酪氨酸激酶ETK/BMX在miR-495的影响下通过上皮向间质的转化参与小细胞肺癌耐药机制的调节
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摘要
(1)研究意义
肺癌已经成为恶性肿瘤患者死亡的主要疾病,全球每年由肺癌导致的死亡人数超过100万,约15%的患者为小细胞肺癌。SCLC分化程度低,生长迅速,较早发生转移,预后很差,一般2年存活率不足5%,90%的患者在确诊的5年内死亡。由于患者对化疗敏感,所以化疗是SCLC的主要治疗方式。但是SCLC很容易发生耐药,导致化疗失败,因此,SCLC的化疗耐药性已成为肺癌临床治疗急需解决的重要问题之一。
上皮及内皮酪氨酸激酶(Etk/Bmx)是Btk家族(Btk, Itk, Etk/Bmx, Txk和Tec)中的重要成员,是Btk家族中唯一除分布于淋巴造血细胞外,还在上皮细胞性瘤细胞和血管内皮细胞中表达的非受体类酪氨酸激酶。研究发现Etk/Bmx的异常表达参与肿瘤细胞增殖、凋亡、血管生成等方面的调控,与肿瘤细胞恶性生物学行为密切相关。有少量研究结果提示Etk/Bmx参与肿瘤细胞耐药的调节。
上皮细胞向间质细胞的转化(epithelial to mesenchymal transition, EMT)是指上皮细胞在一些因素的作用下,失去细胞极性,丢失细胞间紧密连接和黏附连接,获得了浸润性和游走迁移能力,变成了具有间质细胞形态和特性的细胞。研究发现EMT与肿瘤耐药密切相关。
微小RNA (microRNA, miRNAs)是一类广泛存在与生物体内,由19-25个核苷酸组成的非编码RNA,参与基因转录后水平的调控,在机体的生长发育过程中发挥重要的作用。大量的研究表明miRNAs与肿瘤发生、肿瘤干细胞的分化、血管生成等密切相关。近年来研究表明miRNAs可通过调节与药物代谢、细胞增殖、凋亡等相关基因影响肿瘤细胞的耐药。
本课题组长期以来从事小细胞肺癌耐药机制的研究,前期在国家自然基金的资助下已经发现Etk/Bmx可能参与小细胞肺癌耐药的调控,但是具体机制尚未清楚。而前期利用cDNA基因芯片对比小细胞肺癌耐药细胞株H69/AR和亲本细胞H69,结果发现与EMT相关的主要间质标志物(Zeb-2, Twist, Vim)在H69/AR中的表达明显高于母细胞。因此,我们推测EMT可能参与小细胞肺癌耐药的调节。我们还利用miRNA表达谱芯片发现miR-495在H69/AR中的表达明显低于母细胞,提示miR-495可能参与小细胞肺癌耐药的调节。本课题组拟在前期研究工作基础上,采用分子生物学和细胞生物学技术,利用小细胞肺癌细胞株H69,H69/AR和H446和H446/AR,探讨：Etk/Bmx在niR-495的调节下,可通过透导EMT,参与细胞肺癌耐药机制的调节。研究内容包括以下几个方面：①、利用临床标本分析Etk/Bmx与患者病理分、预后的相关性。②、进一步确定Etk/Bmx在小细胞肺癌耐药中的作用：分别上调或者下调H69,H69/AR和H446, H446/AR中Etk/Bmx的表达,观察对细胞药物敏感性、凋亡的影响,③、观察Etk/Bmx对小细胞肺癌恶性生物学行为的影响：(增殖、迁移、侵袭、成瘤能力的影响)④、确定EMT在小细胞肺癌耐药中的作用:分别下调H69,H69/AR和H446, H446/AR中EMT主要间质标志物Zeb-2, TWIST的表达,观察下调Zeb-2, TWIST的表达后,对细胞药物敏感性的影响。⑤、确定Etk/Bmx对EMT的靶向调节作用：用Etk/Bmx的干扰或过表达质粒下调或者上调H69,H69/AR和H446中Etk/Bmx (?)勺表达,观察对EMT主要间质标志物Zeb-2, Twist, Vim和上皮标志物(3-catenin的调节作用。⑥、确定miR-495小细胞肺癌药物敏感性的影响以及对Etk/Bmx的靶向调节作用。通过本项目研究,进一步丰富小细胞肺癌耐药的分子机制,期望能筛选出预测小细胞肺癌耐药的分子标志物,为下一步开展基于Etk/Bmx通路逆转小细胞肺癌耐药提供依据。
(2)研究目的
在课题组前期研究的基础上,采用细胞生物学和分子生物学技术,利用小细胞肺癌耐药细胞H69/AR, H446/AR和相应的母细胞H69,H446,探讨Etk/Bmx在miR-495的调节下,可通过诱导EMT,参与小细胞肺癌耐药机制的调节。通过本项目研究,进一步丰富小细胞肺癌耐药的分子机制,期望能筛选出预测小细胞肺癌耐药的分子标志物,为下一步开展基于Etk/Bmx通路逆转小细胞肺癌耐药的研究提供依据。
(3)研究方法
<1>细胞培养
人小细胞肺癌细胞株H69, H69/AR, H446, H446/AR在37℃5%C02条件下,培养于含新生牛血清的RPMI-1640中,每周传代2-3次。<2>实时荧光定量PCR检测mRNA水平
用Trizol提取对数生长期细胞中总RNA,按实时荧光定量RT-PCR试剂盒说明进行实验。合成cDNA的反应条件为：15分钟37。C,5秒85°C。 PCR的反应条件为：预变性95℃30秒,PCR反应95℃5s,60℃20s(40个循环),溶解曲线分析95℃0秒,65℃15秒,95℃O秒。计算出2-△△α,代表每组mRNA的相对表达量。反应结束后确认Real Time PCR的扩增曲线和融解曲线,进行PCR定量时制作标准曲线等。目的基因的表达量=2-△△α△△ct=(待测样品的目的基因的Ct的平均值—待测样本的看家基因的Ct的平均值)—(对照样品的目的基因的Ct的平均值一对照样本的看家基因的Ct的平均值)
每组重复例数为3。由Invitrogen公司设计合成引物如下表：Name Forward primer (5'-3') Reverse primer (5'-3') Etk/BMX CATAGTGGGTTCTTCGTGGAC TGCCCGAGGTATCTTCAGC Zeb-2GGTCCAGATCGAAGCAGCTCA GTGACTATGTTTGTTCACAT AT T Twist CAGTCTTTACGAGGAGCTG GCTTGAGGGTCTGAATCT Vim TGCTAACTACCAAGACACT GTAGGTGCAATCTCAATG β-catenin ACAAACTGTTTTGAAAATCCA CGAGTCATTGCATACTGTCC GAPDH AGAAGGCTGGGGCTCATTTG AGGGGCCATCCACAGTCTT C
<3>免疫印迹(Western blot)检测蛋白表达
提取总蛋白,测定蛋白浓度,吸取总蛋白,去离子水补至20μL,加入上样buffer煮沸5min,离心后吸取25μL上样,经不连续SDS-PAGE电泳分离后,用半干法电转移至PVDF膜上,7%脱脂奶粉+3%的BSA在37℃封闭2h, TBST洗膜后用相应的一抗4℃孵育过夜,洗膜后分别加入HRP标记的兔抗羊IgG(1:4000),IgG(1：6000),室温下摇动1h,洗膜后用ECL化学发光系统检测,暗室曝光x线片,冲洗胶片,UVI凝胶成像系统摄像,Quantity One软件分析条带灰度值,用GnT-V/GAPDH代表GnT-V蛋白的相对表达量。每组重复例数为3。<4>细胞转染
调整细胞浓度为1×105/ml,按2mL/孔接种入6孔板,用无抗生素培养液培养16-24h以上至细胞密度达到85%左右。吸出丢弃原培养液,1×PBS轻轻冲洗2遍,按2m1/孔加入Opti-MEM I溶液。将Lipofectamin2000轻柔混匀,用Opti-MEMI240μL稀释10μL Lipofectamin2000,室温静置5min。将pEGFP-N1质粒和Etk/BMX-pEGFP-N1表达质粒各0.8g用Opti-MEM I稀释至终体积为250μL。将稀释后Lipofectamin2000和稀释后质粒轻柔混匀(总体积500μL),室温静置20min,以形成质粒DNA/Lipofectamin2000复合物。每孔对应加入500μL转染混合液,轻摇混匀。置于37℃,5%C02、饱和湿度的培养箱中培养。24小时后用含筛选浓度G418的20%胎牛血清的RPMI-1640培养液筛选。待形成阳性单细胞克隆群落后,用尖吸管吸取单克隆阳性细胞,连续培养4周后得到稳定转染细胞株,以含维持剂量的G418培养基维持培养。稳定转染细胞的鉴定：由于所转染质粒标记有eGFP,荧光激发后可显示绿色荧光,故可首先应用荧光显微镜观察质粒是否已转染入细胞。采用实时荧光定量PCR技术和Western Blot技术检测ETK/BMX表达情况。获得稳定转染细胞株H69/Etk和H446/Etk(过表达ETK/BMX), H69/AR/sh和H446/AR/sh (低表达ETK/BMX)。通过瞬时转染得到的细胞株有H69/AR/Zeb-2siRNA1、H69/AR/Zeb-2siRNA2, H446/AR/Zeb-2siRNA1、 H446/AR/Zeb-2siRNA2、H69/AR/Twist siRNA1、 H69/AR/Twist siRNA2、 H446/AR/Twist siRNA1、H446/AR/Twist siRNA2、 H69/AR/mimics,H446/AR/mimics、H69/antagomir, H446/antagomir。
<5>CCK-8法检测细胞药物敏感性
细胞处理后按5×103个/孔接种于96孔培养板中,每孔总体积100μ1。置于37℃,5%CO2细胞培养箱中培养24h后,将化疗药物阿霉素ADM,顺铂DDP,依托泊苷VP-16分别加入培养基中并进行倍比稀释,按不同浓度分别加入指定的孔中,并设不加药物的阳性对照孔。置于37℃,5%C02细胞培养箱中培养24h,加入CCK-8反应液,在37℃,5%CO2环境下培养4h,混匀后在酶标仪上检测450nm吸光度。测得的每个药物浓度的OD值的平均值-空白孔平均值／不含药物的阳性对照组平均值-空白孔平均值即为每个药物浓度下细胞的存活率。求得细胞50%生存率时的药物浓度(IC50)。实验分组如下：
①上调或者下调Etk/BMX对IC50的影响,分组如下：
H69类细胞分为：H69组、H69/NC组、H69/Etk组；
H446类细胞分为：H446组、H446/NC组、H446/Etk组；
H69/AR类细胞分为：H69/AR组、H69/AR/NC组、H69/AR/sh1组、H69/AR/sh2组；
H446/AR类细胞分为：H446/AR组、H446/AR/NC组、H446/AR/shl组、H446/AR/sh2组;
每组重复例数为3。
②下调Zeb-2或者Twist对IC50的影响,分组如下：
H69/AR类细胞分为：H69/AR、H69/AR/NC.H69/AR/Zeb-2siRNA1、H69/AR/Zeb-2siRNA2组;
H69/AR类细胞分为:H69/AR、H69/AR/NC、H69/AR/Twist siRNAl、H69/AR/Twist siRNA2组;
H446/AR类细胞分为：H446/AR、H446/AR/NC、H446/AR/Zeb-2siRNA1、 H446/AR/Zeb-2siRNA2组;
H446/AR类细胞分为：H446/AR、H446/AR/NC. H446/AR/Twist siRNAl、 H446/AR/Twist siRNA2组；
每组重复例数为3。
③上调或者下调miR-495对IC50的影响,分组如下：
H69类细胞分为：H69组、H69/NC组、H69/antagomir组;
H446类细胞分为：H446组、H446/NC组、H446/antagomir组;
H69/AR类细胞分为：H69/AR组、H69/AR/NC组、H446/AR/mimics组；
H446/AR类细胞分为：H446/AR组、H446/AR/NC组、H446/AR/mimics组；
每组重复例数为3。
<6>流式细胞术检测化疗药物诱导的细胞凋亡率
将细胞接种于6孔培养板。置于37℃,5%C02细胞培养箱中培养24h,加入化疗药物DDP、ADM、VP-16。置于37℃,5%C02细胞培养箱中培养48h,收集细胞,用PBS洗涤细胞两次。加入5ul Annexin V-FITC混匀后,加入5u1Propidium Iodide混匀。室温、避光反应15min。流式细胞仪检测。
实验分组
①ADM诱导的细胞凋亡率
H69类细胞分为：H69组、H69/NC组、H69/Etk组；
H446类细胞分为：H446组、H446/NC组、H446/Etk组；
H69/AR类细胞分为：H69/AR组、H69/AR/NC组、H69/AR/sh1组、H69/AR/sh2组；
H446/AR类细胞分为：H446/AR组、H446/AR/NC组、H446/AR/sh1组、H446/AR/sh2组；
每组重复例数为3。
DDP、VP-16诱导的细胞凋亡率分组同上。<7>CCK-8细胞增殖能力
取对数生长期的待测细胞,CCK-8法测量不同时间点的OD450nm值(Oh、24h、48h、72h、96h)。计算出细胞生长分数,绘制生长曲线。<8>划痕愈合实验检测细胞迁移能力
取对数生长期的待测细胞,接种于24孔板,培养至细胞呈单层贴壁生长状态。用100ul的枪头分别在各组单细胞层上划痕,用含10%FBS的培养基继续培养以使划痕愈合。分别于划痕后0h、12h、24h拍照,观察划痕愈合能力,用愈合率代表愈合能力。愈合率=[(愈合前两侧细胞层距离-愈合后两侧细胞层距离)/愈合前两侧细胞层距离]×100%。<9>细胞侵袭实验
