巨噬细胞移动抑制因子对细胞增殖、迁移、粘附的机理研究
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摘要
动脉粥样硬化(atherosclerosis,AS)是动脉血管壁受损后的慢性炎症性疾病,尽管采取了积极的血管干预、控制血压以及调脂等治疗手段,目前仍是许多国家发病和死亡的主要原因。动脉粥样硬化是一种多因子疾病,涉及基因、环境、代谢等诸多因素,但最近人们发现动脉硬化和冠心病等心血管疾病与慢性牙周感染、幽门螺旋杆菌或衣原体肺炎感染有关。来自动物和人类的资料表明,炎症不仅在动脉粥样硬化发生、发展和演变过程中起着重要作用,而且在不同的临床过程中也与炎症的各个阶段有关。如在一些患有冠状动脉、颈动脉或外周动脉硬化的患者中看到,炎症导致血管局部产生中性粒细胞和单核细胞,动脉粥样硬化帽出现活化的巨噬细胞,进而导致硬化的斑块破裂脱落,从而导致心脑血管的堵塞。动脉粥样硬化以动脉内皮细胞功能障碍为起点,进展到众多炎性细胞因子参与,并伴有细胞增生的慢性炎症。经过脂纹细胞形成期、中间病变期、成熟斑块期,最终演进至不稳定粥样斑块破裂、出血及局部血栓的迅速形成,上述病理过程如发生在冠状动脉中即导致急性冠脉综合征(acute coronarysyndromes,ACS)。其中,单核细胞粘在损伤部位进行聚集、粘附、迁移和穿过血管内皮,进而活化、分化成为巨噬细胞;斑块内血管平滑肌细胞(vascularsmooth muscle cell,VSMC)趋化、迁移至血管内膜及局部过度增殖,在冠脉AS的病理演进及其介入治疗后冠脉再狭窄的病变中起着关键作用。目前的研究表明,细胞周期作为各种因素刺激细胞增殖的最后信号通路,在上述病理改变的VSMC迁移和过度增殖中占据中心地位。
更重要的是,炎症在血管的再狭窄过程中也起着重要作用,如血管成形术后管腔损失的严重性与炎症细胞活化有关。最近,在动物模型中,通过抗炎措施已成功限制了损伤后新生内皮的增生。如对兔进行球囊损伤和支架置入后,通过抗炎手段就可降低单核细胞活性,阻断白细胞黏附,减少新生内皮生成。
慢性炎症是动脉粥样硬化的标志,促炎因子在动脉硬化进展过程中起关键作用。在动脉壁不规则部位或受血液动力学因素影响,如血流紊乱的分叉部位或血流增快部位易产生斑块,而斑块也可聚积在曾受感染或受免疫侵袭部位。
细胞因子与许多细胞(如淋巴细胞、单核细胞、巨噬细胞、内皮细胞、平滑肌细胞和心肌细胞等)的功能活化有关。这些细胞表达分泌一系列细胞因子如巨噬细胞移动抑制因子(macrophage migration inhibitory factor,MIF)、白细胞介素1(interleukin-1,IL-1)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、细胞粘附分子1(intercellular adhesion molecule-1,ICAM-1)、血管细胞粘附分子1(vascular adhesion molecule-1,VCAM-1)、P选择素等,而这些因子在调节心脏功能、心血管重塑和功能恢复、血栓和动脉粥样硬化的形成和发展等方面具有重要作用。
巨噬细胞移动抑制因子(MIF)是一种在急慢性炎症和癌症有关的新的炎症标记物。MIF能广泛的调节多种炎性反应,在动脉粥样硬化斑块进展中起重要作用。许多人体细胞可以分泌这种物质,这些细胞主要有:血管平滑肌细胞,活性巨噬细胞,分化晚期的巨噬细胞,经过炎症和细胞外组织改造过的活体组织上发现的巨噬细胞的亚型,像粥样硬化斑块上的巨噬细胞等。MIF能提高趋化性,细胞附着性和VSMCs的迁移及小血管分支的形成。
动脉粥样硬化时血液中的单核细胞在损伤部位进行聚集、粘附、迁移和穿过血管内皮,进而活化、分化成为巨噬细胞。而巨噬细胞吞噬脂质成为巨噬—泡沫细胞,形成脂纹。然而目前对单核细胞移动、粘附迁移和血管平滑肌迁移,并在局部增殖的确切分子机制仍不清楚。MIF由动脉粥样硬化斑块中的多种细胞产生,在早期斑块的发生和斑块进展中起重要作用。因此,寻找和鉴定能控制动脉粥样硬化的靶蛋白对治疗冠心病来说非常重要。本研究通过RNA干扰技术特异性沉默MIF蛋白的表达并观察MIF对细胞增殖、迁移、粘附中所起的作用并探讨其作用机制。
本研究分为以下二部分:第一部分
siRNA靶向MIF重组逆转录病毒载体构建及稳定表达细
胞株的筛选
1.目的:
构建并鉴定MIF特异性siRNA重组逆转录病毒载体,将其导入phoenix细胞中,筛选出分泌MIF-siRNA病毒的包装细胞,进而鉴定该病毒上清可抑制MIF蛋白的表达。
2.方法:
2.1 pSuper.retroRNAi的设计及合成
根据GenBank提供的MIF基因cDNA序列,通过www.oligoengine.com网站提供的工具软件设计,所选择MIF的靶点序列分别为:
pSRP/MIF-siRNA1
5'-CTATTACGACATGAACGCG-3'
pSRP/MIF-siRNA2
5'-CAACTCCACCTTCGCCTAA-3
2.2逆转录病毒载体的构建
将合成的正、反义链经过变性、复性后形成双链MIF基因siRNA,采用双链定向亚克隆入逆转录病毒载体SUPER.retro,转化大肠杆菌菌株E.coli DH5α,氨苄青霉素(100mg/L)筛选出阳性克隆,提取质粒。
2.3重组质粒MIFsiRNA的鉴定
重组质粒用EcoRⅠ与HindⅢ酶进行双酶切,酶切产物行1%琼脂糖凝胶电泳。阳性克隆分别命名为pSRP/MIF1、pSRP/MIF2,纯化后送到上海博尚生物公司测序鉴定。
2.4重组质粒转染包装病毒细胞株
将包装病毒细胞phoenix接种到6孔板中,分别使用pSRP(空载体pSuper.retro)、pSRP/MIF1、pSRP/MIF2质粒各4μg,脂质体Lipofectamin2000 10μl/孔转染phoenix。转染后嘌呤霉素筛选3-4周获得阳性克隆。细胞株命名为phoenix-pSRP、phoenix-pSRP/MIF1 siRNA、phoenix-pSRP/MIF2 siRNA。
2.5重组逆转录病毒感染HeLa细胞株
将筛选获得的phoenix阳性克隆培养至90%融合后换成无嘌呤霉素的完全培养基,24小时后收集细胞上清,滤液-70℃保存。HeLa细胞株接种到6孔板中,加入1ml phoenix细胞上清滤液并加入2μg/ml的聚凝胺,更换含嘌呤霉素的完全培养基筛选培养,约20天筛选完成。所产生的细胞株分别命名为HeLa-pSRP、HeLa-pSRP/MIF1 siRNA、HeLa-pSRP/MIF2 siRNA。
2.6 Western-blot检测MIF蛋白的沉默
将筛选后的HeLa细胞株接种到6孔板中,培养生长至90%时,收集细胞。用细胞裂解液裂解,15%SDS聚丙烯酰胺凝胶电泳,将蛋白质转移到PVDF膜,用含5%脱脂奶粉的TTBS(0.1 mol/LTris-HCL,pH7.5,0.2%NaCl;0.05%Tween20)37℃封闭1-2h,分别加入兔抗人MIF多克隆抗体(1:1000稀释),或小鼠抗人GAPDH单克隆抗体(1:1000稀释)4℃孵育过夜。TTBS漂洗5min×3次;兔抗辣根过氧化物酶标记二抗(1:5000稀释)37℃孵育1h;TTBS漂洗5min×3次;SuperSignal Weste Femto(Pierce公司)敏感曝光试剂盒曝光底片显影。
3.结果
3.1酶切鉴定重组质粒
逆转录病毒载体pSuper.retro由本室改造:构建后重组质粒BglⅡ酶切位点应该消失,阳性克隆经EcoRⅠ与HindⅢ酶切后可产生280 bp大小的片段。阴性克隆经EcoRⅠ与HindⅢ酶切后则产生230bp、680bp两条带。本实验中重组质粒经EcoRⅠ与HindⅢ酶切后显示出约280bp左右的条带,表明载体构建成功。将重组质粒pSRP/MIF1、pSRP/MIF2扩增纯化后送上海博尚生物有限公司测序,测序结果均与目的序列完全相同,表明构建载体成功。
3.2 Western-blot检测MIF蛋白的沉默
通过western-blot检测MIF蛋白的沉默效果。结果显示:细胞株HeLa-pSRP/MIF1 siRNA,HeLa-pSRP/MIF2 siRNA细胞株内源性MIF均被不同程度沉默,而空病毒载体感染的细胞株HeLa-pSRP内源性MIF水平没有变化。说明细胞株的筛选是成功的。并且本研究之后选取的MIF沉默的细胞株均为HeLa-pSRP/MIF1 siRNA标记为HeLa-pSRP/MIF。第二部分
MIF在细胞生长、迁移、粘附中的作用研究
1.目的:探讨MIF对细胞生长、迁移、粘附功能的影响和机制。
2.方法:
2.1细胞培养
将HeLa-pSRP和HeLa-pSRP/MIF用含1μg/ml嘌呤霉素的完全培养基37℃5%CO2条件下培养,细胞生长至90%左右更换为完全培养基。将细胞消化至12孔板,起始细胞数为0.1×10~6/孔,连续计数6天。同时观察HeLa-pSRP和HeLa-pSRP/MIF siRNA细胞表型变化,显微镜200倍下拍摄相片。
2.2流式细胞仪检测HeLa-pSRP、HeLa-pSRP/MIF细胞周期及凋亡
