( )2017 57 5JournalofNanchangUniversity(MedicalSciences)2017,Vol.57No.5 11 LY294002, (, 330006) : LY294002 PI3K/AKT 0 10 15 20 25 30μmol L -1 LY294002(LY294002 ) A549,PBS CCK8 LY294002 A549 ; Westernblot 2 A549 Beclin-1 LC3B(Ⅱ Ⅰ) PI3K p-akt AKT ; WoundhealingTranswelinvasion 2A549 LY294002 A549, 24h,24h IC5019.348μmol L -1 LY294002 Beclin-1 PI3K p-akt LC3B(Ⅱ/Ⅰ) (P<0.05) LY294002 PI3K/AKT A549 : LY294002 ; A549;PI3K/AKT ; ; : R392 ;R734.2 : A : 2095-4727 (2017)05-0011-05 DOI:10.13764/j.cnki.ncdm.2017.05.003 :2017-05-31 LY294002SuppressesAutophagy,Proliferation,Migration andinvasionoflungcancercelsinvitro : (20151BAB215026) : (1990 ),,, :,,E-mail:zgm7977@163.com WU Hong-shan,LIUZhi-li (Departmentof Orthopaedics,the FirstAffiliated Hospitalof Nanchang University,Nanchang330006,China) ABSTRACT:Objective ToinvestigatewhetherLY294002inhibitstheautophagy,proliferation, migrationandinvasionoflungcancercelsthroughpi3k/aktsignalingpathway.methods Hu- manlungcancera549celsweretreated withly294002atconcentrationsof10,15,20,25and 30μmol L -1.Controlcelsweretreated withpbs.theproliferationofcelswasdetectedby CCK8assay.TheexpressionofBeclin-1,LC3B(Ⅱ,Ⅰ),PI3K,p-AKTandAKTinA549celswas measuredby Western blotanalysis.the migrationandinvasion ofcels weredetermined by WoundhealingandTranswelinvasionassays,respectively.Results LY294002treatmentinhibi- tedtheproliferationofa549celsinadose-dependentmanner,andthe24-houric 50 was19.348 μmol L -1.Comparedwithcontrolgroup,theexpressionofBeclin-1,PI3Kandp-AKT,ratioof LC3B Ⅱ/Ⅰ,cel migrationandinvasionsignificantlydecreasedin LY294002treatmentgroup (P<0.05).Conclusion LY294002treatmentsuppressestheautophagy,proliferation,migration andinvasionofa549celsthroughinhibitingpi3k/aktsignalingpathway. KEY WORDS:LY294002;humanlungcancerA549cels;PI3K/AKTsignalingpathway; autophagy;migration;invasion
12 (autophagy),imagej,,, 3 [1-4] (self-eating) 1.2.4 Transwelinvasion, 20μmol L -1 LY294002 A549 [5] PI3K/AKT 24h,PBS, [6-8],, 1 10 5 ml -1 ( ) 600μL wel -1 10%, LY294002 A549, PI3K/AKT, 200μL wel -1 37 24h,, 800μL wel -1 95%, 30 min,,800μl 1 wel -1 PBS3,800μL 1.1 wel -1 0.1% 30min, A549, ; ddh 2O, DMEM( ) ( ), 3 (TransGen ) Celcultureinsert(8.0μm,ImageJ, PETtrck-etched membrane, Corning ) 3 Westernblot MarkerPVDF ( 1.2.5 Westernblot Formentas ) (Abcam ) ( ) LY294002( abmole) 1.2 1.2.1 ( )201710,57 5 RIPA, 10%SDS-PAGE,5% 1h, ( PI3KIgG,1 2000; p-aktigg,1 DMEM 15% ( 2000; AKTIgG,1 2000; Beclin-1 100kU L -1 ) A549, IgG,1 1000; LC3BIgG,1 3000; 37 5%CO 2 β-actinigg,1 2500)4,TBST 1.2.2 CelCountingKit-8(CCK8) PVDF3, (,1 5000) 2h,TBST 3, 10min,ECL,, 3, 5 10 3 ml -1 1.3 96, 100μL, 12h SPSS19.0 x±s,, 0 10 15 20 25 30 μmol L -1,P<0.05 LY294002,CCK8,, 450nm 2 (OD), 3 2.1 LY294002A549 1.2.3 Woundhealing CCK8 LY294002, A549, 24h (1) 5 10 5 ml -1, 6 24hIC,,20μmol L -1 50 19.348μmol L -1 20μmol L -1 LY294002 LY294002 A549 (PBS 2.2 LY294002A549 ),24h 100μL, Transwel,LY294002,PBS 2 (95±7) -1, ; DMEM (130±16) -1 (P<0.05), 2 24h,
伍宏山等 ＬＹ２９４００２ 抑制肺癌细胞自噬 增殖 侵袭和迁徙的体外研究 １３ ２ ３ ＬＹ２９４００２ 对 Ａ５４９ 细胞迁移能力的影响 Ｗｕｎｄｈ ａ ｎｇ 实验结果 显 示 ＬＹ２９４００２ 处 理 组细胞 ２４ｈ 迁 徙 率 为 ２２ ４±２ ６ 显 著 低 于 对 照组 ３９ １±３ ２ Ｐ ０ ０５ 见图 ３ ２ ４ ＬＹ２９４００２ 对 Ａ５４９ 细胞自噬相关蛋白 Ｂ ｎ １ 及 ＬＣ３Ｂ Ⅱ Ⅰ 的影响 Ｗ ｒｎｂ 检测结果显示 ＬＹ２９４００２ 处理的 Ａ５４９ 细胞中 Ｂ ｎ １ 蛋 白 表 达 和 ＬＣ３Ｂ Ⅱ Ⅰ 比 率比对照组显著降低 均 Ｐ ０ ０５ 见图 ４ ＬＹ２９４００２ 浓度 Ｌ １ μｍ 图 １ ＬＹ２９４００２ 对 Ａ５４９ 细胞增殖的影响 Ａ Ａ５４９ 细胞侵袭数量 Ｂ Ａ５４９ 细胞 ２４ｈ 穿膜细胞数 图 ２ Ｔｒ ａｎｗ 侵袭实验 Ａ Ａ５４９ 细胞迁移数 Ｂ Ａ５４９ 细胞 ２４ｈ 细胞迁移率 图 ３ 划痕实验检测细胞的迁徙能力 Ａ Ｗ ｒ ｎｂ 电泳图 Ｂ 各蛋白与 β ａ ｎ 蛋白灰度值之比 图 ４ Ｗ ｒ ｎｂ 检测蛋白的表达
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