Matrigel胶4℃过夜融化,每个Transwell上室加入50μL,从边缘轻轻加入,避免出现气泡,轻轻摇晃铺平Marigel胶,37℃10min凝固。取对数生长期的细胞,用无血清RPMI1640培养过夜。用0.25%胰酶消化细胞,1xPBS洗涤细胞,800r/min离心5min沉淀细胞,弃上清,将细胞重悬于氯化钙1mmol/L,氯化镁1mmol/L,氯化锰0.2mmol/L及5g/L BSA的RPMI1640中,调整细胞浓度为1×106/mL,上室每室加入200/μL细胞悬液,下室每室加500μL含10%新生牛血清的RPMI-1640培养液,37℃5%C02培养48h。取出上室,用棉签擦去膜上室面的Matrigel胶和细胞,4%多聚甲醛固定膜下室面的细胞15min,1×PBS清洗3次,每次5min,结晶紫染色3min,1×PBS清洗干净,小心用刀片将膜从小室中分离出来,将膜下室面朝上置于载玻片上,吹风机干燥后,中性树脂封固。细胞侵袭性结果表示为同时穿过Matrigel胶和聚碳酸酯膜的细胞数。
实验分组
H446类细胞分为：H446组、H446/NC组、H446/Etk组;
H69/AR类细胞分为：H69/AR组、H69/AR/NC组、H69/AR/shl组、H69/AR/sh2组;
H446/AR类细胞分为：H446/AR组、H446/AR/NC组、H446/AR/sh1组、H446/AR/sh2组;
每组重复例数为3。<10>裸鼠皮下成瘤实验
6周龄Balb/c裸鼠,雄性,体重(18±2)g,购自广东省动物实验中心,均置于SPF环境下饲养。将细胞培养至对数生长期,胰酶消化孵育后制作细胞悬液,用0.4%台盼蓝染色后计数活细胞比率>90%,PBS洗涤3遍后,用生理盐水重悬细胞,调整其浓度为107个/mL。置于冰上备用。医用碘伏消毒局部皮肤后,用1ml注射器,每组接种100μL癌细胞悬液于臀部皮下。注射后定期测量瘤体的长径、宽径,瘤体体积=长径X短径2/2；24天后,断颈处死裸鼠,剥离瘤体,液氮保存备用,检测组织中EMT相关标志物蛋白表达水平变化。同法建立裸鼠皮下成瘤模型,观察裸鼠存活天数。<11>荧光素酶实验
将psiCHECK-2载体转化至DH5aαTM, Ampicillin (100μg/ml)筛选,挑取单克隆。培养时间不超过14h,挑取三个克隆扩大培养,试剂盒提取质粒,酶切鉴定。采用CaCl2法,42℃/90sec热击转化入DH5αTM克隆菌株,经Ampicillin (100μg/ml)抗性选择。挑取三个克隆扩大培养。试剂盒提取质粒,Not I和Xho I双酶切鉴定psiCHECK-2-ETK/BMX-3'UTR-R.挑取酶切鉴定正确克隆进行测序鉴定。试剂盒(QIAGEN)提取质粒以进行荧光素酶报告基因检测。将psiCHECK-2-ETK/BMX-3'UTR-R、psiCHECK-2-ETK/BMX-S'UTR-mut或psiCHECK-2转染H69/AR细胞；共转染miRNA mimics、antagomir或miR-NC;裂解后检测荧光素酶活性。
<12>免疫组织化学检测Etk/BMX在SCLC中的表达
86例小细胞肺癌石蜡包埋组织取自2005年1月至2009年1月于南方医科大学珠江医院手术或支气管镜活检的小细胞肺癌患者。所有组织均经病理学确诊为小细胞肺癌,全部病例获取前均未行化疗和放疗,临床资料完整。其中男性69例,女性17例；局限期患者66例,广泛期患者20例.3瓶100%二甲苯脱蜡,3×10分钟；2瓶100%酒精脱水,2×8分钟；1瓶95%酒精脱水,5分钟1瓶85%酒精脱水,5分钟；1瓶75%酒精脱水,5分钟；1瓶50%酒精脱水,5分钟。1×PBS冲洗3次,每次3min。脱蜡后玻片放入盛有1%柠檬酸溶液中在微波炉内用中进行抗原修复高火7min,中火15min。冷却至室温后,1×PBS冲洗3次,间隔5min。每张玻片滴加1滴过氧化氢酶阻断剂,室温下孵育12min,1×PBS冲洗3次,间隔5min。每张玻片滴加1滴正常非免疫动物血清,室温下孵育10min。除去血清,阳性对照组每张玻片滴加1滴Etk/BMX一抗(1：100,抗体稀释液稀释),阴性对照组则加入1×PBS,放入湿盒内4℃孵育12h。1×PBS冲洗3次,每次5min,每张玻片滴加1滴生物素标记的羊抗兔二抗,室温下孵育40min,1×PBS冲洗3次,间隔5min。每张玻片滴加1滴链霉素抗生物素-过氧化物酶溶液,室温下孵育30min,1×PBS冲洗3次,间隔5min。每张玻片滴加2滴新鲜配制的DAB溶液.显微镜下观察5min。自来水冲洗去DAB溶液,苏木素复染10s,盐酸酒精分化,氨水返蓝。梯度酒精脱水干燥,吹风机吹干玻片,中性树胶封固。结果判定Etk/BMX阳性染色在胞浆,呈棕黄色。<13>、统计学分析
所有结果均经SPSS20.0统计软件统计。数据以均数±标准差(x±s)表示,两组间的比较采用独立样本t检验。多组间的比较采用完全随机设计的方差分析,进一步的多重比较采用LSD法(方差齐同时)或Dunnett's T3法(方差不齐)。以P<0.05为差异有统计学意义。Etk/BMX表达与临床病理特征分析采用列联表资料分析。多因素对SCLC患者生存时间的影响采用逐步Cox回归模型分析,各因素对SCLC患者生存时间的影响采用Kaplain-Meier分析。以P<0.05为差异有统计学意义。
(4)结果
<1> Etk/Bmx与小细胞肺癌病人临床分期、生存期的关系①、在局限期SCLC中,Etk/Bmx的阳性表达率为63.6%,而在广泛期SCLC中Etk/Bmx的阳性表达率为100%,表明Etk/Bmx的表达量与SCLC的临床分期呈正相关。②、生存期小于1年的病例Etk/Bmx的阳性表达率为81.6%,而大于1年的病例阳性表达率为0%;Kaplan-Meier分析研究结果表明Etk/Bmx表达量越高,患者总生存期越短,预后越差。③、Etk/Bmx与患者性别、年龄无关。
<2> Etk/Bmx、Zeb-2、Twist、Vim、p-catenin在SCLC多药耐药细胞株和相应敏感细胞中的表达差异
①cDNA芯片检测分析SCLC细胞株H69与其多药耐药细胞株H69/AR中基因表达差异,发现Etk/Bmx、EMT间质标志物Zeb-2、Twist、Vim多药耐药细胞株H69/AR中的表达高于H69,上皮标志物β-catenin的表达低于H69,实时荧光定量PCR验证了以上结果,差异有统计学意义(详见正文)。
②实时荧光定量PCR和免疫印迹检测H69、H69/AR中上述各基因mRNA和蛋白的表达量,也发现多药耐药细胞株H69/AR中的表达高于H69,上皮标志物β-catenin的表达低于H69(详见正文)。
③实时荧光定量PCR和免疫印迹检测H446、H446/AR中上述各基因mRNA和蛋白的表达量,也发现多药耐药细胞株H446/AR中的表达高于H446,上皮标志物β-catenin的表达低于H446(详见正文)。
<3> Etk/Bmx通过抑制化疗药物诱导的细胞凋亡调节小细胞肺癌细胞的药物敏感性
①、将Etk/Bmx shRNA转染H69/AR和H446/AR细胞,Etk/Bmx过表达质粒转染H69和H446细胞,通过实时荧光定量PCR (qRT-PCR)和免疫印迹法证明本课题组成功建立了稳定低表达(H69/AR/sh、H446/AR/sh)和稳定高表达(H69/Et、H446/Etk) Etk/Bmx的细胞株；
②、通过CCK-8法检测下调或者上调Etk/Bmx对小细胞肺癌药物敏感性的影响,结果表明H69/AR/sh和H446/AR/sh对化疗药物的IC50值降低,药物敏感性升高,而H69/Etk和H446/Etk对化疗药物的IC50值增高,药物敏感性降低；
③、流式细胞仪的结果显示下调Etk/Bmx的表达后化疗药物(阿霉素ADM、顺铂DDP、依托泊苷VP-16)诱导的细胞凋亡率增加,反之,上调Etk/Bmx的表达后化疗药物(阿霉素ADM、顺铂DDP、依托泊苷VP-16)诱导的细胞凋亡率增加。
<4> ETK/BMX促进小细胞肺癌细胞的增殖、迁移、侵袭和成瘤能力
①、CCK-8增殖实验、划痕愈合实验、transwell侵袭实验证明：H69/AR/sh和H446/AR/sh细胞的增殖、迁移、侵袭能力明显低于相应的母细胞,而H69/Etk和H446/Etk的增殖、迁移、侵袭能力显著高于对照组细胞。表明增加Etk/Bmx的表达可促进肿瘤细胞的增殖、迁移、侵袭能力,降低Etk/Bmx的表达则相反；②、利用稳定低表达(H69/AR/sh、H446/AR/sh)和高表达Etk/Bmx (H69/Etk、H446/Etk)的细胞株建立裸鼠皮下成瘤模型,观察对裸鼠成瘤能力和生存期的影响。结果显示：高表达Etk/Bmx的细胞株其成瘤能力明显增加,裸鼠生存时间缩短；而低表达Etk/Bmx的细胞株其成瘤能力明显降低,裸鼠生存时间延长。
<5>EMT参与小细胞肺癌耐药机制的调节
①、用eb-2siRNA和Twist siRNA转染小细胞肺癌耐药细胞株H69/AR和H446/AR,通过qRT-PCR和免疫印迹法证明Zeb-2siRNA和Twist siRNA成功下调H69/AR和H446/AR中Zeb-2和Twist mRNA和蛋白的表达,上皮标志物β-catenin mRNA和蛋白表达量增加,间质标志物Vim mRNA和蛋白表达量降低,逆转EMT。
②、CCK-8药物敏感性实验表明下调Zeb-2或Twist的表达后细胞对化疗药物(阿霉素ADM、顺铂DDP、依托泊苷VP-16)的IC50值降低,药物敏感性升高。提示EMT参与SCLC耐药机制的调节
<6>ETK/BMX通过EMT途径参与小细胞肺癌耐药机制的调节
①、 H69/AR/sh和H446/AR/sh细胞株中EMT间质标志物(Zeb-2, Twist, Vim)mRNA和蛋白表达量降低,上皮标志物β-catenin mRNA和蛋白表达量升高,而H69/Etk和H446/Etk则相反。从而表明Etk/Bmx参与调节EMT过程。
以上研究结果表明Etk/Bmx可能通过EMT参与SCLC耐药机制的调节。
<7>miR-495通过靶向ETK/BMX参与小细胞肺癌耐药机制的调节
①、miR-495mimics显著上调H69/AR和H446/AR中miR-495的表达,而miR-495antagomir则抑制H69和H446中miR-495的表达。且上调miR-495的表达后肿瘤细胞的IC50值降低,药物敏感性升高,而下调miR-495肿瘤细胞的IC50值升高,药物敏感性降低。表明miR-495参与调节SCLC的药物敏感性。
②、生物信息学软件分析表明Etk/Bmx可能是miR-495的靶基因之一,为确定两者之间的关系,我们通过qRT-PCR和免疫印迹法发现上调miR-495的表达后Etk/Bmx mRNA和蛋白表达量降低,下调miR-495则相反。
③、荧光素酶实验表明上调miR-495可引起Etk/Bmx荧光素酶活性降低,下调miR-495可引起Etk/Bmx荧光素酶活性升高,进一步证明了Etk/Bmx是miR-495的靶基因之一。以上研究结果表明Etk/Bmx在miR-495的调控下参与SCLC耐药机制的调节。
以上研究结果表明Etk/Bmx可能在miR-495的调节下参与SCLC耐药机制的调节。
(5)结论
1、Etk/Bmx的表达量与SCLC的临床分期呈正相关；Etk/Bmx表达量越高,患者总生存期越短,预后越差。
2、降低或增加Etk/Bmx表达可以引起细胞的药物敏感性以及化疗药物诱导的细胞凋亡率的变化,表明Etk/Bmx可能通过抑制肿瘤细胞凋亡从而增加对化疗药物的抵抗性。
3、降低或增加Etk/Bmx表达可以引起细胞增殖、迁移、侵袭、成瘤能力相应的降低或者升高。
4、下调Zeb-2或Twist的表达后细胞对化疗药物的IC50值降低,药物敏感性升高。提示EMT参与SCLC耐药机制的调节
5、动物实验结果与细胞学结果表明Etk/Bmx可调节EMT相关标志物的改变,表明Etk/Bmx可能通过EMT参与SCLC耐药机制的调节。
6、miR-495可调节SCLC的药物敏感性；Etk/Bmx是miR-495的靶基因之一。表明Etk/Bmx在miR-495的调控下参与SCLC耐药机制的调节。
综上所述,本课题组的研究结果表明Etk/Bmx在miR-495的调控下,通过诱导EMT过程参与SCLC耐药机制的调节。Etk/Bmx可能是预测SCLC药物敏感性的可靠标志物之一,下调Etk/Bmx的表达可抑制SCLC的药物抵抗性。
INTRODUCTION
Lung cancer is one of the leading causes for cancer death throughout the world at present. Small cell lung cancer (SCLC) accounted for around15%of all lung cancers1,2. The2-year survival rate is generally less than5%in patients with SCLC and90%of patients often die within5years after final diagnosis. Chemotherapy remains a major treatment for SCLC. Though it shows remarkable sensitivity to chemotherapeutic drugs, SCLC is characterized by high relapse rates and subsequent poor prognosis. The aggressiveness of SCLC could, in part, be due to their intrinsic and extrinsic chemoresistance3,4. Therefore, it is increasingly challenging to understand the molecular mechanism of chemoresistance and develop effective therapeutic strategies to overcome drug resistance of SCLC.
