【作者】 刘换新；

【导师】 郭琳琅；

【作者基本信息】 南方医科大学 ， 肿瘤学， 2014， 博士

【摘要】 研究背景肺癌的发病率已居世界恶性肿瘤首位,近年来,我国肺癌发病率及病死率也逐年升高。根据其组织学特点,肺癌可分为小细胞肺癌(small cell lung cancer, SCLC)和非小细胞肺癌(nonsmall cell lung cancer, NSCLC)两种,SCLC发病率占原发性肺癌的15%左右。目前,人们对非小细胞肺癌(NSCLC)的研究取得诸多进展,但迄今为止对SCLC的研究却较少有重大突破。SCLC的治疗手段主要为药物治疗,但由于其极易发生多药耐药性(multidrug resistance, MDR),从而导致化疗失败,复发率高,临床预后差,两年存活率不足20%,90%的患者多在确诊后5年内死亡。因此,SCLC的化疗耐药性成为近年来SCLC I临床治疗亟需解决的问题之一。目前研究发现肿瘤耐药是一个涉及多基因和多因素共同参与的复杂过程,离子通道可能在化疗耐药产生过程中发挥着重要作用。1984年,DeCoursey等人发现钾离子通道与肿瘤的发生之间存在相关性,随后Pancrazio等人报道钾离子通道与小细胞肺癌耐药之间有相关性,之后还有一些研究提示内向整流钾离子通道与小细胞肺癌耐药相关,但这些研究均未涉及机制方面的探讨。内向整流钾通道(Kir, Inward rectifier potassium channel)是钾离子通道的一种,有7个亚家族,在体内维持可兴奋组织的细胞兴奋和静息膜电位的稳定,在调节神经递质释放、心率、胰岛素分泌、神经细胞分泌、上皮细胞电传导、骨骼肌收缩以及细胞容积的过程中发挥了重要作用。其中Kir2家族主要存在于心脏和神经组织中,具有强大的内向整流作用,研究发现,一些肿瘤细胞膜上也存在着不同的钾离子通道,其中Kir2.1/KCNJ2钾离子通道(以下简称KCNJ2)可通过影响细胞膜电位,导致胞外钙离子内流进入胞内,影响细胞信号转导通路,从而影响整个肿瘤细胞的发生和发展；钾离子通道阻断剂能诱导肿瘤细胞凋亡,与肿瘤多药耐药密切相关。那么,KCNJ2是通过何种途径调节小细胞肺癌多药耐药呢?我们发现KCNJ2及ABCC1在小细胞肺癌耐药细胞株H69AR中显著高表达,前期研究显示二者有显著正相关性,临床组织标本检测结果亦提示二者呈显著正相关,提示KCNJ2可能通过与ABCC1的相互作用调节小细胞肺癌多药耐药。我们注意到,信号通路如Ras-MAPK途径参与对钾离子通道蛋白Kir2.1的调节。另外,锚定蛋白如细丝蛋白A结合位点,psd93δ或SAP97被认为在稳定Kir2.1离子通道在细胞膜的表达起重要作用。最近的研究表明,激酶和磷酸酶可以形成单一的调节蛋白复合物,从而调节钾离子通道的活性。另有研究表明PKA及PKC均可以调节Kir2.1,因此,Ras-MAPK-PKC信号通路可以调节KCNJ2在细胞中的表达,并通过KCNJ2参与小细胞肺癌多药耐药。microRNA (miRNA)是一类高度保守的,非编码的小RNA分子,可以调节基因的表达,在各种真核生物发育,分化,代谢,免疫,细胞的增殖、凋亡等生理和病理过程中扮演关键的角色。越来越多的证据表明,miRNA与肿瘤的发展及预后密切相关的。有部分文献提示与小细胞肺癌多药耐药相关。其机制是通过抑制某些信号通路,如FZD7/β-catenin,或者抑制膜转运蛋白ABCB1及ZNRD1的表达,但其机制尚未完全阐明。目前尚未有文献报道microRNA通过调节钾离子通道蛋白的表达来调节小细胞肺癌多药耐药。我们前期通过基因表达谱芯片对小细胞肺癌耐药细胞株H69AR和非耐药细胞株H69中21,522个基因进行分析,H69细胞中miR-7显著高表达。同时发现,H69AR细胞中的1252个基因表达上调,1131个基因表达下调,其中KCNJ2及ABCC1基因在H69AR细胞株中显著高表达。提示KCNJ2、ABCC1及miR-7均与小细胞肺癌多药耐药有关,生物信息学显示KCNJ2是miR-7的靶基因,miR-7可以抑制KCNJ2的表达,那么,miR-7可能通过抑制KCNJ2的表达调节小细胞肺癌多药耐药。本研究旨在研究钾离子通道蛋白Kir2.1/KCNJ2在小细胞肺癌细胞株及组织标本中的表达,探讨Kir2.1/KCNJ2在小细胞肺癌多药耐药中的作用及其与临床预后的相关性,并进一步探讨Kir2.1/KCNJ2的调节机制,为研究小细胞肺癌多药耐药机制提供新的思路,为临床治疗及预后提供理论依据。目的分析Mir-7、KCNJ2及ABCC1在小细胞肺癌组织标本及细胞株中的表达,探讨KCNJ2在小细胞肺癌多药耐药中的作用以及KCNJ2参与小细胞肺癌多药耐药的机制及其调控机制,并深入研究KCNJ2与ABCC1的相互作用,同时,分析Mir-7、KCNJ2及ABCC1与临床预后的关系,进一步丰富SCLC多药耐药性的分子机制理论,为临床治疗提供理论依据。内容与方法一、分析SCLC多药耐药细胞株H69AR、H446AR及H446AR和敏感细胞株H69及H446中KCNJ2基因表达差异性以SCLC多药耐药细胞株H69AR、H446AR和敏感细胞株H69、H446作为研究对象,采用实时荧光定量PCR和Western Blot技术检测四株细胞中KCNJ2mRNA及蛋白的表达,验证KCNJ2的mRNA和蛋白在四株细胞中的表达差异；二、KCNJ2表达对小细胞肺癌多药耐药性的影响采用KCNJ2过表达质粒和siRNA增加或降低SCLC多药耐药细胞株H69AR、H446AR和敏感细胞株H69、H446中KCNJ2的表达水平,CCK8法分析细胞对小细胞肺癌常用化疗药物顺铂(Cisplatin, DDP)、足叶乙甙(Etoposide,VP-16)及阿霉素(Doxorubicin, ADM)敏感性的变化,流式细胞术分析细胞凋亡和周期的变化；三、KCNJ2的调节机制在H69、H69AR、H446及H446AR细胞株中同时通过抑制PKC-MEK-KCNJ2信号通路中关键蛋白激酶PKC和MEK,检测KCNJ2的表达,探讨KCNJ2的调控机制；四、KCNJ2与ABCC1相关性分析同时运用免疫组化分析组织标本中二者表达相关性,运用免疫共沉淀实验探讨KCNJ2与ABCC1的相互作用,探讨KCNJ2调控小细胞肺癌多药耐药的可能机制五、动物实验：分别把下列细胞：1:H69AR,2:H69AR-NC,3: H69AR-KCNJ2-homo-628,4:H69AR-KCNJ2-homo-1073,5:H69,6:H69-NC,7:H69-KCNJ2-PEX-1以及1:H446AR,2:H446AR-NC,3: H446AR-KCNJ2-homo-628,4:H446AR-KCNJ2-homo-1073,5:H446,6:H446-NC,7:H446-KCNJ2-PEX-1注射到裸鼠皮下,裸鼠成瘤的大小3个星期后处死。分别测量肿物大小、PCNA免疫细胞检测并分析。六、H69AR、H446AR和H69、H446细胞中miR-7表达差异性采用miRNA芯片分析四株细胞中microRNA表达差异,利用miRNA靶基因分析软件(miRanda、TagetScan和PicTar)分析KCNJ2相关miRNA,选取表达差异显著的miR-7进行研究,实时荧光定量PCR技术验证miR-7的表达差异。七、miR-7对KCNJ2的靶向调节作用1、采用miR-7抑制物(inhibitor)和模拟物(mimic)抑制或增加H69AR、 H446AR和H69、H446细胞中miR-7的表达水平,实时荧光定量PCR和、Vestern Blot技术分析KCNJ2mRNA和蛋白水平的变化。2、构建KCNJ23’-非翻译区(3’-untranslated region,3’-UTR)的psiCHECK-2荧光素酶载体(psiCHECK-2-KCNJ2-3’UTE),与miR-7mimic或inhibitor共转染H69、H69AR、H446及H446AR细胞,采用双荧光素酶报告基因测定方法检测KCNJ2荧光素酶活性。八、miR-7对SCLC多药耐药性的影响采用miR-7inhibitor和mimic抑制或增加H69AR/H446AR及H69/H446细胞中miR-7的表达,CCK8法分析细胞药物敏感性的变化,确定miR-7对SCLC耐药性的影响。九、niR-7、KCNJ2及ABCC1在SCLC组织中的表达及临床病理意义收集52例SCLC患者石蜡包埋组织,随访临床资料,免疫组化染色观察组织中KCNJ2及ABCC1的表达,QRT-PCR检测组织中miR-7的表达,分析耐药组(3个月后复发)与敏感组(半年未复发)以及局限期、进展期标本之间KCNJ2、 ABCC1及miR-7的表达有无统计学差异；分析KCNJ2、ABCC1及miR-7的表达与SCLC患者临床特征及生存率的关系。