CN116479130A - 卷曲螺旋形成蛋白激酶rock1作为胰腺癌肿瘤标志物及其应用 - Google Patents
卷曲螺旋形成蛋白激酶rock1作为胰腺癌肿瘤标志物及其应用 Download PDFInfo
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Abstract
本发明涉及卷曲螺旋形成蛋白激酶ROCK1的应用,具体涉及ROCK1作为胰腺癌肿瘤标志物及其应用;ROCK1作为胰腺癌肿瘤标志物,在胰腺癌细胞中高表达;ROCK1作为胰腺癌潜在靶点,敲低ROCK1在体内外均使胰腺癌细胞生长受到抑制;敲低ROCK1增加胰腺癌对吉西他滨的化疗敏感性。本发明提供了一种新的胰腺癌肿瘤标志物ROCK1,并在ROCK1敲低后肿瘤的生长缓慢,提示ROCK1抑制剂对胰腺癌有潜在的治疗作用及意义,并且可以提高吉西他滨的敏感性。
Description
技术领域
本发明涉及卷曲螺旋形成蛋白激酶ROCK1新应用,具体涉及ROCK1作为胰腺癌肿瘤标志物及与吉西他滨联用提高化疗敏感性的应用
背景技术
胰腺癌(Pancreaticcancer)是一种顽固的癌症,是5年生存率最低的癌症之一。胰腺癌是全球癌症死亡的主要原因,其全球负担在过去25年中增加了一倍以上。胰腺癌发病率最高的地区包括北美,欧洲和澳大利亚,尽管这一增长大部分是由于全球人口老龄化,但胰腺癌存在关键的可改变危险因素,如吸烟,肥胖,糖尿病和酒精摄入。由于胰腺癌早期临床症状不明显,大部分病人确诊时已无手术机会,需要通过全身化疗来改善预后。因此放化疗对于治疗胰腺癌至关重要。有许多用于胰腺癌治疗的化疗药物可以提高患者的总体生存率。化疗是多模式PC治疗的重要组成部分,而吉西他滨(GEM)是批准用于治疗PC患者的一线化疗药物,且吉西他滨化疗是不适合手术的胰腺癌患者的首选治疗方法,因而吉西他滨是晚期胰腺癌新辅助治疗、辅助治疗和姑息治疗的基石。
随着靶向治疗和免疫治疗等多种治疗手段的不断涌现,胰腺癌患者的总生存期相对延长,但胰腺癌因其早期低检出率和后期高转移性,晚期胰腺癌患者的5年生存率仍然很低。对胰腺癌肿瘤标志物进行了大量的研究,至今仍未找到一种对胰腺癌特别是早期胰腺癌有足够敏感性、特异性的肿瘤标志物,由于单项肿瘤标志物阳性率不高,故需与其他肿瘤标志物联合检测,因此胰腺癌目前没有可靠的靶向的生物标志物。并且,胰腺癌的不良预后主要是由于对包括吉西他滨在内的治疗产生耐药性。
肿瘤标志物是指肿瘤组织产生并可以反映肿瘤细胞存在于宿主体内的化学分子。胰腺癌为高度恶性的消化系统肿瘤,其起病隐匿,一旦发现多属晚期,预后差,目前改善预后的主要干预方针仍集中在早期诊断,医学界非常重视寻找胰腺癌早期诊断的肿瘤标志物。研究发现CA19-9、CA242、CA50和CA724是诊断胰腺癌最敏感的指标,它们的灵敏度有的为89.2%,特异性有的达92.3%,是胰腺癌诊断首选的肿瘤标志物,但需多种肿瘤标志物联合检测对临床早期诊断胰腺癌方具有一定意义。
ROCK1-Rho相关卷曲螺旋形成蛋白激酶(Rho-associatedcoiled-coilcontainingkinases,ROCKs)是目前十分热门的药物研究靶点。ROCKs家族包含ROCK1和ROCK2,研究显示,ROCKs主要参与肌动蛋白细胞骨架的组成和相关的动态事件:如细胞收缩和迁移等。实验发现,ROCKs介导肌球蛋白磷酸酶靶亚基1磷酸化引起肌球蛋白轻链磷酸化的增加,从而引发细胞收缩行为。ROCKs如何参与肿瘤进程仍旧是目前的研究热点,特别是在肿瘤的发生、发展和转移等阶段。ROCK1(GeneID:6093)在多种实体瘤中均高表达,并且与肿瘤细胞的生长和侵袭密切相关,但未见在胰腺癌中报道。
这使我们将ROCK1与胰腺癌联系起来,将ROCK1作为胰腺癌的一个新的肿瘤标志物,为胰腺癌的早期诊断、预后判断等提供新的思路。
