ELOVL2 Txhawb Kev Loj Hlob Cancer Los Ntawm Inhibiting Cell Apoptosis hauv Renal Cell Carcinoma
Mar 20, 2022
Abstract. Lub raumcell carcinoma (RCC) yog ib tug aggressive genitourinary malignancy uas tau txuam nrog ib tug tsis zoo prognosis, tshwj xeeb tshaj yog nyob rau hauv cov neeg mob nrog metastasis, nws loj subtypes yog ntshiab cell RCC (ccRCC), papillary PCC (pRCC), thiab chromophobe RCC (chRCC). Kev muaj cov lipid droplets (LDs) yog suav tias yog ib qho tseem ceeb ntawm ccRCC.Qhov tseem ceeb ntawm kev hloov lipid metabolism hauv ccRCC tau pom dav dav. Lub elongation ntawm heev-ntev-cov saw fatty acid (ELOVL) catalyzes lub elongation ntawm fatty acids (FAs), modulating lipid muaj pes tsawg leeg, thiab yuav tsum tau rau ib txwm lub cev ua hauj lwm. Txawm li cas los xij, kev koom tes ntawm elongases hauv RCC tseem tsis paub meej. Hauv kev tshawb fawb tam sim no, kev qhia ntawm ELOVL2 hauv ccRCC raug kuaj xyuas; tshwj xeeb, qib siab ntawm xya ELOVLisozymes tau pom hauv thawj cov qog. Ntawm qhov kev ceeb toom, nce qib ELOVL2 qhia tau pom hauv ccRCC, nrog rau hauv pRCC thiab chRCC. Tsis tas li ntawd, qib siab dua ntawm ELOVL2 tau cuam tshuam nrog kev nce ntxiv ntawm qhov tsis zoo ntawm cov neeg mob ccRCC thiab pRCC. Lub CRISPR/Cas9-kev kho kom haum xeeb knockdown ntawm ELOVL2 ua rau muaj kev cuam tshuam ntawm qhov elongation ntawm cov saw ntev polyunsaturated FAs thiab nce LD ntau lawm hauvlub raummob qog noj ntshav. Ntxiv mus, ELOVL2 ablation ua rau muaj kev cuam tshuam ntawm cellular proliferation los ntawm induction ntawm apoptosis hauv vitro thiab txo qis ntawm cov qog loj hlob hauv vivo. Ntawm qhov tod tes, txoj kev tshawb fawb tam sim no muab kev nkag siab tshiab rau hauv cov qog nqaij hlav proliferation mechanisms cuam tshuam nrog lipid metabolism thiab qhia tias ELOVL2 yuav yog lub hom phiaj tshiab zoo nkauj rau RCC txoj kev kho.
Ntsiab lus:Lub raum cell carcinoma, lipid droplet, cellular proliferation, raum, raum
Taw qhia
Lub raum cell carcinoma (RCC) yog ib qho uas feem ntau ntsib thiab tuag malignancy, suav txog ~ 2 feem pua ntawm tag nrho cov mob qog noj ntshav thiab cov neeg tuag thoob ntiaj teb (1), nws cov subtypes loj yog ntshiab cell RCC (ccRCC), papillary PCC (pRCC), thiab chromophobe. RCC (RCC). Ntawm tag nrho cov RCC subtypes, ccRCC yog qhov tshwm sim ntau tshaj plaws histological thiab nws cov kab mob tshwm sim los ntawm kev ua haujlwm ntawm hypoxia-inducible yam (HIFs) vim qhov poob-ntawm-ua haujlwm hauv von Hippel-Lindau (VHL) qog nqaij hlav. (1). Ntau hom phiaj kho mob tiv thaiv vascular endothelial kev loj hlob zoo tshaj plaws (VEGF) lossis mammalian lub hom phiaj ntawm rapamycin (mTOR) qhia thiab tiv thaiv kab mob tiv thaiv kab mob rau cov kab mob metastatic tau tsim. Txawm li cas los xij, txoj kev loj hlob ntawm tus kab mob yog qhov tsis tuaj yeem nyob hauv feem ntau ntawm cov neeg mob (1,2).
CISTANCHE yuav txhim kho lub raum / raum mob ntshav qab zib
Ib qho piv txwv ntawm ccRCC yog qhov ntau ntawm cov lipid droplets (LDs), uas muaj cov lipid nruab nrab uas muaj triglycerides (TGs) thiab cov roj cholesterol-esters (CEs) nyob ib puag ncig los ntawm phospholipid monolayer (3). thiab khaws cia thiab siv los tswj cov homeostasis, pab txhawb kev tsim hluav taws xob thiab tiv thaiv ntau hom kev ntxhov siab lossis txhawb nqa membrane biogenesis thaum cov qog cell loj hlob sai hauv ntau hom mob qog noj ntshav (4).Qhov tseeb, cov kev tshawb fawb yav dhau los tau pom tias LDs pab txhawb ccRCC. kev loj hlob (5,6).
Cov roj ntsha fatty acids (FAs) uas suav nrog cov ntsiab lus tseem ceeb ntawm lipids tau raug tshaj tawm los ua cov substrates rau lub zog cia, membrane synthesis, thiab tsim cov teeb liab molecules. Elongation thiab desaturation yog cov kauj ruam tseem ceeb ntawm thede Novo synthesis ntawm cov saw ntev FAs (LC-FAs), qhov ntev thiab qib ntawm unsaturation yog tus txiav txim siab ntawm FA muaj nuj nqi thiab txoj hmoo metabolic (7). Stearoyl-CoA desaturase 1 (SCD1), ib tug tswv cuab ntawm tsev neeg fatty acyl desaturase, tau kawm dav hauv ccRCC (6.8.9). tau pom tias yuav txwv cov cellular proliferation hauv ccRCC(8.9). Ntxiv mus. Yang et al (10) tau ua pov thawj tias sterol regulatory element-binding protein 1 (SREBP1) txhawb lipid desaturation los ntawm FA acid desat-urase 1 (FADSI) ua ntej ua kom muaj NF-xB signaling rau kev txhawb nqa ntawm cellular proliferation hauv ccRCC. Txawm li cas los xij, txhua lub luag haujlwm ntawm elongases hauv RCCremain tsis meej.
