tsis muaj ntawv tshaj tawm txog qhov cuam tshuam ntawm ECH ntawm kev mob qog noj ntshav siab.
MiRNAs ua lub luag haujlwm tseem ceeb hauv kev tswj hwm kev hloov pauv ntawm noob caj noob ces (10). Cov kev tshawb fawb dav dav tau pom tias miRNAs muaj feem cuam tshuam nrog mob qog noj ntshav [11] thiab Ntau yam Sclerosis. MiR-503-3p yog ib qho miRNA uas tau tshawb pom tshiab uas koom nrog hauv kev loj hlob, kev tsiv teb tsaws, thiab ntxeem tau ntawm cov qog hlwb [12]. Txawm li cas los xij, nws txoj haujlwm hauv daim siab mob cancer tsis paub meej. Tam sim no, kev tshawb fawb ntawm miRNA downstream lub hom phiaj noob yog nyob rau hauv ib qho kev paub tab, tab sis ntau lub hom phiaj gene loci tseem tsis tau pom. Te TGF- 1/Smad3 teeb liab transduction txoj kev yog qhov tseem ceeb tshwj xeeb rau cov qog nqaij hlav thiab kev loj hlob [13]. Nws tau pom tias txoj kev TGF- 1/Smad3 tau qhib rau hauv tus qauv hepatoma, qhia tias TGF- 1/Smad3 txoj hauv kev tau ua lub luag haujlwm tseem ceeb hauv kev txhim kho mob qog noj ntshav [14]. Kev qhia siab ntawm TGF- 1/Smad3 txoj hauv kev yog suav tias yog ib qho ntawm cov laj thawj tseem ceeb rau qhov tshwm sim ntawm daim siab mob cancer [15]. Txhawm rau txhawm rau tshawb xyuas cov txheej txheem hauv qab uas kho cov teebmeem ntawm ECH ntawm daim siab mob qog noj ntshav, miRNA / TGF- 1/Smad3 txoj kev tau tshawb nrhiav.
Hauv txoj kev tshawb no, peb tau soj ntsuam qhov cuam tshuam ntawm ECH ntawm kev loj hlob ntawm cov qog nqaij hlav cancer hauv lub siab thiab pom tias ECH tau ua haujlwm los tiv thaiv qog nqaij hlav ntawm miR-503-3p/TGF- 1/Smad axis hauv daim siab mob cancer, thiab muab kev nyab xeeb thiab siv tau zoo los tiv thaiv qog nqaij hlav cancer rau daim siab.
ceg tcmyog anti-cancer.
Cov txheej txheem thiab cov khoom siv
Khoom siv
Echinacoside (ECH) (purity>98 feem pua, monohydrate) tau los ntawm Hetian Dichen sashing Pharmaceutical Development Co., Ltd (Xinjiang, Suav). Cov qauv qauv tau qhia hauv daim duab 1.
Cell culture thiab transfection
Tib neeg lub siab mob qog noj ntshav kab Huh7 thiab HepG2 tau yuav los ntawm FENGHUISHENGWU (Hunan, Suav). Cells tau coj mus rau hauv DMEM nruab nrab ntxiv nrog 10 feem pua fetal bovine serum (FBS, Gibco, USA) ntawm 37 degree hauv 5 feem pua CO2 cell incubator. MiR-503-3p mimic thiab miR-NC tau txais los ntawm Shanghai Gene Pharmaceutical Co., Ltd. (Shanghai, Suav). Txhua qhov kev hloov pauv tau ua tiav siv Lipofectamine 2000 reagent (Invitrogen) ua raws li cov chaw tsim khoom cov lus qhia. Tom qab 24-48 teev ntxiv, cov kab mob sib kis tau raug sau thiab ua tiav rau kev tshawb fawb ntxiv.
