CN114480653B - 人mrgprf基因在肿瘤临床诊疗中的应用 - Google Patents
人mrgprf基因在肿瘤临床诊疗中的应用 Download PDFInfo
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Abstract
本发明公开了人MRGPRF的新用途,即人MRGPRF基因表达量检测试剂在制备黑色素瘤临床诊断试剂中的应用;实验结果显示人MRGPRF基因在黑色素瘤细胞系中较人永生化表皮细胞表达低;MRGPRF基因过表达后,黑色素瘤细胞系的增殖受到显著抑制,细胞周期阻滞在G0/G1期;此外,内皮细胞增殖抑制剂AMG 706可作为MRGPRF的潜在激动剂,在体内外阻碍肿瘤生长,MrgprF蛋白的274‑343肽段竞争结合p110‑γ、p101蛋白,降低PI3K/AKT信号通路活性作用,从而抑制黑色素瘤发生发展;本发明揭示了人MRGPRF基因是黑色素瘤的肿瘤抑制因子,并筛选到了靶向MRGPRF的潜在抗癌化合物或多肽,为未来的黑色素瘤治疗提供治疗药物。
Description
技术领域
本发明涉及一种基因的新用途,尤其是人MRGPRF基因在肿瘤临床诊疗中的新用途,具体涉及人MRGPRF基因在黑色素瘤临床诊疗中的应用。
背景技术
皮肤黑色素瘤(cutaneous melanoma:CM)是皮肤癌死亡的主要原因,与其他类型的人类癌症相比,近年来在世界范围内迅速增加。根据美国流行病学数据,高龄和高加索种族是黑色素瘤的2个关键危险因素,另外,室内晒黑(尤其是年轻女性)也被明确为危险因素。S100钙结合蛋白-p,MLANA(T细胞1识别的黑色素瘤抗原)和MITF(小眼畸形相关转录因子)经常被用作显示低特异性的模糊病例的标志物。BRAF(B-Raf原癌基因)、CDKN2a(细胞周期蛋白依赖性激酶抑制剂2A)和NRAS(神经母细胞瘤RAS病毒癌基因同源物)中基因突变的鉴定可以提高诊断的准确性。前期的研究表明,有利于肿瘤侵袭和浸润的信号通路的激活证明黑色素瘤进展是由基因突变和肿瘤微环境改变引起的。例如,黑色素瘤中包括MMP-2和MMP-9在内的基质金属蛋白酶(MMPs)已被证明是由NF-κB的信号通路诱导产生。
除肿瘤微环境改变外,BRAF、CDKN2a、NRAS、NF1、TP53、PTEN和TERT的体细胞突变主要导致丝裂原活化蛋白激酶(MAPK)途径和磷酸肌醇-3-激酶信号(PI3K/Akt)通路的激活。参与生长因子和激素转导的MAPK途径,已被证明在不同类型的癌症中被激活。PI3K/Akt途径通常被受体酪氨酸激酶(RTKs)和G蛋白偶联受体(GPCRs)激活,导致磷脂酰肌醇-(3,4)-P2(PIP2)向磷脂酰肌醇-(3,4,5)-P3(PIP3)的转化增加,以及Akt蛋白上的高水平磷酸化。然而,由于缺乏可靠的诊断生物标志物,要改进致死的黑色素瘤的早期诊断,仍有很多工作要做。
大多数黑色素瘤患者应每年接受皮肤科检查,手术切除仍然是大多数治愈性病例的首选,然而处于更晚期的患者可能无法切除或已经转移。近年来,随着靶向和/或免疫疗法的应用,包括BRAF和MEK(MAP-ERK激酶)抑制剂以及免疫检查点抑制剂(抗细胞毒性T淋巴细胞相关抗原4(CTLA-4)抗体和抗程序化细胞死亡蛋白1(PD-1)抗体),黑色素瘤治疗发生了革命性变化。
我们最近的研究确定了多种人类癌症中表达失去管控的泛癌生物标志物,通过使用综合生物信息学分析,结合其他网络来源可用的黑色素瘤相关数据集,我们确定MRGPRF(一种与MAS相关的GPR家族成员)在多种类型的人类癌症(包括黑色素瘤)中减少。Mas相关基因(Mrgs)属于GPCR基因家族,主要在背根神经节(DRGs)和三叉神经节(TG)的感觉神经元亚群中表达,之前的研究主要集中在瘙痒和疼痛感上。然而,Mrgs在神经系统外的表达失调尚未得到深入研究,只有少数报道。例如,MrgprD被证明在非小细胞肺癌和小鼠肠道癌中上调表达。MRGX2已被证明在被抗菌肽基因家族杀菌素LL-37激活后参与癌症进展。这些发现表明,Mrgs在其他正常的人类发育过程和病理状况(如人类癌症)中起着关键作用。MRGPRF在黑色素瘤中的功能作用和分子机制尚不可知。
发明内容
本发明的目的在于提供人MRGPRF基因的新用途,即将人MRGPRF基因表达量检测试剂应用在制备黑色素瘤临床诊断试剂中;人MRGPRF基因作为黑色素瘤关联基因,应用于黑色素瘤检测,人MRGPRF基因表达量检测试剂是检测人MRGPRF基因低表达量的试剂,即将人MRGPRF基因低表达作为诊断黑色素瘤的标志。
检测人MRGPRF基因表达量可以利用人MRGPRF基因序列设计人MRGPRF的RNA引物序列,并通过实时定量PCR法检测人MRGPRF的RNA的水平;所述RNA的引物序列为:
