SNAT2介导蛋氨酸对牛乳腺上皮细胞增殖与自噬的调节作用
(东北农业大学 黑龙江省高校农业生物功能基因重点实验室,哈尔滨 150030)
摘要:钠离子依赖性氨基酸转运体2(the sodium-dependent neutral amino acid transporter 2,SNAT2/SLC38A2)是一种氨基酸转运蛋白,广泛分布于多种细胞中。但SNAT2是否介导氨基酸调节细胞自噬尚未见报道。本研究利用CASY细胞计数和Western blotting技术测定SNAT2过表达和siRNA干扰后奶牛乳腺上皮细胞(bovine mammary epithelial cells,BMECs)细胞增殖情况。Western blotting检测SNAT2对自噬标志蛋白LC3-I /II表达量的影响,并利用免疫荧光检测细胞自噬斑点(LC3-II)变化。结果显示,SNAT2过表达时,p-PI3K、p-mTOR、Cyclin D1表达量增加,反之,p-PI3K、p-mTOR、Cyclin D1表达量下降。SNAT2抑制时,LC3-II表达量增加,免疫荧光检测自噬斑点增多。添加自噬增强剂海藻糖(trehalose,Tre)和蛋氨酸(methionine,Met)后,Met 组同Met + Tre 组比较,p-mTOR表达量增加,LC3-II表达量降低;添加Tre和Met并抑制SNAT2时,p-mTOR表达量下降,LC3-II表达量增多。以上结果表明SNAT2可介导Met通过调控PI3K-mTOR/Cyclin D1信号通路调节BMECs的细胞增殖与自噬。
关键词:SNAT2;BMECs;mTOR;增殖;自噬
SNAT2 mediates Met regulation of proliferation and autophagy of bovine mammary epithelial cells
Hao Qi, Chunyu Meng, Xuejun GAO, Zhen Zhen
(Key Laboratory of Agricultural Biological Functional Gene of Heilongjiang Province, Northeast Agricultural University, Harbin 150030, China)
Abstract:The sodium-dependent neutral amino acid transporter 2 (sodium-dependent neutral amino acid transporter 2, SNAT2 / SLC38A2) is an amino acid transporter and is widely distributed in a variety of cells. However, it is unknown whether SNAT2 mediates the regulatory effects of amino acids on autophagy. In this study, we determined the proliferation of bovine mammary epithelial cells (BMECs) after cells were transfected with SNAT2 overexpression vector or siRNA. Western blotting was used to detect the effects of SNAT2 on the expression of autophagic biomarker LC3-II, and the change of autophagic spots (LC3-II) were detected by immunofluorescence. Our data reveal that when SNAT2 was overexpressed, the expression levels of p-PI3K, p-mTOR and cyclin D1 were increased, whereas the expression levels of p-PI3K, p-mTOR and Cyclin D1 were decreased when SNAT2 was inhibited. When SNAT2 was inhibited, the expression of LC3-II was increased, and the autophagy spots was increased. Cells were treated with the autophagy enhancer trehalose dihydrate (Tre), and methionine (Met) were further added, the expression of p-mTOR was increased and the expression of LC3-II was decreased when cells were further treated with Met. Cells treated with Tre and Met were further transfected with SNAT2 siRNA, the expression of p-mTOR was decreased, and the expression of LC3-II was increased when SNAT2 was knockdown, compared with the Tre and Met group. Thus, our work elucidates that SNAT2 mediates the regulatory effects of Met on cell proliferation and autophagy of BMECs by regulating PI3K-mTOR/Cyclin D1 signaling pathway.