Luciferase tus neeg sau xov xwm soj ntsuam
Tag nrho RNAs tau muab rho tawm los ntawm cov hlwb, thiab TGF- tau amplifed siv WT thiab MUT primers. Ib feem ntawm TGF- 1 tau muab tso rau hauv pGL3-Bashc luciferase reporter vector npe TGF- -WT-Luc. Kev khi thaj tsam ntawm 1 thiab miR-503-3p tau mutagenized los ua ib qho mutant plasmid uas yog TGF- 1-Mut-Luc. TGF 1-WT-Luc thiab TGF- 1-Mut-Luc tau koom ua ke rau hauv cov hlwb nrog miR-503-3p mimic thiab pRLTK. Cells tau sau 6 teev tom qab hloov pauv. Dual-Luciferase Reporter Assay Kit (Promega, Madison, WI, USA) tau siv los txiav txim siab txog kev ua haujlwm ntawm luciferase.
Kev ntsuam xyuas kev loj hlob ntawm tes
Huh7 thiab HepG2 hlwb (2 × 105) tau muab tso rau hauv 96-cov phiaj zoo. Cells raug kho nrog ECH rau 24, 36, thiab 48 teev. Tom qab ntawd, 10 mL MTT tov yog ntxiv rau txhua lub qhov dej thiab cov hlwb raug incubated rau 4 h. Tom qab ntawd, 150 μL ntawm DMSO tau ntxiv rau hauv qhov dej. Thiab qhov nqus ntawm 570 nm tau ntsuas los ntawm ELISA nyeem ntawv (Aoweiya, Beijing, Suav).
Kev tsim ua pawg
Ib leeg-cell raug tshem tawm tau npaj los ntawm cov kab mob sib kis thiab coj mus rau hauv qhov chaw tsim khoom rau 7 hnub. Tom qab muab pov tseg qhov nruab nrab, cov hlwb raug stained nrog Wright's stain rau 5 min thiab tom qab ntawd stained nrog Giemsa dye tov thiab Sorensen phosphomolybdate bufer tov (1: 9) rau 10 min. Tom qab ntxuav, colonies raug suav nyob rau hauv lub tshuab tsom.
Kev ntsuam xyuas Transwell
Kev tsiv teb tsaws chaw thiab kev cuam tshuam tau ua nyob rau hauv Transwell chambers (Costar, Badhoevedorp, Te Nether av) coated nrog lossis tsis muaj Maribel (BD Biosciences) nyob rau saum npoo ntawm {{0}}μm (pore loj) daim nyias nyias. Rau kev soj ntsuam kev tsiv teb tsaws chaw, transfected Huh7 thiab HepG2 hlwb tau rov raug ncua hauv 200 µL ntawm cov tshuaj dawb DMEM nruab nrab thiab noob tau cog rau hauv cov chav sab saud. Rau qhov kev ntsuam xyuas ntxeem tau, Matrigel (30 ug / qhov dej) tau ua ntej coated rau ntawm daim nyias nyias ntawm cov chav tsev sab saud thiab lwm cov kauj ruam yog tib yam li qhov kev ntsuam xyuas tsiv teb tsaws. Tom qab incubation ntawm 24 h, cov hlwb uas tau tsiv teb tsaws los yog invaded los ntawm daim nyias nyias mus rau hauv qab nto yog tsau nrog ethanol thiab stained nrog 0.2 feem pua pob zeb siv lead ua violet. Thaum kawg, cov hlwb raug soj ntsuam thiab suav hauv tsib qhov chaw uas siv lub tshuab tsom.
Kev txheeb xyuas lub voj voog ntawm tes
Huh7 thiab HepG2 hlwb tau muab noob rau hauv 6-cov phiaj zoo ntawm qhov ntom ntawm 1 × 105 hlwb/mL. Cells tau sau tom qab kho nrog ECH rau 0, 24, 48 h. Tese hlwb tau ntxiv nrog 1 mL ntawm dej khov precooled 75 feem pua ethanol, tsau ntawm 20 degree rau 2 h. Tom qab ntawd, cov hlwb tau ntxiv nrog PI staining tov thiab incubated hauv maub rau 15 min. Lub voj voog ntawm tes tau txheeb xyuas los ntawm kev ntws cytometry.