Nws tau raug tshaj tawm tias, hauv cov tsiaj nyeg, thawj zaug thiab tswj tus nqi FA condensation cov tshuaj tiv thaiv yog catalyzed los ntawm ib tsev neeg ntawm elongase enzymes hu ua elongation ntawm heev-ntev-ntev FAs (ELOVL). Txog niaj hnub no, xya tus tswv cuab ELOVL tau raug txheeb xyuas, uas feem ntau tuaj yeem muab faib ua cov tshwj xeeb rau saturated thiab monounsaturated FAs (ELOVL1, ELOVL3, ELOVL6 thiab ELOVL7) lossis rau polyunsaturated FAs (PUFAs; ELOVL2, ELOVL4 thiab ELOVL5). Lucarelli et al (9) qhia txog qhov tseem ceeb ntawm PUFAs thiab kev nthuav qhia ntawm ELOVL2 thiab ELOVL5 hauv ccRCC. Ntawm qhov kev ceeb toom, nws tau pom tias systemic docosahexaenoic acid (DHA) yog endogenously tsim thiab tswj de novo lipogenesis, thaum tseem tswj cov lipid cia nyob rau hauv ib tug sterol regulatory element-binding transcription factor 1 (SREBPF1) - ywj siab yam, vim ELOVL2 ablation hauv nas (11). Ntxiv mus, ELOVL2 overexpression tau pom tias yuav txhawb nqa LD txuam nrog kev txhim kho FA uptake nyob rau hauv ob qho tib si preadipocyte cell kab 3T3-LI thiab F442A hlwb(12). Yav dhau los hauv vitro kev tshawb fawb, nrog DHA, exogenously muab rau cov qog nqaij hlav cancer. tau ua pov thawj tias DHA tawm dag zog los tiv thaiv qog nqaij hlav, tiv thaiv kab mob, thiab pro-apoptotic cuam tshuam rau cov qog nqaij hlav cancer (13-15). Txawm li cas los xij, txhua lub luag haujlwm ntawm ELOVL2 hauv ccRCC kev nce qib los ntawm kev hloov pauv ntawm lipid metabolism tseem tsis tau tshawb nrhiav.
Hauv txoj kev tshawb fawb tam sim no, lub luag haujlwm ntawmELOVL2inccRCCprogres-sion tau tshawb nrhiav los ntawm kev ua qhov kev hloov pauv ntawm thawj ccRCC thiab ib txwm muaj.raumcov qauv, qhia tias ELOVL2 yog overexpressed nyob rau hauv ccRCC thiab yog overrepresented ntawm ELOVL isozymes. Ntawm qhov kev ceeb toom, ELOVL2 kuj tau hais ntau dhau hauv ccRCC, nrog rau hauv pRCCand RCC, qhov no tau cuam tshuam nrog kev pom tsis zoo ntawm cov neeg mob CCR CCR pRCC. Ntxiv mus, nws tau pom tias ELOVL2 inhibition cuam tshuam rau lipid metabolism thiab suppresses cellular proliferation los ntawm kev txhawb nqa ntawm apoptosis, tsawg kawg yog ib feem los ntawm kev cuam tshuam ntawm endoplasmic reticulum (ER) homeostasis hauvmob qog noj ntshavhlwb. Cov txiaj ntsig ntawm txoj kev tshawb fawb tam sim no qhia tias ELOVL2 tuaj yeem yog lub hom phiaj kho tshiab rau RCC.
CISTANCHE yuav txhim kho lub raum / raum tsis ua haujlwm
Cov ntaub ntawv thiab cov txheej txheem
Cell kab thiab kab lis kev cai. 293T (RCB2202) thiab OS-RC-2 (RCB0735) xov tooj ntawm tes tau yuav los ntawm RIKEN BioResource Center thaum 786-O thiab ACHN xov tooj ntawm tes tau yuav los ntawm ATCC; SK-RC-52 hlwb yog khoom plig zoo los ntawm DrJ.G.Old (Memorial Sloan Kettering Cancer Center). RPTEC cell kab, uas yog muab los ntawm epithelial hlwb ntawm tib neeglub raumproximal tubule, tau yuav los ntawm Lonza Group, Ltd. (CC-2553). Lub ACHN, 786-O, SK-RC-52 thiab OS-RC-2 hlwb tau coj los rau hauv RPMI-1640 nruab nrab ntxiv nrog 10 feem pua fetal bovine serum (FBS) ntawm 37˚C nrog 5 feem pua ntawm cov cua sov CO2. Lub 293T hlwb tau coj mus rau hauv Dulbecco's modified Eagle nruab nrab supplemented nrog 10 feem pua fetal bovine serum (FBS) ntawm 37˚C nrog 5 feem pua ntawm CO2 cua. Cov hlwb RPTEC tau coj mus rau hauvlub raumepithelial kev loj hlob nruab nrab (REGM ™) Bulletkit ™ (CC-3190; Lonza Bioscience) ntawm 37˚C nrog 5 feem pua humidified CO2 cua.
Cov neeg mob thiab ccRCC cov qauv. RCC cov ntaub so ntswg thiab ib txwm nyob ib sabraumcov ntaub so ntswg los ntawm 46 tus neeg mob uas tau txais radical lossis ib nrab nephrectomies tau txais los ntawm University of Tsukuba Tsev Kho Mob, ntawm 2006 thiab 2015 raws li cov txheej txheem pom zoo los ntawm Pawg Saib Xyuas Kev Ncaj Ncees ntawm University of Tsukuba (kev pom zoo no. H28-104). Txhua tus neeg mob tau sau ntawv tso cai ua ntej kev phais. Cov kab mob qog tau raug xaiv raws li TNM theem ntawm Union for International Cancer Control (16). Cov qib pathological tau muab cais, raws li plaub-tiered Fuhrman grading system (17). Tag nrho cov yam ntxwv ntawm tus neeg mob tau sau tseg hauv Table SI RNA extraction thiab cDNA synthesis. Tag nrho RNA tau muab rho tawm los ntawm cov ntaub so ntswg khov thiablub raummob qog noj ntshav siv TRIzol® Reagent (Thermo Fisher Scientific, Inc.). Tom qab RNA purification, RNA yog tom qab ntawd thim rov qab rau hauv cDNA siv cov khoom siv siab-Capacity cDNA Reverse Transcription kit (cat. no. 4368814; Thermo Fisher Scientific, Inc.), raws li cov chaw tsim khoom cov lus qhia.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gene qhia qib tau suav nrog siv 7500 Fast Real-Time PCR tshuab nrog Fast SYBR-Green Master Mix (Thermo Fisher Scientific, Inc.). Cov kev caij tsheb kauj vab yog raws li nram no: Thawj tuav ntawm 95˚C rau 20 sec, 40 cycles ntawm 95˚C rau 3 sec thiab 60˚C rau 30 sec, ua raws li los ntawm ib tug melting nkhaus li ntawm 95˚C rau 15 sec rau 60˚ C rau 1 min. Hypoxanthine phosphoribosyltransferase 1 (HPRT1) tau siv los ua kev tswj hwm sab hauv. Tag nrho cov txheej txheem primer tau teev tseg hauv Table SII. Txhawm rau soj ntsuam cov noob caj noob ces, qhov txheeb ze qhia theem ntawm cov noob pro-apoptotic, Bcl-2-associated X protein (BAX), Bcl-2 homologous antagonist/killer (BAK), phorbol-12-myristate-13- acetate-induced protein 1 (PMAIP1/NOXA) thiab p53 upregulated modulator of apoptosis (BBC3/PUMA) thiab ntawm anti-apoptotic gene BCL2 thiab induced myeloid leukemia cell differentiation protein gene (MCL1) tau soj ntsuam. Cov theem ntawm cov noob txheeb ze tau suav nrog siv 2-ΔΔCq txoj kev (18).