Key word:SNAT2; BMECs; mTOR; autophagy;proliferation
钠离子依赖性中性氨基酸转运体2(the sodium-dependent neutral amino acid transporter 2,SNAT2/SLC38A2)属于SLC38基因家族System A亚型,由504个氨基酸组成,分子质量约56kDa。SNAT2在运转中性氨基酸(亮氨酸、异亮氨酸、蛋氨酸等)的同时会同向转运一个Na十,其运转过程受Na十的浓度及pH影响[1]。
泌乳是由各种激素作用于已发育的乳腺而引起的,乳腺分泌乳汁的过程称为泌乳。随着泌乳生物学的广泛研究,已经对泌乳调控有了一定的认识,但仍还有部分机理尚不清楚。奶牛乳腺上皮细胞(bovine mammary epithelial cells,BMECs)在生理条件下分泌乳的同时会进行细胞增殖。已有研究报道,在哺乳期间,0.3%的BMECs会在24小时内增殖[2]。这种增殖作用主要是为了实现哺乳期结束后细胞的自我更新。细胞周期蛋白D1(Cyclin D1)是细胞周期调控的关键蛋白,可促进细胞增殖。研究发现,Gankyrin可通过PI3K/HIF-1α/Cyclin D1途径促进卵巢癌细胞增殖[3]。
自噬(autophagy)是一种由溶酶体介导的细胞内蛋白程序化降解的过程[4]。在奶牛乳腺中,自噬在泌乳周期的各个阶段都能观察到,在干奶期最盛,自噬体和自噬溶酶体数量此时出现最多。氨基酸不仅作为蛋白质合成的底物,也是调节细胞新陈代谢的关键因子。在缺乏氨基酸等营养物质的条件下,会引起细胞自噬,且不同的氨基酸效应不同。例如:添加丙氨酸或脯氨酸可抑制IEC-18细胞中的自噬;添加谷氨酰胺可抑制小鼠胚胎成纤维细胞中的自噬;添加亮氨酸、酪氨酸、脯氨酸、蛋氨酸可抑制离体灌流肝脏中的自噬[5-7]。
哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)是蛋白质和脂质合成、细胞增殖和自噬的中心调节器[8]。已有研究表明氨基酸可以通过mTOR信号传导途径影响BMECs中的乳蛋白、乳脂肪合成和细胞增殖[9, 10],蛋氨酸通过PI3K-mTOR信号通路途径调节BMECs细胞自噬[11]。SNAT2在奶牛乳腺上皮细胞中高度表达,并介导氨基酸调控mTOR信号通路正向调节BMECs乳蛋白和乳脂肪合成[12]。但SNAT2在BMECs中是否介导氨基酸调节BMECs增殖和自噬,尚未见报道。本试验通过Western blotting技术、CASY细胞活力分析技术结合免疫荧光技术检测SNAT2调节BMECs增殖和自噬及可能的分子机理,为利用氨基酸调控乳腺泌乳提供基础理论依据。
1 材料与方法
1.1 主要试剂与仪器
DMEM/F12粉末、胎牛血清(FBS)和Opti-MEM®(1×)Reduced Serum Medium购于Gibco公司(美国);Lipofectamine®3000 Reagent(L3000001)购于Invitrogen公司(美国);蛋氨酸(methionine,Met)和海藻糖(trehalose,Tre)购于Sigma公司(美国);Cytokeratin 18(1:100, sc-51582)、β-actin (1:200, sc-47778)等多克隆抗体都购买于Santa Cruz Biotechnology公司(美国);mTOR(1:1000, #2983)、p-mTOR(1:1000,Ser2448,#5536)等多克隆抗体都购买于Cell Signaling Technology公司(美国);SNAT2(1:500,bs-12125R)Cyclin D1(1:500,bs-0623R)、PI3K(1:500,bs-2067R)、p-PI3K(1:500,Tyr317,bs-5570R)等多克隆抗体都购买于博奥森公司(北京);LC3B(1:1000,ab51520)抗体购于Abcam Technology公司(美国);兔抗山羊IgG(ZB-2306)和山羊抗小鼠IgG(ZB-2305)标记二抗都购买于中杉金桥公司(北京);SNAT2 siRNA干扰片段和阴性对照RNA均购买于苏州吉玛公司(中国)。
CO2培养箱购于Thermo公司(美国),倒置荧光生物显微镜购自于MOTIC CHINA GROUP CO.,LTD公司(中国);激光扫描共聚焦显微镜购于LEICA公司(Leica TCS SP2,德国)。CASY TT细胞计数分析仪购买于SCHARFE SYSTEM公司(德国)
1.2 原代细胞的培养