Epithelial and endothelial tyrosine kinase (Etk), also known as bone marrow X kinase (Bmx), is one of the important Tec family members of non-receptor tyrosine kinase5"7. It has been reported that Etk/BMX is involved in various biological processes, including proliferation, differentiation, apoptosis and cell migration8-10. Besides, our previous studies found that Etk/BMX participated in the chemoresistance. We knockdown Etk/BMX expression in multi-drug resistant SCLC H69/AR cells by Etk/BMX specific small interfering RNA, leading to increased sensitivity of H69/AR cells to chemotherapeutic drug11. Thus far, the mechanism of chemoresistance mediated by Etk/BMX in SCLC remains to be elucidated.
Accumulating studies have revealed that EMT is linked with drug resistance such as in bladder cancer and non-small cell lung caner12>13. The hallmark of EMT is loss of the epithelial markers, such as E-cadherin and P-catenin, and gain of mesenchymal molecules, such as N-cadherin and Vimentin (Vim). The factors containing Snail, Slug, Twist, and so on, which interact with E-box elements, are significant inducers of EMT by repressing E-cadherin expression14,15. Whether EMT plays an important role in drug resistance of SCLC has not been reported yet. We analyzed the data from cDNA microarray in cellular models of SCLC which were widely used as sensitive (H69) and resistant (H69/AR) cell lines to chemotherapy in previous study. The results showed that EMT related markers (Zeb-2, Twist, Vim and P-catenin) were significantly different (>3-fold)(Supplementary figure1). But it remains unclear whether aberrant expression of these EMT-associated genes is directly responsible for chemoresistance in SCLC and regulated by Etk/BMX.
It is becoming clear that miRNAs play a pivotal role in the drug resistance16,17. For example, up-regulation of miR-214induces cell survival and cisplatin resistance primarily through targeting the PTEN/Akt pathway in human ovarian cancer'. Over-expression of miR-216a/217in hepatocellular carcinoma cells results in an acquired resistance to sorafenib by activating the PI3K/Akt and TGF-p pathways19. We also found miR-495was differentially expressed and closely associated with chemoresistance of SCLC. Furthermore, the sensitivity of H69/AR cells to anti-cancer drugs greatly increased following transfection with the miR-495mimics20. Besides, the bioinformation analysis implicated that Etk/BMX may be a targeted gene of miR-495. Whether miR-495directly influenced the expression of Etk/BMX should be further confirmed.
As mentioned above, we proposed the hypothesis that Etk/BMX involving the chemoresistance of SCLC may be under the regulation of Etk/BMX and through EMT process. To prove this hypothesis, we performed two drug resistant cells H69/AR and H446/AR for a series of assays. Loss-and gain-of-function experiments showed Etk/BMX can regulate cancer cells proliferation, migration, invasion and tumor growth. Moreover, we found that up-expression of Etk/BMX in SCLC specimens correlated with poor pathologic stage and survival time.
MATERIALS AND METHODS
Tissue preparation and Immunohistochemical staining
Eighty-six SCLC samples were obtained by biopsy in our department since2005.1-2009.1. All patients had not received chemotherapy. This study was approved by the Ethics Committee of the Southern Medical University. Each case was independently reviewed by two pathologists according to the WHO criteria.
Tissues sections were deparaffinized and rehydrated routinely. Before adding the primary antibody, antigen was retrieved by heating sections. After blocking with0.3%H2O2and goat serum, the slides were then incubated with a primary antibody (Etk,1:100; BD Biosciences, USA) at4℃overnight. Following three washings in PBS, an avidin biotin complex (Vector Labs, Burlingame, USA) and alkaline phosphatase mixture were applied. Reaction products were visualized by3'diaminobenzidine (DAB),5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) and3-amino-9-ethylcarbazole (AEC)(MaximBiotech, Inc CA, USA). All slides were subsequently counterstained with hematoxylin. Brown-yellow granules in cytoplasm were considered positive staining for Etk/BMX. Negative controls were performed by replacing the primary antibodies stated above with PBS.
Cell culture and transfection
The human small lung cancer cell line NCI-H446, NCI-H69and the corresponding drug resistant subline H69/AR were purchased from the American
Type Culture Collection (ATCC) and maintained in RPMI1640medium supplemented with10%new born bovine serum (GIBCO) at37℃with5%CO2. H446/AR cell had been developed as described previously28. The two resistant cell lines were tested regularly for maintained resistance to the selected drugs. Growth and morphology of all cell lines were monitored on a weekly basis.
The plasmid encoding the full-length Etk was kindly provided by Dr Hsing-Jien Kung29. The short hairpin RNA (shRNA) targeted Etk, the small interfering RNA (siRNA) targeted for Zeb-2and Twist, and the mimics and antagomir of miR-495were all supplied by Shanghai GenePharma Co, Ltd. The transfection process was according to the instruction of Lipofectamine2000(Invitrogen). Sequences for RNAs oligo were shown as following:
Name Sense sequence (5'-3') Antisense sequence (5'-3') miR-495AAACAAACAUGGUGCACUUC GAAGUGCACCAUGUUUGUU mimics UU UUU miR-495AAGAAGUGCACCAUGUUUG antagomir UUU Etk/BMX GGGAAATGGAATCTGGGAAC shl T Etk/BMX GCCGAAGTCAGTGGTTGAAA sh2G Zeb-2si1GGCAAGGCCUUCAAAUAUAT UAUAUUUGAAGGCCUUGCC T TT Zeb-2si2GACCACUCCAGGAGUAAUAT UAUUACUCCUGGAGUGGUC T TT Twist si1GAUGGCAAGCUGCAGCUAUT AUAGCUGCAGCUUGCCAUC T TT Twist si2GCAAGAUUCAGACCCUCAAT UUGAGGGUCUGAAUCUUGC T TT
Real-time quantitative reverse transcriptase-PCR (qRT-PCR)
Total RNA was extracted using Trizol reagent (Invitrogen), and quantified by NanoDrop2000. qRT-PCR analysis was performed to validate the mRNA expression levels. Reverse transcription reactions were performed for15min at37℃, followed by5s at85℃for complementary DNA synthesis. Real time reactions were performed using the SYBR(?) PrimeScriptTM RT-PCR Kit (Takara Biotechnology Co, Ltd. Dalian, China). Glyceraldehyde3-phosphate dehydrogenase (GAPDH) or U6snRNA was used as the housekeeping gene. The mRNA expression levels of all samples were normalized to the housekeeping gene and analyzed using the comparative expression level2"△△Ct method. The all reactions were performed at least three times. Sequences for primers were shown as following: Name Forward primer (5'-3') Reverse primer (5'-3') Etk/BMX CATAGTGGGTTCTTCGTGGAC TGCCCGAGGTATCTTCAGC Zeb-2GGTCCAGATCGAAGCAGCTCA GTGACTATGTTTGTTCACAT AT T Twist CAGTCTTTACGAGGAGCTG GCTTGAGGGTCTGAATCT Vim TGCTAACTACCAAGACACT GTAGGTGCAATCTCAATG β-catenin ACAAACTGTTTTGAAAATCCA CGAGTCATTGCATACTGTCC GAPDH AGAAGGCTGGGGCTCATTTG AGGGGCCATCCACAGTCTT C
Western blot analysis
Cell proteins were extracted with RIPA lysis buffer and determined by the standard BCA method (BCATM Protein Assay Kit, Pierce, USA). Each protein sample was homogenized in the loading buffer and boiled for5min, then separated on8%SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Etten-leur, Netherlands). The membranes were blocked with5%non-fat dried milk for2h, then treated with primary antibodies including Etk, Zeb-2, Twist, Vim, and β-catenin overnight at4℃. The membranes were washed again with TBST (10mM Tris, pH8.0,150mM NaCl, and0.1%Tween20), followed by incubation with horseradish peroxidase-labeled secondary antibody (Beijing Biosynthesis Biotechnology Co. Ltd.) for45min at room temperature. Finally, after treatment with super echochemiluminescence (ECL) plus detection reagents, the protein bands of membranes were visualized by exposure to X-ray film. The intensity of protein bands were quantified by Quantity One software.
In vitro drug resistance assay
The ranges of drug concentrations were based on earlier studies and aimed at obtaining half maximal (50%) inhibitory concentration of a substance (IC50)11.30. A total of three anticancer drugs Cisplatin (DDP; Shangdong, China), Etoposide (VP-16; Jiangshu, China), and Adriamycin (ADM; Jiangshu, China) were obtained from commercial sources and were dissolved according to the manufacturer's instructions. Anti-cancer drugs induced cell death was quantified using the Cell Counting Kit-8assay (CCK-8). Cells were seeded into96-well plates and then treated for24h in200 μl of medium with anti-cancer drugs. CCK-8reagent (Dojindo, Kumamoto, Japan) was then added and the cells were incubated at37℃for2h before reading the absorbency using a micro-plate reader at450nm. The assay was conducted in six replicate wells for each sample and three parallel experiments were performed.
Cell proliferation assay
The cells were seeded in96-well plates at2×103cells/well. At the indicated times (0h,24h,48h,72h, and96h),10μl cck-8and100μl RPMI1640were added to each well. The cells were incubated for2h and absorbance at450nm was measured to calculate cell growth rates. Growth rate=(absorbance at450nm at x h-absorbance at450nm at0h)/(absorbance at450nm at0h).
Cell scratch-wound healing assay
The cells were seeded on24-well plates and grown to monolayer. Wound areas were scraped using100μl plastic tips. At the indicated times (0h,12h, and24h), wound areas were photographed and the wound healing rate was calculated. Healing rate=(width of wound at x h-width of the wound at0h)/width of wound at0h.
Cell invasion assay
Using24-well transwell units with8μm pore size polycarbonate insert, matrigel (50μl) as a basement membrane was spread on the polycarbonate membrane and allowed to solidify for1h at room temperature. Cells suspended in RPMI1640containing5g/L BSA were added to each upper compartment of the transwell units. After being cultured for48h, cells migrating through the matrigel-coated polycarbonate membrane were fixed by paraformaldehyde, stained with crystal violet and counted in five different fields randomly.
Flow cytometry assay for cell apoptosis
The cells treated with or without chemotherapeutic drugs including DDP, VP-16and ADM were harvested, washed with phosphate buffered saline (PBS), and resuspended in binding buffer containing7-AAD for10minutes, followed by the addition of Annexin V-PE. Cell apoptosis analysis was carried out using a flow cytometer (BD Biosciences, Oxford, United Kingdom).
In vivo tumorigenicity assay
Male nude mice (six weeks old, weighing18-20g, from the Medical Experimental Animal Center of Guangdong Province, China) were used for in vivo assay. The mice were raised under pathogen-free conditions. All procedures were performed according to guidelines of Association for Assessment and Accreditation of Laboratory Animal Care International.