Western blot tsom xam. Western blot tsom xam tau ua raws li tau piav qhia yav dhau los (19). Cells tau lysed nyob rau hauv lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 feem pua SDS thiab protease inhibitor) thiab sonicated rau dej khov. Cov lysates tau centrifuged ntawm 1, 000 xg rau 20 min ntawm 4˚C thiab cov supernatants tau sau ua qauv. Kev ntsuas cov protein ntau ntawm cov qauv tau ua los ntawm Quick Start Bradford Protein assay (cat. no. 5000202JA; Bio-Rad Laboratories, Inc.). Cov qauv raug xa mus rau 10 feem pua SDS-PAGE thiab cov khoom sib cais tau xa mus rau PVDF membranes.
CISTANCHE yuav txhim kho lub raum / raum kab mob
Cov membranes raug thaiv nrog ECL Prime thaiv tus neeg sawv cev (cat. RPN418V; Cytiva) rau 60 min ntawm chav tsev kub. Cov daim nyias nyias tau muab tso rau ib hmos ntawm 4˚C nrog cov tshuaj tiv thaiv hauv qab no: Anti-serine/threonine-protein kinase/endoribonuclease inositol-yuav tsum tau enzyme 1 (anti-IRE1; 1:200; #3294, Cell Signaling Technology, Inc.), Anti-phosphorylated IRE1 (anti-p-IRE1; 1:200; NB100-2323, Novus Biologicals, LCC), anti-C/EBP homologous protein (anti-CHOP; 1:200; MA1-250, Thermo Fisher Scientific, Inc. .). Cov tshuaj tiv thaiv kab mob los yog tshuaj tiv thaiv nas immunoglobulin G HRP-txuas, tag nrho cov qaib Ab (cat. no. NA934V, NA931VS; GE Healthcare; Cytiva) tau siv ntawm 1:10, 000 ua cov tshuaj tiv thaiv kab mob thib ob. Cov blots sab hnub poob tau pom nrog ImmunoStar® Zeta (FUJIFILM Wako Pure Chemical Corporation) siv Fujifilm LAS‑4000 imager thiab LAS400IR (FUJIFILM Wako Pure Chemical Corporation). -actin tau siv los ua kev tswj hwm sab hauv (1: 10,000, cat. no. A5316; Sigma-Aldrich).
Bioinformatics tsom xam ntawm cov noob qhia. Clinical thiab RNA-sequencing (RNA-seq) cov ntaub ntawv ntawm cov qog thawj zaug sau los ntawm cov neeg mob uas muaj RCC nyob rau hauv The Cancer Genome Atlas (TCGA) database tau rub tawm los ntawm Genomic Data Commons (GDC) Data Portal (20). Tag nrho ntawm 1,005 (880 tus kab mob thiab 125 kev tswj) tau kuaj xyuas, suav nrog 530 tus kab mob thiab 71 tus qauv tswj hwmlub raumClear cell carcinoma cohort (KIRC; ccRCC), 286 tus kab mob thiab 31 cov qauv noj qab haus huv rau lub ncauj tsev menyuamlub raumpapillary cell carcinoma cohort (KIRP; pRCC), thiab 64 kab mob thiab 23 cov qauv noj qab haus huv rau lub raum chromophobe cohort (KICH; chRCC). ELOVL2 gene qhia nyob rau hauv cov qauv qog nqaij hlav genitourinary tau soj ntsuam siv cov Gene Expression database ntawm cov nqaij mos thiab qog nqaij hlav (GENT2). Cov ntaub ntawv qhia txog cov noob tau rub tawm los ntawm cov noob qhia omnibus (GEO) cov chaw khaws ntaub ntawv pej xeem ntawm U133 Plus 2 (GPL570) platform (http://gent2.appex.kr/gent2/) (21).
Plasmids thiab lentiviral transduction. Cov plaub hau me me RNAs (shRNAs) rau ELOVL2 tus yam ntxwv hauv kev tshawb fawb yav dhau los (22,23) tau subcloned rau hauv lentiviral vector pLKO.1 (plasmid #8453; Addgene, Inc.). Cov kab ke oligonucleotide siv hauv kev tsim kho ntawm shRNA vector yog teev nyob rau hauv Table SIII. Lentiviruses tau tsim nyob rau hauv 293T hlwb los ntawm kev sib koom ua plaub plasmids hauv trans, suav nrog lentiviral vector (pLKO-shControl lossis pLKO-shELOVL2), pMDLg / pRRE (plasmid #12251; Addgene, Inc.), pRSV-Rev (plasmid #12253). ; Addgene, Inc.), thiab pMD2.G (plasmid #12259; Addgene, Inc.) siv Lipofectamine 2000® transfection reagent (Thermo Fisher Scientific, Inc.). Thaum 48 teev tom qab kis tau tus kab mob, cov kab mob uas muaj cov kab mob supernatants tau sau thiab lim los ntawm 0.45μm lim rau kev kis kab mob. Rau kev kis kab mob, pLKO-shControl lossis pLKO-shELOVL2 lentiviruses tau tsim nrog 786-O, ACHN thiab SK-RC-52 hlwb thaum hmo ntuj ntawm 37˚C hauv qhov chaw humidified cell culture incubator. Cells raug xaiv nyob rau hauv lub xub ntiag ntawm puromycin ntawm 24 teev tom qab kis kab mob. Txhawm rau kom ruaj khov subclones, cov hlwb tau muab coj los ua hauv nruab nrab nrog puromycin rau 1 lub hlis ntawm 37˚C nyob rau hauv ib qho chaw humidified cell culture incubator thiab cov pas dej uas muaj sia nyob tau siv rau kev qhia thiab kev txheeb xyuas kev loj hlob.
Rau kev sim ntau dhau, OSRC-2 hlwb tau hloov pauv nrog pCMV6 khoob vector (plasmid #PS100001; Origene Technologies, Inc.) lossis pCMV6-ELOVL2 (plasmid #RC209232; Origene Technologies, Inc.) ntawm 37˚C hauv cov kab lis kev cai humidified. incubator, siv Lipofectamine 3000® transfection reagent (Thermo Fisher Scientific, Inc.), raws li cov chaw tsim khoom cov lus qhia. Cov hlwb tau siv rau qhov kev tshuaj ntsuam xyuas ntawm 48 teev tom qab hloov pauv. CRISPR/Cas9 tsim thiab cloning. Lub pX330-U6-Chi‑ meric{12}}BB-CBh-hSpCas9 (pX330) plasmid yog khoom plig los ntawm Feng Zhang (Addgene, Inc.; plasmid #42230) (24). CRISPR ib leeg-qib qhia (sg) ib ntus rau ELOVL2 tau tshwj xeeb rau exon 2 (sgELOVL2 #1) thiab exon 3 (sgELOVL2 #2 thiab #3) ntawm ELOVL2 siv CRISPR Tsim Tool (GE Healthcare Dharmacon, Inc.;‑discovery.com/gene-editing/crispr-cas9/crispr-design-tool/), ua ntej cloning rau pX330. CRISPR cov lus qhia ib ntus rau kev tswj hwm ib leeg (sgControl) tau tsim yav dhau los (25). Cov kab ke oligonucleotide ntawm ib leeg-qhia RNAs (sgRNAs) tau teev nyob rau hauv Table SIII
Tom qab ntawd cov ACHN hlwb tau muab cog rau hauv 6-zoo daim hlau (1.4x105 hlwb/zoo) thiab co-transfected 2 h tom qab nrog 2 µg ntawm pX330-expressed sgRNAs thiab 0.2 µg pCI-neo vector (Promega Corporation; cat. No. E1841) ntawm 37˚C nyob rau hauv lub tshuab nqus tsev vacuum kab lis kev cai, siv FuGENE HD (Promega Corporation; cat. no. E2312). Thaum 24 teev tom qab hloov pauv, 400 µg / ml G418 tau thov rau 7 mus rau 10 hnub thiab cov hlwb raug tso cai rov qab rau 2 lossis 3 hnub. Thaum cov cell tau nce mus txog qhov tsis sib xws, lawv tau rov ua dua tshiab hauv 10 cm lauj kaub tais diav. Tom qab 2 lossis 3 lub lis piam, cov cheeb tsam pom tau raug cais los ntawm kev siv cloning discs (MilliporeSigma; cat. no. Z374431-100EA). Cov tib neeg clones tau nthuav dav thiab ntsuas rau qhov xwm txheej knockout los ntawm Sanger sequencing rau thaj tsam lub hom phiaj.