十、统计学分析所有结果均经SPSS13.0统计软件统计。数据以均数士标准差(x±s)表示,两株细胞间KCNJ2或miR-7表达的比较采用独立样本t检验；多组间miR-7、KCNJ2或ABCC1表达的比较采用one-way ANOVA(方差齐同时)或Welch法进行矫正(方差不齐时),多重比较采用SNK法(方差齐同时)或Dunnett’s T3法(方差不齐时)；miR-7、KCNJ2及ABCC1表达与临床病理特征分析采用配对样本的卡方分析(McNemar检验)；采用多元Logistic回归模型分析年龄,性别,药物敏感性,临床分期等因素对Mir7、ABCC1和KCNJ2表达的影响；采用Cox比例风险模型分析以上变量及Mir7蛋白、ABCC1和KCNJ2表达水平对对SCLC患者生存时间的影响；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。结果一、KCNJ2在SCLC多药耐药细胞株H69AR和H446AR中表达显著增高KCNJ2在H69,H69AR中差异有统计学意义(t=16.880,p=0.003),且H69AR组显著高于H69组；KCNJ2在H446,H466AR中差异有统计学意义(t=27.798,p<0.001),且H446AR组显著高于H446组。二、上调KCNJ2表达能显著增加H69和H446细胞的耐药性1、根据人cDNA文库中KCNJ2基因组序列,结合Ambion’S siRNA Target Finder提供的方法及参考文献设计出一对过表达质粒KCNJ2-PEX-1(由上海吉玛基因股份有限公司合成),经测序和序列对比显示与人KCNJ2编码区(coding sequence,CDS)完全一致。2、建立稳定高表达细胞株H69-KCNJ2-PEX-1及H446-KCNJ2-PEX-1将KCNJ2-PEX-1分子体外经脂质体介导转染细胞,以通用scrambled negative control(NC)做阴性对照,H69和H446分别转染NC、KCNJ2-PEX-1,经G418筛选后建立稳定转染细胞株H69-KCNJ2-PEX-1和H446-KCNJ2-PEX-1,荧光定量PCR和Western Blot检测显示H69-KCNJ2-PEX-1及H446细胞中KCNJ2的nRNA实时荧光定量PCR结果(4.89倍,t=10.184,P=-0.006；7.23倍,t=13.562,P=0.003)。3、H69-KCNJ2-PEX-1和H446-KCNJ2-PEX-1细胞对ADM、DDP和VP-16的生存率较H69、H446细胞的生存率显著升高(P<0.001),50%抑制浓度(half maximal inhibitory concentration of a substance,IC50)亦升高。4、流式细胞术分析显示ADM, DDP和VP-16诱导的H69-KCNJ2-PEX-1及H446-KCNJ2-PEX-1细胞凋亡率较H69和H446细胞降低,而G0-G1期和G2-M期细胞数增加。三、下调KCNJ2表达能显著增加H69AR和H446AR细胞的药物敏感性1、KCNJ2-homo-628、KCNJ2-homo-1073显著降低KCNJ2在H69AR及H446AR细胞中的表达,同时ABCC1的表达亦显著降低根据人cDNA文库中KCNJ2基因组序列,结合Ambion’S siRNA Target Finder提供的方法及参考文献设计出靶向人KCNJ2基因的四对shRNAg干扰质粒KCNJ2-homo-514、KCNJ2-homo-628、KCNJ2-homo-1073、KCNJ2-homo-1357,分别通过荧光定量PCR和Western Blot方法检测细胞中KCNJ2蛋白的表达水平变化,筛选出对KCNJ2表达抑制效果最显著的两对shRNA (KCNJ2-homo-628, KCNJ2-homo-1073)进行后续实验。2、CCK-8法分析结果显示,转染KCNJ2-homo-628和KCNJ2-homo-1073的H69AR及H446AR细胞对药物的生存率较阴性对照(]negative control, NC)组和空白对照组显著降低(P<0.001),IC50值亦显著降低。3、流式细胞术分析结果显示,ADM、DDP和VP-16处理后,转染KCNJ2-homo-628和KCNJ2-homo-1073的H69AR及H446AR细胞的凋亡率较阴性对照组和空白对照组增加(P<0.001),G0-G1期和G2-M期细胞数降低。四、KCNJ2能促进小细胞肺癌体内肿瘤生长动物实验：分别把下列细胞：1:H69AR,2:H69AR-NC,3: H69AR-kcnj2-homo-628,4:H69AR-kcnj2-homo-1073,5:H69,6:H69-NC,7: H69-KCNJ2-PEX-1注射到裸鼠皮下,裸鼠成瘤的大小3个星期后处死。分别测量肿物大小、PCNA免疫细胞检测并分析。结果提示KCNJ2可以增强肿瘤细胞增殖能力。五、PKC-MEK信号通路调节KCNJ2的表达在H69AR及H446AR细胞株中同时通过抑制PKC-MEK信号通路中关键蛋白激酶PKC和MEK, RT-PCR和Western blot检测细胞中KCNJ2的变化,发现同时抑制PKC和MEK, H69AR及H446AR细胞株中KCNJ2mRNA水平分别降低45.8%(P<0.001)、51.6%(P<0.001), Western Blot显示蛋白水平分别下降62.3%(P<0.001)、45.2%(P<0.001),单独抑制PKC, KCNJ2mRNA水平分别降低26.2%(P=0.023)和38.6%(P=0.003), Western Blot显示蛋白水平分别下降23.5%(P=0.026)、29.6%(P=0.022),单独抑制MEK, KCNJ2mRNA水平分别降低31.3%(P=0.017)、25.8%(P=0.029), Western Blot显示蛋白水平分别下降29.4%(P=0.034)、21.6%(P=0.036)。说明PKC-MEK信号通路可以调节KCNJ2的表达。六、KCNJ2与ABCC1相关性分析1、增加KCNJ2表达,ABCC1的表达明显增加；2、同时运用免疫组化分析组织标本中二者表达相关性,发现二者呈显著正相关(χ2=19.396,P<0.001)3、运用免疫共沉淀实验检测KCNJ2与ABCC1的相互作用,发现二者在H69AR细胞株内有相互作用七、miR-7在SCLC多药耐药细胞株H69AR和H446AR中表达与敏感株H69及H446之间无显著差异miRNA芯片结果显示miR-7在SCLC多药耐药细胞株H69AR中的表达降低,敏感细胞株H69细胞中miR-7的表达是H69AR细胞中的4.64倍,然而实时荧光定量PCR结果显示miR-7在H69、H446细胞中的表达是H69AR、446AR细胞中的表达的1.09倍、1.03倍(t=2.148, P=0.098;t=0.478,P=0.657)。差异无统计学意义。八、KCNJ2是miR-7的靶基因1、miRNA靶基因预测软件(PicTar,TarScan和miRBase)分析显示,miR-7与KCNJ23’-UTR具有互补结合位点。2、miR-7对KCNJ2mRNA及蛋白表达的调节作用(1)将mir-7agomir转染H69、H69AR、H446及H446AR细胞,以升高miR-7表达后,结果显示KCNJ2mRNA水平的表达均显著降低(F=57.780,P=<0.001), KCNJ2蛋白表达显著降低(F=254.520,P=<0.001)。ABCC1mRNA水平及ABCC1蛋白水平表达亦显著降低(F=381.121,P<0.001；F=365.439,P<0.001)。(2)将mir-7antagomir转染H69、H69AR、H446及H446AR细胞,以降低miR-7表达后,结果显示KCNJ2mRNA水平的表达均显著升高(F=152.419,P<0.001). KCNJ2蛋白表达显著增高(F=87.124, P<.0.001)。 ABCC1mRNA水平及ABCC1蛋白水平表达亦显著升高(F=3.385, P<0.001; F=786.360, P<0.001)。3、miR-7对KCNJ2具有靶向调节作用(1)成功构建psiCHECK-2-KCNJ2-3’UTR和psiCHECK-2-KCNJ2-3’UTR-R载体。通过PCR扩增KCNJ23’-UTR,产物长度为1250bp,经转化、抗性筛选、挑取单克隆等步骤后试剂盒提取质粒,Not I和Xho I双酶切,产物大小约为6365bp/1235bp。