SEQ ID NO:1:CGTCACTGACCTGTGCATCT;
SEQ ID NO:2:CTCCATGGTGACTGTGTTGG。
针对黑色素瘤中MRGPRF低表达的情况,本发明另一目的是将以激活或提高人MRGPRF基因的表达为目的,筛选能促进MRGPRF高表达的药物,用于治疗黑色素瘤。
基于人MRGPRF基因对PI3K/AKT信号通路的抑制作用,筛选模拟人MrgprF抑制PI3K/AKT信号通路的小肽,阻断或降低p110-γ与p101的相互结合,进而抑制PI3K/AKT信号通路,实现治疗黑色素瘤的目的。
所述小肽为人MrgprF蛋白第274-343位的肽段,其氨基酸序列如SEQ ID NO:3所示,
该小肽段是人MrgprF蛋白与人p110-γ蛋白相互作用的区段,通过该小肽段竞争结合p110-γ蛋白,降低PI3K/AKT信号通路活性作用,从而抑制黑色素瘤发生发展。
本发明在研究中发现人MRGPRF基因与黑色素瘤的发生发展的相关性,通过肿瘤芯片染色结果及网络数据库分析发现,人MrgprF在黑色素瘤中低表达,且其表达量高低与预后呈正相关。因此,我们通过NCBI数据库,找到人MRGPRF基因的序列,人MRGPRF基因的核苷酸序列见genebank中116535,位于11号染色体的69004395-69013382位。Description:MRGPRF(from geneSymbol);Gencode Transcript:ENST00000441623.1;Gencode Gene:protein coding。根据人MRGPRF基因的序列,我们利用实时定量PCR发现人MRGPRF基因确实在多个黑色素瘤细胞系中低表达,因此我们将靶向人MRGPRF的shRNAs转染正常永生化人角质细胞及表达量相对较高的黑色素瘤细胞系,将靶向人MRGPRF基因的过表达质粒转染低表达的黑色素瘤细胞系,构建稳转细胞系,观察其对肿瘤细胞增殖和迁移能力的影响。我们首先评估了所设计的shRNA的敲低效率,实时定量PCR结果显示,shRNAs显著降低了MRGPRF基因的表达(P<0.001),与对照相比,黑色素瘤细胞的增殖和迁移能力都显著加强。而过表达MRGPRF基因,则反之。进一步检测其细胞周期分布,发现过表达MRGPRF基因,可以将细胞阻滞在G0/G1期,促进黑色素瘤细胞的凋亡并抑制黑色素瘤细胞的裸鼠移植瘤形成能力,降低黑色素瘤细胞体内增殖和迁移,促进黑色素瘤在体内凋亡。
深入的研究发现人MRGPRF基因过表达的细胞或者用AMG 706药物处理促进MRGPRF基因表达的细胞,在顺铂处理时,这些黑色素瘤细胞表现出对顺铂更加敏感,细胞凋亡更加显著,裸鼠移植瘤体内实验进一步证实促进人MRGPRF基因的表达,黑色素瘤对顺铂更加敏感,表明促进人MRGPRF基因的表达的方法或药物在未来可与顺铂联合使用,用于黑色素瘤患者的治疗。
进一步的研究发现,受体酪氨酸激酶(RTKs)和G蛋白偶联受体(GPCRs)激活,导致磷脂酰肌醇-(3,4)-P2(PIP2)向磷脂酰肌醇-(3,4,5)-P3(PIP3)的转化增加,以及Akt蛋白上的高水平磷酸化。而MrgprF的274-343肽段能与p101竞争结合p110-γ,从而影响PIP2向PIP3的转化,进而抑制PI3K/AKT信号通路的活性,发挥抑制黑色素瘤发生发展的作用。
综上所述,人MRGPRF基因在体外对黑色素瘤细胞的增殖、凋亡和化疗耐受有调节作用;人MRGPRF基因过表达后,肿瘤细胞的增殖受到显著抑制,凋亡显著增加,对化疗药物顺铂更加敏感。本发明首次揭示了MRGPRF基因是黑色素瘤发生发展的潜在风险基因,为黑色素瘤的临床诊断提供新的生物标志物;明确了可以通过促进人MRGPRF基因的表达与顺铂联合用于黑色素瘤的治疗,并明确了人MrgprF蛋白第274-343位的肽段通过抑制PI3K/AKT信号通路的活性在治疗黑色素瘤中的潜在功能。
本发明明确了人MRGPRF基因的表达与黑色素瘤发生发展的相关性;并建立了通过促进MRGPRF基因的表达与顺铂联合用于黑色素瘤细胞的治疗,解锁了AMG 706的新功能,发现其可以促进MRGPRF基因的表达,明确了MrgprF蛋白的第274-343位的肽段在治疗黑色素瘤中的潜在功能,具有较大的应用价值和前景。
附图说明
图1为利用不同网络数据库GSE100050、GSE83583、GSE15605和TCGA-CVAA整合生物信息学分析筛选到MRGPRF基因;
图2为MRGPRF在CCLE数据库中不同基因型的黑色素瘤细胞中的表达情况;
图3为MRGPRF在皮肤正常痣、原发性黑色素瘤和高级别黑色素瘤中的表达情况,数据来源于GSE12391和GSE15605;