Apoptotic cell tsom xam
Cells tau plated rau hauv 6-cov phiaj zoo ntawm qhov ntom ntawm 5 × 105 hlwb/qhov zoo. Tom qab kev sau, cov hlwb raug centri fuged thiab 195 μL ntawm Annexin V-FITC binding tov tau ntxiv rau resuspend lub hlwb. Tom ntej no, 5 μL Annexin V-FITC thiab 10 μL propidium iodide staining tov tau ntxiv. Cells tau incubated nyob rau hauv qhov tsaus ntuj nti thiab ces muab tso rau hauv ib tug dej khov da dej.
Cistanche tiv thaiv daim siab.
Quantitative reverse transcription PCR (qRT-PCR)
Tag nrho RNAs tau muab rho tawm los ntawm TRIzol reagent (Haigene, Haerbin, Suav). Te viiA™ 7 real-time PCR system tau siv rau qRT-PCR. RNA tau thim rov qab rau hauv cDNA siv BeyoRT™ First Strand cDNA Synthesis Kit (Beyotime Institute of Biotechnology). U6 tau siv los ua kev tswj hwm endogenous [16]. Lub primer sequences yog raws li nram no:
miR-503-3p: pem hauv ntej: 5′-CTGATGGTTAAGGAATGT-3′,
miR-503-3p: thim rov qab: 5′-GTCCTTGGACATCCGGCCG-3′,
U6: pem hauv ntej: 5′-GACAGATTCGGTCTGTGGCAC-3′,
U6: rov qab: 5′-GATTACCCGTCGGCCATCGATC-3′.
Western blot tsom xam
Cov kab mob kis tau raug sau thiab tag nrho cov protein tau muab rho tawm. Protein concentrations tau txiav txim siab siv BCA Protein Assay Kit. Cov qauv protein raug cais los ntawm 10 feem pua SDS-PAGE thiab xa mus rau polyvinylidene fluoride membrane. Tom qab ntawd lub membrane tau incubated nrog anti-TGF- 1 (1: 1000, Shifeng, Shanghai, Tuam Tshoj), SMAD3 (1: 1000, Shifeng, Shanghai, Tuam Tshoj), SMAD 7 (1: 1000, Shifeng, Shanghai, Tuam Tshoj), Bcl -2 (1: 1000, Shifeng, Shanghai, Tuam Tshoj), Bax (1: 1000, Shifeng, Shanghai, Tuam Tshoj), Cyto C (1: 1000, Shifeng, Shanghai, Tuam Tshoj), caspase{ {19}} (1: 1000, Shifeng, Shanghai, Tuam Tshoj), caspase -8 (1: 1000, Shifeng, Shanghai, Tuam Tshoj) thiab anti- -actin antibody (1: 1000, Shifeng, Shanghai , Suav teb) hmo ntuj. Kaum daim nyias nyias tau incubated nrog cov tshuaj tiv thaiv tus luav theem nrab antibody (1: 1000, Shifeng, Shanghai, Tuam Tshoj) [17].