酶切鉴定psiCHECK-2-KCNJ2-3’UTR克隆1-4和psiCHECK-2-KCNJ2-3’UTR-R克隆3正确,挑起psiCHECK-2-KCNJ2-3’UTR-1号克隆和psiCHECK-2-KCNJ2-3’UTR-R-3号克隆进行测序。测序结果与理论序列一致,试剂盒提取质粒进行后续实验。(2)KCNJ2荧光素酶活性受miR-7mimic和inhibitor调节将psiCHECK-2-KCNJ2-3’UTR-R或psiCHECK-2-KCNJ2-3’UTR-mut载体与miR-7mimic或inhibitor共转染H69AR细胞,检测KCNJ2荧光素酶活性显示,与miR-NC组相比, miR-7mimic可显著抑制转染KCNJ23’-UTR的H69AR细胞中KCNJ2荧光素酶活性(F=437.572, P<0.001), miR-7inhibitor可显著增加转染KCNJ23’-UTR的H69AR细胞中KCNJ2荧光素酶活性(多重比较P<0.05),miR-7mimic和niR-7inhibitor对转染KCNJ23’UTR-mut的H69AR细胞中KCNJ2荧光素酶活性无显著影响(多重比较P>0.05)。九、miR-7对H69/H69AR和H446/H446AR细胞的药物敏感性无显著相关转染miR-7inhibitor及mimic的H69、H69AR、H446及H446AR细胞对ADM、CDDP和VP-16的生存率较阴性对照组和空白对照组无显著升高(多重比较P>0.005),IC50值亦无显著升高(多重比较P>0.05)。十、niR-7、KCNJ2及ABCC1在SCLC组织中的表达与临床病理特征的关系Mir-7表达为临床分期保护因素(OR=0.258,P=0.043), KCNJ2(?)ABCC1表达为临床分期危险因素(OR=11.124, P=0.001; OR=10.710,P=0.001);其余指标对上述指标的表达影响无统计学意义(P均>0.05)。十一、SCLC患者生存分析1、COX比例风险回归模型采用逐步COX回归模型分析,选入的变量分别为“性别”、“年龄”、“分期”、“敏感性”、"Mir-7"、"KCNJ2"及"ABCC1",其中变量“Mir-7”的相对危险度为0.75(P=0.026),即Mir-7高表达的患者死亡风险为Mir-7低表达患者的0.75倍。性别、分期、药物敏感性以及KCNJ2和ABCC1的表达与相对危险性无显著相关。2、生存时间估计采用Kaplan-Meier法比较不同因素对患者生存时间的影响,结果发现男性患者与女性患者的生存时间无统计学差异(χ2=0.473,P=0.492)。不超过55岁的患者的生存时间显著高于55岁以上患者(χ2=6.867,P=0.009)。局限期患者的生存时间显著高于广泛期患者(χ2=4.065,P=0.0231)。Mir-7表达高患者生存时间显著高于低表达患者(χ2=5.825, P=0.016); KCNJ2及ABCC1表达与患者的生存时间无显著性差异(χ2=1.016,P=0.313; χ2=1.696,P=0.193)。结论1、KCNJ2在SCLC多药耐药细胞株H69AR、H446AR中表达显著升高,增加或降低细胞中KCNJ2表达可以引起细胞的耐药性变化,表明KCNJ2参与了SCLC的多药耐药。2、KCNJ2与ACC1的表达在SCLC细胞株及临床组织标本中均呈显著正相关,免疫共沉淀显示二者在H69AR细胞中存在相互作用,提示KCNJ2可能通过与ABCC1的相互作用参与小细胞肺癌多药耐药；3、单独或同时抑制PKC或MEK可以降低KCNJ2在SCLC细胞株中的表达,提示PKC-MEK信号通路参与了对KCNJ2的调控；4、KCNJ2是miR-7的靶基因,miR-7可以调节KCNJ2的表达,降低或增加miR-7表达可以引起细胞中KCNJ2变化,表明miR-7参与了对KCNJ2的表达调控；5、miR-7、KCNJ2及ABCC1在SCLC组织中的表达与患者的临床分期有关,而miR-7与患者生存时间及临床分期均显著相关,提示miR-7有望成为SCLC患者的临床预后指标及治疗靶标。更多还原

【Abstract】 BackgroundThe incidence of lung cancer is the top1all over the world. According the cell type, lung cancer is grouped into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The incidence of SCLC covers15%of primary lung cancer. The treatment of SCLC is mainly in medicine, but because of multidrug resistance (MDR), the lost chemotherapy, or a higher recurrence, a weak prognosis is often occurred in clinic. A two-year survival rate is less than20%and90%of patients is dead in5years.Several kinds of ion channels play important roles in cell development and differentiation, i.e. Kir2.1encoded by KCNJ2gene. In1984, DeCoursey et al discovered the co-relationship between the K channel and tumorigenesis. Pancrazio et al reported that the K channel plays important role in SCLC resistance. Some publications reported that the inner-current K channel had a close relevance with the MDR. All the publications did not answer the mechanism. We discover that the Ras-MAPK signal pathway participates in the regulation of Kir2.1. Additionally some binding sites for anchoring proteins such as the filamin A, psd935, or SAP97, promote the Kir2.1channel expression in cell membrane. Recently, it has been suggested that kinases and phosphatases can be intimately associated to channels in a single regulatory protein complex that modulates channel activity and PKA and PKC regulate the Kir2.1expression. So the Ras-MAPK-PKC signal pathway regulates the KCNJ2expression in SCLC cells.MicroRNAs (miRNAs) are a class of small non-coding RNAs of18-24nucleotides which regulate protein expressions of specific mRNAs by either translational inhibition or mRNA degradation. Recent evidence has shown that more than50%of miRNAs are located in cancer-associated genomic break points, and can function as tumor suppressor genes or oncogenes depending on their targets. The differential expression profiles of miRNAs from healthy tissue across cancers, and the surprising