CISTANCHE yuav txhim kho lub raum / raum mob
Kev ntsuam xyuas cellular proliferation. Cellular proliferation raug soj ntsuam siv MTT assay nrog Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) raws li cov chaw tsim khoom cov lus qhia. Luv luv, cov hlwb raug muab cog rau hauv 96-zoo daim hlau thiab 10 µl ntawm WST-8 tau ntxiv tom qab 72 h.OD460 tau ntsuas tom qab 1-h incubation ntawm 37˚C. Subcutaneous xenografting. BALB/c liab qab (nu/nu) nas poj niam (n=12; 6-8 lub lis piam) tau yuav los ntawm Charles River Laboratories, Inc.. Cov nas tau nyob hauv cov kab mob tshwj xeeb tsis muaj kab mob, hauv qab 12- h lub teeb / tsaus voj voog, nrog ad libitum nkag mus rau zaub mov thiab dej. Rau kev soj ntsuam subcutaneous xeno-graft, 1x107 hlwb raug tshem tawm hauv 100 µl PBS tau txhaj subcutaneously rau hauv txoj cai flank los ntawm kev siv 24G koob thiab cov qog ntim tau ntsuas ib zaug ib lub lim tiam. Cov qog ntim tau suav los ntawm cov mis: mm3=ntev x dav x qhov siab x 0.52 (26). Tom qab 90 hnub, tag nrho cov tsiaj raug txi, thiab cov qog xenograft raug tshem tawm. Tsiaj txhu tau tshuaj loog nrog 2 feem pua isoflurane ua ntej euthanized los ntawm ncauj tsev menyuam dislocation
Formalin-fixed thiab paraffin-embedded (FFPE) cov qauv ntawm cov qog xenograft raug txiav rau hauv 4-µm-thick seem, ua ntej deparaffinization thiab rehydration. Rau antigen retrieval, cov seem tau pretreated los ntawm microwave rau 21 min nyob rau hauv ib tug citric acid tsis. Tom qab cov txheej txheem antigen retrieval, endogenous peroxidase kev ua haujlwm raug thaiv nrog 3 feem pua H2O2 rau 25 min thiab cov slides tau incubated nrog Ki‑67 antibody (1:200; Dako; Agilent Technologies, Inc.; M7240) at4˚C thaum hmo ntuj. Cov tshuaj tiv thaiv immunohistochemical tau pom los ntawm kev siv cov tshuaj tiv thaiv thib ob Histofine Simple Stain MAX PO® (Nichirei Biosciences, Inc,; cat. no. 424151) los ntawm kev siv diaminobenzidine ua cov chromogen. Tag nrho cov kev tshawb fawb tsiaj tau pom zoo los ntawm Pawg Saib Xyuas Tsiaj Kev Tshawb Fawb ntawm University of Tsukuba thiab txhua qhov kev sim tau ua raws li cov lus qhia ntawm University of Tsukuba Cov Kev Cai Tsiaj Txhaum Cai (kev pom zoo no. 20-370). Kev ntsuam xyuas Apoptosis. Apoptosis raug soj ntsuam siv Caspase-Glo® 3/7 Assay System (Promega Corporation; cat. no. G8090), Annexin-V-FLUOS staining kit (Roche Diagnostics; cat. no. 11858777001), thiab JC-1 Mitochondrial Potential Membrane Assay kit (Cayman Chemical Company; cat. no. 10009172), raws li cov chaw tsim khoom cov lus qhia.
Staining ntawm LDs. Cov noob tau cog rau ntawm cov iav npog hauv 60mm cov tais diav thiab coj los ua ke nrog oleic acid (200 µM)-muaj nruab nrab ntawm 37˚C thaum hmo ntuj hauv 5 feem pua CO 2 incubator. Qhov nruab nrab yog nqus tau, thiab cov hlwb raug ntxuav ob zaug nrog PBS ua ntej kho nrog 4 feem pua formaldehyde (FUJIFILM Wako Pure Chemical Corporation) rau 5 min ntawm chav tsev kub. Cov hlwb raug ntxuav ob zaug nrog PBS, thiab incubated nrog 0.5 µM Lipi-Green (Dojindo Molecular Technologies, Inc.) rau 30 min nyob rau hauv qhov tsaus ntuj ntawm 37˚C. Tom qab ntawd, cov hlwb raug ntxuav ob zaug nrog PBS thiab cov ntawv npog tau muab tso rau ntawm cov iav slides siv VECTASHIELD® Antifade Mounting Medium nrog DAPI (Vector Laboratories, Inc.; cat. no. H-1200). Cov duab ntawm cov stained hlwb tau txais nrog BZ-X710 fluorescence microscope (Keyence Corporation). Cov hlwb nyob tau incubated nrog 0.5 µM Lipi-Green nyob rau hauv Hanks 'Balanced Salt Solution (HBSS) rau 30 min ua ntej ntxuav ob zaug hauv HBSS, resuspended nyob rau hauv 1 mM EDTA / PBS (pH 8.0) nrog 0.5 feem pua BSA, thiab dhau los ntawm lub cell strainer . Cells tau txheeb xyuas ntawm Gallios flow cytometer (Beckman-Coulter, Inc.) thiab cov ntaub ntawv raug tshuaj xyuas siv FlowJo v10 software (FlowJo LLC).