Cells were harvested, washed with PBS and re-suspended in normal culture medium at a concentration of1x107/0.1ml. Cells in RPMI1640were subcutaneously inoculated into the legs of nude mice to establish the tumor model, respectively. The tumor volume was determined three times a week by direct measurement with sliding caliper and calculated by the following equation:V=(4/3)×71×R12×R2, where Rl is radius1and R2is radius2and R1 Luciferase reporter assay
The wild and mutational3'UTR segments of Etk/BMX predicted to interact with miR-495were amplified by PCR from human genomic DNA and inserted into psiCHECK-2vector immediately downstream from the stop codon of luciferase (Promega) to develop psiCHECK2-Etk/BMX-3'UTR and psiCHECK2-Etk/BMX-mut-3'UTR. Cells in24-well plates were transfected with psiCHECK2-Etk/BMX-3'UTR, psiCHECK2-Etk/BMX-mut-3'UTR or psiCHECK-2, respectively. Besides, miR-495mimics and antagomir was also con-transfected into the cells, respectively. Luciferase activity was assayed at48h post-transfection using a dual-luciferase reporter assay system (Promega).
Statistical analysis
Data were analyzed using SPSS20.0software. Results are presented using means±SD. Comparison of means between two samples was performed using Student's t test. Statistical comparisons of more than two groups were performed using one-way analysis of variance (ANOVA). and then multiple comparisons were performed using least-significant difference (LSD). The association between Etk/BMX expression and clinical features were analyzed by Pearson Chi-Square test. Survival curves were obtained by Kaplain-Meier analysis. In all cases, P<0.05was considered statistically significant.
RESULTS
Relationship between ETK/BMX expression and clinicopathological characteristics in SCLC patients
Table1summarized the relationship between Etk/Bmx expression and clinical characteristics in SCLC patients. Of the66cases with limited stage small cell lung cancer,42(63.6%) were expressed for Etk/Bmx, while all20(100.0%) cases were positive in patients with extensive stage. The expression of Etk/Bmx revealed obvious correlation with the pathologic stage (P=0,001). Positive expression for Etk/Bmx was more frequent in cases with survival rate<1year (62/76,81.6%) than that in cases with survival rate>1year (0/10,0.0%)(P<0.001). The expression of Etk/Bmx exhibited no significant relationship with gender (P=0.448) and age (P=0.106). The Kaplan-Meier analysis revealed that Etk/Bmx expression was closely associated with overall survival rate of the SCLC patients (Figure1).
Etk/Bmx was involved in chemoresistance of SCLC by inhibiting apoptosis induced by chemotherapeutic drugs
The drug resistant cells H69/AR and H446/AR showed greatly elevated mRNA and protein expression of Etk/Bmx compared to their parental cells by qRT-PCR and Western blot assay (Figure2A). To further study the role of Etk/Bmx in SCLC cells, we developed Etk/Bmx stable over-expression cells (H69/Etk and H446/Etk) and down-expression cells (H69/AR/sh and H446/AR/sh)(Figure2B). Then, we analyzed the viabilities of SCLC cells when exposed to chemotherapeutic drugs. The IC50values to chemotherapeutic drugs were greatly decreased in H69/AR/sh and H446/AR/sh cells, while notably increased in H69/Etk and H446/Etk cells (Table2). These data indicated that Etk/Bmx modulated SCLC cells to chemotherapeutic drugs.
Subsequently, the number of apoptotic rate was analyzed following the cells treated with chemotherapeutic drugs including ADM, DDP and VP-16using flow cytometry assay. The cell apoptosis was significantly suppressed in H69/Etk and H446/Etk cells. However, it was obviously increased in H69/AR/sh and H446/AR/sh cells (Figure2C and Supplementary figure2). These data provided strong indication that Etk/Bmx was involved in chemoresistance of SCLC by inhibiting apoptosis induced by chemotherapeutic drugs.
ETK/BMX promoted cancer cells proliferation, migration, invasion, tumor growth and shortened the survival time
To investigate the biological roles of Etk/Bmx in SCLC cells, the cell proliferation was assessed by CCK-8assay. Comparing with the results from the control cells, H69/Etk and H446/Etk cells presented a significant increase of the cell growth rates, while H69/AR/sh and H446/AR/sh cells showed obviously decreased cell growth rates (Figure3A). We also detected the effects of Etk/Bmx on migration and invasion ability by scratch-wound healing and transwell-invasion assay. As shown in Figure3B and C, Etk/Bmx over-expression enhanced the healing rates of H446cells. However, knockdown of Etk/Bmx in H69/AR and H446/AR cells inhibited the healing rates (Figure3B). For transwell-invasion assay, more H446/Etk cells penetrated the matrigel-coated membrane than the control H446cells did. By contrast, H69/AR/sh and H446/AR/sh cells showed a notably reduced number of the cells penetrating the matrigel-coated membrane (Figure3C).
The tumorigenic properties of Etk/Bmx in vivo were conducted in nude mice xenograft. The nude mice were subcutaneously inoculated with Etk/Bmx over-or down-expressing SCLC cells. The tumors of H69/Etk and H4467Etk cells grew significantly rapidly compared with that of H69and H446cells. But the tumors grew slowly in H69/AR/sh and H446/AR/sh cells. The null mice presented longer survival time accompanied by knockdown of Etk/Bmx, while shorter survival time with elevation of Etk/Bmx (Figure3D).
Taken together, both loss-and gain-of-function experiments demonstrated that Etk/Bmx promoted the SCLC proliferation, migration, invasion and tumor growth, but shorten the survival time.
Reversing EMT reduced the chemoresistance of SCLC
Our previous studies had showed that the EMT related markers (epithelial molecule P-catenin, and the mesenchymal markers including Zeb-2, Twist, and Vim) expressed significantly between H69and H69/AR cells by cDNA microarray (Supplementary figure1). In the current study, we further examined the expression of related markers in H69/AR and H446/AR cells at mRNA and protein level. The results were consistent with the data from cDNA microarray (Figure2A). Zeb-2and Twist have been documented as the important transcriptional repressors of EMT12. Knockdown of Zeb-2and Twist can cause a reversal of EMT21,22. Then, the Zeb-2and Twist expression were inhibited with Zeb-2siRNA or Twist siRNA in H69/AR and H446/AR cells. The expression of Vim was subsequently suppressed while β-catenin was increased accompany with the decreased Zeb-2and Twist. The results indicated that down-regulation of Zeb-2and Twist reversed the EMT process (Figure4). We further investigated whether reversing EMT can influence the drug sensitivity of SCLC cells. The results showed the IC50values of the cells down-expression Zeb-2and Twist were notably reduced when cells were exposed to chemotherapeutic drugs including ADM, DDP and VP-16(Table3). It suggested that the drug resistance to chemotherapeutic drugs was inhibited with reversing EMT in SCLC.
Etk/BMX regulated chemoresistance of SCLC through EMT
As mentioned above, the EMT process participated in the regulation of drug resistance of SCLC. We further studied whether Etk/BMX modulated the sensitivity of SCLC cells to chemotherapeutic drugs through EMT. The expression of Zeb-2, Twist, Vim and β-catenin was evaluated after up or down-regulation of Etk/BMX. The results revealed that the expression of Zeb-2, Twist and Vim at mRNA and protein level was remarkably increased while β-catenin was inhibited by over-expression of Etk/BMX (Figure5A). In contrast, suppression of Etk/BMX reduced the expression of Zeb-2, Twist and Vim, but promoted the expression of β-catenin (Figure5B). We also analyzed the expression of the EMT markers stated above in xenograft tumors. The results were consistent with the data from the cell lines (Figure5C).
Our observations provided valid evidence that Etk/BMX may influence SCLC chemoresistance through EMT process.
miR-495modulated chemoresistance by targeting Etk/BMX
The sensitivity of the H69/AR cells to anti-cancer drugs was greatly increased following transfection with the miR-495mimics as described previously20. To better understand the roles of miR-495in the drug sensitivity of SCLC cells, both H69/AR and H446/AR cells were used to quantify the expression of miR-495by qRT-PCR. The H69/AR and H446/AR cells presented lower level of miR-495than their corresponding control groups. Then, the miR-495expression was up-regulated in H69/AR and H446/AR cells by miR-495mimics and down-regulated in H69and H446cells by antagomir, respectively (Figure6A). Furthermore, the effect of miR-495on the drug sensitivity of SCLC cells was analyzed. We found that H69and H446cells treated with miR-495antagomir exhibited higher IC50values to chemotherapeutic drugs including ADM, DDP and VP-16. In contrast, the IC50values were obviously declined in H69/AR and H446/AR cells by miR-495mimics (Table4). These findings demonstrated that the miR-495was related to chemoresistance of SCLC cells.
Considering on Etk/BMX as a potential targeted gene of miR-495by the bioinformation analysis (Figure6C), we examined the Etk/BMX expression after modulation of miR-495by qRT-PCR and Western blot. The result showed that the Etk/BMX mRNA and protein expression were elevated in H69and H446cells by inhibition of miR-495, while decreased in H69/AR and H446/AR cells as a result of up-regulation of miR-495(Figure6B). The correlation between miR-495and Etk/BMX was further detected by luciferase reporter assay. The Etk/BMX luciferase activity was significantly declined in H69/AR cells treated with miR-495mimics, but obviously elevated by treatment with miR-495antagomir (Figure6C).
For the reasons mentioned above, all these results suggested that the influence of Etk/BMX on SCLC chemoresistance may be regulated by miR-495.
DISCUSSION
It has been reported that Etk/BMX is associated with various cellular processes including proliferation, differentiation, apoptosis and tumorigenicity8"10. In this study, we investigated the influence of Etk/BMX on the biological behaviors of SCLC by loss-and gain-of-function experiments. The proliferation, migration and invasion abilities were decreased after suppression of Etk/BMX, while they were all elevated following the up-regulation of Etk/BMX. The in vivo data revealed that over-expression of Etk/BMX can promote tumor growth and prolong survival time. By clinical samples, we observed that expression of Etk/BMX showed closely relationship with poor pathologic stage and survival time. In sum, our findings indicated that Etk/BMX played an important role in the biological behaviors of SCLC, suggesting that Etk/Bmx may serve as a predictor for poor prognosis in SCLC.
Our previous studies had showed that suppression of Etk/BMX expression reduced the chemoresistance of H69/AR cells. In this study, we further confirmed the effect of Etk/BMX on chemoresistance of SCLC using two multi-drug resistant cells H69/AR and H446/AR cells. The H69/AR/sh and H446/AR/sh cells showed much more sensitivity to chemotherapeutic drugs. In contrast, over-expression of Etk/BMX in H69and H446cells reduced the drug sensitivity. Besides, our research revealed that the cell apoptosis induced by chemotherapeutic drugs decreased significantly after up-regulation of Etk/Bmx, but it increased obviously with knockdown of Etk/Bmx. There was a negative correlation between Etk/BMX and cell apoptosis. These results provided sufficient proof to demonstrate that Etk/Bmx affected the chemoresistance of SCLC by inhibiting apoptosis induced by chemotherapeutic drugs.
Studies have shown that EMT process confers resistance to chemotherapy in ovarian carcinoma, colorectal cancer and so on23-25. Base on our previous study, the role of EMT in SCLC chemoresistance was firstly clarified in this study. Inhibition of Zeb-2and Twist in H69/AR and H446/AR cells led to declined Vim while elevated β-catenin. The results indicated that suppression of Zeb-2and Twist can reverse the EMT process. Furthermore, the IC50values to chemotherapeutic drugs were suppressed after knockdown of Zeb-2and Twist. It suggested that EMT was associated with chemoresistance of SCLC. Subsequently, the relationship between Etk/BMX and EMT was explored. The H69/AR/sh and H446/AR/sh cells exhibited a significant decrease in the expression of mesenchymal markers, while increased epithelial markers. However, the H69and H446cells presented an opposite results by elevation of Etk/BMX. The expression of the EMT markers stated above in xenograft tumors was consistent with the data from the cell lines. Taken together, Etk/BMX modulated the chemoresistance of SCLC possibly through EMT process.
miR-495has been reported as a tumor suppressor by inhibiting gastric cancer cell migration and invasion26. Ectopic expression of miR-495in breast cancer cells promotes tumorigenesis in the mice27. Our previous study firstly reported that transfection of the H69/AR cells with the miR-495mimics significantly reduced the chemoresistance. In the present study, we used two drug resistant cells to further investigate the role of miR-495in drug sensitivity of SCLC. Up-or down-regulation of miR-495can modulate IC50values to chemotherapeutic drugs, indicating miR-495was involved in the chemoresistance of SCLC cells. Elevation of miR-495resulted in decreased expression of Etk/BMX. On the contrary, Etk/BMX was over expressed following the reduced expression of miR-495. The result from luciferase reporter assay confirmed that Etk/BMX was one of the directly targeted genes of miR-495. All these data presented that miR-495may participate in the chemoresistance and regulate the expression of Etk/BMX in SCLC.
Taken together, our study supported for the first time that elevation of Etk/BMX can led to chemoresistance of SCLC by EMT process and was under the regulation of miR-495. Therefore, our research provided a new insight into the mechanism of SCLC chemoresistance. Etk/BMX can be a useful predictor for drug sensitivity, raising the possibility of Etk/BMX depletion as a promising strategy for interfering with chemoresistance in SCLC.