图4为TCGA-和黑色素瘤数据库中MrgprF、S100、MITF和MLANA的ROC曲线及AUC数值;
图5为MRGPRF在肿瘤样本中的高低表达与病人生存情况分析结果;
图6为利用LinkedOmics数据库,通过KEGG信号通路分析发现MRGPRF参与了PI3K/Akt信号通路;
图7为使用GDSC数据库在MRGPRF mRNA表达和药物IC50之间进行Pearson相关性分析结果;
图8为利用GSCALite数据库分析黑色素瘤中MRGPRF mRNA表达水平与其启动子甲基化状态相关性分析;
图9为DNMIVD数据库中MRGPRF启动子区域的甲基化水平高低(高:高风险;低:低风险)与各种类型人类癌症的生存概率相关性分析;
图10为MRGPRF基因与DNA甲基转移酶3A(DNMT3A)及DNA甲基转移酶3B(DNMT3B)的关联性分析结果;
图11为MRGPRF基因的mRNA在不同的人正常组织中的表达情况;
图12为通过GEPIA网站分析MRGPRF在泛癌中的表达情况;
图13为通过CCLE数据库分析MRGPRF在各种的不同癌细胞系中MRGPRF mRNA表达情况;
图14为A375和A875细胞在5-Aza处理后MRGPRF的mRNA和蛋白表达情况;
图15为不同的黑色素瘤细胞系中5-Aza处理或不处理(DMSO对照组)后,甲基化特异性PCR(MSP)实验检测MRGPRF启动子的甲基化状态;
图16为MRGPRF在正常人表皮细胞及黑色素瘤细胞系(A375、A875、SK-MEL-5和SK-MEL-1)中的RNA(上)和蛋白(下)的表达情况;
图17为在黑色素瘤细胞系A375和A875中构建MRGPRF过表达稳转细胞株中MRGPRF的RNA(上)和蛋白(下)验证结果,其中A图为A375细胞系,B图为A875细胞系;
图18为在黑色素瘤细胞系A375和人永生化表皮细胞(HaCaT)中构建MRGPRF敲低的稳转细胞株中MRGPRF的RNA(上)和蛋白(下)验证结果,其中A图为A375细胞系,B图为HaCaT细胞系;
图19为在小鼠黑色素瘤细胞系B16中构建MRGPRF敲低的稳转细胞株中MRGPRF的RNA(上)和蛋白(下)验证结果;
图20为用不同浓度的AMG-706处理黑色素瘤细胞系A375和A875后MRGPRF的RNA(上)和蛋白(下)表达检测结果,其中A图为A375细胞系,B图为A875细胞系;
图21为在A375和A875中过表达MRGPRF后,细胞周期相关蛋白CDK2、CDK4、CDK6、Cyclin D1和p27的蛋白表达结果检测,其中A图为A375细胞系,B图为A875细胞系,β-actin作为内参;
图22为在A375和A875中过表达MRGPRF后,迁移侵袭相关蛋白E-cadherin、N-cadherin、Vimentin的蛋白表达结果检测,其中A图为A375细胞系,B图为A875细胞系,β-actin作为内参;
图23为在A375和A875细胞中过表达MRGPRF以及在A375和HaCaT中敲低MRGPRF表达后,相关信号蛋白p-Akt、Akt、p-GSK3β、GSK3β、p-S6K、S6K、p-mTOR和mTOR的蛋白表达结果检测,A图为过表达细胞系,B图为敲低细胞系,β-actin作为内参;
图24为A375(A)和A875(B)不同的稳转株细胞用SC79处理后,检测p-Akt、Akt蛋白表达水平结果,β-actin作为内参;
图25为A375和A875细胞中用AMG 706处理后,细胞周期相关蛋白CDK2、CDK4、CDK6、Cyclin D1的蛋白表达结果检测,其中A图为A375细胞系,B图为A875细胞系,β-actin作为内参;
图26为A375和A875细胞中用AMG 706处理后,迁移侵袭相关蛋白E-cadherin、N-cadherin、Vimentin的蛋白表达结果检测,其中A图为A375细胞系,B图为A875细胞系,β-actin作为内参;
图27为A375和A875细胞中用AMG 706处理后,相关信号蛋白p-GSK3β、GSK3β、p-mTOR、mTOR、p-S6K、S6K、p-Akt和Akt的蛋白表达结果检测,其中左图为A375细胞系,右图为A875细胞系,β-actin作为内参;
图28为在黑色素瘤细胞系A375和A875中过表达MRGPRF与对照组细胞生长曲线实验结果,其中A图为A375细胞系,B图为A875细胞系;
图29为在黑色素瘤细胞系A375和A875中过表达MRGPRF与对照组细胞BrdU掺入实验结果展示图(左)和统计结果(右),其中A图为A375细胞系,B图为A875细胞系;
图30为在黑色素瘤细胞系A375和人永生化表皮细胞(HaCaT)中敲低MRGPRF的稳转细胞株与对照组细胞株中BrdU掺入实验结果,其中A图为HaCaT细胞系,B图为A375细胞系;