ER tracker staining. ACHN hlwb (ACHN/sgControl, ACHN/sgELOVL2‑1 thiab ACHN/sgELOVL2‑2) tau noob rau ntawm cov iav npog hauv 6{{20}}-hli cov tais diav thiab kab lis kev cai ntawm 37˚C rau 48 teev hauv 5% CO 2 incubator. Qhov nruab nrab yog cov aspi-rated, thiab cov hlwb raug ntxuav ob zaug nrog HBSS ua ntej kho nrog 4 feem pua formaldehyde (FUJIFILM Wako Pure Chemical Corporation) rau 5 min ntawm chav tsev kub. Cov hlwb raug ntxuav ob zaug nrog HBSS thiab incubated nrog 500 nM ER Tracker Liab (Thermo Fisher Scientific, Inc.; cat. No. E34250) rau 2 teev nyob rau hauv qhov tsaus ntuj ntawm 37˚C. Tom qab ntawd, cov hlwb raug ntxuav ob zaug nrog PBS thiab cov ntawv npog tau muab tso rau ntawm cov iav swb. Cov duab ntawm cov stained hlwb tau txais nrog BZ-X710 fluorescence microscope (Keyence Corporation). Cov hlwb nyob tau incubated nrog 500 nM ER Tracker hauv HBSS rau 30 feeb ua ntej ntxuav ob zaug hauv HBSS, resuspended hauv 1 mM EDTA / PBS (pH 8.0) nrog 0.5 feem pua BSA, thiab dhau los ntawm lub cell strainer. Cells tau txheeb xyuas ntawm Gallios flow cytometer thiab cov ntaub ntawv raug tshuaj xyuas siv FlowJo v10 software.
Gas chromatography-mass spectrometry (GC-MS). Kev rho tawm tag nrho cov lipids los ntawm cov cell pellets tau ua raws li tau piav qhia dhau los (27). Lub hydrolysis thiab derivatization ntawm cov extracts rau FA methyl esters (FAMEs) tau ua. Rau cov qauv sab hauv ntawm conjugated FAs, C20: 4 (ω‑6), C20:5 (ω‑3), C22:5 (ω‑6), C22:5 (ω‑3) thiab C22 :6 (ω‑3) FAMEs tau yuav los ntawm MilliporeSigma lossis Cayman Chemical Company. Kev tshuaj xyuas GC-MS tau ua tiav siv GCMS-TQ8040 (Shimadzu Corporation) nrog Omegawax® Capillary GC Kab (MilliporeSigma; cat. no. 24136). Qhov kub gradient tau pib nce los ntawm 70 txog 150˚C ntawm tus nqi ntawm 20˚C ib feeb thiab los ntawm 150 txog 280˚C ntawm tus nqi ntawm 8˚C ib feeb ua ntej yuav txo los ntawm 280 txog 200˚C ntawm tus nqi ntawm 15 ˚C ib min. Cov qauv txhaj tshuaj tau ua nyob rau hauv hom sib cais nrog 1.0 µl ntawm cov qauv txhaj. Rau MS tsom xam, ion qhov chaw thiab lub ntsej muag kub tau teem rau 200˚ thiab 270˚C, raws li. Cov ntaub ntawv tau txais nyob rau hauv tag nrho scan hom (30-600 m / z) nyob rau hauv 70 eV ntawm ionization voltage. FAMEs raug txheeb xyuas raws li lub sijhawm khaws cia thiab electron ionization-MS (EI-MS) spectrum txuam nrog cov qauv siv FAME. Tus nqi txheeb ze ntawm FAMEs tau suav los ntawm kev sib piv qhov nruab nrab qhov siab tshaj plaws ntawm ib qho piv txwv loj. Txhua tus qauv tau ntsuas nws tus kheej peb zaug.
Kev txheeb cais. Cov ntaub ntawv raug nthuav tawm raws li qhov nruab nrab ± SD. Txhua qhov kev txheeb cais tau ua tiav siv JMP 10 software (SAS Institute, Inc.), GraphPad Prism8 (GraphPad Software, Inc.) lossis R pob (version 4.0.2; RStudio, Inc.). Qhov tseem ceeb ntawm qhov sib txawv ntawm ob pab pawg tau raug soj ntsuam los ntawm kev sib tw Student's t-test lossis Mann-Whitney test. Qhov tseem ceeb ntawm qhov sib txawv ntawm peb lossis ntau pawg tau soj ntsuam siv ib txoj kev ANOVA nrog kev sib piv tom qab siv Dunnett's test lossis Kruskal-Wallis test ua raws li Dunn's post hoc test nrog Bonferroni kho. Cov kab nkhaus ciaj sia tau tsim los siv txoj hauv kev Kaplan-Meier thiab qhov sib txawv ntawm cov nkhaus tau raug soj ntsuam los ntawm kev ntsuas ntsuas ntsuas. Cov neeg mob tau muab faib ua ob pawg, ib pawg neeg hais lus qis lossis pab pawg qhia siab, siv kev txiav tawm ntawm qhov nruab nrab ntawm qhov kev qhia qhov tseem ceeb. P<0.05 was="" considered="" to="" indicate="" a="" statistically="" significant="">
Cov txiaj ntsig
ELOVL2 tau hais ntau dhau hauv RCC. Qhov ntau ntawm xya ELOVL isozymes nyob rau hauv cov ntaub so ntswg sib xws thiab qog nqaij hlav tau kuaj thawj zaug hauv pawg neeg tam sim no, qhia tias ELOVL1, ELOVL2, ELOVL5 thiab ELOVL7 qhia qib tau nce siab hauv ccRCC cov ntaub so ntswg (Fig. 1A). Txhawm rau txheeb xyuas cov txiaj ntsig no, ELOVL isozyme gene qhia hauv cov ntaub so ntswg thiab cov qog nqaij hlav raug tshuaj xyuas siv TCGA database (KIRC cohort). Nws tau lees paub tias ELOVL2 mRNA kev qhia yog qhov siab tshaj plaws thiab nce siab hauv ccRCC cov ntaub so ntswg (Fig. 1B; P<0.0001). subsequently,="" elovl2="" mrna="" expression="" was="" investigated="" further="" among="" three="" major="" histological="" subtypes="" of="" rcc="" using="" tcga="" database="" (kirc,="" kirp="" and="" kich="" cohorts).="" this="" revealed="" that="" elovl2="" was="" overexpressed="" in="" the="" prcc="" and="" chrcc="" tissues;="" however,="" its="" expression="" in="" ccrcc="" tissues="" was="" the="" highest="" among="" the="" histological="" subtypes="" (fig.="" 1c).="" moreover,="" the="" elovl2="">
Kev qhia theem tau nce siab nyob rau hauv ACHN, 786-O thiab SK-RC-52 cell kab, txawm hais tias mRNA qhia qib hauv OS-RC-2 kab xov tooj ntawm tes tau qis dua li piv nrog cov hauv RPTEC cell kab (Fig. 1D) . Tom qab ntawd, kev hloov kho mob qog noj ntshav ntawm ELOVL2 gene qhia hauv ntau hom mob qog noj ntshav tau tshawb xyuas siv GENT2 database (Fig. 1E). Piv nrog rau cov ntaub so ntswg ib txwm, ELOVL2 qhia tau pom tias muaj ntau dua nyob rau hauvraum, prostate, mob ntsws thiab mob qog noj ntshav, thaum ELOVL2 qhia tau zoo ib yam hauv cov qog nqaij hlav zais zis lossis lub mis. Cov txiaj ntsig no tau qhia tias lub luag haujlwm ntawm ELOVL isozymes txawv ntawm hom mob qog noj ntshav thiab qhov ELOVL2 overexpression tuaj yeem cuam tshuam nrog cov carcinogenesis lossis kab mob kev loj hlob ntawm RCC, tshwj xeeb tshaj yog ccRCC.