Cov txheej txheem txheeb cais
Cov ntaub ntawv tau nthuav tawm raws li qhov nruab nrab ± tus qauv sib txawv (SD). Txhua qhov kev txheeb cais tau ua nrog SPSS 13.0 software. Cov tub ntxhais kawm qhov t-test tau siv rau kev sib piv. p-value < 0.05="" tau="" suav="" tias="" yog="" qhov="" tseem="">
Cov txiaj ntsig
Kev cuam tshuam ntawm ECH rau tib neeg lub siab mob qog noj ntshav ntawm tes thiab kev loj hlob
Txhawm rau tshawb xyuas cov txiaj ntsig ntawm ECH rau tib neeg lub siab mob qog noj ntshav ntawm tes, MTT kev soj ntsuam tau ua thiab cov txiaj ntsig tau pom tias lub cell viability ntawm HepG2 (Fig. 2a) thiab Huh7 (Fig. 2b) tau maj mam txo nrog kev nce koob tshuaj thiab lub sijhawm ntawm Kev kho ECH piv nrog pawg tswj hwm (p < 0="" 01).="" tom="" qab="" 48="" teev="" raug="" ech,="" lub="" cell="" viability="" yog="" qhov="" qis="" tshaj,="" yog="" li="" 48="" h="" raug="" ech="" raug="" xaiv="" rau="" kev="" sim="" ntxiv.="" cov="" txiaj="" ntsig="" ntawm="" colony="" formation="" assay="" tau="" pom="" tias="" tus="" naj="" npawb="" ntawm="" cov="" cheeb="" tsam="" ntawm="" hepg2="" (fig.="" 2c)="" thiab="" huh7="" (daim="" duab="" 2d)="" cov="" hlwb="" maj="" mam="" txo="" qis="" los="" ntawm="" kev="" kho="" ech="" nyob="" rau="" hauv="" ib="" koob="" tshuaj="" raws="" li="" piv="" nrog="" rau="" pawg="" tswj="" hwm="" (p="">< 0="" .01,="" p=""><0.001). cov="" txiaj="" ntsig="" no="" qhia="" tau="" tias="" ech="" ua="" lub="" luag="" haujlwm="" tseem="" ceeb="" hauv="" hcc="" cov="" haujlwm="" ntawm="" tes="" thiab="" kev="" loj="">
Kev cuam tshuam ntawm ECH rau tib neeg lub siab mob qog noj ntshav cell migration thiab ntxeem tau
Txhawm rau tshawb xyuas ntxiv cov txiaj ntsig ntawm ECH ntawm tib neeg lub siab mob qog noj ntshav ntawm tes, kev txav chaw ntawm tes thiab kev ntxeem tau raug kuaj pom. Raws li qhia hauv daim duab 3a, b, piv nrog cov pab pawg uas tsis muaj kev kho mob ntawm ECH, kev tsiv teb tsaws thiab inva sion ntawm Huh7 thiab HepG2 hlwb tau txo qis tom qab kev kho ECH nyob rau hauv ib koob tshuaj (p<0.01,><0.001), suggesting="" that="" ech="" inhibited="" the="" migration="" and="" invasion="" of="" huh7="" and="" hepg2="">
ECH tswj tib neeg lub siab mob qog noj ntshav cell faib thiab apoptosis
Txhawm rau tshawb nrhiav cov txiaj ntsig ntawm ECH ntawm daim siab mob qog noj ntshav cell apoptosis, ntws cytometry tau ua tiav. Raws li pom hauv daim duab 4a, piv nrog rau pawg tswj hwm, feem pua ntawm Huh7 thiab HepG2 hlwb hauv G0/G1 theem tau txo qis heev tom qab ECH kev kho mob raws li koob tshuaj (p<0.01), and="" the="" percentage="" of="" huh7="" and="" hepg2="" cells="" in="" the="" s="" phase="" was="" gradually="" increased="" after="" ech="" treatment="" in="" a="" dose-dependent="" manner=""><0.01), but="" there="" was="" no="" change="" of="" the="" percentage="" of="" huh7="" and="" hepg2="" cells="" in="" g2/m="" phase="" after="" ech="" treatment="" (p="">0.05), qhia tias ECH ua rau S theem ntes ntawm Huh7 thiab HepG2 hlwb. Tsis tas li ntawd, nrog kev nce ntawm koob tshuaj ntawm ECH, qhov apoptosis tus nqi ntawm Huh7 thiab HepG2 hlwb maj mam nce piv nrog rau pawg tswj hwm (Fig. 4b, p.<0.01,><0.001). these="" results="" indicated="" that="" ech="" induced="" the="" apoptosis="" of="" huh7="" and="" hepg2="">