因此,进一步研究和探索胰腺癌发生发展过程中潜在的分子机制,寻找胰腺癌早期诊断的肿瘤标志物和治疗的新靶点、提高化疗敏感性迫在眉睫。
发明内容
发明目的
基于此,本发明提供一种新的胰腺癌肿瘤标志物卷曲螺旋形成蛋白激酶ROCK1及其应用,有望为胰腺癌的治疗提供新的靶点抑制剂和提高联合吉西他滨的用药效果。
技术方案
ROCK1在制备作为胰腺癌肿瘤标志物的应用,其中ROCK1为GeneID:6093(https://www.ncbi.nlm.nih.gov/gene/6093)
一种qRT-PCR检测试剂盒,其特征在于,含有模板、引物和dNTP,taq酶,PCR缓冲溶液;其中所述的模板为ROCK1;所述的引物为:
ForwardPrimer:TGTTCTGCTTACATCAGCCTG,见SEQ ID NO.1;
ReversePrimer:TGTATCACATCGTACCATGCCT,见SEQ ID NO.2。
shROCK1与吉西他滨联用在制备治疗胰腺癌药物中的应用,其特征在于shROCK1为ROCK1的干扰序列,其核苷酸序列为sense:CCTGGTGGAGATCTTGTAA,见SEQ ID NO.3;antisense:TTACAAGATCTCCACCAGG,见SEQ ID NO.4;。
本发明提供了一种新的胰腺癌肿瘤标志物,所述胰腺癌肿瘤标志物为ROCK1;所述ROCK1在胰腺癌患者胰腺癌组织中的表达水平采用免疫组化检测分析,发现ROCK1在胰腺癌组织中处于显著高表达状态,且其表达水平与胰腺癌患者的临床预后显著相关。本发明通过qRT-PCR与Westernblot实验检测了与正常胰腺导管上皮细胞HPDE6-C7相比六株不同胰腺癌细胞中ROCK1的表达情况。选取一株与HPDE6-C7相比ROCK1高表达的胰腺癌细胞AsPC-1,应用siRNA基因沉默技术构建ROCK1的沉默模型,并对ROCK1在胰腺癌细胞中发挥的生物学功能进行探究:发现敲低ROCK1不仅可以促进胰腺癌细胞的增殖、集落形成、迁移侵袭,而且能够降低胰腺癌细胞凋亡。
所述标志物可通过设计引物序列作为胰腺癌检测试剂盒的关键组分之一,并对胰腺癌患者临床样本进行该肿瘤标志物的检测。胰腺癌标本的收集程序如下:切取新鲜胰腺癌组织保存于-80℃,以备后续用TRIzol试剂进行胰腺癌组织样本总RNA的提取。ROCK1引物序列:
ForwardPrimer:TGTTCTGCTTACATCAGCCTG;
ReversePrimer:TGTATCACATCGTACCATGCCT;
根据引物采用荧光定量PCR(qRT-PCR)对患者的胰腺癌组织进行ROCK1表达水平的检测。
所述的应用可根据ROCK1的表达水平对胰腺癌患者进行早期筛选并进行临床预后的预测指标,具有临床转化价值。
本发明实验研究发现,裸鼠移植瘤模型中,在敲除ROCK1的胰腺癌细胞并给予吉西他滨治疗的组别中,肿瘤的体积明显减少,并且呈剂量依赖性。本发明选用胰腺癌细胞系AsPC-1,采用慢病毒质粒构建稳定沉默ROCK1的细胞系(shROCK1),以shNC作为对照。采用皮下注射的方式,构建AsPC-1(shNC和shROCK1)裸鼠移植瘤模型。体外试验结果表明,基因沉默组的肿瘤体积明显比对照组小,表明是存在ROCK1的促癌作用,并有可以成为胰腺癌肿瘤标志物的前景。并且在此基础上对其进行GEM治疗,大剂量组(50mg/kg)效果显著。本发明结果表明ROCK1是胰腺癌的促癌基因,可作为胰腺癌的肿瘤标志物,并且敲除ROCK1的胰腺癌与GEM联用有明显的肿瘤抑制作用。
本发明的关键点
1、ROCK1作为胰腺癌肿瘤标志物,在胰腺癌细胞中高表达。
2、ROCK1作为胰腺癌潜在靶点,敲低ROCK1在体内外均使胰腺癌细胞生长受到抑制。