ELOVL2 overexpression yog txuam nrog kev nce qib ntawm RCC. Kev koom tes ntawm cov noob qhia ntawm ELOVL2 thiab qog-node-metastasis (TNM) theem hauv KIRC pawg raug kuaj xyuas, txhawm rau txheeb xyuas qhov tseem ceeb ntawm kev kho mob ntawm ELOVL2 hauv ccRCC. ELOVL2 kev qhia tau siab dua hauv cov kab mob hauv zos thiab cov kab mob metastatic, txawm hais tias nws cov lus qhia tsis cuam tshuam nrog cov qog nqaij hlav qog nqaij hlav metastasis (Fig. 2A-C). Ua ke, ELOVL2 kev qhia tau nce raws li theem kho mob (Daim duab 2D). Tom qab ntawd, kev sib koom ua ke ntawm kev nthuav qhia ntawm ELOVL2 thiab kev kwv yees ntawm cov neeg mob RCC tau raug soj ntsuam, txhawm rau txhawm rau txheeb xyuas qhov cuam tshuam ntawm ccRCC. Raws li kev tshuaj xyuas Kaplan-Meier, nws tau nthuav tawm tias qhov kev qhia siab ntawm ELOVL2 tau cuam tshuam nrog kev pom tsis zoo hauv pawg neeg tam sim no (P=0.0015, Fig. 2E). Txhawm rau kom ntseeg tau cov txiaj ntsig no, kev sib koom ua ke ntawm kev qhia ntawm ELOVL2 thiab cov kev mob tshwm sim ntawm cov neeg mob nrog ccRCC tau tshuaj xyuas ntxiv hauv KIRC pawg thiab nws tau pom tias, zoo ib yam, qhov kev qhia siab ntawm ELOVL2 tau cuam tshuam nrog kev ua tsis zoo (P<0,01, fig.="" 2f).="" moreover,="" a="" high="" expression="" of="" elovl2="" was="" significantly="" associated="" with="" a="" poor="" prognosis="" of="" patients="" with="" prcc=""><0.0001, fig.="" 2g).="" however,="" this="" was="" not="" observed="" in="" patients="" with="" chrcc="" (fig.="" 2h).="" in="" total,="" the="" aforementioned="" results="" indicated="" that="" elovl2="" may="" mediate="" ccrcc="" and="" prcc="" disease="" progression,="" at="" least="">
ELOVL2 txhawb kev loj hlob ntawm lub raum mob qog noj ntshav. Txhawm rau ntsuas kev ua haujlwm ntawm ELOVL2 hauv kev loj hlob ntawm RCC hlwb, shRNA knockdown ntawm ELOVL2 hauv 786-O, ACHN thiab SK-RC-52 hlwb tau ua. Cov teebmeem ntawm txhua tus shRNA raug soj ntsuam siv RT-qPCR thiab qhov txo qis hauv ELOVL2 kev qhia tau lees paub (Daim duab 3A). MTT kev ntsuam xyuas tau ua tom qab kuaj xyuas cov teebmeem ntawm ELOVL2 knockdown ntawm cellular proliferation. Nws tau pom tias knockdown ntawm ELOVL2 inhibited kev loj hlob ntawm tag nrho cov hlwb piv nrog cov hlwb transfected nrog tswj shRNA (Fig. 3B).
Los ntawm qhov sib txawv, ELOVL2 overexpression tau raug ntxias hauv OS-RC-2 hlwb, kab xov tooj ntawm tes nrog qis qis qis ntawm ELOVL2. Kev ua tau zoo ntawm kev hloov pauv tau raug ntsuas los ntawm kev siv RT-qPCR thiab nthuav qhia ntau ntxiv ntawm ELOVL2 tau lees paub (Daim duab 3C). MTT kev soj ntsuam tau qhia tias qhov kev loj hlob ntawm ELOVL2-overexpressing hlwb tau nce siab, piv nrog pawg tswj hwm (Daim duab 3D), qhia tias ELOVL2 tuaj yeem yog tus txhawb nqa kev loj hlob ntawmlub raummob qog noj ntshav.
ELOVL2 ablation suppresses qog loj hlob hauv vivo, cov synthesis ntawm PUFAs, thiab tsim cov LDs hauvlub raummob qog noj ntshav. Txhawm rau kom paub meej ntxiv txog lub luag haujlwm ntawm ELOVL2 hauv RCC kev nce qib, ELOVL2-knockout ACHN cell kab tau tsim, los ntawm kev siv CRISPR/Cas9 system (sgELOVL2-1 thiab sgELOVL2-2) (Fig. S1). Hauv vitro, cellular proliferation tau txo qis los ntawm ELVOVL2 ablation hauv ACHN hlwb (Fig. S1). Qhov tseem ceeb tshaj, CRISPR/Cas9-mediated ablation ntawm ELOVL2 suppressed qog loj hlob thaum lub hlwb tau implanted subcutaneously rau nas (Fig. 4A). Qhov siab tshaj plaws ntawm cov qog subcutaneous xenograft nyob rau hauv ACHN / tswj thiab ACHN / sgELOVL2-2 nyob rau hnub ntawm kev txi yog 241 thiab 109 mm3, feem. Nco ntsoov, tag nrho cov qog xenograft nyob rau hauv pawg ACHN / sgELOVL2-1 spontaneously regressed ntawm 14 hnub tom qab hloov pauv thiab tsis tau kuaj pom thaum lub sij hawm extirpation. Lub Ki-67 Performance index kuj tau tshuaj xyuas nyob rau hauv cov qog xenograft kis nrog kev tswj lossis sgELOVL2 thiab nws tau pom tias ACHN / sgELOVL2-2 hlwb pom qhov ntsuas Ki-67 qis dua li ACHN / tswj hlwb (Fig. 4B thiab C). . Cov txiaj ntsig no txhawb ntxiv qhov ua tau tias ELOVL2 augment qog loj hlob hauv vivo. Raws li ELOVL2 nyob rau ntawm chromosome 6p24.2 thiab encodes rau ib qho endoplasmic reticulum transmembrane protein tswj qhov elongation ntawm C20-C24 PUFAs (7), tag nrho cov qib FA tau raug soj ntsuam hauv ACHN / sgControl thiab ACHN / sgELOVL2 cov hlwb siv GIC- (Fig. S2). Kev hloov pauv ntawm qib LC-PUFA tau pom hauv daim duab 4D. ELOVL2 yog ib qho tseem ceeb enzyme nyob rau hauv lub endogenous zus tau tej cov docosahexaenoic acid (DHA, C22:6 n-3) (28,29) thiab, raws li kev cia siab, DHA qib tau txo qis los ntawm ablation ntawm ELOVL2 nyob rau hauv lublub raummob qog noj ntshav. Ntxiv mus, lub
Tus nqi ntawm lwm hom LC-PUFAs, suav nrog arachidonic acid (AA, C20: 4 n-6), eicosapentaenoic acid (EPA) thiab docosapentaenoic acid (DPA), kuj txo qis. Ntawm qhov tod tes, kev tsim DPA tsis tas li hloov pauv hauv ACHN / sgELOVL2 hlwb. Tus nqi ntawm DPA hauv ACHN hlwb yog tsawg heev thiab tsis tau kuaj pom hauv ntau qhov qauv (Fig. S2). Txij li cov kev tshawb fawb yav dhau los tau pom tias ELOVL2 txhawb nqa cov LDs sib sau los ntawm kev tsim DHA (11,12), kev khaws cia ntawm LDs tau soj ntsuam ntxiv siv qhov nruab nrab lipid staining. Nco ntsoov, qhov ntau ntawm LDs hauv ACHN / sgELOVL2 hlwb tau txo qis dua piv nrog ACHN / sgControl hlwb (Fig. 4E-4G), qhia tias kev hloov pauv ntawm lipid metabolism los ntawm ELOVL2 overexpression tuaj yeem cuam tshuam rau RCC cell proliferation.