实验用原代BMECs细胞培养方法参照史琳琳等[13]、陈建辉[14]介绍的组织块法。乳腺细胞取自黑龙江省哈尔滨市连发屠宰场,取泌乳期中国荷斯坦奶牛乳腺组织,用组织块贴壁法培养BMECs。将组织浸泡于75%乙醇5 min后,用D-Hank’s溶液清洗数次,氟康睉清洗数次后,将其剪成约为1mm3的正方体小块并接种于铺好鼠尾胶原的细胞培养瓶中,在37℃、5%的CO2培养箱中倒置培养4 h,向培养瓶中加入含有15%的FBS、10×双抗、5×两性的DMEM/F12培养液4 ml。7 d左右,将组织块附近爬出的细胞进行传代,经过4-5次传代,得到纯化的BMECs。
1.3 免疫荧光鉴定细胞纯度
采用免疫荧光法(immunofluorescence,IF)进行BMECs的角蛋白18(Cytokeratin 18,CK18)、酪蛋白(β-casein)鉴定。将BMECs接种于放入灭菌盖玻片的六孔板中,待细胞长至50%-60%,PBST溶液清洗数次后用4%多聚甲醛固定细胞,再用含5%BSA的PBST封闭液37°C孵育1.5 h。弃废液,加入封闭液稀释的一抗,4℃过夜后清洗数次,加入封闭液稀释的FITC标记的荧光二抗,37°C避光孵育1 h,用封闭液稀释的DAPI进行染核后抗荧光淬灭剂封片,激光共聚焦显微镜下观察。
1.4 N-flag-SNAT2载体转染与CASY细胞计数
构建过表达N-flag-SNAT2载体,参照孟春雨介绍的方法[12]。取生长状态良好纯化后的BMECs,以适当的密度接种于六孔板中,当细胞生长至70%-80%时,通过脂质体Lipofectamine 3000转染试剂进行转染,用120 μl 无血清的 Opti-MEM® I分别稀释5 μl Lipofectamine® 3000和5 μg N-flag-SNAT2载体,静置5min后,将2种稀释液缓慢混合,孵育15min后加至六孔板中,48 h后收样。
试验分三组:对照组、空载体组、转染N-flag-SNAT2载体过表达组。将三组试验样品制成单细胞悬液,取100 μl加入含有10ml缓冲液的CASY杯。混合颠倒CASY杯后,置于CASY外部电极处,进行测量。每次测量体积为200 μl,测量3次。
1.5 小RNA干扰与CASY细胞计数
SNAT2基因干扰片段购于苏州吉码公司,从三条siRNA中筛选出最有效的一条干扰片段(上游:GCAGCAGCUAUAGUUCCAACATT,下游:UGUUGGAACUAUAGCUGCUGCTT)[12]。转染SNAT2 siRNA干扰片段,试验分三组:对照组、NC组、转染SNAT2 siRNA干扰片段组。转染方法同上,24 h收样,并同上述方法操作,进行CASY细胞计数。
收集经过不同处理的细胞样品,参照李冬等[15]介绍的方法将等量的蛋白质样品(每个泳道10-30μg蛋白)在8-10%SDS-聚丙烯酰胺凝胶中电泳,然后将目的蛋白湿转到硝酸纤维素膜上,一抗、二抗孵育后进行Western blotting检测。
1.7 免疫荧光检测细胞自噬
取生长状态良好纯化后的BMECs,以适当的密度接种于六孔板中。试验分三组:NC对照组、干扰组及添加Rapamycin组。参照上述转染方法,转染NC和SNAT2 siRNA,第三组添加8 nM 雷帕霉素(Rapamycin),处理24 h,同时每组需共转染pCMV-C-EGFP-LC3B质粒。pCMV-C-EGFP-LC3B载体的构建方法见Yu M[11]。参照上述方法免疫荧光检测自噬体,并利用Western blotting检测相关蛋白表达。
取生长状态良好纯化后的BMECs,以适当的密度接种于六孔板中。试验分为四组:NC对照组、添加Tre组、添加Tre和Met组与添加Tre、Met及SNAT2干扰片段组。每组需共转染pCMV-C-EGFP-LC3B质粒。待转染细胞培养至70%-80%后,用无血清培养液继续培养12 h,于细胞中添加0.6 mM Met、5 μM Tre继续培养12 h,共处理36-48 h后,常规方法收样或制片,参照上述方法免疫荧光检测自噬体,并利用Western blotting检测相关蛋白。
1.8 统计分析
所得数据以平均值±标准差表示。各组数据之间采用GraphPad Prism 6.0软件进行方差分析,所得结果中*表示P<0.05为差异显著,**表示P<0.01为差异极显著。
2 结果与分析
2.1 SNAT2过表达对BMECs细胞增殖的影响
纯化后的BMECs的CK18及酪蛋白表达呈阳性,如图1A所示,证明获得高纯度的BMECs。细胞转染N-flag-SNAT2载体48 h后,Western blotting检测SNAT2、p-mTOR、mTOR、Cyclin D1、PI3K、p-PI3K、β-actin的表达量。由图1B-1E可知,与其他两组相比,转染N-flag-SNAT2载体过表达组mTOR、PI3K的蛋白表达量不变,p-mTOR、Cyclin D1、p-PI3K的蛋白表达量明显升高。由图1F可知,与空载体组相比,转染N-flag-SNAT2载体过表达组的细胞数明显增多。结果表明,转染N-flag-SNAT2过表达载体可以促进BMECs细胞增殖。