Cov teebmeem ntawm ECH ntawm TGF- 1/Smad txoj kev
Nws paub tias TGF- 1/Smad signaling pathway muaj kev koom tes zoo hauv cell apoptosis. Txhawm rau tshawb xyuas cov txheej txheem ntawm ECH-induced apoptosis hauv HepG2 hlwb. Kev qhia ntawm TGF- 1/Smad signaling pathway tau kuaj pom. Piv nrog rau pawg tswj hwm, peb pom tias ECH kev kho mob tau txo qis kev qhia theem ntawm TGF- 1 thiab Smad3 ntawm ob qho tib si mRNA (Fig. 5a) thiab protein (Fig. 5b) qib, thiab signifcantly up-regulated kev qhia ntawm Smad7 nyob rau hauv ib koob tshuaj ntawm ob qho tib si mRNA thiab protein ntau (p<0.01,><0.001). these="" results="" indicated="" that="" ech="" inhibited="" the="" activation="" of="" the="" tgf-β1/smad="" signaling="">
TGF-1 yog lub hom phiaj ncaj qha ntawm miR-503-3p
Ntxiv rau, cov cuab yeej twv ua ntej online Starbase v2. qhia tias TGF- 1 yog lub hom phiaj muaj peev xwm ntawm miR-503-3p (Fig. 6a). Luciferase reporter gene assay cov txiaj ntsig tau pom tias kev ua haujlwm ntawm luciferase ntawm HepG2 hlwb thiab Huh7 hlwb co-transfected nrog miR-503-3p mimic thiab TGF- 1-WT tau txo qis, tab sis tsis yog cov hlwb sib txuas nrog miR{ {10}}p mimic thiab TGF- 1-MUT (Fig. 6b, p<0.01). similarly,="" the="" luciferase="" activity="" of="" hepg2="" and="" huh7="" cells="" co-transfected="" with="" mir-503-3p="" inhibitor="" and="" tgf-β1-="" mut="" was="" signifcantly="" increased,="" but="" not="" the="" cells="" co-transfected="" with="" mir-503-3p="" inhibitor="" and="" tgf-β1-wt="" (fig.="" 6c,=""><0.01). furthermore,="" the="" expression="" levels="" of="" tgf-β1="" were="" signifcantly="" reduced="" in="" the="" mir-503-3p="" mimic="" group="" compared="" with="" that="" in="" the="" nc="" group="" (fig.="" 6d,=""><0.01), but="" signifcantly="" increased="" in="" the="" mir-503-3p="" inhibitor="" group="" (fig.="" 6d,=""><0.05). these="" results="" suggested="" that="" tgf-β1="" was="" a="" direct="" target="" of="" mir-503-3p.="" to="" further="" investigate="" whether="" mir-503-3p="" mediated="" the="" effect="" of="" ech="" on="" liver="" cancer,="" the="" expression="" of="" mir-503-3p="" was="" detected="" in="" liver="" cancer="" tissues="" and="" it="" showed="" that="" the="" expression="" levels="" of="" mir-503-3p="" were="" decreased="" in="" liver="" cancer="" tissues="" compared="" with="" that="" in="" normal="" liver="" tissues="" (fig.="" 6f,=""><0.01). moreover,="" the="" expression="" levels="" of="" mir503-3p="" were="" further="" decreased="" in="" metastasis="" liver="" cancer="" tissues="" than="" that="" in="" nonmetastatic="" liver="" cancer="" tissues="" (fig.="" 6g,=""><0.01). the="" expression="" levels="" of="" mir-503-3p="" in="" hepg2="" and="" huh7="" cells="" were="" gradually="" increased="" after="" ech="" exposure="" (fig.="" 6e,=""><0.05,><0.01). taken="" together,="" these="" results="" suggest="" that="" mir-503-3p/tgf-β1="" may="" mediate="" the="" effect="" of="" ech="" on="" liver="">