ELOVL2 ablation induces lub apoptosis ntawmlub raummob qog noj ntshav. Cov kev tshawb fawb yav dhau los tau qhia tias kev tsim cov LDs intracellular txhawb kev apoptosis nyob rau hauv kev nyuaj siab qog microenvironments (4). Yog li ntawd, nyob rau hauv txoj kev tshawb no, apoptosis raug soj ntsuam nyob rau hauv ACHN / sgControl los yog ACHN / sgELOVL2 hlwb thiab ib qho kev nce siab ntawm caspase 3/7 tau kuaj pom nyob rau hauv ACHN / sgELOVL2 hlwb (Fig. 5A). Ib yam li ntawd, ntau dua ntawm apoptotic ACHN/sgELOVL2 hlwb piv nrog ACHN/sgControl hlwb raug txheeb xyuas, raws li muaj pov thawj los ntawm Annexin V/PI staining (Fig. 5B). Raws li kev ntxhov siab ntawm cellular, intrinsic apoptosis (30) ua rau poob ntawm mitochondrial membrane peev xwm; Yog li, txoj kev tshawb fawb tam sim no tau soj ntsuam ntxiv ntawm mitochondrial transmembrane hluav taws xob muaj peev xwm ntawm ACHN / sgControl lossis ACHN / sgELOVL2 hlwb siv fluorescent JC-1. Qhov sib xyaw ua ke / monomer piv tau txo qis hauv ACHN / sgELOVL2 hlwb (Fig. 5C), qhia txog kev txhawb nqa ntawm mitochondrial transmembrane polarization thiab kev ua kom lub hauv paus apoptotic txoj hauv kev los ntawm ELOVL2 ablation. Ntau precisely, pro-apoptotic gene (BAX, BAK, PUMA thiab NOXA) qhia theem tau nce, thaum anti-apoptotic gene (BCL2 thiab MCL1) qhia qib tau txo qis vim ELOVL2 ablation (Fig. 5D). Cov txiaj ntsig no tau qhia tias ELOVL2 pab txhawb kom muaj sia nyob ntawm tes los ntawm kev cuam tshuam ntawm apoptosis ntawm lub raum mob qog noj ntshav.
Qhov degradation ntawm lipid cia muaj peev xwm hauv LD daim ntawv tuaj yeem ua rau lub cev qhuav dej ntawm ER homeostasis, ua rau cov lus teb tsis muaj protein ntau (UPR) thiab cellular apoptosis (4,5). Yog li ntawd, txhawm rau txhawb nqa qhov kev xav ntawm kev koom tes ntawm ELOVL2 qhia hauv ER homeostasis, ER tracker staining tau ua hauv ACHN / sgControl lossis ACHN / sgELOVL2 hlwb. Tom qab ER imaging (Fig. 5E) thiab quantification siv flow cytometry (Fig. 5F thiab G) qhia ER expansion nyob rau hauv lub ACHN / sgELOVL2 hlwb, vim ER kev nyuaj siab. Tsis tas li ntawd, ELOVL2 ablation txhawb nqa cov phosphorylation ntawm IRE1, ib qho activator ntawm UPR sensors (Fig. 5H), thaum qhia txogCHOP, uas ua lub luag haujlwm tseem ceeb hauv ER kev ntxhov siab-induced apoptosis, tau tswj hwm los ntawm ELOVL2 ablation (Fig. 5H). Ua ke, cov txiaj ntsig no tau pom tias ELOVL2 mediated-lipid metabolism suppresses cell apoptosis hauvlub raummob qog noj ntshav thiab qhia tias kev cuam tshuam ntawm ER homeostasis tuaj yeem yog lub peev xwm ntawm induction.
Kev sib tham
Txoj kev tshawb fawb tam sim no tau pom tias ELVOL2 tuaj yeem hloov pauv lipid metabolism thiab txhawb cov qog loj hlob los ntawm inhibition ntawm apoptosis ntawm lub raum mob qog noj ntshav. Ntxiv mus, nws tau pom tias ELOVL2 qhia hauv RCC cov ntaub so ntswg tau nce siab thiab qhov ntawd
Kev nthuav qhia ntau dua ntawm ELOVL2 tau cuam tshuam nrog kev pom tsis zoo ntawm cov neeg mob RCC. Muaj qee qhov kev tshawb fawb muaj nyob ntawm lub luag haujlwm ntawm ELOVL2 hauv kev mob qog noj ntshav (22,31,32) thiab lawv cov txiaj ntsig tau muaj teeb meem. Yooj yim thiab al (22) qhia tau tias ELOVL2 txhawb nqa LC-PUFA synthesis, txhawb nqa EGFR signaling thiab kev loj hlob ntawm glioblastoma qia hlwb. Los ntawm qhov sib txawv, Kang et al (31) tau tshaj tawm tias ELOVL2 ablation tuaj yeem txhawb kev tsiv teb tsaws ntawm tes thiab kev tsim cov kab mob qog noj ntshav ntawm lub mis thiab qhia tias qhov txo qis ntawm ELOVL2 qhia tau cuam tshuam nrog cov neeg mob qog noj ntshav tsis zoo. Tsis tas li ntawd, Ding li al (32) tau tshaj tawm tias ELOVL2 inhibits kev loj hlob ntawm cov hlwb neuroblastoma thiab cuam tshuam nrog cov txiaj ntsig zoo. Raws li qhov kev tshawb pom tsis sib haum xeeb tau tshaj tawm hauv cov kev tshawb fawb yav dhau los, ntau lub luag haujlwm rau ELOVL2 hauv kev mob qog noj ntshav tau tshwm sim, uas qhia meej meej txawv ntawm hom mob qog noj ntshav.