将HeLa-pSRP、HeLa-pSRP/MIF细胞分三组铺6cm培养皿,待细胞生长至80%时,一组将HeLa-pSRP、HeLa-pSRP/MIF细胞分别用0.25%胰蛋白酶和0.02%EDTA(1:1)消化细胞,制备单个细胞悬液,70%冷乙醇固定,再用1%TritonX-100、0.01%RNase和0.05%PI处理,通过300目尼龙网备检检测细胞周期。另两组加入TNF-α10ng/ml和CHX10μg/ml处理12小时后,其中一组提取蛋白,另一组将HeLa-pSRP、HeLa-pSRP/MIF细胞分别消化细胞,制备单个细胞悬液,70%冷乙醇固定,0.01%RNase和0.05%PI处理,通过300目尼龙网备检。用ProfileⅡ型流式细胞仪,在488nm激发波长下测定细胞DNA,用Multicycle软件分析细胞周期分布情况。细胞早期凋亡数目通过annexin V-PI检测试剂盒(BD Biosciences)检测。激发波长Ex＝488 nm;发射波长Em＝530 nm。AnnexinV-FITC的绿色荧光通过FITC通道(FL1)检测;PI红色荧光通过PI通道FL3检测。提取蛋白后检测Bax、Bcl-2表达情况。
2.3细胞衰老试验
HeLa-pSRP、HeLa-pSRP/MIF消化后将1×10~5HeLa-pSRP、HeLa-pSRP/MIF细胞悬液分别进行细胞爬片,待细胞生长至80%左右进行实验。0.2%戊二醛固定5分钟后,加入X-gal混合液2ml/孔,放入温箱孵12小时,镜检,阳性细胞为出现蓝绿色。
2.4免疫荧光检测
将HeLa-pSRP、HeLa-pSRP/MIF细胞分别消化成细胞悬液加已加盖玻片的6cm培养皿中,待细胞生长至70%后,去除培养液,PBS清洗3次,加入4%多聚甲醛溶液固定20分钟,后使用0.1%Triton X-100-PBS浸泡10分钟PBS/0.1%Triton X-100渗透10min,PBS洗涤3次,每次5min;5%BSA封闭30min后加一抗:p53抗体(1:100稀释)4℃孵育过夜。PBS清洗3次,每次5分钟,5%BSA再封闭30 min;加二抗:加入Alexa Fluor(?)555 rabbit anti-mouse IgG(H+L)(1:1000稀释)Molecular Probe公司,室温孵育45分钟。PBS洗涤5次,每次5min(避光操作),加入DAPI(1:1000稀释Roche公司)室温染核10分钟。PBS清洗1次,(避光操作)封片,在载玻片上滴一滴水溶性封片剂,然后将盖玻片(培养有细胞的一面朝向水溶性封片剂)从一侧轻轻粘在载玻片上,激光共聚焦显微镜扫描和照相。
2.5细胞迁移实验
在Transwell下室的DMEM培养液中加入10%血清,在Transwell上室分别滴加1×10~5 HeLa-pSRP、HeLa-pSRP/MIF细胞悬液。将上室置于下室之中,在37℃温箱孵育。16h后取出上室,用棉签将残留在上室内表面上的细胞拭去。上室经PBS洗涤后,用4%多聚甲醛固定迁移至上室外表面上的细胞,细胞用含0.1%龙胆紫的20%乙醇染色20分钟,用清水洗3遍,显微镜下(200×)计算迁移细胞数,结果取5个视野细胞计数的平均值。
2.6划痕试验
HeLa-pSRP、HeLa-pSRP/MIF细胞铺6孔板中,当单细胞层生长至95%时,使用10μl枪头对细胞进行划痕。分别于0,24小时,36小时,48小时对细胞进行拍照,比较细胞划痕愈合的速度。
2.7细胞粘附性实验
按照CHEMICON公司Cytomatrix cell adhension strips说明书进行。室温200μl/孔PBS洗涤2遍,胰酶消化HeLa-pSRP、HeLa-pSRP/MIF细胞,制成单细胞悬液,计数,调整细胞浓度为1×10~5/ml。每组分别设3个复孔,每孔加入100μm细胞悬液,37℃孵育1h后取出96孔培养板,用PBS洗涤2-3遍,去除未粘附细胞。含0.2%龙胆紫的10%乙醇染色5分钟,用PBS洗涤3-5遍,每孔加入100μl等体积混匀的0.1MNaH_2PO_4,PH4.5和50%乙醇以去除残余染色。酶标仪在波长570nm读数。
2.8 Western-blot检测
将筛选后的HeLa细胞株接种到6孔板中,培养至每孔0.1×10~6时,收集细胞。用适量细胞裂解液(150mmol/L Tris-HCL,0.2%叠氮钠,0.1%SDS,100μg/mlPMSF,1μg/ml Aprotinin,1%NP40,0.5%去氧胆酸钠,150mmol/L NaCl)裂解,上样,15%SDS聚丙烯酰胺凝胶电泳,将蛋白质转移到PVDF膜,用含5%脱脂奶粉的TTBS(0.1mol/L Tris-HCL,pH7.5,0.2%NaCl;0.05%Tween20)室温封闭1.2小时,分别加入兔抗人Cyclin D1、Bcl-2、Bax、FAK、AKT、ERK1/2、和ICAM-1多克隆抗体(1:1000稀释),鼠抗人p53、p21单克隆抗体1:800鲜?或小鼠抗人GAPDH单克隆抗体(1:1000稀释)4℃孵育过夜。TTBS漂洗5min×3次;兔抗辣根过氧化物酶标记二抗(1:5000稀释)37℃孵育1h;TTBS漂洗5min×3次;SuperSignal Weste Femto(Pierce公司)敏感曝光试剂盒曝光底片显影。
3.统计方法
使用SPSS17.0统计软件,两组数据比较采用两独立样本t检验,结果用平均值±标准差((?)±s)表示,以P＜0.05为有统计学差异。
4.结果
4.1细胞生长变化情况
沉默MIF后的HeLa-pSRP/MIF与对照组HeLa-pSuper相比生长速度明显减慢。达到半数生长期的时间:HeLa pSRP需要30小时,而HeLa-pSRP/MIF需要64小时。沉默MIF后的HeLa-pSRP/MIF在形态上成铺路石样分布,而空病毒对照组细胞多有触突,而且有聚集成团的趋势。
4.2沉默MIF后HeLa细胞生长停留在G0/G1期
流式细胞仪显示HeLa-pSRP/MIF G0/G1期比例为51.887±4.241%,与对照组HeLa-pSRP相比G0/G1期比例为28.453±4.230%,两组比较有统计学差异(P＜0.05)。沉默MIF后HeLa细胞生长停留在G0/G1期。
4.3沉默MIF后HeLa细胞Cyclin D1表达受抑制,p53、p21表达下降
沉默MIF细胞周期出现变化,因此我们检测实验组和对照组之间影响细胞周期的蛋白有无改变。Western结果显示:沉默MIF后HeLa细胞Cyclin D1表达受抑制,并且p53、p21水平较HeLa-pSRP明显下降。
4.4沉默MIF后HeLa细胞迁移活性下降,细胞的粘附性下降
在10%小牛血清趋化作用下,迁移至下室的细胞数目对照组pSRP为86.333±16.166个/视野,pSRP/MIF组为23±10.148个/视野。两组比较有显著性差异(P＜0.01)。另外,划痕实验显示,HeLa-pSRP在48小时已使划痕愈合,而HeLa-pSRP/MIF siRNA迁移能力明显减弱,至48小时仍未能使划痕愈合。胰酶消化HeLa-pSRP、HeLa-pSRP/MIF细胞,使用CHEMICON公司Cytomatrix celladhension strips进行细胞粘附实验。酶标仪在波长570nm读数,OD值结果:HeLa-pSRP 0.509±0.0890,HeLa-pSRP/MIF 0.180±0.031,两组比较有显著性差异(P＜0.01)。
4.5沉默MIF后细胞凋亡减少
TNF-α和放线菌酮(CHX)10μg/ml诱导细胞凋亡后,流式细胞仪检测HeLa-pSRP细胞凋亡比例为54.833±6.443%,HeLa-pSRP/MIF细胞凋亡比例为4.457±1.154%,两组比较有显著性差异(P＜0.05)。western-blot结果显示MIF蛋白的沉默后使HeLa细胞表达凋亡相关蛋白Bax减少而Bcl-2轻度升高。
4.6沉默MIF后细胞衰老减少
使用X-gal实验结果显示,沉默MIF后细胞衰老减少。
4.7 MIF蛋白的沉默后使HeLa细胞Src、FAK、ERKl/2、AKT、ICAM-1表达减少。论文小结
1.成功构建了针对人MIF基因的siRNA重组逆转录病毒载体pSRP/MIF1、pSRP/MIF2。获得稳定抑制MIF表达的细胞株HeLa-pSRP/MIF和表达空载体的对照组细胞HeLa-pSRP。
2.沉默MIF后的HeLa细胞对TNF-α和放线菌酮诱导的凋亡减少,并且沉默MIF后细胞衰老减少。沉默MIF后细胞阻滞在G0/G1期,细胞生长减慢,迁移能力、粘附功能减弱。
3.本研究显示MIF通过影响cyclinD1、p53、p21表达维持细胞正常生长周期;MIF通过促进p53、p21的表达促进细胞衰老。另外,MIF通过增加Bax表达,促进细胞凋亡。MIF能促进细胞Src、FAK、ICAM-1、AKT、ERK1/2的表达,可能是MIF维持细胞的生长、移动、粘附特性的机制之一。本文的创新点:通过HeLa细胞模型证实了MIF在细胞的生长、增殖、黏附、迁移过程中扮演重要作用及可能的分子机制。结合课题组早期的工作,这一创新性结果进一步揭示了MIF可能通过调节多种与细胞粘附、迁移有关的蛋白表达,促进动脉粥样硬化的发生、发展。