图31为在黑色素瘤细胞系A375和A875中过表达MRGPRF与对照组细胞相比抑制BrdU掺入的实验结果可以被Akt的激动剂SC79逆转,其中A图为A375细胞系,B图为A875细胞系;
图32为黑色素瘤细胞系A375和A875用AMG706处理细胞后BrdU掺入的实验结果,其中A图为A375细胞系,B图为A875细胞系;
图33为在黑色素瘤细胞系A375和A875中过表达MRGPRF与空载对照组细胞的细胞周期检测结果,其中A图为A375细胞系,B图为A875细胞系;
图34为黑色素瘤细胞系A375和A875用AMG706处理后细胞周期检测结果,其中A图为A375细胞系,B图为A875细胞系;
图35为用黑色素瘤细胞系A375和人永生化表皮细胞(HaCaT)构建的MRGPRF敲低的稳转细胞株及其对照组(Ctrl shRNA)的克隆球形成实验结果,其中A图为HaCaT细胞系,B图为A375细胞系;
图36为在黑色素瘤细胞系A375和A875中过表达MRGPRF与对照组细胞相比克隆球形成能力降低的表型可以被Akt的激动剂SC79逆转的实验结果,其中A图为A375细胞系,B图为A875细胞系;
图37为黑色素瘤细胞系A375和A875用AMG 706处理细胞后其克隆球形成的实验结果,其中A图为A375细胞系,B图为A875细胞系;
图38为过表达MRGPRF的裸鼠移植瘤实验结果,A图为裸鼠移植瘤的展示图,B图为肿瘤体积随时间增长的统计结果,C图为肿瘤重量的统计结果,D图为不同组别小鼠的重量;
图39为在人永生化表皮细胞HaCaT中敲低MRGPRF的裸鼠移植瘤实验结果,A图为裸鼠移植瘤的展示图,B图为不同稳转敲低株裸鼠移植瘤无肿瘤生成时间统计结果,C图为稳转敲低株裸鼠移植瘤HE染色结果;
图40为裸鼠移植瘤用AMG706处理或与DDP联合用药对肿瘤生长的影响,A图为实验流程示意图,B图为形成的肿瘤大小示意图,C图为肿瘤体积随时间增长的统计结果,D图为肿瘤重量的统计结果,E图为不同组别小鼠的重量统计结果;
图41为免疫组化检测MrgprF在黑色素瘤肿瘤组织和正常皮肤中的表达情况,其中A图为免疫组化展示图,B图为统计结果;
图42为过表达MRGPRF的裸鼠移植瘤免疫组化染色结果,其中A图为免疫组化染色(HE、MrgprF、Ki67和CC3)结果展示图,B图为免疫组化染色(Ki67和CC3)统计结果;
图43为不同药物处理的裸鼠移植瘤免疫组化染色结果,A图为免疫组化染色(HE、MrgprF、Ki67、CC3和p-AKT)结果展示图,B图为免疫组化染色(Ki67、CC3和p-AKT)统计结果;
图44为过表达MRGPRF的裸鼠移植瘤免疫组化染色结果,其中A图为免疫组化染色(MrgprF、E-cadherin、N-cadherin和Ki67)结果展示图,B图为免疫组化染色(E-cadherin、N-cadherin和Ki67)统计结果;
图45为AMG706处理的裸鼠移植瘤的免疫组化染色结果,其中A图为免疫组化染色(HE、MrgprF、E-cadherin、N-cadherin和Ki67)结果展示图,B图为免疫组化染色(Ki67、E-cadherin和N-cadherin)统计结果;
图46为过表达MRGPRF的稳转细胞株在有无DDP处理时细胞的凋亡情况,其中A图为凋亡流式检测结果展示图,左边为无DDP处理时的细胞凋亡情况展示图,右边为DDP处理时的细胞凋亡情况展示图,B图为细胞凋亡的统计结果;
图47为不同的黑色素瘤细胞系在AMG 706、DDP以及AMG 706和DDP共处理时细胞的凋亡情况,其中A图为A375凋亡流式检测结果展示图,左边为不同药物处理时的细胞凋亡情况展示图,右边为各种不同药物处理结果统计图,B图为A875凋亡流式检测结果展示图,左边为不同药物处理时的细胞凋亡情况展示图,右边为各种不同药物处理结果统计图;
图48为过表达MRGPRF的不同稳转细胞株划痕实验的检测结果,其中A图为A375细胞系构建的稳转株的划痕实验结果,左边是示意图,右边是统计结果,B图为A875细胞系构建的稳转株的划痕实验结果,左边是示意图,右边是统计结果;
图49在不同细胞系中过表达MRGPRF的表达,检测相应稳转细胞株的小室迁移能力,其中A图为A375细胞系小室迁移结果展示图(左)和统计结果(右),B图为A875细胞系小室迁移结果展示图(左)和统计结果(右);
图50为在不同敲低MRGPRF表达的稳转细胞株的小室迁移实验检测结果,其中A图为A375细胞系小室迁移结果展示图和统计结果,B图为HaCaT细胞系小室迁移结果展示图和统计结果;