strength of these profiles play an important role in tumor classification and prediction of therapy response. More and more evidences confirm that miRNA has close relevance in tumorigenesis and tumor prognosis, the evidence of the roles for miRNAs in determining drug sensitivity/resistance has been emerging. miRNAs regulate a variety of cellular activities through negatively regulating special target genes. Furthermore, it may also be involved in chemoresistance to cancer therapy. Some miRNAs are involved the MDR in lung tumor. The mechanism may be mediated by inhibiting FZD7/β-catenin signal, or ABCB1, ZNRD1protein. In2012, Goldoni D et al reported that Mi-212down-regulated the KCNJ2expression. We ever screened the gene spectra in the H69AR and H69cells.21,522genes were analyzed, showing that miR-7had higher expression in the H69cells as well as the up-regulations of1252genes in the H69AR cells and the down-regulations of1131genes. Of them, KCNJ2gene showed higher expression in the H69AR cells, suggesting that the KCNJ2gene and miR-7were relevant to SCLC MDR. Biological information show that KCNJ2gene is the target of miR-7and miR-7can inhibit the expression of KCNJ2, then, miR-7may regulate small-cell lung cancer multidrug resistance by inhibiting the expression of KCNJ2.This study aims to investigate the expression of the potassium ion channel protein Kir2.1/KCNJ2in small cell lung cancer cell lines and tissues, and to explore the role of Kir2.1/KCNJ2in small cell lung cancer multidrug resistance and its relationship with clinical prognosis relevance, and to further explore the regulatory mechanisms of Kir2.1/KCNJ2regulating of the multidrug resistance of small cell lung cancer, this study may provide new ideas for investigation of multidrug resistance of small cell lung cancer and provide a theoretical basis for clinical treatment and prognosis.ObjectivesTo analyze the expression of Mir-7, KCNJ2and ABCC1in small cell lung cancer specimens and cell lines, and to explore the role of KCNJ2in multidrug resistance in small cell lung cancer and the mechanisms of KCNJ2involved in multidrug resistance and it’s regulation mechanisms and in-depth study of the interaction between KCNJ2and ABCC1; furthere more, to analyze the correlationship between Mir-7, KCNJ2and ABCC1with clinical outcome. To further enriched theoretical molecular mechanisms of multi-drug resistance in SCLC and to provide a theoretical basis for clinical treatment.Materials and methods1. An analysis of KCNJ2gene expression differences in multi-drug resistant SCLC cell lines H69AR, H446AR and there sensitive cell lines H69and H446Multidrug resistance cell lines H69AR, H446AR and sensitive cell lines H69, H446as the research object, and KCNJ2mRNA and protein expression real-time PCR and Western Blot Detection of four cell lines, verify KCNJ2mRNA and protein in four differential expression of cell lines;2. KCNJ2expression of multidrug resistance in small cell lung cancerUsing KCNJ2overexpression plasmids and siRNA increase or decrease multidrug resistant SCLC cell lines H69AR, H446AR and sensitive cell lines H69, H446in KCNJ2expression levels, CCK8analysis on small cell lung cancer cells commonly used chemotherapy drug cisplatin (Cisplatin, DDP), sensitivity to changes etoposide (Etoposide, VP-16) and adriamycin (Doxorubicin, ADM), flow cytometry analysis of changes in cell cycle distribution and apoptosis.3. KCNJ2regulation mechanismIn H69, H69AR, H446and H446AR cell lines, to detect the expression of KCNJ2mRNA and protein using real-time PCR and Western Bloting by inhibiting the key protein kinase PKC and MEK of PKC-MEK-KCNJ2signaling pathway respectively or both. 4. KCNJ2correlation