The highly conserved and archetypical yet atypical cytokine macrophage migration inhibitory factor(MIF) fulfills pleiotropic immune functions in many acute and chronic inflammatory diseases.Recent evidence has emerged from both expression and functional studies to implicate MIF in various aspects of cardiovascular disease.MIF plays important role in the inflammatory pathogenesis of atherosclerosis and its consequences,namely unstable plaque formation, remodeling after arterial injury,aneurysm formation,myocardial infarction,or ischemia-reperfusion injury.
The functional data are reconciled with recent progress in the identification of heptahelical(CXC chemokine) receptors for MIF,its prototypic role as their noncanonical ligand,and its signal transduction profile operative in atherogenic and inflammatory recruitment of mononuclear cells and in the oxidative damage and apoptosis of cardiomyocytes.Its unique features and functions clearly distinguish MIF from other cytokines implicated in atherogenesis and make it a prime target for achieving therapeutic regression of atherosclerosis.The potential of targeting or exploiting MIF for therapeutic strategies or as a diagnostic marker in the management of cardiovascular diseases or disorders is scrutinized.
In this study,we found inactivation of MIF in HeLa cells resulted in suppression of migration ability.We further show that MIF is required for cells motility through activates Src and FAK signaling and provided a new target for improving the treatment efficiency of atherosclerosis through inactivation of the MIF gene.
EXPERIMENTAL PROCEDURES
Vector construction.
Among different MIF target sequences examined,the 19-nucleotide gene-specific sequence spanning from nucleotides 427 nt to 445 nt downstream of the gene transcription start site was selected to suppress MIF gene expression.After BLAST analysis,to ensure that there was no significant sequence homology with other human genes,the selected sequence was inserted into a BglⅡ/HindⅢ-cut pSuper puro vector to generate the pSuper-MIF vector.All constructs were confirmed by DNA sequence analysis.
Cell culture,treatments,and transfections.
HeLa cells were grown at 37℃in a 5%CO_2 atmosphere in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10%FCS,2 mmol/L glutamine,and antibiotics.phoenix packaging cells and HeLa cells stably transfected with pSuper.puro and pSuper-MIF were maintained in DMEM supplemented with 10% FCS in the presence of puromycin(2 mg/ml).All transfections were done using LipofectAMINE 2000 reagent(Invitrogen) according to the manufacturer's protocols. Transfect phoenix packaging cells were done according to the manufacturer's protocols to produce ecotropic retroviral supernatants.48 hours posttransfection, select infected cells with puromycin(1-3μg/ml).After 20 days in selective medium, three pools,referred as phoenix-pSuper and phoenix- pSuper-MIF1,pSuper-MIF2, were isolated respectaly.
Generation of stable MIF-silenced lines.
To obtain HeLa cell lines stably expressing siRNAs,the ecotropic retroviral supernatants produced by phoenix packaging cells were collected and filter the tissue culture medium through a 0.45μm filter,and use the viral supernatant for infection of HeLa cell lines after addition of 4μg/ml polybrene.Infected HeLa cell lines for at least 6 hr and allow to recover for 24 hr with fresh medium.Select infected cells with puromycin(1-3μg/ml).After 15 days in selective medium,three pools,referred as HeLa-pSuper.puro,HeLa-pSuper-MIF1 SiRNAand HeLa-pSuper-MIF2 SiRNA were isolated.The HeLa- pSuper-MIF1 SiRNA was subjected to further cloning procedures by the limiting dilution method.The clones presenting a＞95%reduction in MIF protein levels compared with control.All stable cell lines obtained were cultured in DMEM supplemented with 2mg/ml puromycin.The puromycin selective pressure was removed 24 hours before experimental procedures.HeLa-pSuper.puro and HeLa-pSuper-MIF cells proliferation was measured by plating equal cell numbers onto six-well dishes in duplicate,followed by direct counting of trypsinized cells using a Coulter counter.Each growth curve represents at least two independent experiments.