图51为在不同细胞系中过表达MRGPRF后用SC79处理后小室迁移实验检测结果,其中A图为A375细胞系小室迁移结果展示图和统计结果,B图为A875细胞系小室迁移结果展示图和统计结果,pCDH-vec为对照细胞株,而MRGPRF ove为过表达MRGPRF细胞株;DMSO为SC79的药物对照;
图52为在不同细胞系中用AMG 706处理后小室迁移实验检测结果,A图为A375细胞系小室迁移实验结果展示图和统计结果,B图为A875细胞系小室迁移实验结果展示图和统计结果;
图53为过表达MRGPRF后细胞形态的变化结果展示,pCDH-vec为对照细胞株,而MrgprF ove为过表达MRGPRF细胞株;
图54为在A375细胞系中过表达MRGPRF后phalloidin染色结果展示图,其中pCDH-vec为对照细胞株,而MrgprF ove为过表达MRGPRF细胞株;
图55为在HaCaT细胞系中敲低MRGPRF后phalloidin染色结果展示图,其中CtrlshRNA为对照细胞株,而MRGPRF sh#1和MRGPRF sh#2为敲低MRGPRF表达细胞株;
图56为过表达MRGPRF细胞系稳转细胞株中用SC79处理后phalloidin染色结果展示,其中pCDH-vec为对照细胞株,DMSO为SC79药物的溶剂对照;
图57为A375细胞株用AMG 706处理后phalloidin染色结果展示图,其中DMSO为AMG706药物的溶剂对照;
图58为黑色素瘤肺转移侵袭实验检测结果,A图为肺转移的结果展示图,B图为肺转移结果统计图;
图59为AMG 706对黑色素瘤侵袭转移的影响,A图为实验流程图,B图为黑色素瘤肺转移结果展示图,C图为肺转移的结果统计;DMSO为AMG 706药物的溶剂对照;
图60为用免疫沉淀的方法,检测p110-γ、p85和MrgprF的相互结合结果;
图61为用免疫共沉淀的方法,检测p110-γ、p101和MrgprF的相互结合结果;
图62为用免疫共沉淀的方法,检测p110-γ、p101和MrgprF的相互竞争结合结果;
图63为在A375和A875细胞中过表达MRGPRF以及在A375和HaCaT中敲低MRGPRF表达后,细胞内PIP3浓度检测,其中A图为A375、A875黑色素瘤细胞的过表达细胞株,B图为A375、HaCaT正常人表皮细胞敲低细胞株;
图64为AMG 706处理不同细胞(A375、A875黑色素瘤细胞和HaCaT人永生化表皮细胞)后细胞的存活情况及药物IC50的值;
图65为IP和GST pull-down实验结果,用不同的截短体分别与互作蛋白的全长相互作用,筛选p110-γ和MrgprF互作的位点,并再进一步验证互作,明确竞争小肽,A图为不同截短体的构建信息图,B图为不同的截短体p110-γ(FL、P1~P6)与MrgprF全长互作及不同的截短体MrgprF(FL、M1~M5)与p110-γ全长互作免疫沉淀(IP)的实验结果图,C图为GSTpull-down实验进一步验证功能互作小肽。
图66为GST pull-down实验结果,用不同剂量的蛋白小肽处理后,检测p110-γ、p101的相互结合的能力变化。
具体实施方式
下面通过实施例对本发明作进一步详细说明,但本发明的保护范围不局限于所述内容,实施例中方法如无特殊说明均采用常规方法,使用试剂如无特殊说明,均为常规市售试剂或采用常规方法配置的试剂。
实施例1:生信分析和数据分析
本研究中使用的所有数据库都采用公共数据库数据,基因表达数据分别使用来自Gene Expression Omnibus(GEO)网站、TCGA数据集、GEPIA数据库、GTEx Portal和CCLE等数据集。为筛选MRGPRF作为黑色素瘤细胞的差异基因,我们通过收集GEO数据集和已发表的数据集,采取NCBI在线GEO2R工具分析GEO数据。为分析MRGPRF启动子的甲基化状态,我们从GSCA、DNMIVD和MethSurv数据集中收集数据,并进行在线分析。从GSCA和cBioportal数据库获得突变数据,并使用GSEA软件进行KEGG通路富集分析。使用GraphPad Prism 5.0应用学生t检验和单向ANOVA检验进行数据处理。P<0.05(*)、P<0.01(**)和P<0.001(***)的表示差异显著,ns=无显着性。