肺癌已经成为恶性肿瘤患者死亡的主要疾病,全球每年由肺癌导致的死亡人数超过100万,约15%的患者为小细胞肺癌。SCLC分化程度低,生长迅速,较早发生转移,预后很差,一般2年存活率不足5%,90%的患者在确诊的5年内死亡。由于患者对化疗敏感,所以化疗是SCLC的主要治疗方式。但是SCLC很容易发生耐药,导致化疗失败,因此,SCLC的化疗耐药性已成为肺癌临床治疗急需解决的重要问题之一。
上皮及内皮酪氨酸激酶(Etk/Bmx)是Btk家族(Btk, Itk, Etk/Bmx, Txk和Tec)中的重要成员,是Btk家族中唯一除分布于淋巴造血细胞外,还在上皮细胞性瘤细胞和血管内皮细胞中表达的非受体类酪氨酸激酶。研究发现Etk/Bmx的异常表达参与肿瘤细胞增殖、凋亡、血管生成等方面的调控,与肿瘤细胞恶性生物学行为密切相关。有少量研究结果提示Etk/Bmx参与肿瘤细胞耐药的调节。
上皮细胞向间质细胞的转化(epithelial to mesenchymal transition, EMT)是指上皮细胞在一些因素的作用下,失去细胞极性,丢失细胞间紧密连接和黏附连接,获得了浸润性和游走迁移能力,变成了具有间质细胞形态和特性的细胞。研究发现EMT与肿瘤耐药密切相关。
微小RNA (microRNA, miRNAs)是一类广泛存在与生物体内,由19-25个核苷酸组成的非编码RNA,参与基因转录后水平的调控,在机体的生长发育过程中发挥重要的作用。大量的研究表明miRNAs与肿瘤发生、肿瘤干细胞的分化、血管生成等密切相关。近年来研究表明miRNAs可通过调节与药物代谢、细胞增殖、凋亡等相关基因影响肿瘤细胞的耐药。
本课题组长期以来从事小细胞肺癌耐药机制的研究,前期在国家自然基金的资助下已经发现Etk/Bmx可能参与小细胞肺癌耐药的调控,但是具体机制尚未清楚。而前期利用cDNA基因芯片对比小细胞肺癌耐药细胞株H69/AR和亲本细胞H69,结果发现与EMT相关的主要间质标志物(Zeb-2, Twist, Vim)在H69/AR中的表达明显高于母细胞。因此,我们推测EMT可能参与小细胞肺癌耐药的调节。我们还利用miRNA表达谱芯片发现miR-495在H69/AR中的表达明显低于母细胞,提示miR-495可能参与小细胞肺癌耐药的调节。本课题组拟在前期研究工作基础上,采用分子生物学和细胞生物学技术,利用小细胞肺癌细胞株H69,H69/AR和H446和H446/AR,探讨：Etk/Bmx在niR-495的调节下,可通过透导EMT,参与细胞肺癌耐药机制的调节。研究内容包括以下几个方面：①、利用临床标本分析Etk/Bmx与患者病理分、预后的相关性。②、进一步确定Etk/Bmx在小细胞肺癌耐药中的作用：分别上调或者下调H69,H69/AR和H446, H446/AR中Etk/Bmx的表达,观察对细胞药物敏感性、凋亡的影响,③、观察Etk/Bmx对小细胞肺癌恶性生物学行为的影响：(增殖、迁移、侵袭、成瘤能力的影响)④、确定EMT在小细胞肺癌耐药中的作用:分别下调H69,H69/AR和H446, H446/AR中EMT主要间质标志物Zeb-2, TWIST的表达,观察下调Zeb-2, TWIST的表达后,对细胞药物敏感性的影响。⑤、确定Etk/Bmx对EMT的靶向调节作用：用Etk/Bmx的干扰或过表达质粒下调或者上调H69,H69/AR和H446中Etk/Bmx (?)勺表达,观察对EMT主要间质标志物Zeb-2, Twist, Vim和上皮标志物(3-catenin的调节作用。⑥、确定miR-495小细胞肺癌药物敏感性的影响以及对Etk/Bmx的靶向调节作用。通过本项目研究,进一步丰富小细胞肺癌耐药的分子机制,期望能筛选出预测小细胞肺癌耐药的分子标志物,为下一步开展基于Etk/Bmx通路逆转小细胞肺癌耐药提供依据。
(2)研究目的
在课题组前期研究的基础上,采用细胞生物学和分子生物学技术,利用小细胞肺癌耐药细胞H69/AR, H446/AR和相应的母细胞H69,H446,探讨Etk/Bmx在miR-495的调节下,可通过诱导EMT,参与小细胞肺癌耐药机制的调节。通过本项目研究,进一步丰富小细胞肺癌耐药的分子机制,期望能筛选出预测小细胞肺癌耐药的分子标志物,为下一步开展基于Etk/Bmx通路逆转小细胞肺癌耐药的研究提供依据。
(3)研究方法
<1>细胞培养
人小细胞肺癌细胞株H69, H69/AR, H446, H446/AR在37℃5%C02条件下,培养于含新生牛血清的RPMI-1640中,每周传代2-3次。<2>实时荧光定量PCR检测mRNA水平
用Trizol提取对数生长期细胞中总RNA,按实时荧光定量RT-PCR试剂盒说明进行实验。合成cDNA的反应条件为：15分钟37。C,5秒85°C。 PCR的反应条件为：预变性95℃30秒,PCR反应95℃5s,60℃20s(40个循环),溶解曲线分析95℃0秒,65℃15秒,95℃O秒。计算出2-△△α,代表每组mRNA的相对表达量。反应结束后确认Real Time PCR的扩增曲线和融解曲线,进行PCR定量时制作标准曲线等。目的基因的表达量=2-△△α△△ct=(待测样品的目的基因的Ct的平均值—待测样本的看家基因的Ct的平均值)—(对照样品的目的基因的Ct的平均值一对照样本的看家基因的Ct的平均值)
每组重复例数为3。由Invitrogen公司设计合成引物如下表：Name Forward primer (5'-3') Reverse primer (5'-3') Etk/BMX CATAGTGGGTTCTTCGTGGAC TGCCCGAGGTATCTTCAGC Zeb-2GGTCCAGATCGAAGCAGCTCA GTGACTATGTTTGTTCACAT AT T Twist CAGTCTTTACGAGGAGCTG GCTTGAGGGTCTGAATCT Vim TGCTAACTACCAAGACACT GTAGGTGCAATCTCAATG β-catenin ACAAACTGTTTTGAAAATCCA CGAGTCATTGCATACTGTCC GAPDH AGAAGGCTGGGGCTCATTTG AGGGGCCATCCACAGTCTT C
<3>免疫印迹(Western blot)检测蛋白表达
提取总蛋白,测定蛋白浓度,吸取总蛋白,去离子水补至20μL,加入上样buffer煮沸5min,离心后吸取25μL上样,经不连续SDS-PAGE电泳分离后,用半干法电转移至PVDF膜上,7%脱脂奶粉+3%的BSA在37℃封闭2h, TBST洗膜后用相应的一抗4℃孵育过夜,洗膜后分别加入HRP标记的兔抗羊IgG(1:4000),IgG(1：6000),室温下摇动1h,洗膜后用ECL化学发光系统检测,暗室曝光x线片,冲洗胶片,UVI凝胶成像系统摄像,Quantity One软件分析条带灰度值,用GnT-V/GAPDH代表GnT-V蛋白的相对表达量。每组重复例数为3。<4>细胞转染
调整细胞浓度为1×105/ml,按2mL/孔接种入6孔板,用无抗生素培养液培养16-24h以上至细胞密度达到85%左右。吸出丢弃原培养液,1×PBS轻轻冲洗2遍,按2m1/孔加入Opti-MEM I溶液。将Lipofectamin2000轻柔混匀,用Opti-MEMI240μL稀释10μL Lipofectamin2000,室温静置5min。将pEGFP-N1质粒和Etk/BMX-pEGFP-N1表达质粒各0.8g用Opti-MEM I稀释至终体积为250μL。将稀释后Lipofectamin2000和稀释后质粒轻柔混匀(总体积500μL),室温静置20min,以形成质粒DNA/Lipofectamin2000复合物。每孔对应加入500μL转染混合液,轻摇混匀。置于37℃,5%C02、饱和湿度的培养箱中培养。24小时后用含筛选浓度G418的20%胎牛血清的RPMI-1640培养液筛选。待形成阳性单细胞克隆群落后,用尖吸管吸取单克隆阳性细胞,连续培养4周后得到稳定转染细胞株,以含维持剂量的G418培养基维持培养。稳定转染细胞的鉴定：由于所转染质粒标记有eGFP,荧光激发后可显示绿色荧光,故可首先应用荧光显微镜观察质粒是否已转染入细胞。采用实时荧光定量PCR技术和Western Blot技术检测ETK/BMX表达情况。获得稳定转染细胞株H69/Etk和H446/Etk(过表达ETK/BMX), H69/AR/sh和H446/AR/sh (低表达ETK/BMX)。通过瞬时转染得到的细胞株有H69/AR/Zeb-2siRNA1、H69/AR/Zeb-2siRNA2, H446/AR/Zeb-2siRNA1、 H446/AR/Zeb-2siRNA2、H69/AR/Twist siRNA1、 H69/AR/Twist siRNA2、 H446/AR/Twist siRNA1、H446/AR/Twist siRNA2、 H69/AR/mimics,H446/AR/mimics、H69/antagomir, H446/antagomir。
<5>CCK-8法检测细胞药物敏感性
细胞处理后按5×103个/孔接种于96孔培养板中,每孔总体积100μ1。置于37℃,5%CO2细胞培养箱中培养24h后,将化疗药物阿霉素ADM,顺铂DDP,依托泊苷VP-16分别加入培养基中并进行倍比稀释,按不同浓度分别加入指定的孔中,并设不加药物的阳性对照孔。置于37℃,5%C02细胞培养箱中培养24h,加入CCK-8反应液,在37℃,5%CO2环境下培养4h,混匀后在酶标仪上检测450nm吸光度。测得的每个药物浓度的OD值的平均值-空白孔平均值／不含药物的阳性对照组平均值-空白孔平均值即为每个药物浓度下细胞的存活率。求得细胞50%生存率时的药物浓度(IC50)。实验分组如下：
①上调或者下调Etk/BMX对IC50的影响,分组如下：
H69类细胞分为：H69组、H69/NC组、H69/Etk组；
H446类细胞分为：H446组、H446/NC组、H446/Etk组；
H69/AR类细胞分为：H69/AR组、H69/AR/NC组、H69/AR/sh1组、H69/AR/sh2组；
H446/AR类细胞分为：H446/AR组、H446/AR/NC组、H446/AR/shl组、H446/AR/sh2组;
每组重复例数为3。
②下调Zeb-2或者Twist对IC50的影响,分组如下：
H69/AR类细胞分为：H69/AR、H69/AR/NC.H69/AR/Zeb-2siRNA1、H69/AR/Zeb-2siRNA2组;
H69/AR类细胞分为:H69/AR、H69/AR/NC、H69/AR/Twist siRNAl、H69/AR/Twist siRNA2组;
H446/AR类细胞分为：H446/AR、H446/AR/NC、H446/AR/Zeb-2siRNA1、 H446/AR/Zeb-2siRNA2组;
H446/AR类细胞分为：H446/AR、H446/AR/NC. H446/AR/Twist siRNAl、 H446/AR/Twist siRNA2组；
每组重复例数为3。
③上调或者下调miR-495对IC50的影响,分组如下：
H69类细胞分为：H69组、H69/NC组、H69/antagomir组;
H446类细胞分为：H446组、H446/NC组、H446/antagomir组;
H69/AR类细胞分为：H69/AR组、H69/AR/NC组、H446/AR/mimics组；
H446/AR类细胞分为：H446/AR组、H446/AR/NC组、H446/AR/mimics组；
每组重复例数为3。
<6>流式细胞术检测化疗药物诱导的细胞凋亡率
将细胞接种于6孔培养板。置于37℃,5%C02细胞培养箱中培养24h,加入化疗药物DDP、ADM、VP-16。置于37℃,5%C02细胞培养箱中培养48h,收集细胞,用PBS洗涤细胞两次。加入5ul Annexin V-FITC混匀后,加入5u1Propidium Iodide混匀。室温、避光反应15min。流式细胞仪检测。
实验分组
①ADM诱导的细胞凋亡率
H69类细胞分为：H69组、H69/NC组、H69/Etk组；
H446类细胞分为：H446组、H446/NC组、H446/Etk组；
H69/AR类细胞分为：H69/AR组、H69/AR/NC组、H69/AR/sh1组、H69/AR/sh2组；
H446/AR类细胞分为：H446/AR组、H446/AR/NC组、H446/AR/sh1组、H446/AR/sh2组；
每组重复例数为3。
DDP、VP-16诱导的细胞凋亡率分组同上。<7>CCK-8细胞增殖能力
取对数生长期的待测细胞,CCK-8法测量不同时间点的OD450nm值(Oh、24h、48h、72h、96h)。计算出细胞生长分数,绘制生长曲线。<8>划痕愈合实验检测细胞迁移能力
取对数生长期的待测细胞,接种于24孔板,培养至细胞呈单层贴壁生长状态。用100ul的枪头分别在各组单细胞层上划痕,用含10%FBS的培养基继续培养以使划痕愈合。分别于划痕后0h、12h、24h拍照,观察划痕愈合能力,用愈合率代表愈合能力。愈合率=[(愈合前两侧细胞层距离-愈合后两侧细胞层距离)/愈合前两侧细胞层距离]×100%。<9>细胞侵袭实验
Matrigel胶4℃过夜融化,每个Transwell上室加入50μL,从边缘轻轻加入,避免出现气泡,轻轻摇晃铺平Marigel胶,37℃10min凝固。取对数生长期的细胞,用无血清RPMI1640培养过夜。用0.25%胰酶消化细胞,1xPBS洗涤细胞,800r/min离心5min沉淀细胞,弃上清,将细胞重悬于氯化钙1mmol/L,氯化镁1mmol/L,氯化锰0.2mmol/L及5g/L BSA的RPMI1640中,调整细胞浓度为1×106/mL,上室每室加入200/μL细胞悬液,下室每室加500μL含10%新生牛血清的RPMI-1640培养液,37℃5%C02培养48h。取出上室,用棉签擦去膜上室面的Matrigel胶和细胞,4%多聚甲醛固定膜下室面的细胞15min,1×PBS清洗3次,每次5min,结晶紫染色3min,1×PBS清洗干净,小心用刀片将膜从小室中分离出来,将膜下室面朝上置于载玻片上,吹风机干燥后,中性树脂封固。细胞侵袭性结果表示为同时穿过Matrigel胶和聚碳酸酯膜的细胞数。