Cov nyhuv ntawm ECH ntawm cytochrome C thiab caspases cov dej num
Raws li qhia hauv daim duab 7a, piv nrog rau pawg tswj hwm, peb pom tias kev kho ECH ua rau muaj kev txo qis hauv kev qhia qib ntawm Bcl-2 nyob rau hauv ib koob tshuaj (p<0.01,><0.001), while="" the="" expression="" levels="" of="" bax="" were="" signifcantly="" increased="" in="" a="" dose-dependent="" manner=""><0.01,><0.001). it="" was="" demonstrated="" that="" bcl-2="" and="" bax="" were="" involved="" in="" ech-induced="" apoptosis="" in="" hepg2="" and="" huh7="" cells.="" in="" addition,="" the="" expression="" levels="" of="" cyto="" c,="" caspase-8,="" and="" caspase-3="" were="" signifcantly="" increased="" in="" hepg2="" and="" huh7="" cells="" (fig.="" 7b,=""><0.01,><0.001), indicating="" that="" the="" release="" of="" cyto="" c="" and="" the="" activation="" of="" caspase-8="" and="" caspase-3="" were="" involved="" in="" ech-induced="" apoptosis="" in="" hepg2="" and="" huh7="">
Kev sib tham
Kev phais phais yog txoj kev kho mob tseem ceeb rau cov neeg mob uas muaj mob qog noj ntshav, thiab qhov rov ua dua thiab metastasis tom qab daim siab txiav tawm siab [18, 19]. Hauv txoj kev tshawb no, peb pom tias ECH muaj qee yam cuam tshuam rau cov qog nqaij hlav hauv siab, thiab muab cov tshuaj tiv thaiv qog nqaij hlav zoo rau lub siab. Kev xyaum kho mob tau pom tias cov tshuaj suav tshuaj tuaj yeem txo cov tsos mob ntawm cov neeg mob qog noj ntshav siab [20]. Rau ECH, kev tshawb fawb tau pom tias ECH muaj cov tshuaj tiv thaiv kab mob antioxidant, tshuaj tiv thaiv kev ntxhov siab, tshuaj tiv thaiv kab mob, tshuaj tua kab mob, tshuaj tua kab mob, thiab tshuaj tua kab mob [21, 22]. Hauv kev mob qog nqaij hlav hauv plab, ECH tuaj yeem tsim cov qog cell apoptosis los ntawm kev tswj hwm caspase-3 thiab tshem tawm DNA kho cov enzymes kom ntxias DNA oxidation [23]. Hauv txoj kev tshawb no, peb pom thawj zaug uas ECH tawm dag zog ua haujlwm ntawm miR 503-3p/TGF- 1/Smad axis hauv daim siab mob cancer.
Lub sijhawm intercellular ntawm lub voj voog ntawm tes ntawm eukaryotic hlwb tuaj yeem muab faib ua G1, S, thiab G2. Cov ntsiab lus tswj hwm ntawm lub voj voog ntawm tes ntawm lub sijhawm sib txawv yog tswj hwm los ntawm cov kev cai sib txawv [24]. Txoj kev tshawb no pom tias feem pua ntawm Huh7 thiab HepG2 hlwb hauv G0/G1 thiab G2/M theem raug txo, thiab feem pua ntawm cov hlwb hauv S theem tau nce ntxiv tom qab ECH, qhia tias ECH ua rau S theem raug ntes. ntawm Huh7 thiab HepG2 hlwb. Imbal ance ntawm apoptosis yog ib qho ntawm cov txheej txheem tseem ceeb ntawm tib neeg cov qog. Nws tau raug tshaj tawm tias cov tshuaj tiv thaiv qog noj ntshav feem ntau ua rau apoptosis ntawm cov qog nqaij hlav cancer thiab tswj kev mob qog noj ntshav. Hauv peb txoj kev tshawb fawb, peb kuj pom tias ECH tuaj yeem ua rau lub siab mob qog noj ntshav ntawm lub hlwb apoptosis nyob rau hauv koob tshuaj, qhia tias inducing cancer cell apoptosis tej zaum yuav yog ib qho ntawm cov hauv paus ntsiab lus ntawm ECH ntawm daim siab mob cancer.