Kev hloov pauv lipid metabolism ua rau muaj kev cuam tshuam rau kev mob qog noj ntshav, ib qho txiaj ntsig tau pom nyob rau hauv cov txiaj ntsig ntawm txoj kev tshawb fawb tam sim no, ua raws li kev tshawb fawb qhia txog lub luag haujlwm ntawm ELOVL2 ua tus thawj tswj hwm ntawm cov txheej txheem elongation ntawm LC-PUFAs thiab cov enzyme tseem ceeb hauv DHA biosynthesis ( 7). Hauv kev tshawb fawb tam sim no, nws tau pom tias endogenous LC-PUFAs,
suav nrog AA thiab DHA, tau txo qis vim ELOVL2 ablation hauv lub raum mob qog noj ntshav. Hauv kev hais txog qhov no, nws tau raug tshaj tawm yav dhau los tias inhibition ntawm AA txoj hauv kev los ntawm lipoxygenase (LOX) inhibitors induces apoptosis thiab txo cov kev muaj peev xwm ntawmlub raummob qog noj ntshav hauv vitro (33,34). Txawm hais tias ELOVL2 overexpression cov txiaj ntsig ntawm txoj kev tshawb fawb tam sim no yog ua raws li cov kev tshawb pom no, nws tau raug tshaj tawm yav dhau los, txawm li cas los xij, DHA cov thawj coj tuaj yeem cuam tshuam kev loj hlob thiab kev cuam tshuam ntawm lub raum mob qog noj ntshav hauv vitro (35,36). Txawm li cas los xij, LC-PUFAs paub tias muaj qhov sib txawv thiab sib txawv ntawm kev mob qog noj ntshav. Yog li, kev hloov pauv ntawm qhov sib npaug ntawm ntau yam ntawm LC-PUFAs, vim qhov inhibition ntawm ELOVL2 tuaj yeem cuam tshuam nrog cov phenotypes sib txawv pom hauv ntau hom mob qog noj ntshav. Cov kev tshawb fawb yav dhau los tau pom tias LDs tau nce los ntawm kev tsim tawm endogenous ntawm DHA los ntawm ELOVL2 hauv cov nas (11,12). Ib yam li ntawd, nws tau tshaj tawm tias ELOVL2 ablation tuaj yeem cuam tshuam kev tsim DHA thiab LDs hauv lub raum mob qog noj ntshav, qhia tias ELOVL2 overexpression tuaj yeem txhawb nqa LD ntau lawm los ntawm endogenous DHA ntau lawm hauv RCC. Cov qog nqaij hlav cancer feem ntau pom muaj nyob rau hauv ib puag ncig hnyav (macro thiab micro), suav nrog hypoxia thiab kev noj zaub mov tsis txaus, tseem tseem, loj hlob sai nyob rau hauv cov kev ntxhov siab hnyav. Kev ua haujlwm ntawm LDs nyob rau hauv cov xwm txheej ntawm kev ntxhov siab ntawm tes, xws li kev saib xyuas lub zog thiab redox homeostasis, kev tswj hwm ntawm autophagy, kev saib xyuas ntawm ER homeostasis, thiab kev tiv thaiv lipotoxicity, tau ua pov thawj (4).
Ackerman li al (6) tau qhia tias LDs tiv thaiv kev sib sau ntawm cov tshuaj lom, cov lipids saturated thaum lub sij hawm hypoxia los ntawm buffering ntawm cellular lipid saturation los ntawm kev sib pauv ntawm TG-tus neeg nyob hauv unsaturated FAs hauv ccRCC. Cellular lipotoxicity vim saturated FAs (SFA), suav nrog palmitate (C16:0), tuaj yeem ua rau apoptosis, ua rau mob qog noj ntshav (37,38). Tsis ntev los no, ELOVL2 tau raug xaiv los ua ib qho tseem ceeb ntawm kev muaj sia nyob enzyme, pab tiv thaiv glucolipotoxicity-induced-cell apoptosis (39). Ntawm kev ceeb toom, ELOVL2 / DHA axis-hloov lipid partitioning ua rau tsis muaj tshuaj lom siv SFA palmitate los ntawm kev nyiam nws thauj mus rau mitochondria rau FA oxidation (FAO) los ntawm CPT1-dependent mechanism. Tsis tas li ntawd, Balaban et al tau qhia tias C4-2B prostate qog nqaij hlav qog noj ntshav thiab MCF-7 cov qog nqaij hlav cancer mis raug tiv thaiv los ntawm palmitate-induced apoptosis los ntawm mitochondrial FAO (37,38). Thaum lub sij hawm palmitate-induced lipotoxicity, cellular theem ntawm anti-apoptotic proteins, xws li BCL-2 los yog MCL-1, tau raug tshaj tawm tias yuav txo qis (40,41) thaum theem ntawm pro-apoptotic proteins, suav nrog PUMA lossis NOXA, tau tshaj tawm. nce (42,43). Nws tau tshwm sim hauv txoj kev tshawb fawb tam sim no tias ELOVL2 ablation tuaj yeem txhawb nqalub raummob qog noj ntshav apoptosis los ntawm kev hloov pauv ntawm pro- thiab anti-apoptotic genes zoo ib yam. Cov kev tshawb pom no qhia tias ELOVL2 tuaj yeem tiv thaiv cov hlwb tiv thaiv lipotoxicity-driven apoptosis los txhawb cov qog loj hlob hauv RCC.
Tsis tas li ntawd, nws tau pom tias ELOVL2 ablation hauv RCC tuaj yeem txhawb ER kev ntxhov siab thiab CHOP upregulation, downregulating BCL-2 thiab MCL-1, thaum upregulating BAK thiab BAX, ntawm txoj kev mitochondria-dependent (44). Tseeb tiag, cov txiaj ntsig ntawm txoj kev tshawb fawb tam sim no tau pom tias cov noob pro- thiab anti-apoptotic tau zoo ib yam hloov pauv los ntawm ELOVL2 ablation. Raws li qhov kev tshawb pom hauv kev tshawb fawb los ntawm Qui et al (5), uas tau tshaj tawm tias HIF2 / PLIN2-dependent LDs txhawb kev tiv thaiv ER kev nyuaj siab thiab cell ciaj sia nyob hauv ccRCC, cov kev tshawb pom no tau txhawb nqa ER kev ntxhov siab txhawb nqa cellular apoptosis los ntawm ELOVL2. ablation hauvlub raummob qog noj ntshav, txo LDs thiab tshem tawm cov cellular tsis tiv thaiv cov lipids lom.
Hauv kev xaus, qhov kev tshawb fawb tam sim no tau nthuav tawm thawj zaug, rau qhov zoo tshaj plaws ntawm peb txoj kev paub, tias ELOVL2 txhawb cov qog loj hlob los ntawm inhibition ntawm apoptosis ntawm qhov elongation ntawm PUFAs, tsawg kawg yog ib feem los ntawm kev tswj ER homeostasis hauvlub raummob qog noj ntshav. Ua ke, nws tau pom tias ELOVL2-induced LDs tuaj yeem ua rau mob qog noj ntshav vim muaj ntau yam kev tiv thaiv tiv thaiv cellular stress hauv RCC. Ntxiv mus, nws tau pom zoo tias ELOVL2 yuav yog lub hom phiaj kho mob zoo rau cov neeg mob RCC.