Cell Cycle Analysis
HeLa-pSuper-MIF and HeLa-pSuper were treated with TNF-a and CHX10μg/ml for 12 hours.Trypsinized HeLa-pSuper.and HeLa-pSuper-MIF were washed with phosphate-buffered saline and fixed in 70%ethanol on ice for at least 24 h.The cells were washed with phosphate-buffered saline containing 1%FBS,resuspended in phosphate-buffered saline containing 1%FBS and 25μg/ml RNase A,and incubated for 30 min at 37℃.Prior to flow cytometry,100μl of propidium iodide solution(50μg/ml propidium iodide and 0.1%sodium citrate) was added to the cell suspension and incubated for 1 h on ice.The staining was assessed using the FACSC alibur flow cytometer and CellQuest software following calibration using DNA QCTM beads(BD Biosciences).Data were analyzed using ModFit software(BD Biosciences). SA-β-gal staining
Cells were stained for SA-β-gal activity(Senescence-associatedβ-galactosidase) as described by Dimri.Briefly,1×10~4 cells were seeded in 6-well plate for 24h and were washed twice with phosphate-buffered saline,fixed with 2%formaldehyde and 0.2%glutaraldehyde in PBS,and washed twice in PBS.Cells were stained at 37℃for 12h in X-gal staining solution(1mg/ml X-gal,40mmol/L citric acid/sodium phosphate(pH 6.0),5 mmol/L potassium ferrocyanide,150 mmol/L NaCl,2 mmol/L MgCl_2).Cells were examined under bright field illumination(Leica)
Adhesion Assay
Coat 96-well-plate with FN at 37℃for 1 hr Leave some wells uncoated as negative control.Wash with washing buffer for 2 times.Block plates with blocking buffer at 37℃in CO_2 incubator for 45-60 minutes.Wash with washing buffer.Chill the plates on ice.Count cell to 0.4×10~6/ml.Add 50μl cells in each well. Incubate in CO_2 incubator at 37 C for 30 minutes.Shake the plate at 2000 rpm for 10-15 seconds.Wash with washing buffer 2-3 times.Fix with 4%paraformaldehyde. Incubate at RT for 10-15 minutes.Wash with washing buffer.Stain with Crystal Violet for 10 minutes.Wash with water.Turn the plates upside down.Let the plates dry up completely.Add 2%SDS.Incubate at RT for 30 min.Read plate at 570nm.
Migration Assays
For migration assays,modified Boyden chambers(Millicell-PCF,8-μm pore size;Millipore,Bedford,MA) were placed in a 24-well plate and coated with 10μg/ml rat tail collagen(Roche Applied Science) for 16 h at 37℃.After removal of collagen and washing with PBS,migration medium(DMEM with 0.5%bovine serum albumin)was added to lower chamber in 0.4 ml.HeLa pSuper or HeLa-pSuper-MIF RNAi cells were added to the upper compartment(0.2×10~6 in 0.3 ml of migration medium).The plates were incubated at 37℃for 16 h for migration. The cells were removed from the upper membrane surface with a cotton tip applicator and washed with PBS,and cells on the lower membrane surface were fixed with 4% formaldehyde.The cells were then stained(0.1%Crystal Violet in 20%ethanol and enumerated by counting four(200x) fields/chamber).
Wound closure assay
A wound was induced on the confluent monolayer cells by scraping a gap using a micropipette tip and the speed of wound closure was monitored every 24 hours. Photographs were taken under 100×magnifications using phase-contrast microscopy immediately after wound incision and at later time points.
Confocal microscopy
Cells grown on coverslips were fixed with 4%paraformaldehyde and permeabilized in 0.1%Triton X-100-PBS for 10 minutes.After blocking with 0.2% bovine serum albumin,cells were incubated with rabbit anti-p53 monoclonal antibody followed by decoration with fluorescein-conjugated anti-mouse IgG.Images were acquired on a Leica confocal microscope.
Western blot analysis
Equal amounts of protein(35μg/sample) from whole-cell extracts were separated by SDS-PAGE and blotted to PVDF membranes.After blocking with 5%skim milk solution,membranes were incubated with rabbit polyclonal anti-MIF, anti-cyclinD1,anti-Bcl-2,anti-Bax,anti-FAK,anti-p53,anti-p21,anti-ICAM-1, anti-Src,anti-AKT and anti-ERK1/2(Santa Cruz Biotechnology,Santa Cruz,USA) followed by decoration with peroxidase-labeled anti-rabbit or anti-mouse IgG, respectively(Super Signal detection kit,Pierce).
Statistics
The results are expressed as the means±S.E.The data were analyzed by one-way or two-way analysis of variance using SPSS17.0 statistical program.P values <0.05 were considered significant.
RESULTS
Growth Properties of HeLa cells knockdown MIF
To test the hypothesis that MIF contributes in HeLa cell growth,we established stable knockdown of MIF cell line HeLa-pSuper-MIF using SiRNA technique, which displayed a significant decrease MIF at protein level compared with vector control.HeLa cells were derived from and assayed in several experimental settings. Under normal growth conditions,HeLa-pSuper-MIF proliferated slightly more slowly than the control HeLa-pSuper.Note morphologic changes induced by downregulation of MIF in HeLa cells.
Silencing of MIF in HeLa Cells Results in the cell stay in G0/G1 phase and decrease the cyelin D1 expression
To further confirm the MIF expression on HeLa cell growth,we then studied if downregulation of MIF could lead to any changes in the cell cycle distribution and the relative protein such as cyclin D1.Cell cycle analysis showed that silencing of MIF in HeLa cells results in the cell stay in G0/G1 phase and decrease the cyclin D1 expression.
Down-regulation of MIF leads to suppression of p53 and p21
As p53 play an important role in cell survivle,we then studied if there were any change in p53 and p21 expression,p53 and p21 were detectable in low level in HeLa-pSuper-MIF cell,whereas p53 protein level was high in HeLa-pSuper cell.In addition,we also used immunoflourecent staining to test p53 expression.These results show that MIF can affect the proappotosis gene p53.
Silencing of MIF does not change the TNF-αinduced apoptosis pathway
HeLa-pSuper-MIF and HeLa-pSuper were treated with TNF-αand CHX 10μg/ml for 12 hours.We found that decreased Bax protein expression was found in the HeLa-pSuper-MIF.These results indicate that downregulation of MIF in HeLa cells has led to increased sensitivity to TNF-αthrough activation of induced apoptosis. Silencing of MIF in HeLa Cells Results in decrease cell senescence
At the normal grown condition,silencing of MIF in HeLa cells results in decrease cell senescence,while the HeLa-pSuper cells showed a senescent phenotype that included flattening of cells and expression of senescence-associatedβ-galaetosidase(SA-βgal)
MIF is required for cell migration potential of HeLa Cell
To further study the association between MIF expression and metastatic ability of HeLa cells,we then tested the migration ability of the HeLa cells expressing.We analyzed the effect of MIF on the migratory capability of the HeLa cells by using several function assays.First,wound-healing assay showed that,24h after a wound was made on the monolayer of cells,the control cells extensively migrated into the denuded area,but this migratory capacity was significantly compromised by depletion of MIF in HeLa cells.Then,migration assay showed that compared with control cells,diminished MIF expression also reduced HeLa cell migration through a membrane by two-chamber assay.Overall,these data suggest that MIF expression is essential for the migratory behavior of HeLa cell.
MIF-induced migration activity is associated with the activation of FAK signaling
Cell migration is a complex event that depends on the coordinated remodeling of the actin cytoskeleton as well as the regulated assembly and turnover of focal adhesions.The FAK acts as a protein-protein adapter critical to the formation of focal adhesions and mediates adhesion and growth factor-dependent signals into the cells. Tyrosine phosphorylation of FAK is one of the key signaling events during cell motility.Consistent with our observation above,HeLa cells with MIF knockdown showed decreased FAK expression.Consequently,Src,one of the upstream FAK, also showed reduced in MIF knockdown cells.These data suggest that MIF-induced migration activity is associated with the activation of FAK signaling through Src. Since ICAM-1 is the most important adhesion molecule involved in the extravasation of leukocytes into the surrounding tissue-it has been demonstrated to be both required as well as sufficient for the process of extravasation-most studies have been focused on the expression of ICAM-1.We also tested the expression of ICAM-1 in HeLa-pSuper-MIF and HeLa-pSuper.The data shows that compared with control cells,diminished MIF expression reduced ICAM-1,ERK1/2 and AKT in HeLa cells.