结果见图1-13,其中图1显示,不同网络数据库GSE100050、GSE83583、GSE15605和TCGA-CVAA整合生物信息学分析筛选到6个差异表达的基因,其中一个是MRGPRF基因;图2显示,在CCLE数据库中,MRGPRF基因在不同基因型的黑色素瘤细胞中的表达量在NRAS和BRAF突变的细胞株中降低。图3显示,根据GSE12391和GSE15605数据库,MRGPRF基因在皮肤正常痣、原发性黑色素瘤和高级别黑色素瘤中的表达逐渐降低。图4显示,TCGA-和黑色素瘤数据库中MRGPRF、S100、MITF和MLANA的ROC曲线及AUC数值表明,MRGPRF是很好的黑色素瘤的独立诊断标志物。图5显示,MRGPRF基因在肿瘤样本中表达量越低,病人生存时间越短,表达量越高,病人生存时间越长。图6显示,通过LinkedOmics数据库,通过KEGG信号通路分析发现MRGPRF参与了PI3K/Akt信号通路。图7显示,使用GDSC数据库在MRGPRF mRNA表达和药物IC50之间进行Pearson相关性分析发现AMG 706的IC50药物浓度与MRGPRF mRNA表达量成反比。图8显示,利用GSCALite数据库分析发现黑色素瘤中MRGPRF mRNA表达水平与其启动子甲基化状态密切相关。如图9所示,通过DNMIVD数据库对MRGPRF启动子区域的甲基化水平高低(高:高风险;低:低风险)与各种类型人类癌症的生存概率相关性分析,发现高风险的生存概率低,低风险的,生存概率高。结果如图10中MRGPRF基因与DNA甲基转移酶3A(DNMT3A)及DNA甲基转移酶3B(DNMT3B)的关联性分析表明,DNMT3A及DNMT3B与MRGPRF的表达呈负相关。图11显示MRGPRF基因的mRNA在不同的人正常组织中的表达情况,在皮肤组织中,MRGPRF基因的mRNA的表达水平相对较高。图12显示,通过GEPIA网站分析MRGPRF在泛癌中的表达较正常组织低。图13显示,通过CCLE数据库分析MRGPRF在不同癌细胞系中MRGPRF mRNA表达较低。
实施例2:甲基化特异性的PCR实验(methylation-specific PCR,MSP)和DNA甲基化测序
消化并收集处理过的及其对照组细胞,用DNA抽提试剂盒(Promega,cat.no.A1125)提取细胞基因组DNA,并按照DNA重亚硫酸盐转化试剂盒(Tiangen,DP215-02)处理转化基因组DNA,并纯化回收进行PCR扩增,配制2%的DNA琼脂糖进行电泳检测。
在DNA甲基化测序实验中,我们采取了BSP克隆测序法(Bisulfite sequencingPCR,BSP)。按照试剂盒实验步骤提取HaCaT、A875、A375、SK-MEL-5、SK-MEL-1及其5μM 5-氮杂胞苷(5-Azacytidine,5-Aza)处理的A375、A875、A375、SK-MEL-5、SK-MEL-1细胞基因组DNA,并通过EZ DNA Methylation-Gold Kit(ZYMO Research,USA)转化,PCR扩增并克隆至pMD19-T载体上,每个样品挑取10个阳性克隆进行Sanger测序,并分析甲基化水平。
结果如图14所示,A375和A875细胞在5-Aza处理后MRGPRF的mRNA和蛋白表达较未处理细胞中显著升高。结果如图15所示,不同的黑色素瘤细胞系中5-Aza处理或不处理(DMSO对照组)后,甲基化特异性PCR(MSP)实验检测MRGPRF启动子的甲基化状态,发现,5-Aza处理后,细胞内的甲基化水平显著降低。
实施例3:载体、病毒包装和稳转细胞系的建立与检测
本文中编码人源和鼠源的MRGPRF全长cDNA均合成于上海捷瑞生物公司,并通过NheⅠ和EcoRⅠ克隆至pCDH-CMV-E2F-eGFP慢病毒载体上;靶向MRGPRFmRNA不同区域的shRNA合成并克隆至pLKO.1载体(Addgene)上;在Co-IP和GST-pulldown实验中,编码MRGPRF及其截短体的DNA序列亚克隆至带有不同标签蛋白的载体pcDNA3.1和pGEX-4T-1载体上。在稳转细胞系的建立中,慢病毒表达载体、包装质粒pMD2.G和psPAX2同时通过磷酸钙转染法转染至HEK-293T中,并在转染后48h和72h收集上清病毒液,过滤后加入4μg/mL polybrene感染目的细胞,感染48h后用相应的嘌呤霉素筛选阳性细胞,其中全长的MRGPRF蛋白序列如SEQID NO:4所示;而其中小肽的氨基酸序列如SEQ ID NO:3。
实施例4:实时定量PCR检测RNA表达水平及蛋白免疫印记检测蛋白表达水平检测实时定量PCR检测RNA表达水平
取足够量的细胞,加入1mL Trizol试剂(Takara)充分吹打裂解细胞沉淀。4℃、12000g下离心10min,转移上清。上清室温放置5min,加0.2mL氯仿,用手或Votex剧振15s,室温放置2~3min。4℃、11000g下离心15min,转移水相。在水相中加入0.25mL异丙醇,室温放置10min。4℃、11000g下离心10min去上清。加1mL75%乙醇,Votex混匀,4℃、7000g下离心5min,去上清。沉淀在空气中干燥5~10min,用100μL DEPC水溶解。电泳及检测OD值,检定RNA的量及完整性。取1μg RNA根据试剂盒(Takara)进行去除基因组DNA和反转成cDNA,并根据实际进行稀释。取适量cDNA根据试荧光定量试剂盒(Vazyme)进行反应,所得数据利用2-ΔΔCt进行处理。