with ABCC1Using immunohistochemical analysis of tissue specimens to express the correlation between KCNJ2and ABCC1,and using co-immunoprecipitation experiments to explore the interaction between KCNJ2and ABCC1in cells, to explore the mechanisms that KCNJ2regulate the small cell lung cancer multidrug resistance.5. In vivo tumor xenograft model: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. H69AR, H69AR-NC, H69AR-KCNJ2-homo-628, H69AR-KCNJ2-homo-1073, H69, H69-NC, H69-KCNJ2-PEX-1and H446AR, H446AR-NC, H446AR-KCNJ2-homo-628, H446AR-KCNJ2-homo-1073,:H446, H446-NC, H446-KCNJ2-PEX-1cells were subcutaneously inoculated into the legs of nude mice to establish the tumor model, respectively. Three weeks later,5mice of each group were sacrificed and tumors were excised, tumor size were measured and PCNA immune cells were to be detected and analyzed.6. Expression differences of miR-7in H69AR, H446AR and H69, H446cells.Using miRNA microarray to analyze the microRNA expression differences in H69AR, H446AR and H69, H446cells, using miRNA target gene analysis software (miRanda, TagetScan and PicTar)to analyye KCNJ2related miRNA, then select differentially expressed miRNA (miR-7) to study and the miR-7expression difference were validated by real-time quantitative PCR.7. KCNJ2is the target genes of miR-7(1). using miR-7inhibitor (antangomir) and mimics (angomir)to inhibit or increase the expression level of miR-7in H69AR, H446AR and H69, H446cells, using real-time quantitative PCR and Western Bloting to analyze KCNJ2mRNA and protein expression changes.(2)..To construct the psiCHECK-2luciferase vector (psiCHECK-2-KCNJ2-3’UTE) of KCNJ23’-untranslated region (3’-untranslated region,3’-UTR), and to transfect the miR-7mimic or inhibitor into H69, H69AR, H446and H446AR cells, KCNJ2luciferase activity were determined by the method dual luciferase reporter gene assay.8. miR-7involve in multidrug resistance of SCLCmiR-7mimic or inhibitor was transfected into cells to increase or inhibit miR-7expression. Chemosensitivity to drugs were determined by using CCK-8assay.9.expression and clinicopathological significance miR-7, KCNJ2and ABCC1in SCLC tissues52cases of paraffin-embedded tissues of SCLC patients were collected, follow-up clinical data, KCNJ2and ABCC1expression are to be detected by immunohistochemical staining, the expression of miR-7is to be detected by QRT-PCR, to analyze the statistical difference betwteen the expression of KCNJ2, ABCC1and miR-7in resistance group (relapse after three months) and sensitive group (six months without recurrence), limited disease group(LD) and extensive disease group(ED), and further to analyze expression of KCNJ2, miR-7and ABCC1with the clinical characteristics of SCLC patients and survival relationship.10. statistical analysisAll results were confirmed by statistical software SPSS13.0statistics. Data are expressed as mean±standard deviation (x±s), diferences of KCNJ2or miR-7expression between two cells were used independent samples t test to compare; miR-7, KCNJ2or ABCC1expression in multiple groups were compared using one-way ANOVA (homogeneity of variance simultaneously) or Welch method correction (when heterogeneity of variance), multiple comparisons using the SNK method (homogeneity of variance simultaneously) or Dunnett’s T3method (when heterogeneity of variance); miR-7, the relationship between KCNJ2and ABCC1expression with clinicopathological characteristics using paired sample chi-square analysis (McNemar test); using multivariate Logistic regression analysis the effects of age, sex, drug sensitivity, clinical stage and other factors on Mir-7, ABCC1and KCNJ2expression; using Cox proportional hazards model