Innovation of this study In this study,we used siRNA to knockdown MIF for evaluation of its function in HeLa cell line,which showed that MIF plays an important role in proliferation,migration and adhesion in vitro.We also investigated the potential molecular mechanism of the phenomenon.Together with prior studies of our group,these results strongly indicate that MIF may possibly promote the atherosclerotic lesions development through regulating the proteins that controlling cell adhesion,migration and proliferation.The present data suggest that MIF may represent an important new target for human atherosclerosis therapeutics.
更重要的是,炎症在血管的再狭窄过程中也起着重要作用,如血管成形术后管腔损失的严重性与炎症细胞活化有关。最近,在动物模型中,通过抗炎措施已成功限制了损伤后新生内皮的增生。如对兔进行球囊损伤和支架置入后,通过抗炎手段就可降低单核细胞活性,阻断白细胞黏附,减少新生内皮生成。
慢性炎症是动脉粥样硬化的标志,促炎因子在动脉硬化进展过程中起关键作用。在动脉壁不规则部位或受血液动力学因素影响,如血流紊乱的分叉部位或血流增快部位易产生斑块,而斑块也可聚积在曾受感染或受免疫侵袭部位。
细胞因子与许多细胞(如淋巴细胞、单核细胞、巨噬细胞、内皮细胞、平滑肌细胞和心肌细胞等)的功能活化有关。这些细胞表达分泌一系列细胞因子如巨噬细胞移动抑制因子(macrophage migration inhibitory factor,MIF)、白细胞介素1(interleukin-1,IL-1)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、细胞粘附分子1(intercellular adhesion molecule-1,ICAM-1)、血管细胞粘附分子1(vascular adhesion molecule-1,VCAM-1)、P选择素等,而这些因子在调节心脏功能、心血管重塑和功能恢复、血栓和动脉粥样硬化的形成和发展等方面具有重要作用。
巨噬细胞移动抑制因子(MIF)是一种在急慢性炎症和癌症有关的新的炎症标记物。MIF能广泛的调节多种炎性反应,在动脉粥样硬化斑块进展中起重要作用。许多人体细胞可以分泌这种物质,这些细胞主要有:血管平滑肌细胞,活性巨噬细胞,分化晚期的巨噬细胞,经过炎症和细胞外组织改造过的活体组织上发现的巨噬细胞的亚型,像粥样硬化斑块上的巨噬细胞等。MIF能提高趋化性,细胞附着性和VSMCs的迁移及小血管分支的形成。
动脉粥样硬化时血液中的单核细胞在损伤部位进行聚集、粘附、迁移和穿过血管内皮,进而活化、分化成为巨噬细胞。而巨噬细胞吞噬脂质成为巨噬—泡沫细胞,形成脂纹。然而目前对单核细胞移动、粘附迁移和血管平滑肌迁移,并在局部增殖的确切分子机制仍不清楚。MIF由动脉粥样硬化斑块中的多种细胞产生,在早期斑块的发生和斑块进展中起重要作用。因此,寻找和鉴定能控制动脉粥样硬化的靶蛋白对治疗冠心病来说非常重要。本研究通过RNA干扰技术特异性沉默MIF蛋白的表达并观察MIF对细胞增殖、迁移、粘附中所起的作用并探讨其作用机制。
本研究分为以下二部分:第一部分
siRNA靶向MIF重组逆转录病毒载体构建及稳定表达细
胞株的筛选
1.目的:
构建并鉴定MIF特异性siRNA重组逆转录病毒载体,将其导入phoenix细胞中,筛选出分泌MIF-siRNA病毒的包装细胞,进而鉴定该病毒上清可抑制MIF蛋白的表达。
2.方法:
2.1 pSuper.retroRNAi的设计及合成
根据GenBank提供的MIF基因cDNA序列,通过www.oligoengine.com网站提供的工具软件设计,所选择MIF的靶点序列分别为:
pSRP/MIF-siRNA1
5'-CTATTACGACATGAACGCG-3'
pSRP/MIF-siRNA2
5'-CAACTCCACCTTCGCCTAA-3
2.2逆转录病毒载体的构建
将合成的正、反义链经过变性、复性后形成双链MIF基因siRNA,采用双链定向亚克隆入逆转录病毒载体SUPER.retro,转化大肠杆菌菌株E.coli DH5α,氨苄青霉素(100mg/L)筛选出阳性克隆,提取质粒。
2.3重组质粒MIFsiRNA的鉴定
重组质粒用EcoRⅠ与HindⅢ酶进行双酶切,酶切产物行1%琼脂糖凝胶电泳。阳性克隆分别命名为pSRP/MIF1、pSRP/MIF2,纯化后送到上海博尚生物公司测序鉴定。
2.4重组质粒转染包装病毒细胞株
将包装病毒细胞phoenix接种到6孔板中,分别使用pSRP(空载体pSuper.retro)、pSRP/MIF1、pSRP/MIF2质粒各4μg,脂质体Lipofectamin2000 10μl/孔转染phoenix。转染后嘌呤霉素筛选3-4周获得阳性克隆。细胞株命名为phoenix-pSRP、phoenix-pSRP/MIF1 siRNA、phoenix-pSRP/MIF2 siRNA。
2.5重组逆转录病毒感染HeLa细胞株
将筛选获得的phoenix阳性克隆培养至90%融合后换成无嘌呤霉素的完全培养基,24小时后收集细胞上清,滤液-70℃保存。HeLa细胞株接种到6孔板中,加入1ml phoenix细胞上清滤液并加入2μg/ml的聚凝胺,更换含嘌呤霉素的完全培养基筛选培养,约20天筛选完成。所产生的细胞株分别命名为HeLa-pSRP、HeLa-pSRP/MIF1 siRNA、HeLa-pSRP/MIF2 siRNA。
2.6 Western-blot检测MIF蛋白的沉默
将筛选后的HeLa细胞株接种到6孔板中,培养生长至90%时,收集细胞。用细胞裂解液裂解,15%SDS聚丙烯酰胺凝胶电泳,将蛋白质转移到PVDF膜,用含5%脱脂奶粉的TTBS(0.1 mol/LTris-HCL,pH7.5,0.2%NaCl;0.05%Tween20)37℃封闭1-2h,分别加入兔抗人MIF多克隆抗体(1:1000稀释),或小鼠抗人GAPDH单克隆抗体(1:1000稀释)4℃孵育过夜。TTBS漂洗5min×3次;兔抗辣根过氧化物酶标记二抗(1:5000稀释)37℃孵育1h;TTBS漂洗5min×3次;SuperSignal Weste Femto(Pierce公司)敏感曝光试剂盒曝光底片显影。
3.结果
3.1酶切鉴定重组质粒
逆转录病毒载体pSuper.retro由本室改造:构建后重组质粒BglⅡ酶切位点应该消失,阳性克隆经EcoRⅠ与HindⅢ酶切后可产生280 bp大小的片段。阴性克隆经EcoRⅠ与HindⅢ酶切后则产生230bp、680bp两条带。本实验中重组质粒经EcoRⅠ与HindⅢ酶切后显示出约280bp左右的条带,表明载体构建成功。将重组质粒pSRP/MIF1、pSRP/MIF2扩增纯化后送上海博尚生物有限公司测序,测序结果均与目的序列完全相同,表明构建载体成功。
3.2 Western-blot检测MIF蛋白的沉默
通过western-blot检测MIF蛋白的沉默效果。结果显示:细胞株HeLa-pSRP/MIF1 siRNA,HeLa-pSRP/MIF2 siRNA细胞株内源性MIF均被不同程度沉默,而空病毒载体感染的细胞株HeLa-pSRP内源性MIF水平没有变化。说明细胞株的筛选是成功的。并且本研究之后选取的MIF沉默的细胞株均为HeLa-pSRP/MIF1 siRNA标记为HeLa-pSRP/MIF。第二部分
MIF在细胞生长、迁移、粘附中的作用研究
1.目的:探讨MIF对细胞生长、迁移、粘附功能的影响和机制。
2.方法:
2.1细胞培养
将HeLa-pSRP和HeLa-pSRP/MIF用含1μg/ml嘌呤霉素的完全培养基37℃5%CO2条件下培养,细胞生长至90%左右更换为完全培养基。将细胞消化至12孔板,起始细胞数为0.1×10~6/孔,连续计数6天。同时观察HeLa-pSRP和HeLa-pSRP/MIF siRNA细胞表型变化,显微镜200倍下拍摄相片。
2.2流式细胞仪检测HeLa-pSRP、HeLa-pSRP/MIF细胞周期及凋亡