A:奶牛乳腺上皮细胞的CK18和β-casein免疫荧光鉴定(×800):蓝色为DAPI染色的细胞核,绿色荧光为CK18、β-casein; B:Western blotting检测SNAT2过表达后相关信号分子蛋白的表达(EV,空载体;SNAT2 OE,SNAT2 overexpression); C:SNAT2过表达后p-PI3K/PI3K蛋白灰度值扫描结果; D:SNAT2过表达后Cyclin D1与SNAT2蛋白灰度值扫描结果; E:SNAT2过表达后p-mTOR/mTOR蛋白灰度值扫描结果; F:CASY细胞计数检测SNAT2过表达的细胞数
A: Identification of bovine mammary epithelial cell by immunofluorescence observation of CK18 and β-casein (×800):Blue means nuclei stained with DAPI , green immunofluorescence means CK18 or β-casein; B: Western blotting was used to detect the expression of related signal molecules after SNAT2 overexpression (SNAT2 OE).Cells transfected with empty vector (EV) was used as a control; C: Gray value scan results of p-PI3K/PI3K protein after SNAT2 overexpression; D: Cyclin D1 and SNAT2 protein gray value scan results after SNAT2 overexpression; E: Gray value scan results of p-mTOR/mTOR protein after SNAT2 overexpression; F: CASY cell counting to detect the number of cells overexpressing SNAT2
图1 SNAT2过表达对乳腺上皮细胞增殖的影响
Fig.1 Effect of SNAT2 overexpression on the proliferation of bovine mammary epithelial cells
2.2 SNAT2抑制对BMECs细胞增殖的影响
细胞转染SNAT2 siRNA干扰片段24 h后,Western blotting检测SNAT2、p-mTOR、 mTOR、Cyclin D1、PI3K、p-PI3K、β-actin的表达,结果见图2。由图2A-2D可知,与其他两组相比,转染SNAT2 siRNA干扰片段组mTOR、PI3K的蛋白表达量不变,p-mTOR、Cyclin D1、p-PI3K的蛋白表达量明显下降。由图2E可知,与NC组相比,转染SNAT2 siRNA干扰片段的细胞数明显下降。结果表明,转染SNAT2 siRNA干扰片段可以抑制BMECs细胞增殖。
A:Western blotting检测SNAT2抑制后相关信号分子蛋白的表达(NC,阴性对照;SNAT2 KD,SNAT2 knocked);B:SNAT2抑制后p-PI3K/PI3K蛋白灰度值扫描结果;C:SNAT2抑制后Cyclin D1与SNAT2蛋白灰度值扫描结果;D:SNAT2抑制后p-mTOR/mTOR蛋白灰度值扫描结果; E:CASY细胞计数检测SNAT2抑制的细胞数
A: Western blotting was used to detect the expression of related signal molecules after SNAT2 was knocked down by siRNA .Cells transfected with negative control RNA (NC) was used as a control; B: Gray value scan results of p-PI3K/PI3K protein after SNAT2 inhibition; C: Cyclin D1 and SNAT2 protein gray value scan results after SNAT2 inhibition; D: Gray value scan results of p-mTOR/mTOR protein after SNAT2 inhibition; E: CASY cell counting to detect the number of cells inhibited by SNAT2