MiRNAs ua lub luag haujlwm tseem ceeb hauv kev tswj hwm ntawm HCC cell proliferation, voj voog, apoptosis, tsiv teb tsaws los ntawm kev sib cuam tshuam nrog cov kev taw qhia sib txawv thiab cov molecules [25, 26]. Piv txwv li, nws tau pom tias miR-26a tau qhia ntau heev nyob rau hauv lub siab lub cev, tab sis nws yog ib qho tseem ceeb downregulated nyob rau hauv daim siab mob hlwb thiab inhibits proliferation [27]. Overexpression ntawm miR-503-3p induces apoptosis ntawm lub ntsws cancer hlwb thiab inhibits cell invasion thiab tsiv teb tsaws [28]. Tsis tas li ntawd, ntau qhov kev tshawb fawb tau pom tias TGF- 1/Smad signaling txoj hauv kev ua lub luag haujlwm tseem ceeb hauv kev loj hlob ntawm daim siab mob cancer [7]. Tsis tas li ntawd, thaum mob qog noj ntshav txuas ntxiv mus, kev qhia ntawm TGF- 1 tau tshaj tawm tias yuav maj mam nce [29]. Hauv txoj kev tshawb no, peb pom tias ECH down-regulated TGF- 1 thiab Smad3, thiab up-regulated Smad7, qhia tias ECH inhibited qhov ua kom TGF{15}}/Smad signaling pathway. Hauv txoj kev tshawb no, peb pom tias qhov kev qhia ntawm miR-503-3p tau pom tias txo qis hauv daim siab qog nqaij hlav, tab sis kev kho ECH tau nce qhov kev qhia ntawm miR-503-3p hauv Huh7 thiab HepG2 hlwb. Ntxiv rau, peb pom tias TGF- 1 raug txheeb xyuas tias yog lub hom phiaj muaj peev xwm ntawm miR-503-3p. ECH txhawb kev ua kom TGF- 1/Smad signaling pathway thiab nce qib kev qhia ntawm Bax/Bcl-2. Cov txiaj ntsig no qhia tias miR-503-3p/ TGF- 1/Smad signaling txoj kev tuaj yeem kho qhov cuam tshuam ntawm ECH ntawm daim siab mob cancer. Nws tau raug paub tias qhov nce Bax/Bcl-2 piv yuav ua rau Cyto C hauv mitochondria tso rau hauv cytoplasm. Cyto C cuam tshuam nrog Caspase-8 thiab tsim lub cev apoptotic uas tom qab ntawd nrhiav thiab qhib Caspase-3, uas ua rau Caspases cascade thiab txhawb kev tuag ntawm tes [30]. Hauv txoj kev tshawb no, peb pom tias ECH txo qhov kev qhia ntawm Bcl-2 thiab nce qhov kev qhia ntawm Bax, Cyto C, caspase-8, thiab caspase-3. Cov txiaj ntsig no qhia tias ECH tuaj yeem kho qhov raug mob mitochondrial rau qee yam.
Kev xaus hauv txoj kev tshawb no, peb pom tias kev kho ECH inhibited qhov kev loj hlob, kev ntxeem tau, thiab kev tsiv teb tsaws, thiab ua rau apoptosis ntawm daim siab mob hlwb. Lub hauv paus txheej txheem ntawm ECH kev kho mob ntawm daim siab mob hlwb tau pom tias muaj feem xyuam nrog miR-503-3p/TGF- 1/Smad signaling pathway. Txoj kev tshawb no yuav muab qee qhov kev sim hauv paus rau kev tshawb fawb ntawm ECH ntawm kev tiv thaiv qog. Nws yog tsim nyog sau cia tias qhov kev tshawb fawb tam sim no raug txwv los ntawm qhov tsis muaj kev sim tsiaj. Yog li ntawd, peb txoj kev tshawb fawb yav tom ntej yuav ua cov kev sim tsiaj kom paub meej ntxiv txog peb qhov kev tshawb pom.