3、敲低ROCK1增加胰腺癌对吉西他滨的化疗敏感性。
有益效果
1.本发明所述的ROCK1在胰腺癌细胞中高表达,并根据GEPIA数据库提示与患者生存率呈现负相关性,通过qRT-PCR实验检测了与正常胰腺导管上皮细胞HPDE6-C7相比六株不同胰腺癌细胞中基因的表达情况,以确定ROCK1高表达的胰腺癌细胞系AsPC-1。
2.利用选定胰腺癌细胞系AsPC-1,采用慢病毒质粒构建稳定沉默ROCK1的细胞系,以shNC作为对照。采用皮下注射的方式,构建AsPC-1(shNC和shROCK1)裸鼠移植瘤模型,通过评估基因沉默组与对照组相比肿瘤的大小,发现敲低ROCK1后,有一定的抑癌作用,并且在敲低ROCK1的胰腺癌细胞并给予吉西他滨(GEM)治疗的组别中,肿瘤的体积明显减小。所以本发明的意义在于提供了一种新的胰腺癌肿瘤标志物ROCK1,并在ROCK1敲低后肿瘤的生长缓慢,提示我们ROCK1抑制剂可能对胰腺癌有治疗的作用,并且可以提高吉西他滨的敏感性。
附图说明
图1:GEPIA数据库预测ROCK1在胰腺癌组织和癌旁组织中的mRNA表达情况,其中红色代表癌组织,黑色代表癌旁组织(*p<0.05)(A)。GEPIA数据库分析ROCK1的表达量与患者总生存率的关系(*p<0.05)(B);
图2:免疫组化实验分析胰腺癌临床样本中ROCK1的蛋白表达量(n=5,左:40×;右:100×)。通过临床样本的免疫组化结果分析ROCK1在胰腺癌组织与癌旁组织的蛋白表达水平,可得到与数据库一致的结果。
图3:图3qRT-PCR检测ROCK1在六株胰腺癌细胞中的相对表达量(以HPDE6-C7组为参照,n=4,**p<0.01,***p<0.001)。
图4:WB检测ROCK1在六株胰腺癌细胞中的相对表达量,其中A为WB结果照片,B为统计结果。
图5:MTT法检测敲低ROCK1对胰腺癌细胞增殖的影响(A)。平板克隆法检测敲低ROCK1对胰腺癌细胞集落形成的影响(B),统计结果(C)。
图6:Transwell实验检测敲低ROCK1对细胞迁移的影响,其中照片(A),统计结果(B)。
图7:流式细胞术检测敲低ROCK1对细胞凋亡的影响,其中流式(A),统计结果(B)。
图8:敲低ROCK1对裸鼠胰腺癌移植瘤的影响以及敲低ROCK1在体内对胰腺癌吉西他滨敏感性的影响,WB照片(A),统计结果(B)。
图9:移植瘤后测量体积,照片(A),统计结果(B)。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。
为了使胰腺癌患者的诊断更为简单、便捷、快速及准确,本发明提供了一种新的胰腺癌肿瘤标志物,即卷曲螺旋形成蛋白激酶ROCK1,进一步的,ROCK1作为肿瘤标志物,我们设计了其荧光定量PCR的引物,并以此进行ROCK1在胰腺癌细胞系中表达水平的检测,本发明还验证了ROCK1表达水平与胰腺癌患者临床预后之间的相关性,在肿瘤细胞功能学方面,本发明还进一步验证了ROCK1可以在胰腺癌组织中起到促癌作用,并且还可以降低胰腺癌细胞对吉西他滨的敏感性。本发明最终提供了ROCK1作为胰腺癌肿瘤标志物的检测应用。
实施例1:数据库分析ROCK1在胰腺癌组织中的表达以及与患者预后关系
本实施例的一种新的胰腺癌肿瘤标志物,所述胰腺癌肿瘤标志物为卷曲螺旋形成蛋白激酶ROCK1;运用GEPIA数据库对ROCK1在胰腺癌组织中的表达量进行研究,比较ROCK1在胰腺癌组织和癌旁组织中表达量的差异;以及利用GEPIA数据库预测ROCK1表达量与胰腺癌患者总生存率的关系,发现ROCK1的表达量与患者预后密切相关。
结果:如图1所示,GEPIA数据库(http://gepia.cancer-pku.cn/)提示:胰腺癌组织中,ROCK1的表达要显著高于癌旁组织(p<0.05,图1A),并进一步接着,利用GEPIA数据库预测ROCK1表达量与胰腺癌患者总生存率的关系,发现ROCK1的表达量与患者预后密切相关,随着ROCK1表达量的升高,胰腺癌患者总生存率显著降低(p=0.025,图1B),患者预后差。