Fig.2 Effect of SNAT2 inhibition on the proliferation of bovine mammary epithelial cells
2.3 SNAT2抑制对BMECs自噬的影响
将质粒pCMV-C-EGFP-LC3B与筛选出的SNAT2最佳干扰片段共转染至细胞中,36 h后收样,Western blotting检测抑制SNAT2后,细胞自噬相关信号分子蛋白的表达变化。如图3A-3C可知,SNAT2抑制后,p-mTOR蛋白表达量减少,自噬标记性蛋白LC3-II表达量增多。添加Rapamycin的细胞作为阳性对照组,实验结果说明,干扰SNAT2可以促进奶牛乳腺上皮细胞发生自噬。
转染分组同上,36 h后制片,在激光共聚焦显微镜下观察结果如图3D和3E所示。NC组未发生自噬,胞浆内出现弥漫性绿光,干扰SNAT2组与添加Rapamycin组,胞浆内均出现绿色聚集性荧光斑点,与Western blotting结果一致。实验结果进一步说明,SNAT2是 BMECs自噬的重要抑制蛋白。
A:Western blotting检测SNAT2抑制后细胞自噬相关信号分子蛋白的表达,rapamycin处理组作为阳性对照;B:SNAT2抑制后LC3-II与SNAT2蛋白灰度值扫描结果;C:SNAT2抑制后p-mTOR/mTOR蛋白灰度值扫描结果D:免疫荧光染色法检测SNAT2抑制后细胞自噬斑点的发生;E:统计分析细胞LC3荧光点,3次独立实验,t检验,*P<0.05,**P<0.01
A: Detection of the indicated protein levels related to autophagy by Western blotting analysis after SNAT2 inhibition, rapamycin-treated cells were used as a positive control; B: LC3-II and SNAT2 protein gray value scan results after SNAT2 inhibition; C: Gray value scan results of p-mTOR/mTOR protein after SNAT2 inhibition; D: Detection of autophagy spots by immunofluorescence assay; E: Statistical analysis of cell LC3 fluorescence points, 3 independent experiments, T test, * P <0.05, ** P <0.01
Fig.3 Effects of SNAT2 inhibition on autophagy of bovine mammary epithelial cells
2.4 SNAT2介导蛋氨酸对BMECs自噬的影响
将质粒pCMV-C-EGFP-LC3B与筛选出SNAT2最佳干扰片段共转染至细胞中,并按上述分组实验,Western blotting检测各实验组中和细胞自噬相关的信号分子蛋白表达变化。如图4A-4D可知,添加自噬增强剂Tre可以促进细胞发生自噬;当同时添加Tre与Met后,自噬标记性蛋白LC3-II表达量降低,说明Met能够有效抑制Tre引起的自噬;添加Tre与Met并抑制SNAT2表达,自噬标记性蛋白LC3-II表达量增多,实验结果表明Met通过SNAT2抑制自噬,干扰SNAT2对Met抑制自噬的作用具有阻断作用。
分组转染方法同上,36-48 h后制片,激光共聚焦观察检测LC3-II自噬斑点。如图4E和4F所示,Blank组未发生自噬,胞内呈弥漫性绿光,添加自噬增强剂Tre组胞内出现绿色聚集性荧光斑点,Tre作为自噬增强剂可增强细胞自噬;同时添加Tre与Met后,胞浆内绿色自噬斑点减少,说明Met能够抑制Tre引起的自噬;添加Tre与Met并抑制SNAT2表达,胞浆内自噬斑点增多,进一步表明Met通过SNAT2抑制细胞自噬。
A:Western blotting检测SNAT2介导蛋氨酸对细胞自噬相关基因蛋白表达的影响;B:p-mTOR/mTOR蛋白灰度值扫描结果;C:SNAT2蛋白灰度值扫描结果;D:LC3-II蛋白灰度值扫描结果;E:免疫荧光染色法检测SNAT2介导Met对细胞自噬斑点的影响;F:统计分析细胞LC3荧光点,3次独立实验,t检验,*P<0.05,**P<0.01