实验分组
H446类细胞分为：H446组、H446/NC组、H446/Etk组;
H69/AR类细胞分为：H69/AR组、H69/AR/NC组、H69/AR/shl组、H69/AR/sh2组;
H446/AR类细胞分为：H446/AR组、H446/AR/NC组、H446/AR/sh1组、H446/AR/sh2组;
每组重复例数为3。<10>裸鼠皮下成瘤实验
6周龄Balb/c裸鼠,雄性,体重(18±2)g,购自广东省动物实验中心,均置于SPF环境下饲养。将细胞培养至对数生长期,胰酶消化孵育后制作细胞悬液,用0.4%台盼蓝染色后计数活细胞比率>90%,PBS洗涤3遍后,用生理盐水重悬细胞,调整其浓度为107个/mL。置于冰上备用。医用碘伏消毒局部皮肤后,用1ml注射器,每组接种100μL癌细胞悬液于臀部皮下。注射后定期测量瘤体的长径、宽径,瘤体体积=长径X短径2/2；24天后,断颈处死裸鼠,剥离瘤体,液氮保存备用,检测组织中EMT相关标志物蛋白表达水平变化。同法建立裸鼠皮下成瘤模型,观察裸鼠存活天数。<11>荧光素酶实验
将psiCHECK-2载体转化至DH5aαTM, Ampicillin (100μg/ml)筛选,挑取单克隆。培养时间不超过14h,挑取三个克隆扩大培养,试剂盒提取质粒,酶切鉴定。采用CaCl2法,42℃/90sec热击转化入DH5αTM克隆菌株,经Ampicillin (100μg/ml)抗性选择。挑取三个克隆扩大培养。试剂盒提取质粒,Not I和Xho I双酶切鉴定psiCHECK-2-ETK/BMX-3'UTR-R.挑取酶切鉴定正确克隆进行测序鉴定。试剂盒(QIAGEN)提取质粒以进行荧光素酶报告基因检测。将psiCHECK-2-ETK/BMX-3'UTR-R、psiCHECK-2-ETK/BMX-S'UTR-mut或psiCHECK-2转染H69/AR细胞；共转染miRNA mimics、antagomir或miR-NC;裂解后检测荧光素酶活性。
<12>免疫组织化学检测Etk/BMX在SCLC中的表达
86例小细胞肺癌石蜡包埋组织取自2005年1月至2009年1月于南方医科大学珠江医院手术或支气管镜活检的小细胞肺癌患者。所有组织均经病理学确诊为小细胞肺癌,全部病例获取前均未行化疗和放疗,临床资料完整。其中男性69例,女性17例；局限期患者66例,广泛期患者20例.3瓶100%二甲苯脱蜡,3×10分钟；2瓶100%酒精脱水,2×8分钟；1瓶95%酒精脱水,5分钟1瓶85%酒精脱水,5分钟；1瓶75%酒精脱水,5分钟；1瓶50%酒精脱水,5分钟。1×PBS冲洗3次,每次3min。脱蜡后玻片放入盛有1%柠檬酸溶液中在微波炉内用中进行抗原修复高火7min,中火15min。冷却至室温后,1×PBS冲洗3次,间隔5min。每张玻片滴加1滴过氧化氢酶阻断剂,室温下孵育12min,1×PBS冲洗3次,间隔5min。每张玻片滴加1滴正常非免疫动物血清,室温下孵育10min。除去血清,阳性对照组每张玻片滴加1滴Etk/BMX一抗(1：100,抗体稀释液稀释),阴性对照组则加入1×PBS,放入湿盒内4℃孵育12h。1×PBS冲洗3次,每次5min,每张玻片滴加1滴生物素标记的羊抗兔二抗,室温下孵育40min,1×PBS冲洗3次,间隔5min。每张玻片滴加1滴链霉素抗生物素-过氧化物酶溶液,室温下孵育30min,1×PBS冲洗3次,间隔5min。每张玻片滴加2滴新鲜配制的DAB溶液.显微镜下观察5min。自来水冲洗去DAB溶液,苏木素复染10s,盐酸酒精分化,氨水返蓝。梯度酒精脱水干燥,吹风机吹干玻片,中性树胶封固。结果判定Etk/BMX阳性染色在胞浆,呈棕黄色。<13>、统计学分析
所有结果均经SPSS20.0统计软件统计。数据以均数±标准差(x±s)表示,两组间的比较采用独立样本t检验。多组间的比较采用完全随机设计的方差分析,进一步的多重比较采用LSD法(方差齐同时)或Dunnett's T3法(方差不齐)。以P<0.05为差异有统计学意义。Etk/BMX表达与临床病理特征分析采用列联表资料分析。多因素对SCLC患者生存时间的影响采用逐步Cox回归模型分析,各因素对SCLC患者生存时间的影响采用Kaplain-Meier分析。以P<0.05为差异有统计学意义。
(4)结果
<1> Etk/Bmx与小细胞肺癌病人临床分期、生存期的关系①、在局限期SCLC中,Etk/Bmx的阳性表达率为63.6%,而在广泛期SCLC中Etk/Bmx的阳性表达率为100%,表明Etk/Bmx的表达量与SCLC的临床分期呈正相关。②、生存期小于1年的病例Etk/Bmx的阳性表达率为81.6%,而大于1年的病例阳性表达率为0%;Kaplan-Meier分析研究结果表明Etk/Bmx表达量越高,患者总生存期越短,预后越差。③、Etk/Bmx与患者性别、年龄无关。
<2> Etk/Bmx、Zeb-2、Twist、Vim、p-catenin在SCLC多药耐药细胞株和相应敏感细胞中的表达差异
①cDNA芯片检测分析SCLC细胞株H69与其多药耐药细胞株H69/AR中基因表达差异,发现Etk/Bmx、EMT间质标志物Zeb-2、Twist、Vim多药耐药细胞株H69/AR中的表达高于H69,上皮标志物β-catenin的表达低于H69,实时荧光定量PCR验证了以上结果,差异有统计学意义(详见正文)。
②实时荧光定量PCR和免疫印迹检测H69、H69/AR中上述各基因mRNA和蛋白的表达量,也发现多药耐药细胞株H69/AR中的表达高于H69,上皮标志物β-catenin的表达低于H69(详见正文)。
③实时荧光定量PCR和免疫印迹检测H446、H446/AR中上述各基因mRNA和蛋白的表达量,也发现多药耐药细胞株H446/AR中的表达高于H446,上皮标志物β-catenin的表达低于H446(详见正文)。
<3> Etk/Bmx通过抑制化疗药物诱导的细胞凋亡调节小细胞肺癌细胞的药物敏感性
①、将Etk/Bmx shRNA转染H69/AR和H446/AR细胞,Etk/Bmx过表达质粒转染H69和H446细胞,通过实时荧光定量PCR (qRT-PCR)和免疫印迹法证明本课题组成功建立了稳定低表达(H69/AR/sh、H446/AR/sh)和稳定高表达(H69/Et、H446/Etk) Etk/Bmx的细胞株；
②、通过CCK-8法检测下调或者上调Etk/Bmx对小细胞肺癌药物敏感性的影响,结果表明H69/AR/sh和H446/AR/sh对化疗药物的IC50值降低,药物敏感性升高,而H69/Etk和H446/Etk对化疗药物的IC50值增高,药物敏感性降低；
③、流式细胞仪的结果显示下调Etk/Bmx的表达后化疗药物(阿霉素ADM、顺铂DDP、依托泊苷VP-16)诱导的细胞凋亡率增加,反之,上调Etk/Bmx的表达后化疗药物(阿霉素ADM、顺铂DDP、依托泊苷VP-16)诱导的细胞凋亡率增加。
<4> ETK/BMX促进小细胞肺癌细胞的增殖、迁移、侵袭和成瘤能力
①、CCK-8增殖实验、划痕愈合实验、transwell侵袭实验证明：H69/AR/sh和H446/AR/sh细胞的增殖、迁移、侵袭能力明显低于相应的母细胞,而H69/Etk和H446/Etk的增殖、迁移、侵袭能力显著高于对照组细胞。表明增加Etk/Bmx的表达可促进肿瘤细胞的增殖、迁移、侵袭能力,降低Etk/Bmx的表达则相反；②、利用稳定低表达(H69/AR/sh、H446/AR/sh)和高表达Etk/Bmx (H69/Etk、H446/Etk)的细胞株建立裸鼠皮下成瘤模型,观察对裸鼠成瘤能力和生存期的影响。结果显示：高表达Etk/Bmx的细胞株其成瘤能力明显增加,裸鼠生存时间缩短；而低表达Etk/Bmx的细胞株其成瘤能力明显降低,裸鼠生存时间延长。
<5>EMT参与小细胞肺癌耐药机制的调节
①、用eb-2siRNA和Twist siRNA转染小细胞肺癌耐药细胞株H69/AR和H446/AR,通过qRT-PCR和免疫印迹法证明Zeb-2siRNA和Twist siRNA成功下调H69/AR和H446/AR中Zeb-2和Twist mRNA和蛋白的表达,上皮标志物β-catenin mRNA和蛋白表达量增加,间质标志物Vim mRNA和蛋白表达量降低,逆转EMT。
②、CCK-8药物敏感性实验表明下调Zeb-2或Twist的表达后细胞对化疗药物(阿霉素ADM、顺铂DDP、依托泊苷VP-16)的IC50值降低,药物敏感性升高。提示EMT参与SCLC耐药机制的调节
<6>ETK/BMX通过EMT途径参与小细胞肺癌耐药机制的调节
①、 H69/AR/sh和H446/AR/sh细胞株中EMT间质标志物(Zeb-2, Twist, Vim)mRNA和蛋白表达量降低,上皮标志物β-catenin mRNA和蛋白表达量升高,而H69/Etk和H446/Etk则相反。从而表明Etk/Bmx参与调节EMT过程。
以上研究结果表明Etk/Bmx可能通过EMT参与SCLC耐药机制的调节。
<7>miR-495通过靶向ETK/BMX参与小细胞肺癌耐药机制的调节
①、miR-495mimics显著上调H69/AR和H446/AR中miR-495的表达,而miR-495antagomir则抑制H69和H446中miR-495的表达。且上调miR-495的表达后肿瘤细胞的IC50值降低,药物敏感性升高,而下调miR-495肿瘤细胞的IC50值升高,药物敏感性降低。表明miR-495参与调节SCLC的药物敏感性。
②、生物信息学软件分析表明Etk/Bmx可能是miR-495的靶基因之一,为确定两者之间的关系,我们通过qRT-PCR和免疫印迹法发现上调miR-495的表达后Etk/Bmx mRNA和蛋白表达量降低,下调miR-495则相反。
③、荧光素酶实验表明上调miR-495可引起Etk/Bmx荧光素酶活性降低,下调miR-495可引起Etk/Bmx荧光素酶活性升高,进一步证明了Etk/Bmx是miR-495的靶基因之一。以上研究结果表明Etk/Bmx在miR-495的调控下参与SCLC耐药机制的调节。
以上研究结果表明Etk/Bmx可能在miR-495的调节下参与SCLC耐药机制的调节。
(5)结论
1、Etk/Bmx的表达量与SCLC的临床分期呈正相关；Etk/Bmx表达量越高,患者总生存期越短,预后越差。
2、降低或增加Etk/Bmx表达可以引起细胞的药物敏感性以及化疗药物诱导的细胞凋亡率的变化,表明Etk/Bmx可能通过抑制肿瘤细胞凋亡从而增加对化疗药物的抵抗性。
3、降低或增加Etk/Bmx表达可以引起细胞增殖、迁移、侵袭、成瘤能力相应的降低或者升高。
4、下调Zeb-2或Twist的表达后细胞对化疗药物的IC50值降低,药物敏感性升高。提示EMT参与SCLC耐药机制的调节
5、动物实验结果与细胞学结果表明Etk/Bmx可调节EMT相关标志物的改变,表明Etk/Bmx可能通过EMT参与SCLC耐药机制的调节。
6、miR-495可调节SCLC的药物敏感性；Etk/Bmx是miR-495的靶基因之一。表明Etk/Bmx在miR-495的调控下参与SCLC耐药机制的调节。
综上所述,本课题组的研究结果表明Etk/Bmx在miR-495的调控下,通过诱导EMT过程参与SCLC耐药机制的调节。Etk/Bmx可能是预测SCLC药物敏感性的可靠标志物之一,下调Etk/Bmx的表达可抑制SCLC的药物抵抗性。
INTRODUCTION
Lung cancer is one of the leading causes for cancer death throughout the world at present. Small cell lung cancer (SCLC) accounted for around15%of all lung cancers1,2. The2-year survival rate is generally less than5%in patients with SCLC and90%of patients often die within5years after final diagnosis. Chemotherapy remains a major treatment for SCLC. Though it shows remarkable sensitivity to chemotherapeutic drugs, SCLC is characterized by high relapse rates and subsequent poor prognosis. The aggressiveness of SCLC could, in part, be due to their intrinsic and extrinsic chemoresistance3,4. Therefore, it is increasingly challenging to understand the molecular mechanism of chemoresistance and develop effective therapeutic strategies to overcome drug resistance of SCLC.