实施例2:免疫组化分析ROCK1在胰腺癌组织中的表达情况
本实施例样本人胰腺癌组织以及癌旁组织来自于上海交通大学医学院附属瑞金医院,进一步通过临床样本的免疫组化结果分析ROCK1在胰腺癌组织与癌旁组织的蛋白表达水平。
结果:如图2所示,通过临床样本的免疫组化结果分析ROCK1在胰腺癌组织的蛋白表达水平高于癌旁组织,可得到与数据库一致的结果。
实施例3:ROCK1在胰腺癌细胞系中mRNA的表达情况
本实施例样本人胰腺癌细胞(AsPC-1、PANC-1、MIAPaCa-2、Capan-1、BxPC3、SW1990)和正常胰腺导管上皮细胞HPDE6-C7均购自上海中科院细胞所,用TRIzol试剂进行各细胞样本总RNA的提取。
本实施例的ROCK1引物序列:
ForwardPrimer:TGTTCTGCTTACATCAGCCTG;
ReversePrimer:TGTATCACATCGTACCATGCCT;
根据引物采用荧光定量PCR(qRT-PCR)对患者的胰腺癌细胞进行ROCK1mRNA表达水平的检测。
结果:从图3中可以发现与正常胰腺导管上皮细胞相比,ROCK1的mRNA水平在AsPC-1和PANC-1细胞中呈现显著高表达的趋势,在Capan-1、MIAPaCa-2、BxPC3、SW1990细胞中呈现显著低表达的趋势。
实施例4:ROCK1在胰腺癌细胞系中蛋白的表达情况
本实施例样本人胰腺癌细胞(AsPC-1、PANC-1、Capan-1、BxPC3、SW1990)和正常胰腺导管上皮细胞用由RIPA裂解液和PMSF配制的细胞裂解液(二者体积比100:1),冰上裂解时间为10min,将细胞刮下并收集于1.5mLEP管中,4℃,12000g离心时间8min,吸取上清液,BCA法测量蛋白浓度,而后加入PBS、5×loadingbuffer调整样品蛋白浓度一致,将样品管放入沸水中煮10min,蛋白样品放-80℃低温冰箱保存。
根据所制得的样品进行WesternBlot检测ROCK1在胰腺癌细胞系中的蛋白表达情况。
结果:如图4所示,同时与正常胰腺导管上皮细胞相比,ROCK1的蛋白质水平在AsPC-1和PANC-1细胞中呈现显著高表达的趋势,在BxPC3、SW1990细胞中呈现显著低表达的趋势(图4A),相对表达量如(图4B)。
实施例5:ROCK1对胰腺癌细胞增殖的影响
本实施例选择了ROCK1高表达的细胞株AsPC-1转染siRNA-ROCK1的序列构建ROCK1的沉默模型。在转染了ROCK1的沉默质粒的AsPC-1细胞,用胰酶消化细胞,细胞混悬液铺于96孔板,细胞密度为5×104个/mL,每孔100μL细胞悬液,每组4个复孔,避免细胞聚团现象,分别继续培养6、24、48、72h,用MTT实验检测细胞的增殖情况。
同样取转染沉默质粒后的AsPC-1细胞,胰酶消化,将其接种在6孔板中,细胞密度为600个/mL,每孔2mL细胞悬液,静置培养10天,通过结晶紫染色方法考察AsPC-1细胞的集落形成能力。
结果:发现在沉默了ROCK1继续培养72h可显著抑制AsPC-1胰腺癌细胞的生长活力,抑制率达到了29.8%(图5A),由此可见沉默ROCK1后可显著抑制胰腺癌细胞的增殖能力。在沉默了ROCK1后可显著抑制AsPC-1胰腺癌细胞的集落形成,抑制率达到了51.3%(图5B)。
实施例6:ROCK1对胰腺癌细胞迁移的影响