analysis above variables and Mir-7, ABCC1and KCNJ2protein expression levels of survival time for patients with SCLC; influence of various factors on the survival time of patients with SCLC using Kaplain-Meier analysis. P<0.05was considered as statistically significant.Result1. KCNJ2expression increased significantly in multi-drug resistant cell line H69AR and H446AR.Real-time quantitative PCR showed KCNJ2mRNA expression in multidrug resistant SCLC cell lines H69AR, H446AR was significantly higher than the sensitive strain H69and H446(t=16.88, P=0.003; t=27.798, P<0.001), Western Blot detection showed KCNJ2protein was also significantly increased;.2. Forced KCNJ2expression enhanced chemosenresistance of H69and H446cells significantly, while the expression of ABCC1was also increased.(1) We succesfully constructed KCNJ2expression plasmid KCNJ2-PEX-1, by sequencing and comparison showed consistent with human KCNJ2coding (coding sequence, CDS).(2) Estabishement of stably transfected cell line H69-KCNJ2-PEX-1and H446-KCNJ2-PEX-1.KCNJ2-PEX-1or negtive control GM scrambled negative control (NC) were transfected into H69and H446cell, after G418selection to establish stable transfected cell line H69-KCNJ2-PEX-1and H446-KCNJ2-PEX-1, Elevation of KCNJ2mRNA and protein level in H69-KCNJ2-PEX-1and H446-KCNJ2-PEX-1were verified by using using quantitive real-time PCR (4.89times, t=10.184, P=0.006;7.23times, t=13.562, P=0.003);(3) After treatment of ADM, DDP and VP-16, survival and half maximal inhibitory concentration of a substance (IC50) value of H69-KCNJ2-PEX-1and H446-KCNJ2-PEX-1cells increased significantly compared with H69-NC and H446-NC cells(P<0.001).(4) flow cytometry analysis showed that apoptosis of H69-KCNJ2-PEX-1and H446-KCNJ2-PEX-1cell H69AR-KCNJ2cell decreased compared with H69-NC and H446-NC cells(P<0.001) after treatment of ADM, DDP and VP-16, while G0-G1and G2-M phase cells increased. 3. Downregulation of KCNJ2expression significantly increased drug sensitivity of H69AR and H446AR cells(1).KCNJ2-homo-628, KCNJ2-homo-1073significantly reduced the expression of KCNJ2in H69AR and H446AR cells, while ABCC1expression was also significantly reduced;According KCNJ2genome sequence of a human cDNA library, the combination of methods Ambion’S siRNA Target Finder provided and references to design targeting human KCNJ2gene four pairs shRNAg interference plasmid: KCNJ2-homo-514, KCNJ2-homo-628, KCNJ2-homo-1073, KCNJ2-homo-1357, were used to detect expression levels of KCNJ2mRNA and protein changes by quantitative PCR and Western Blot method, the two pairs of shRNA (KCNJ2-homo-628, KCNJ2-homo-1073) which had the most significantly inhibitory effection were chosen to be used in following experiments.(2). CCK-8analysis showed that survival of H69AR and H446AR cell transfeced with KCNJ2-homo-628and KCNJ2-homo-1073were significantly lower compared with negative control (NC) group and the control group (P<0.001), IC50values were also significantly reduced.(3). flow cytometry analysis showed that the apoptosis rate of H69AR and H446AR cells transfeced with KCNJ2-homo-628and KCNJ2-homo-1073after treatment of ADM, DDP and VP-16treatment increased significantly compared with the negative control group and blank control group(P<0.001), while the number of G0-G1phase and G2-M phase cells decreased significantly.4. In vivo tumor xenograft model:following cells:H69AR, H69AR-NC, H69AR-kcnj2-homo-628, H69AR-kcnj2-homo-1073, H69, H69-NC and H69-KCNJ2-PEX-1injected into nude mice, three weeks later,5mice of each group were sacrificed and tumors were excised, tumor size were measured, PCNA was detected and analyzed. The results suggest KCNJ2can enhance tumor cell