将HeLa-pSRP、HeLa-pSRP/MIF细胞分三组铺6cm培养皿,待细胞生长至80%时,一组将HeLa-pSRP、HeLa-pSRP/MIF细胞分别用0.25%胰蛋白酶和0.02%EDTA(1:1)消化细胞,制备单个细胞悬液,70%冷乙醇固定,再用1%TritonX-100、0.01%RNase和0.05%PI处理,通过300目尼龙网备检检测细胞周期。另两组加入TNF-α10ng/ml和CHX10μg/ml处理12小时后,其中一组提取蛋白,另一组将HeLa-pSRP、HeLa-pSRP/MIF细胞分别消化细胞,制备单个细胞悬液,70%冷乙醇固定,0.01%RNase和0.05%PI处理,通过300目尼龙网备检。用ProfileⅡ型流式细胞仪,在488nm激发波长下测定细胞DNA,用Multicycle软件分析细胞周期分布情况。细胞早期凋亡数目通过annexin V-PI检测试剂盒(BD Biosciences)检测。激发波长Ex＝488 nm;发射波长Em＝530 nm。AnnexinV-FITC的绿色荧光通过FITC通道(FL1)检测;PI红色荧光通过PI通道FL3检测。提取蛋白后检测Bax、Bcl-2表达情况。
2.3细胞衰老试验
HeLa-pSRP、HeLa-pSRP/MIF消化后将1×10~5HeLa-pSRP、HeLa-pSRP/MIF细胞悬液分别进行细胞爬片,待细胞生长至80%左右进行实验。0.2%戊二醛固定5分钟后,加入X-gal混合液2ml/孔,放入温箱孵12小时,镜检,阳性细胞为出现蓝绿色。
2.4免疫荧光检测
将HeLa-pSRP、HeLa-pSRP/MIF细胞分别消化成细胞悬液加已加盖玻片的6cm培养皿中,待细胞生长至70%后,去除培养液,PBS清洗3次,加入4%多聚甲醛溶液固定20分钟,后使用0.1%Triton X-100-PBS浸泡10分钟PBS/0.1%Triton X-100渗透10min,PBS洗涤3次,每次5min;5%BSA封闭30min后加一抗:p53抗体(1:100稀释)4℃孵育过夜。PBS清洗3次,每次5分钟,5%BSA再封闭30 min;加二抗:加入Alexa Fluor(?)555 rabbit anti-mouse IgG(H+L)(1:1000稀释)Molecular Probe公司,室温孵育45分钟。PBS洗涤5次,每次5min(避光操作),加入DAPI(1:1000稀释Roche公司)室温染核10分钟。PBS清洗1次,(避光操作)封片,在载玻片上滴一滴水溶性封片剂,然后将盖玻片(培养有细胞的一面朝向水溶性封片剂)从一侧轻轻粘在载玻片上,激光共聚焦显微镜扫描和照相。
2.5细胞迁移实验
在Transwell下室的DMEM培养液中加入10%血清,在Transwell上室分别滴加1×10~5 HeLa-pSRP、HeLa-pSRP/MIF细胞悬液。将上室置于下室之中,在37℃温箱孵育。16h后取出上室,用棉签将残留在上室内表面上的细胞拭去。上室经PBS洗涤后,用4%多聚甲醛固定迁移至上室外表面上的细胞,细胞用含0.1%龙胆紫的20%乙醇染色20分钟,用清水洗3遍,显微镜下(200×)计算迁移细胞数,结果取5个视野细胞计数的平均值。
2.6划痕试验
HeLa-pSRP、HeLa-pSRP/MIF细胞铺6孔板中,当单细胞层生长至95%时,使用10μl枪头对细胞进行划痕。分别于0,24小时,36小时,48小时对细胞进行拍照,比较细胞划痕愈合的速度。
2.7细胞粘附性实验
按照CHEMICON公司Cytomatrix cell adhension strips说明书进行。室温200μl/孔PBS洗涤2遍,胰酶消化HeLa-pSRP、HeLa-pSRP/MIF细胞,制成单细胞悬液,计数,调整细胞浓度为1×10~5/ml。每组分别设3个复孔,每孔加入100μm细胞悬液,37℃孵育1h后取出96孔培养板,用PBS洗涤2-3遍,去除未粘附细胞。含0.2%龙胆紫的10%乙醇染色5分钟,用PBS洗涤3-5遍,每孔加入100μl等体积混匀的0.1MNaH_2PO_4,PH4.5和50%乙醇以去除残余染色。酶标仪在波长570nm读数。
2.8 Western-blot检测
将筛选后的HeLa细胞株接种到6孔板中,培养至每孔0.1×10~6时,收集细胞。用适量细胞裂解液(150mmol/L Tris-HCL,0.2%叠氮钠,0.1%SDS,100μg/mlPMSF,1μg/ml Aprotinin,1%NP40,0.5%去氧胆酸钠,150mmol/L NaCl)裂解,上样,15%SDS聚丙烯酰胺凝胶电泳,将蛋白质转移到PVDF膜,用含5%脱脂奶粉的TTBS(0.1mol/L Tris-HCL,pH7.5,0.2%NaCl;0.05%Tween20)室温封闭1.2小时,分别加入兔抗人Cyclin D1、Bcl-2、Bax、FAK、AKT、ERK1/2、和ICAM-1多克隆抗体(1:1000稀释),鼠抗人p53、p21单克隆抗体1:800鲜?或小鼠抗人GAPDH单克隆抗体(1:1000稀释)4℃孵育过夜。TTBS漂洗5min×3次;兔抗辣根过氧化物酶标记二抗(1:5000稀释)37℃孵育1h;TTBS漂洗5min×3次;SuperSignal Weste Femto(Pierce公司)敏感曝光试剂盒曝光底片显影。
3.统计方法
使用SPSS17.0统计软件,两组数据比较采用两独立样本t检验,结果用平均值±标准差((?)±s)表示,以P＜0.05为有统计学差异。
4.结果
4.1细胞生长变化情况
沉默MIF后的HeLa-pSRP/MIF与对照组HeLa-pSuper相比生长速度明显减慢。达到半数生长期的时间:HeLa pSRP需要30小时,而HeLa-pSRP/MIF需要64小时。沉默MIF后的HeLa-pSRP/MIF在形态上成铺路石样分布,而空病毒对照组细胞多有触突,而且有聚集成团的趋势。
4.2沉默MIF后HeLa细胞生长停留在G0/G1期
流式细胞仪显示HeLa-pSRP/MIF G0/G1期比例为51.887±4.241%,与对照组HeLa-pSRP相比G0/G1期比例为28.453±4.230%,两组比较有统计学差异(P＜0.05)。沉默MIF后HeLa细胞生长停留在G0/G1期。
4.3沉默MIF后HeLa细胞Cyclin D1表达受抑制,p53、p21表达下降
沉默MIF细胞周期出现变化,因此我们检测实验组和对照组之间影响细胞周期的蛋白有无改变。Western结果显示:沉默MIF后HeLa细胞Cyclin D1表达受抑制,并且p53、p21水平较HeLa-pSRP明显下降。
4.4沉默MIF后HeLa细胞迁移活性下降,细胞的粘附性下降
在10%小牛血清趋化作用下,迁移至下室的细胞数目对照组pSRP为86.333±16.166个/视野,pSRP/MIF组为23±10.148个/视野。两组比较有显著性差异(P＜0.01)。另外,划痕实验显示,HeLa-pSRP在48小时已使划痕愈合,而HeLa-pSRP/MIF siRNA迁移能力明显减弱,至48小时仍未能使划痕愈合。胰酶消化HeLa-pSRP、HeLa-pSRP/MIF细胞,使用CHEMICON公司Cytomatrix celladhension strips进行细胞粘附实验。酶标仪在波长570nm读数,OD值结果:HeLa-pSRP 0.509±0.0890,HeLa-pSRP/MIF 0.180±0.031,两组比较有显著性差异(P＜0.01)。
4.5沉默MIF后细胞凋亡减少
TNF-α和放线菌酮(CHX)10μg/ml诱导细胞凋亡后,流式细胞仪检测HeLa-pSRP细胞凋亡比例为54.833±6.443%,HeLa-pSRP/MIF细胞凋亡比例为4.457±1.154%,两组比较有显著性差异(P＜0.05)。western-blot结果显示MIF蛋白的沉默后使HeLa细胞表达凋亡相关蛋白Bax减少而Bcl-2轻度升高。
4.6沉默MIF后细胞衰老减少
使用X-gal实验结果显示,沉默MIF后细胞衰老减少。
4.7 MIF蛋白的沉默后使HeLa细胞Src、FAK、ERKl/2、AKT、ICAM-1表达减少。论文小结
1.成功构建了针对人MIF基因的siRNA重组逆转录病毒载体pSRP/MIF1、pSRP/MIF2。获得稳定抑制MIF表达的细胞株HeLa-pSRP/MIF和表达空载体的对照组细胞HeLa-pSRP。
2.沉默MIF后的HeLa细胞对TNF-α和放线菌酮诱导的凋亡减少,并且沉默MIF后细胞衰老减少。沉默MIF后细胞阻滞在G0/G1期,细胞生长减慢,迁移能力、粘附功能减弱。
3.本研究显示MIF通过影响cyclinD1、p53、p21表达维持细胞正常生长周期;MIF通过促进p53、p21的表达促进细胞衰老。另外,MIF通过增加Bax表达,促进细胞凋亡。MIF能促进细胞Src、FAK、ICAM-1、AKT、ERK1/2的表达,可能是MIF维持细胞的生长、移动、粘附特性的机制之一。本文的创新点:通过HeLa细胞模型证实了MIF在细胞的生长、增殖、黏附、迁移过程中扮演重要作用及可能的分子机制。结合课题组早期的工作,这一创新性结果进一步揭示了MIF可能通过调节多种与细胞粘附、迁移有关的蛋白表达,促进动脉粥样硬化的发生、发展。