A: Western blotting analysis of the indicated protein levels related to autophagy in cells treated with Tre and Met and transfected with SNAT2 siRNA; B: Gray value scan results of p-mTOR/mTOR protein levels in (A); C: Gray value scan results of SNAT2 protein levels in (A); D: Gray value scan results of LC3-II protein levels in (A); E: Detection of autophagy spots by immunofluorescence assay; F: Statistical analysis of cell LC3 fluorescence points, 3 independent experiments, T test, * P <0.05, ** P <0.01
Fig.4 Effect of SNAT2-mediated Met on autophagy of bovine mammary epithelial cells
3 讨论
SNAT2是一种在哺乳动物组织中广泛表达的氨基酸转运蛋白,它在谷氨酸-谷氨酰胺循环、肝脏糖异生和其他生物途径中起着关键作用。研究发现,SNAT2可通过mTOR信号通路调控细胞生长与增殖[16]。本实验结果表明,SNAT2可正向调节PI3K/mTOR信号转导通路。SNAT2的过表达显著增加p-PI3K水平和p-mTOR水平,而SNAT2 siRNA的敲低具有相反的效果。这些数据表明SNAT2是PI3K/mTOR途径的关键上游信号分子,与先前的报道一致[17]。实验结果显示SNAT2增加Cyclin D1的蛋白质水平,并可调节体外培养的BMECs增殖。已知,Cyclin D1可调控细胞周期的G1期至S期,是细胞增殖的关键调节因子。先前的报道显示PI3K信号的激活可以促进Cyclin D1的合成[18],SNAT2可介导Met调节PI3K信号通路[19]。以上的研究结果表明,SNAT2通过PI3K-Cyclin D1途径调节细胞增殖。
细胞自噬是一个复杂多步骤的过程。海藻糖可通过线粒体途径诱导细胞自噬从而消除肝脏脂肪变性及保护细胞免受氧化刺激[4, 20]。在本实验中,Tre作为自噬增强剂促进细胞自噬。调控细胞自噬的分子机制主要包括:mTOR信号通路对细胞自噬的调控、泛素样蛋白系统对细胞自噬的调控、P53信号通路对细胞自噬的调控及miRNA对细胞自噬的调控等。其中,mTOR信号通路对细胞自噬的调控研究的较为广泛。研究发现,牛磺酸通过ROS / AMPK / mTOR信号通路来调节细胞自噬;GAS5通过激活mTOR信号通路抑制过度自噬来减弱胶质瘤细胞对顺铂的抗性[21, 22]。通过Western blotting和免疫荧光实验,发现SNAT2的抑制导致p-mTOR蛋白水平降低,且通过所检测到的与对照组相比LC3-II / LC3-I比率和EGFP-LC3-II斑点数的增加,表明自噬在SNAT2抑制或雷帕霉素处理的细胞中被激活。这些数据说明,SNAT2通过PI3K-mTOR信号通路抑制细胞自噬。
氨基酸可以通过钠偶联中性氨基酸转运蛋白(SNAT或SLC38)家族进入细胞,增加雷帕霉素复合物1(mTORC1)的活性,以促进细胞生长和代谢,且氨基酸的缺乏可引起强烈的细胞自噬现象。研究发现,氨基酸和激素通过mTOR途径调节乳腺中乳合成和BMECs的增殖[23, 24];SLCTA5可介导谷氨酰胺通过mTOR信号通路调节胚胎成纤维细胞自噬;维生素C调节氨基酸通过溶酶体途径调控小鼠肝脏细胞自噬[25, 26];IFN-γ介导精氨酸通过GCN2/eIF2a信号途径调节BMECs细胞自噬[27]。SNAT2作为重要的氨基酸转运体可以通过调节氨基酸间接调控mTOR信号通路来影响细胞的生长情况。研究发现,SNAT2抑制导致亮氨酸的消耗并减少肌肉细胞中PI3K/AKT/mTOR信号传导途径的激活[28-30]。本研究发现,添加Met后,SNAT2可促进BMECs增殖,并可以消除海藻糖引起的细胞自噬。这些结果表明,SNAT2介导Met通过PI3K-mTOR/Cyclin D1信号通路调节BMECs的增殖和自噬。
4 结论
SNAT2介导Met激活PI3K-mTOR/Cyclin D1信号通路调控BMECs增殖与自噬。
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