所用检测RNA表达水平的引物序列如下:
蛋白免疫印记检测蛋白表达水平检测
按照特定实验处理后的细胞,弃去上清培养基,用PBS洗涤1次;根据细胞沉淀的量加入相应的细胞裂解液,于冰上反复冻融裂解3次,期间不停吹打;4℃、12000rpm离心10min,取上清弃沉淀用于后续实验。根据BCA试剂盒说明书的步骤测定总的蛋白浓度。按照需要,配制7.5%-12%不同浓度的分离胶,缓缓加入适量的无水乙醇封平胶面,待胶充分凝固后,吸出无水乙醇,按配方加入浓缩胶,插入样品槽梳子;待胶凝固后,取出胶,拔出梳子,蒸馏水清洗表面胶;把玻璃板和凝胶一同固定在电泳架上,加入适量的Tris-甘氨酸电泳缓冲液,使电泳缓冲液在短玻璃板上方约0.5cm;每孔加入蛋白量10-30μg,首先80V电压下电泳跑完浓缩胶,再用120V电压电泳;把转膜架放在转膜液中,黑色塑料板面向下,白色在上,在黑色面上方放一块海绵垫,海绵垫上方放两张滤纸;轻轻把凝胶从电泳玻璃板上取下来后放到滤纸上,把PVDF膜放到凝胶上方,再放两张滤纸,滤纸上方放一块海绵垫,把黑白两块塑料板固定牢固,放入已经预冷的转膜缓冲液中;冰浴条件下,83V电压转膜3h;将PVDF膜置于TBST(含5%脱脂奶粉)中,室温封闭2h;配制一抗,4℃缓慢摇动中孵育过夜;TBST洗膜10min,重复3次;加入二抗(抗鼠:1∶2000,抗兔1∶2000稀释),置于室温1h;TBST洗膜10min,共计3次;把PVDF膜转移到发光板上,避光条件下加入ECL试剂(用前把A,B液进行等量混合),显影,定影后拍照。
结果如图16所示,MRGPRF基因在黑色素瘤细胞系(A375、A875、SK-MEL-5和SK-MEL-1)中的RNA(上)和蛋白(下)的表达较人永生化表皮细胞(HaCaT)中显著降低。
图17结果显示,在黑色素瘤细胞系A375和A875中构建MRGPRF过表达稳转细胞株中MRGPRF的RNA(上)和蛋白(下)表达水平显著升高;图18结果显示,在黑色素瘤细胞系A375和人永生化表皮细胞(HaCaT)中构建MRGPRF敲低的稳转细胞株中MRGPRF的RNA(上)和蛋白(下)表达水平显著降低;图19结果显示,在小鼠黑色素瘤细胞系B16中构建MRGPRF过表达的稳转细胞株中MRGPRF的RNA(上)和蛋白(下)表达水平显著升高。
用不同浓度的AMG-706处理黑色素瘤细胞系A375和A875后,结果如图20所示,MRGPRF蛋白表达水平随着药物浓度的升高显著升高。
图21结果显示,在A375和A875中过表达MRGPRF后,细胞周期相关蛋白CDK2、CDK4、CDK6和Cyclin D1的蛋白表达较对照组细胞相比显著降低,而p27的蛋白表达显著升高。图22结果显示,在A375和A875中过表达MRGPRF后,迁移侵袭相关蛋白E-cadherin的蛋白表达水平显著升高,而N-cadherin、Vimentin蛋白表达水平显著降低;
图23结果显示,在A375和A875细胞中过表达MRGPRF以及在A375和HaCaT中敲低MRGPRF表达后,相关信号蛋白p-Akt、Akt、p-GSK3β、GSK3β、p-S6K、S6K、p-mTOR和mTOR的蛋白表达结果检测显示,过表达MRGPRF,细胞内p-Akt、p-GSK3β、p-S6K、p-mTOR均较对照组有显著降低,反之亦然。图24结果显示,A375和A875不同的稳转株细胞用SC79处理后,检测p-Akt的蛋白表达水平较对照组得到明显回复,β-actin作为内参;
图25结果显示,A375和A875细胞中用AMG 706处理后,细胞周期相关蛋白CDK2、CDK4、CDK6、Cyclin D1的蛋白表达较对照组细胞相比显著降低;图26结果显示,A375和A875细胞中用AMG 706处理后,迁移侵袭相关蛋白E-cadherin的蛋白表达水平显著升高,而N-cadherin、Vimentin蛋白表达水平显著降低,A图为A375细胞系,B图为A875细胞系,β-actin作为内参;图27结果显示,A375和A875细胞中用AMG 706处理后,相关信号蛋白p-Akt、Akt、p-GSK3β、GSK3β、p-S6K、S6K、p-mTOR和mTOR的蛋白表达结果与对照组相比,细胞内p-Akt、p-GSK3β、p-S6K、p-mTOR均较对照组有显著降低。
实施例5:细胞增殖、BrdU掺入实验