本实施例在AsPC-1细胞中转染ROCK1沉默质粒后继续培养24h,胰酶消化,1300rpm,离心5min,吸弃上清,PBS洗涤细胞一次,1300rpm,离心5min,用基础培养基重悬细胞,调整细胞密度为2.5×105个/mL,取200μL加入小室的上室,下室加入500μL完全培养基,静置培养24h;取出小室,弃去小室中的液体,PBS洗涤两次;24孔板内,用500μL4%多聚甲醛固定时间为30min,PBS洗涤两次后适当风干;300μL0.1%结晶紫染色液染色时间为20min,PBS洗涤两次,用棉签擦净小室上室残留的细胞,PBS洗涤小室3遍,室温风干5min。分别在40、100、200倍显微镜下观察细胞迁移数并拍照。
结果:如图6所示,Transwell实验结果表明沉默ROCK1后显著阻抑了AsPC-1细胞的迁移(图6A),抑制分别达到了61.6%(图6B)。
实施例7:ROCK1对胰腺癌细胞凋亡的影响
在AsPC-1转染ROCK1沉默质粒后继续培养48h,后用胰酶消化细胞(不含EDTA),弃去胰酶,PBS洗涤两次,每次2000rpm,离心5min,弃上清,避光条件下,每管加入BindingBuffer500μL重悬细胞,随后加入AnnexinV-FITC5μL,轻缓混匀,最后加入5μLPropidiumIodide。室温条件下避光反应10min后用流式细胞仪检测凋亡变化。
结果:如图7所示,在沉默ROCK1后,促进了AsPC-1胰腺癌细胞的凋亡(图7A),与对照组相比,沉默ROCK1后AsPC-1细胞的总凋亡率从1.1%上升到25.0%(图7B),结果显示ROCK1能够阻遏胰腺癌细胞凋亡。
实施例8:ROCK1在体外影响胰腺癌对吉西他滨的敏感性
本实施例由上海吉玛制药技术有限公司合成靶向ROCK1的shRNA慢病毒包装质粒,转染AsPC-1细胞构建稳定敲低ROCK1的胰腺癌细胞。
研究ROCK1是否在体内也有致癌活性。通过Westernblot实验验证了shRNA的沉默效率。然后,我们研究了ROCK1缺失是否增强了胰腺癌细胞对吉西他滨的敏感性。用shROCK1和shNC慢病毒质粒转染AsPC-1细胞,用不同浓度梯度吉西他滨(0、1、2.5、5、7.5μM)培养48h,用MTT检测细胞毒性。
结果:转染shROCK1慢病毒质粒后,ROCK1蛋白的表达明显被抑制,说明shROCK1沉默质粒已成功转染(图8A),沉默ROCK1显著提高了AsPC-1细胞对吉西他滨的敏感性(图8B)。
实施例9:ROCK1在体内影响胰腺癌对吉西他滨的敏感性
本实施例由上海吉玛制药技术有限公司合成靶向ROCK1的shRNA慢病毒包装质粒,转染AsPC-1细胞构建稳定敲低ROCK1的胰腺癌细胞。
BALB/c雄性裸鼠被饲养于动物实验中心SPF级层流柜内,每5只为一笼分开饲养。取shNC、shROCK1慢病毒质粒转染过的稳定传代的AsPC-1胰腺癌细胞,胰酶消化后PBS洗涤细胞三次,每次1300rpm,离心5min。收集细胞沉淀,生理盐水重悬细胞,计数,将密度调整为6×106个/mL,采用皮下注射方式,每只裸鼠接种200μL细胞混悬液,每组5只裸鼠。在可以明显看到肿瘤时候,每周一、周四腹腔注射吉西他滨(25mg/kg、50mg/kg),24天后脱颈处死裸鼠,取出移植瘤。测量肿瘤大小和体积。
结果:如图9所示,取出移植瘤后测量体积,结果发现,shROCK1组肿瘤体积显著小于shNC组(图9A,B),说明敲除ROCK1后,显著增加了异种移植瘤对吉西他滨的敏感性(p<0.05)。
Claims (2)
1.卷曲螺旋形成蛋白激酶ROCK1在制备作为胰腺癌肿瘤标志物的应用。
2.ROCK1抑制剂shROCK1与吉西他滨联用在制备治疗胰腺癌药物中的应用,其特征在于shROCK1为ROCK1的干扰序列,其核苷酸序列为sense:CCTGGTGGAGATCTTGTAA,antisense:TTACAAGATCTCCACCAGG。
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