The highly conserved and archetypical yet atypical cytokine macrophage migration inhibitory factor(MIF) fulfills pleiotropic immune functions in many acute and chronic inflammatory diseases.Recent evidence has emerged from both expression and functional studies to implicate MIF in various aspects of cardiovascular disease.MIF plays important role in the inflammatory pathogenesis of atherosclerosis and its consequences,namely unstable plaque formation, remodeling after arterial injury,aneurysm formation,myocardial infarction,or ischemia-reperfusion injury.
The functional data are reconciled with recent progress in the identification of heptahelical(CXC chemokine) receptors for MIF,its prototypic role as their noncanonical ligand,and its signal transduction profile operative in atherogenic and inflammatory recruitment of mononuclear cells and in the oxidative damage and apoptosis of cardiomyocytes.Its unique features and functions clearly distinguish MIF from other cytokines implicated in atherogenesis and make it a prime target for achieving therapeutic regression of atherosclerosis.The potential of targeting or exploiting MIF for therapeutic strategies or as a diagnostic marker in the management of cardiovascular diseases or disorders is scrutinized.
In this study,we found inactivation of MIF in HeLa cells resulted in suppression of migration ability.We further show that MIF is required for cells motility through activates Src and FAK signaling and provided a new target for improving the treatment efficiency of atherosclerosis through inactivation of the MIF gene.
EXPERIMENTAL PROCEDURES
Vector construction.
Among different MIF target sequences examined,the 19-nucleotide gene-specific sequence spanning from nucleotides 427 nt to 445 nt downstream of the gene transcription start site was selected to suppress MIF gene expression.After BLAST analysis,to ensure that there was no significant sequence homology with other human genes,the selected sequence was inserted into a BglⅡ/HindⅢ-cut pSuper puro vector to generate the pSuper-MIF vector.All constructs were confirmed by DNA sequence analysis.
Cell culture,treatments,and transfections.
HeLa cells were grown at 37℃in a 5%CO_2 atmosphere in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10%FCS,2 mmol/L glutamine,and antibiotics.phoenix packaging cells and HeLa cells stably transfected with pSuper.puro and pSuper-MIF were maintained in DMEM supplemented with 10% FCS in the presence of puromycin(2 mg/ml).All transfections were done using LipofectAMINE 2000 reagent(Invitrogen) according to the manufacturer's protocols. Transfect phoenix packaging cells were done according to the manufacturer's protocols to produce ecotropic retroviral supernatants.48 hours posttransfection, select infected cells with puromycin(1-3μg/ml).After 20 days in selective medium, three pools,referred as phoenix-pSuper and phoenix- pSuper-MIF1,pSuper-MIF2, were isolated respectaly.
Generation of stable MIF-silenced lines.
To obtain HeLa cell lines stably expressing siRNAs,the ecotropic retroviral supernatants produced by phoenix packaging cells were collected and filter the tissue culture medium through a 0.45μm filter,and use the viral supernatant for infection of HeLa cell lines after addition of 4μg/ml polybrene.Infected HeLa cell lines for at least 6 hr and allow to recover for 24 hr with fresh medium.Select infected cells with puromycin(1-3μg/ml).After 15 days in selective medium,three pools,referred as HeLa-pSuper.puro,HeLa-pSuper-MIF1 SiRNAand HeLa-pSuper-MIF2 SiRNA were isolated.The HeLa- pSuper-MIF1 SiRNA was subjected to further cloning procedures by the limiting dilution method.The clones presenting a＞95%reduction in MIF protein levels compared with control.All stable cell lines obtained were cultured in DMEM supplemented with 2mg/ml puromycin.The puromycin selective pressure was removed 24 hours before experimental procedures.HeLa-pSuper.puro and HeLa-pSuper-MIF cells proliferation was measured by plating equal cell numbers onto six-well dishes in duplicate,followed by direct counting of trypsinized cells using a Coulter counter.Each growth curve represents at least two independent experiments.