Epithelial and endothelial tyrosine kinase (Etk), also known as bone marrow X kinase (Bmx), is one of the important Tec family members of non-receptor tyrosine kinase5"7. It has been reported that Etk/BMX is involved in various biological processes, including proliferation, differentiation, apoptosis and cell migration8-10. Besides, our previous studies found that Etk/BMX participated in the chemoresistance. We knockdown Etk/BMX expression in multi-drug resistant SCLC H69/AR cells by Etk/BMX specific small interfering RNA, leading to increased sensitivity of H69/AR cells to chemotherapeutic drug11. Thus far, the mechanism of chemoresistance mediated by Etk/BMX in SCLC remains to be elucidated.
Accumulating studies have revealed that EMT is linked with drug resistance such as in bladder cancer and non-small cell lung caner12>13. The hallmark of EMT is loss of the epithelial markers, such as E-cadherin and P-catenin, and gain of mesenchymal molecules, such as N-cadherin and Vimentin (Vim). The factors containing Snail, Slug, Twist, and so on, which interact with E-box elements, are significant inducers of EMT by repressing E-cadherin expression14,15. Whether EMT plays an important role in drug resistance of SCLC has not been reported yet. We analyzed the data from cDNA microarray in cellular models of SCLC which were widely used as sensitive (H69) and resistant (H69/AR) cell lines to chemotherapy in previous study. The results showed that EMT related markers (Zeb-2, Twist, Vim and P-catenin) were significantly different (>3-fold)(Supplementary figure1). But it remains unclear whether aberrant expression of these EMT-associated genes is directly responsible for chemoresistance in SCLC and regulated by Etk/BMX.
It is becoming clear that miRNAs play a pivotal role in the drug resistance16,17. For example, up-regulation of miR-214induces cell survival and cisplatin resistance primarily through targeting the PTEN/Akt pathway in human ovarian cancer'. Over-expression of miR-216a/217in hepatocellular carcinoma cells results in an acquired resistance to sorafenib by activating the PI3K/Akt and TGF-p pathways19. We also found miR-495was differentially expressed and closely associated with chemoresistance of SCLC. Furthermore, the sensitivity of H69/AR cells to anti-cancer drugs greatly increased following transfection with the miR-495mimics20. Besides, the bioinformation analysis implicated that Etk/BMX may be a targeted gene of miR-495. Whether miR-495directly influenced the expression of Etk/BMX should be further confirmed.
As mentioned above, we proposed the hypothesis that Etk/BMX involving the chemoresistance of SCLC may be under the regulation of Etk/BMX and through EMT process. To prove this hypothesis, we performed two drug resistant cells H69/AR and H446/AR for a series of assays. Loss-and gain-of-function experiments showed Etk/BMX can regulate cancer cells proliferation, migration, invasion and tumor growth. Moreover, we found that up-expression of Etk/BMX in SCLC specimens correlated with poor pathologic stage and survival time.
MATERIALS AND METHODS
Tissue preparation and Immunohistochemical staining
Eighty-six SCLC samples were obtained by biopsy in our department since2005.1-2009.1. All patients had not received chemotherapy. This study was approved by the Ethics Committee of the Southern Medical University. Each case was independently reviewed by two pathologists according to the WHO criteria.
Tissues sections were deparaffinized and rehydrated routinely. Before adding the primary antibody, antigen was retrieved by heating sections. After blocking with0.3%H2O2and goat serum, the slides were then incubated with a primary antibody (Etk,1:100; BD Biosciences, USA) at4℃overnight. Following three washings in PBS, an avidin biotin complex (Vector Labs, Burlingame, USA) and alkaline phosphatase mixture were applied. Reaction products were visualized by3'diaminobenzidine (DAB),5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) and3-amino-9-ethylcarbazole (AEC)(MaximBiotech, Inc CA, USA). All slides were subsequently counterstained with hematoxylin. Brown-yellow granules in cytoplasm were considered positive staining for Etk/BMX. Negative controls were performed by replacing the primary antibodies stated above with PBS.
Cell culture and transfection
The human small lung cancer cell line NCI-H446, NCI-H69and the corresponding drug resistant subline H69/AR were purchased from the American
Type Culture Collection (ATCC) and maintained in RPMI1640medium supplemented with10%new born bovine serum (GIBCO) at37℃with5%CO2. H446/AR cell had been developed as described previously28. The two resistant cell lines were tested regularly for maintained resistance to the selected drugs. Growth and morphology of all cell lines were monitored on a weekly basis.
The plasmid encoding the full-length Etk was kindly provided by Dr Hsing-Jien Kung29. The short hairpin RNA (shRNA) targeted Etk, the small interfering RNA (siRNA) targeted for Zeb-2and Twist, and the mimics and antagomir of miR-495were all supplied by Shanghai GenePharma Co, Ltd. The transfection process was according to the instruction of Lipofectamine2000(Invitrogen). Sequences for RNAs oligo were shown as following:
Name Sense sequence (5'-3') Antisense sequence (5'-3') miR-495AAACAAACAUGGUGCACUUC GAAGUGCACCAUGUUUGUU mimics UU UUU miR-495AAGAAGUGCACCAUGUUUG antagomir UUU Etk/BMX GGGAAATGGAATCTGGGAAC shl T Etk/BMX GCCGAAGTCAGTGGTTGAAA sh2G Zeb-2si1GGCAAGGCCUUCAAAUAUAT UAUAUUUGAAGGCCUUGCC T TT Zeb-2si2GACCACUCCAGGAGUAAUAT UAUUACUCCUGGAGUGGUC T TT Twist si1GAUGGCAAGCUGCAGCUAUT AUAGCUGCAGCUUGCCAUC T TT Twist si2GCAAGAUUCAGACCCUCAAT UUGAGGGUCUGAAUCUUGC T TT
Real-time quantitative reverse transcriptase-PCR (qRT-PCR)
Total RNA was extracted using Trizol reagent (Invitrogen), and quantified by NanoDrop2000. qRT-PCR analysis was performed to validate the mRNA expression levels. Reverse transcription reactions were performed for15min at37℃, followed by5s at85℃for complementary DNA synthesis. Real time reactions were performed using the SYBR(?) PrimeScriptTM RT-PCR Kit (Takara Biotechnology Co, Ltd. Dalian, China). Glyceraldehyde3-phosphate dehydrogenase (GAPDH) or U6snRNA was used as the housekeeping gene. The mRNA expression levels of all samples were normalized to the housekeeping gene and analyzed using the comparative expression level2"△△Ct method. The all reactions were performed at least three times. Sequences for primers were shown as following: Name Forward primer (5'-3') Reverse primer (5'-3') Etk/BMX CATAGTGGGTTCTTCGTGGAC TGCCCGAGGTATCTTCAGC Zeb-2GGTCCAGATCGAAGCAGCTCA GTGACTATGTTTGTTCACAT AT T Twist CAGTCTTTACGAGGAGCTG GCTTGAGGGTCTGAATCT Vim TGCTAACTACCAAGACACT GTAGGTGCAATCTCAATG β-catenin ACAAACTGTTTTGAAAATCCA CGAGTCATTGCATACTGTCC GAPDH AGAAGGCTGGGGCTCATTTG AGGGGCCATCCACAGTCTT C
Western blot analysis
Cell proteins were extracted with RIPA lysis buffer and determined by the standard BCA method (BCATM Protein Assay Kit, Pierce, USA). Each protein sample was homogenized in the loading buffer and boiled for5min, then separated on8%SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Etten-leur, Netherlands). The membranes were blocked with5%non-fat dried milk for2h, then treated with primary antibodies including Etk, Zeb-2, Twist, Vim, and β-catenin overnight at4℃. The membranes were washed again with TBST (10mM Tris, pH8.0,150mM NaCl, and0.1%Tween20), followed by incubation with horseradish peroxidase-labeled secondary antibody (Beijing Biosynthesis Biotechnology Co. Ltd.) for45min at room temperature. Finally, after treatment with super echochemiluminescence (ECL) plus detection reagents, the protein bands of membranes were visualized by exposure to X-ray film. The intensity of protein bands were quantified by Quantity One software.
In vitro drug resistance assay
The ranges of drug concentrations were based on earlier studies and aimed at obtaining half maximal (50%) inhibitory concentration of a substance (IC50)11.30. A total of three anticancer drugs Cisplatin (DDP; Shangdong, China), Etoposide (VP-16; Jiangshu, China), and Adriamycin (ADM; Jiangshu, China) were obtained from commercial sources and were dissolved according to the manufacturer's instructions. Anti-cancer drugs induced cell death was quantified using the Cell Counting Kit-8assay (CCK-8). Cells were seeded into96-well plates and then treated for24h in200 μl of medium with anti-cancer drugs. CCK-8reagent (Dojindo, Kumamoto, Japan) was then added and the cells were incubated at37℃for2h before reading the absorbency using a micro-plate reader at450nm. The assay was conducted in six replicate wells for each sample and three parallel experiments were performed.
Cell proliferation assay
The cells were seeded in96-well plates at2×103cells/well. At the indicated times (0h,24h,48h,72h, and96h),10μl cck-8and100μl RPMI1640were added to each well. The cells were incubated for2h and absorbance at450nm was measured to calculate cell growth rates. Growth rate=(absorbance at450nm at x h-absorbance at450nm at0h)/(absorbance at450nm at0h).
Cell scratch-wound healing assay
The cells were seeded on24-well plates and grown to monolayer. Wound areas were scraped using100μl plastic tips. At the indicated times (0h,12h, and24h), wound areas were photographed and the wound healing rate was calculated. Healing rate=(width of wound at x h-width of the wound at0h)/width of wound at0h.
Cell invasion assay
Using24-well transwell units with8μm pore size polycarbonate insert, matrigel (50μl) as a basement membrane was spread on the polycarbonate membrane and allowed to solidify for1h at room temperature. Cells suspended in RPMI1640containing5g/L BSA were added to each upper compartment of the transwell units. After being cultured for48h, cells migrating through the matrigel-coated polycarbonate membrane were fixed by paraformaldehyde, stained with crystal violet and counted in five different fields randomly.
Flow cytometry assay for cell apoptosis
The cells treated with or without chemotherapeutic drugs including DDP, VP-16and ADM were harvested, washed with phosphate buffered saline (PBS), and resuspended in binding buffer containing7-AAD for10minutes, followed by the addition of Annexin V-PE. Cell apoptosis analysis was carried out using a flow cytometer (BD Biosciences, Oxford, United Kingdom).
In vivo tumorigenicity assay
Male nude mice (six weeks old, weighing18-20g, from the Medical Experimental Animal Center of Guangdong Province, China) were used for in vivo assay. The mice were raised under pathogen-free conditions. All procedures were performed according to guidelines of Association for Assessment and Accreditation of Laboratory Animal Care International.