proliferation,5. PKC-MEK signaling pathway may regulate the expression of KCNJ2In H69AR and H446AR cells, both inhibited the PKC-MEK signaling pathway critical protein kinase PKC and MEK, KCNJ2expression was detected by RT-PCR and Western blot, KCNJ2mRNA levels were reduced by45.8%(P<0.001),51.6%(P <0.001), protein levels decreased by62.3%(P<0.001),45.2%(P<0.001), inhibited PKC alonely, KCNJ2mRNA levels were reduced by26.2%(P=0.023) and38.6%(P=0.003), protein levels decreased by23.5%,(P=0.026),29.6%(P<0.022), respectively, inhibited MEK alonely, KCNJ2mRNA levels were reduced31.3%(P=0.017),25.8%(P=0.029), protein levels decreased29.4%,(P=0.034),21.6%(P=0.036). respectively. Surgesting PKC-MEK signaling pathway may regulate the expression of KCNJ2.6. correlation between KCNJ2and ABCC1(1). An increase KCNJ2expression significantly increased the expression of ABCC1;(2).immunohistochemical analysis of tissue samples showed significantly positive correlation of KCNJ2and ABCCl(χ2=19.396, P<0.001);(3). Co-immunoprecipitation assay verified the interaction of KCNJ2with ABCC1in H69AR cells7. There are no statistically significance between miR-7expression in multidrug resistant SCLC cell lines H69AR, H446AR and sensitive cell lines H69,H446miRNA microarray results showed miR-7expression in multidrug resistant SCLC cell lines H69AR was4.64times lower than that in sensitive cell lines H69, while real-time PCR results suggested that. miR-7expression in H69and H446cells are higher than that in H69AR and H446AR cell. But the defferrenes have no statistically significance (1.09times,2=2.148, P=0.098;1.03times,t=0.478, P=0.657).8. KCNJ2is the target genes of miR-7(1) According to miRNA target gene prediction software (PicTar, TarScan and miRBase), there are complementary binding sites between miR-7and KCNJ23’-UTR.(2) regulation of KCNJ2mRNA and protein expression by miR-7 ①After transfection of mir-7agomir H69, H69AR, H446and H446AR cells to increase the expression of miR-7, showed KCNJ2mRNA expression levels were significantly lower (F=57.780, P=<0.001), KCNJ2expression was significantly reduced (F=254.520, P=<0.001). ABCC1mRNA levels and protein levels was also significantly decreased (F=381.121,P<0.001; F=365.439, P<0.001).②After transfection of mir-7antagomir to reduce the expression of miR-7in H69, H69AR, H446and H446AR cells, the results showed that the expression KCNJ2mRNA levels and protein expression were significantly elevated(F=152.419, P<0.001; F=87.124, P<0.001.); ABCC1mRNA levels and protein levels were also significantly increased (F=3.385, P<0.001; F=786.360, P<0.001).(3) KCNJ2is the target gene of miR-7①successfully constructed psiCHECK-2-KCNJ2-3’UTR and psiCHECK-2-KCNJ2-3’UTR-R carrier.KCNJ2-3’-UTR was amplified by PCR, the product length is1250bp, After it was converted, resistance screened and selected for monoclone, we extracted plasmid. The plasmid was cleaved with Not I and Xho I, and the product of6365bp/1235bp were obtained. Clone1-4of psiCHECK-2-KCNJ2-3’UTE and clone3of psiCHECK-2-KCNJ2-3’UTR-R were verified to be correct by using restriction enzyme digestion, so we selected them to be sequenced. The sequences were consistent with the theoretical sequence. Plasmid was extracted for following experiments.