Cell Cycle Analysis
HeLa-pSuper-MIF and HeLa-pSuper were treated with TNF-a and CHX10μg/ml for 12 hours.Trypsinized HeLa-pSuper.and HeLa-pSuper-MIF were washed with phosphate-buffered saline and fixed in 70%ethanol on ice for at least 24 h.The cells were washed with phosphate-buffered saline containing 1%FBS,resuspended in phosphate-buffered saline containing 1%FBS and 25μg/ml RNase A,and incubated for 30 min at 37℃.Prior to flow cytometry,100μl of propidium iodide solution(50μg/ml propidium iodide and 0.1%sodium citrate) was added to the cell suspension and incubated for 1 h on ice.The staining was assessed using the FACSC alibur flow cytometer and CellQuest software following calibration using DNA QCTM beads(BD Biosciences).Data were analyzed using ModFit software(BD Biosciences). SA-β-gal staining
Cells were stained for SA-β-gal activity(Senescence-associatedβ-galactosidase) as described by Dimri.Briefly,1×10~4 cells were seeded in 6-well plate for 24h and were washed twice with phosphate-buffered saline,fixed with 2%formaldehyde and 0.2%glutaraldehyde in PBS,and washed twice in PBS.Cells were stained at 37℃for 12h in X-gal staining solution(1mg/ml X-gal,40mmol/L citric acid/sodium phosphate(pH 6.0),5 mmol/L potassium ferrocyanide,150 mmol/L NaCl,2 mmol/L MgCl_2).Cells were examined under bright field illumination(Leica)
Adhesion Assay
Coat 96-well-plate with FN at 37℃for 1 hr Leave some wells uncoated as negative control.Wash with washing buffer for 2 times.Block plates with blocking buffer at 37℃in CO_2 incubator for 45-60 minutes.Wash with washing buffer.Chill the plates on ice.Count cell to 0.4×10~6/ml.Add 50μl cells in each well. Incubate in CO_2 incubator at 37 C for 30 minutes.Shake the plate at 2000 rpm for 10-15 seconds.Wash with washing buffer 2-3 times.Fix with 4%paraformaldehyde. Incubate at RT for 10-15 minutes.Wash with washing buffer.Stain with Crystal Violet for 10 minutes.Wash with water.Turn the plates upside down.Let the plates dry up completely.Add 2%SDS.Incubate at RT for 30 min.Read plate at 570nm.
Migration Assays
For migration assays,modified Boyden chambers(Millicell-PCF,8-μm pore size;Millipore,Bedford,MA) were placed in a 24-well plate and coated with 10μg/ml rat tail collagen(Roche Applied Science) for 16 h at 37℃.After removal of collagen and washing with PBS,migration medium(DMEM with 0.5%bovine serum albumin)was added to lower chamber in 0.4 ml.HeLa pSuper or HeLa-pSuper-MIF RNAi cells were added to the upper compartment(0.2×10~6 in 0.3 ml of migration medium).The plates were incubated at 37℃for 16 h for migration. The cells were removed from the upper membrane surface with a cotton tip applicator and washed with PBS,and cells on the lower membrane surface were fixed with 4% formaldehyde.The cells were then stained(0.1%Crystal Violet in 20%ethanol and enumerated by counting four(200x) fields/chamber).
Wound closure assay
A wound was induced on the confluent monolayer cells by scraping a gap using a micropipette tip and the speed of wound closure was monitored every 24 hours. Photographs were taken under 100×magnifications using phase-contrast microscopy immediately after wound incision and at later time points.
Confocal microscopy
Cells grown on coverslips were fixed with 4%paraformaldehyde and permeabilized in 0.1%Triton X-100-PBS for 10 minutes.After blocking with 0.2% bovine serum albumin,cells were incubated with rabbit anti-p53 monoclonal antibody followed by decoration with fluorescein-conjugated anti-mouse IgG.Images were acquired on a Leica confocal microscope.
Western blot analysis
Equal amounts of protein(35μg/sample) from whole-cell extracts were separated by SDS-PAGE and blotted to PVDF membranes.After blocking with 5%skim milk solution,membranes were incubated with rabbit polyclonal anti-MIF, anti-cyclinD1,anti-Bcl-2,anti-Bax,anti-FAK,anti-p53,anti-p21,anti-ICAM-1, anti-Src,anti-AKT and anti-ERK1/2(Santa Cruz Biotechnology,Santa Cruz,USA) followed by decoration with peroxidase-labeled anti-rabbit or anti-mouse IgG, respectively(Super Signal detection kit,Pierce).
Statistics
The results are expressed as the means±S.E.The data were analyzed by one-way or two-way analysis of variance using SPSS17.0 statistical program.P values <0.05 were considered significant.
RESULTS
Growth Properties of HeLa cells knockdown MIF
To test the hypothesis that MIF contributes in HeLa cell growth,we established stable knockdown of MIF cell line HeLa-pSuper-MIF using SiRNA technique, which displayed a significant decrease MIF at protein level compared with vector control.HeLa cells were derived from and assayed in several experimental settings. Under normal growth conditions,HeLa-pSuper-MIF proliferated slightly more slowly than the control HeLa-pSuper.Note morphologic changes induced by downregulation of MIF in HeLa cells.
Silencing of MIF in HeLa Cells Results in the cell stay in G0/G1 phase and decrease the cyelin D1 expression
To further confirm the MIF expression on HeLa cell growth,we then studied if downregulation of MIF could lead to any changes in the cell cycle distribution and the relative protein such as cyclin D1.Cell cycle analysis showed that silencing of MIF in HeLa cells results in the cell stay in G0/G1 phase and decrease the cyclin D1 expression.
Down-regulation of MIF leads to suppression of p53 and p21
As p53 play an important role in cell survivle,we then studied if there were any change in p53 and p21 expression,p53 and p21 were detectable in low level in HeLa-pSuper-MIF cell,whereas p53 protein level was high in HeLa-pSuper cell.In addition,we also used immunoflourecent staining to test p53 expression.These results show that MIF can affect the proappotosis gene p53.
Silencing of MIF does not change the TNF-αinduced apoptosis pathway
HeLa-pSuper-MIF and HeLa-pSuper were treated with TNF-αand CHX 10μg/ml for 12 hours.We found that decreased Bax protein expression was found in the HeLa-pSuper-MIF.These results indicate that downregulation of MIF in HeLa cells has led to increased sensitivity to TNF-αthrough activation of induced apoptosis. Silencing of MIF in HeLa Cells Results in decrease cell senescence
At the normal grown condition,silencing of MIF in HeLa cells results in decrease cell senescence,while the HeLa-pSuper cells showed a senescent phenotype that included flattening of cells and expression of senescence-associatedβ-galaetosidase(SA-βgal)
MIF is required for cell migration potential of HeLa Cell
To further study the association between MIF expression and metastatic ability of HeLa cells,we then tested the migration ability of the HeLa cells expressing.We analyzed the effect of MIF on the migratory capability of the HeLa cells by using several function assays.First,wound-healing assay showed that,24h after a wound was made on the monolayer of cells,the control cells extensively migrated into the denuded area,but this migratory capacity was significantly compromised by depletion of MIF in HeLa cells.Then,migration assay showed that compared with control cells,diminished MIF expression also reduced HeLa cell migration through a membrane by two-chamber assay.Overall,these data suggest that MIF expression is essential for the migratory behavior of HeLa cell.
MIF-induced migration activity is associated with the activation of FAK signaling
Cell migration is a complex event that depends on the coordinated remodeling of the actin cytoskeleton as well as the regulated assembly and turnover of focal adhesions.The FAK acts as a protein-protein adapter critical to the formation of focal adhesions and mediates adhesion and growth factor-dependent signals into the cells. Tyrosine phosphorylation of FAK is one of the key signaling events during cell motility.Consistent with our observation above,HeLa cells with MIF knockdown showed decreased FAK expression.Consequently,Src,one of the upstream FAK, also showed reduced in MIF knockdown cells.These data suggest that MIF-induced migration activity is associated with the activation of FAK signaling through Src. Since ICAM-1 is the most important adhesion molecule involved in the extravasation of leukocytes into the surrounding tissue-it has been demonstrated to be both required as well as sufficient for the process of extravasation-most studies have been focused on the expression of ICAM-1.We also tested the expression of ICAM-1 in HeLa-pSuper-MIF and HeLa-pSuper.The data shows that compared with control cells,diminished MIF expression reduced ICAM-1,ERK1/2 and AKT in HeLa cells.
Innovation of this study In this study,we used siRNA to knockdown MIF for evaluation of its function in HeLa cell line,which showed that MIF plays an important role in proliferation,migration and adhesion in vitro.We also investigated the potential molecular mechanism of the phenomenon.Together with prior studies of our group,these results strongly indicate that MIF may possibly promote the atherosclerotic lesions development through regulating the proteins that controlling cell adhesion,migration and proliferation.The present data suggest that MIF may represent an important new target for human atherosclerosis therapeutics.
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