Cells were harvested, washed with PBS and re-suspended in normal culture medium at a concentration of1x107/0.1ml. Cells in RPMI1640were subcutaneously inoculated into the legs of nude mice to establish the tumor model, respectively. The tumor volume was determined three times a week by direct measurement with sliding caliper and calculated by the following equation:V=(4/3)×71×R12×R2, where Rl is radius1and R2is radius2and R1
The wild and mutational3'UTR segments of Etk/BMX predicted to interact with miR-495were amplified by PCR from human genomic DNA and inserted into psiCHECK-2vector immediately downstream from the stop codon of luciferase (Promega) to develop psiCHECK2-Etk/BMX-3'UTR and psiCHECK2-Etk/BMX-mut-3'UTR. Cells in24-well plates were transfected with psiCHECK2-Etk/BMX-3'UTR, psiCHECK2-Etk/BMX-mut-3'UTR or psiCHECK-2, respectively. Besides, miR-495mimics and antagomir was also con-transfected into the cells, respectively. Luciferase activity was assayed at48h post-transfection using a dual-luciferase reporter assay system (Promega).
Statistical analysis
Data were analyzed using SPSS20.0software. Results are presented using means±SD. Comparison of means between two samples was performed using Student's t test. Statistical comparisons of more than two groups were performed using one-way analysis of variance (ANOVA). and then multiple comparisons were performed using least-significant difference (LSD). The association between Etk/BMX expression and clinical features were analyzed by Pearson Chi-Square test. Survival curves were obtained by Kaplain-Meier analysis. In all cases, P<0.05was considered statistically significant.
RESULTS
Relationship between ETK/BMX expression and clinicopathological characteristics in SCLC patients
Table1summarized the relationship between Etk/Bmx expression and clinical characteristics in SCLC patients. Of the66cases with limited stage small cell lung cancer,42(63.6%) were expressed for Etk/Bmx, while all20(100.0%) cases were positive in patients with extensive stage. The expression of Etk/Bmx revealed obvious correlation with the pathologic stage (P=0,001). Positive expression for Etk/Bmx was more frequent in cases with survival rate<1year (62/76,81.6%) than that in cases with survival rate>1year (0/10,0.0%)(P<0.001). The expression of Etk/Bmx exhibited no significant relationship with gender (P=0.448) and age (P=0.106). The Kaplan-Meier analysis revealed that Etk/Bmx expression was closely associated with overall survival rate of the SCLC patients (Figure1).
Etk/Bmx was involved in chemoresistance of SCLC by inhibiting apoptosis induced by chemotherapeutic drugs
The drug resistant cells H69/AR and H446/AR showed greatly elevated mRNA and protein expression of Etk/Bmx compared to their parental cells by qRT-PCR and Western blot assay (Figure2A). To further study the role of Etk/Bmx in SCLC cells, we developed Etk/Bmx stable over-expression cells (H69/Etk and H446/Etk) and down-expression cells (H69/AR/sh and H446/AR/sh)(Figure2B). Then, we analyzed the viabilities of SCLC cells when exposed to chemotherapeutic drugs. The IC50values to chemotherapeutic drugs were greatly decreased in H69/AR/sh and H446/AR/sh cells, while notably increased in H69/Etk and H446/Etk cells (Table2). These data indicated that Etk/Bmx modulated SCLC cells to chemotherapeutic drugs.
Subsequently, the number of apoptotic rate was analyzed following the cells treated with chemotherapeutic drugs including ADM, DDP and VP-16using flow cytometry assay. The cell apoptosis was significantly suppressed in H69/Etk and H446/Etk cells. However, it was obviously increased in H69/AR/sh and H446/AR/sh cells (Figure2C and Supplementary figure2). These data provided strong indication that Etk/Bmx was involved in chemoresistance of SCLC by inhibiting apoptosis induced by chemotherapeutic drugs.
ETK/BMX promoted cancer cells proliferation, migration, invasion, tumor growth and shortened the survival time
To investigate the biological roles of Etk/Bmx in SCLC cells, the cell proliferation was assessed by CCK-8assay. Comparing with the results from the control cells, H69/Etk and H446/Etk cells presented a significant increase of the cell growth rates, while H69/AR/sh and H446/AR/sh cells showed obviously decreased cell growth rates (Figure3A). We also detected the effects of Etk/Bmx on migration and invasion ability by scratch-wound healing and transwell-invasion assay. As shown in Figure3B and C, Etk/Bmx over-expression enhanced the healing rates of H446cells. However, knockdown of Etk/Bmx in H69/AR and H446/AR cells inhibited the healing rates (Figure3B). For transwell-invasion assay, more H446/Etk cells penetrated the matrigel-coated membrane than the control H446cells did. By contrast, H69/AR/sh and H446/AR/sh cells showed a notably reduced number of the cells penetrating the matrigel-coated membrane (Figure3C).
The tumorigenic properties of Etk/Bmx in vivo were conducted in nude mice xenograft. The nude mice were subcutaneously inoculated with Etk/Bmx over-or down-expressing SCLC cells. The tumors of H69/Etk and H4467Etk cells grew significantly rapidly compared with that of H69and H446cells. But the tumors grew slowly in H69/AR/sh and H446/AR/sh cells. The null mice presented longer survival time accompanied by knockdown of Etk/Bmx, while shorter survival time with elevation of Etk/Bmx (Figure3D).
Taken together, both loss-and gain-of-function experiments demonstrated that Etk/Bmx promoted the SCLC proliferation, migration, invasion and tumor growth, but shorten the survival time.
Reversing EMT reduced the chemoresistance of SCLC
Our previous studies had showed that the EMT related markers (epithelial molecule P-catenin, and the mesenchymal markers including Zeb-2, Twist, and Vim) expressed significantly between H69and H69/AR cells by cDNA microarray (Supplementary figure1). In the current study, we further examined the expression of related markers in H69/AR and H446/AR cells at mRNA and protein level. The results were consistent with the data from cDNA microarray (Figure2A). Zeb-2and Twist have been documented as the important transcriptional repressors of EMT12. Knockdown of Zeb-2and Twist can cause a reversal of EMT21,22. Then, the Zeb-2and Twist expression were inhibited with Zeb-2siRNA or Twist siRNA in H69/AR and H446/AR cells. The expression of Vim was subsequently suppressed while β-catenin was increased accompany with the decreased Zeb-2and Twist. The results indicated that down-regulation of Zeb-2and Twist reversed the EMT process (Figure4). We further investigated whether reversing EMT can influence the drug sensitivity of SCLC cells. The results showed the IC50values of the cells down-expression Zeb-2and Twist were notably reduced when cells were exposed to chemotherapeutic drugs including ADM, DDP and VP-16(Table3). It suggested that the drug resistance to chemotherapeutic drugs was inhibited with reversing EMT in SCLC.
Etk/BMX regulated chemoresistance of SCLC through EMT
As mentioned above, the EMT process participated in the regulation of drug resistance of SCLC. We further studied whether Etk/BMX modulated the sensitivity of SCLC cells to chemotherapeutic drugs through EMT. The expression of Zeb-2, Twist, Vim and β-catenin was evaluated after up or down-regulation of Etk/BMX. The results revealed that the expression of Zeb-2, Twist and Vim at mRNA and protein level was remarkably increased while β-catenin was inhibited by over-expression of Etk/BMX (Figure5A). In contrast, suppression of Etk/BMX reduced the expression of Zeb-2, Twist and Vim, but promoted the expression of β-catenin (Figure5B). We also analyzed the expression of the EMT markers stated above in xenograft tumors. The results were consistent with the data from the cell lines (Figure5C).
Our observations provided valid evidence that Etk/BMX may influence SCLC chemoresistance through EMT process.
miR-495modulated chemoresistance by targeting Etk/BMX
The sensitivity of the H69/AR cells to anti-cancer drugs was greatly increased following transfection with the miR-495mimics as described previously20. To better understand the roles of miR-495in the drug sensitivity of SCLC cells, both H69/AR and H446/AR cells were used to quantify the expression of miR-495by qRT-PCR. The H69/AR and H446/AR cells presented lower level of miR-495than their corresponding control groups. Then, the miR-495expression was up-regulated in H69/AR and H446/AR cells by miR-495mimics and down-regulated in H69and H446cells by antagomir, respectively (Figure6A). Furthermore, the effect of miR-495on the drug sensitivity of SCLC cells was analyzed. We found that H69and H446cells treated with miR-495antagomir exhibited higher IC50values to chemotherapeutic drugs including ADM, DDP and VP-16. In contrast, the IC50values were obviously declined in H69/AR and H446/AR cells by miR-495mimics (Table4). These findings demonstrated that the miR-495was related to chemoresistance of SCLC cells.
Considering on Etk/BMX as a potential targeted gene of miR-495by the bioinformation analysis (Figure6C), we examined the Etk/BMX expression after modulation of miR-495by qRT-PCR and Western blot. The result showed that the Etk/BMX mRNA and protein expression were elevated in H69and H446cells by inhibition of miR-495, while decreased in H69/AR and H446/AR cells as a result of up-regulation of miR-495(Figure6B). The correlation between miR-495and Etk/BMX was further detected by luciferase reporter assay. The Etk/BMX luciferase activity was significantly declined in H69/AR cells treated with miR-495mimics, but obviously elevated by treatment with miR-495antagomir (Figure6C).
For the reasons mentioned above, all these results suggested that the influence of Etk/BMX on SCLC chemoresistance may be regulated by miR-495.
DISCUSSION
It has been reported that Etk/BMX is associated with various cellular processes including proliferation, differentiation, apoptosis and tumorigenicity8"10. In this study, we investigated the influence of Etk/BMX on the biological behaviors of SCLC by loss-and gain-of-function experiments. The proliferation, migration and invasion abilities were decreased after suppression of Etk/BMX, while they were all elevated following the up-regulation of Etk/BMX. The in vivo data revealed that over-expression of Etk/BMX can promote tumor growth and prolong survival time. By clinical samples, we observed that expression of Etk/BMX showed closely relationship with poor pathologic stage and survival time. In sum, our findings indicated that Etk/BMX played an important role in the biological behaviors of SCLC, suggesting that Etk/Bmx may serve as a predictor for poor prognosis in SCLC.
Our previous studies had showed that suppression of Etk/BMX expression reduced the chemoresistance of H69/AR cells. In this study, we further confirmed the effect of Etk/BMX on chemoresistance of SCLC using two multi-drug resistant cells H69/AR and H446/AR cells. The H69/AR/sh and H446/AR/sh cells showed much more sensitivity to chemotherapeutic drugs. In contrast, over-expression of Etk/BMX in H69and H446cells reduced the drug sensitivity. Besides, our research revealed that the cell apoptosis induced by chemotherapeutic drugs decreased significantly after up-regulation of Etk/Bmx, but it increased obviously with knockdown of Etk/Bmx. There was a negative correlation between Etk/BMX and cell apoptosis. These results provided sufficient proof to demonstrate that Etk/Bmx affected the chemoresistance of SCLC by inhibiting apoptosis induced by chemotherapeutic drugs.
Studies have shown that EMT process confers resistance to chemotherapy in ovarian carcinoma, colorectal cancer and so on23-25. Base on our previous study, the role of EMT in SCLC chemoresistance was firstly clarified in this study. Inhibition of Zeb-2and Twist in H69/AR and H446/AR cells led to declined Vim while elevated β-catenin. The results indicated that suppression of Zeb-2and Twist can reverse the EMT process. Furthermore, the IC50values to chemotherapeutic drugs were suppressed after knockdown of Zeb-2and Twist. It suggested that EMT was associated with chemoresistance of SCLC. Subsequently, the relationship between Etk/BMX and EMT was explored. The H69/AR/sh and H446/AR/sh cells exhibited a significant decrease in the expression of mesenchymal markers, while increased epithelial markers. However, the H69and H446cells presented an opposite results by elevation of Etk/BMX. The expression of the EMT markers stated above in xenograft tumors was consistent with the data from the cell lines. Taken together, Etk/BMX modulated the chemoresistance of SCLC possibly through EMT process.
miR-495has been reported as a tumor suppressor by inhibiting gastric cancer cell migration and invasion26. Ectopic expression of miR-495in breast cancer cells promotes tumorigenesis in the mice27. Our previous study firstly reported that transfection of the H69/AR cells with the miR-495mimics significantly reduced the chemoresistance. In the present study, we used two drug resistant cells to further investigate the role of miR-495in drug sensitivity of SCLC. Up-or down-regulation of miR-495can modulate IC50values to chemotherapeutic drugs, indicating miR-495was involved in the chemoresistance of SCLC cells. Elevation of miR-495resulted in decreased expression of Etk/BMX. On the contrary, Etk/BMX was over expressed following the reduced expression of miR-495. The result from luciferase reporter assay confirmed that Etk/BMX was one of the directly targeted genes of miR-495. All these data presented that miR-495may participate in the chemoresistance and regulate the expression of Etk/BMX in SCLC.
Taken together, our study supported for the first time that elevation of Etk/BMX can led to chemoresistance of SCLC by EMT process and was under the regulation of miR-495. Therefore, our research provided a new insight into the mechanism of SCLC chemoresistance. Etk/BMX can be a useful predictor for drug sensitivity, raising the possibility of Etk/BMX depletion as a promising strategy for interfering with chemoresistance in SCLC.
引文
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