②miR-7mimic and inhibitor regulated luciferase activity of KCNJ2PsiCHECK-2-KCNJ2-3’UTR or psiCHECK-2-KCNJ2-3’UTR-mut was transfected into H69AR cell. Co-transfection of miR-7mimic or inhibitor was performed. Compared with H69AR cell transfeected with miR-NC, luciferase activity of KCNJ2in H69AR cell co-transfected with miR-7mimic and KCNJ23’-UTR reduced significantly (F=437.572, P<0.001), miR-7inhibitor can be significantly increased transfection KCNJ23’-H69AR cells KCNJ2UTR of luciferase activity (multiple comparison P<0.05), miR-7mimic and miR-7inhibitor had no significant effect on transfection KCNJ23’UTR-mut in H69AR cells KCNJ2 luciferase activity (multiple comparison of P>0.05).9. miR-7have no significant correlation with drug sensitivity in H69/H69AR and H446/H446AR cellsAfter treatment of ADM, DDP and VP-16, the differences between survival of H69, H446,H69AR and H446AR cells transfected with miR-7mimic or miR-7inhibitor were no statistically significance (multiple comparisons, P>0.05)..10. Relationships between miR-7, KCNJ2and ABCC1expression and clinicopathological features in SCLC tissues.Mir-7expression was the protective factors for clinical stages of SCLC patients (OR=0.258, P=0.043), while KCNJ2and ABCC1expression were the risk factors for the clinical stages of SCLC patients (OR=11.124, P=0.001; OR=10.710, P=0.001), other indicators were not statistically significance (P>0.05).11. survival analysis of SCLC patients(1) COX proportional hazards regression modelUsing COX stepwise regression analysis, the variables were selected into the " sex "," age"," staging "," sensitive ","Mir-7","KCNJ2" and "ABCC1", the relative risk of the variable "Mir-7" was0.75(P=0.026), i.e.,suggest that death risk of patients with higher Mir-7expression was0.75times compared with patient with lower Mir-7expression, there were no significant correlation between gender, stage, drug sensitivity, KCNJ2and ABCC1expression with the relative risk of SCLC patients.(2). The estimated survival timeUsing the Kaplan-Meier method compares the impact of different factors on the survival time of patients and found that male patients and female patients with no significant difference in survival time (x2=0.473, P=0.492). survival time of patients younger than55years was significantly longer than that of patients over55years of age (χ2=6.867, P=0.009). survival time of patients with limited-stage was significantly longer than patients with extensive stage (x,2=4.065, P=0.0231). survival time of patients with higher Mir-7expression was significantly longer than patients with lower Mir-7expression (χ2=5.825, P=0.016); there were no significant difference between the survival time of patients with KCNJ2and ABCC1positive expression and that of the patients with KCNJ2and ABCC1negative expression (x2=1.016, P=0.313; x2=1.696, P=0.193).Conclusion1. KCNJ2expression of multidrug resistance in SCLC cell lines H69AR, H446AR significantly increased cell KCNJ2increase or decrease the expression of resistance can cause cell changes, indicating KCNJ2involved in multi-drug resistant of SCLC.2. KCNJ2and ABCC1expression in SCLC cell lines and clinical tissue samples showed a significant positive correlation, co-immunoprecipitation display verified the interaction of KCNJ2and ABCC1in H69AR cells, suggesting KCNJ2involved in multi-drug resistant of SCLC possibly through interaction with ABCC1;3.Separately or simultaneously inhibiting PKC or MEK can reduce the expression of KCNJ2in SCLC cell lines, suggesting that PKC-MEK signaling pathway involved in the regulation of KCNJ2;4. KCNJ2is a target gene of miR-7, miR-7expression can regulate the expression of KCNJ2, decrease or increase the expression of miR-7in cells may change the expression of KCNJ2, suggesting that miR-7involved in regulation of KCNJ2;5. The expression of miR-7, KCNJ2and ABCC1in SCLC tissues related to the clinical stages of SCLC patients, whereas miR-7expression was significantly associated with the patient’s survival time as well, suggesting that miR-7is expected to be a clinical prognostic indicator in patients with SCLC and therapeutic target.更多还原