为检测MrgprF蛋白对黑色素瘤细胞增殖的影响,我们采取了生长曲线、BrdU掺入实验探究过表达MRGPRF后黑色素瘤细胞增殖能力的变化。对于生长曲线实验,将目的细胞种在12-well板中(2×104每个孔),培养七天,每天胰酶消化,利用Countstar(ShanghaiRuiyu Biotech Co.)进行计数。在BrdU实验中,将目的细胞种于8-well板中(3-5×104每个孔),培养24h(SC79拯救实验中,SC79预先处理24h),加入10μM BrdU(Abcam,ab142567)处理20min,室温下4%多聚甲醛(PFA)固定20min,PBST(PBS+2N HCL+0.5%Trion X-100)破膜30min,加入相应NaHCO3中和,室温用含有10%山羊血清(NGS)的PBST封闭1h,按照说明书加入BrdU一抗,4℃摇床孵育过夜,清洗一抗并孵育相应的二抗,拍照并计数。
结果如图28所示,在黑色素瘤细胞系A375和A875中过表达MRGPRF与对照组细胞相比,其生长显著受到抑制;图29结果显示,在黑色素瘤细胞系A375和A875中过表达MRGPRFBrdU掺入与对照组细胞相比显著减少;图30结果显示,在黑色素瘤细胞系A375和人永生化表皮细胞(HaCaT)中敲低MRGPRF的稳转细胞株中BrdU掺入与对照组细胞株相比显著增多。图31结果显示,在加入了Akt的激动剂SC79后,黑色素瘤细胞系A375和A875中过表达MRGPRF与对照组细胞相比抑制BrdU掺入的实验结果可以被Akt的激动剂SC79逆转,其BrdU掺入显著增加;图32结果显示,在加入AMG 706处理黑色素瘤细胞系A375和A875后,用AMG706处理的细胞BrdU掺入显著降低。
实施例6:细胞周期实验
在流式细胞术检测细胞周期中,待稳转细胞生长到80%覆盖度时,去掉培养基上清,PBS清洗一次,0.25%胰酶1mL消化3min,然后加入5mL培养基终止,吹打成单细胞悬液,用Countstar进行计数,按照每孔2mL培养基体积,细胞总量4×105-6×105个,接种于6孔板中,每个样品设置三个重复孔,轻轻混匀以保证细胞分布均匀,然后置于37℃、5%二氧化碳培养箱中过夜。24h后,去除培养基上清,加2mL无血清培养基进行饥饿,置于37℃、5%二氧化碳培养箱中过夜。去除无血清培养基,加入含血清的完全培养基进行释放6-8h。去掉培养基上清,PBS清洗一次,0.25%胰酶1mL消化3min,然后加入2mL培养基终止,轻柔地吹打成单细胞悬液。收集细胞到离心管中,1500rpm离心5min。加入5mL PBS洗一次,1500rpm离心5min,重复一次。去除PBS液体,用500μLPBS重悬细胞沉淀,注意轻柔。将4.5mL预冷的75%的乙醇加入到新的离心管中,标注清楚样品名称。将重悬好的细胞悬液逐滴加入到预冷的75%乙醇中,注意滴入后装有乙醇的管及时摇匀,以保证充分固定,另外样品须一一对应。盖紧离心管盖,于4℃固定。上机前将样品从4℃取出,1500rpm离心5min。去除固定液,加入5mL PBS清洗,1500rpm离心5min,重复一次。每管加入500μL含有RNAase(1:500)的PBS+0.1%Trion X-100溶液,去除RNA酶。然后每管加入10μLPI进行染色。15-30min后使用流式细胞仪进行检测细胞周期的变化。数据进行整理分析,绘制统计图。
结果如图33所示,在黑色素瘤细胞系A375和A875中过表达MRGPRF与空载对照组细胞相比,细胞周期阻滞在G0/G1期;图34结果显示,黑色素瘤细胞系A375和A875用AMG706处理后,细胞周期阻滞在G0/G1期。
实施例7:克隆球形成实验
在克隆球形成实验中,待稳转细胞生长到80%覆盖度时,去掉培养基上清,PBS清洗一次,0.25%胰酶1mL消化3min,然后加入5mL培养基终止,吹打成单细胞悬液,用Countstar进行计数,按照每孔2mL培养基体积,细胞总量2000个/孔,种于6孔板中,每个样品设置三个重复孔,轻轻混匀以保证细胞分布均匀,然后置于37℃、5%二氧化碳培养箱中过夜。培养7-12天,PBS洗一次,4%PFA固定后,0.5%结晶紫染色1h,ddH2O洗3次,拍照后统计分析。
用人永生化表皮细胞(HaCaT)和黑色素瘤细胞系A375构建的MRGPRF敲低的稳转细胞株及其对照组(Ctrl shRNA)的克隆球形成实验,结果如图35所示,结果显示敲低组的克隆球形成数量显著高于对照组;图36结果显示,在黑色素瘤细胞系A375和A875中过表达MRGPRF与对照组细胞相比克隆球形成能力降低,这一表型可以被Akt的激动剂SC79逆转,图37结果显示,黑色素瘤细胞系A375和A875用AMG 706处理细胞后其克隆球形成能力受到显著的抑制。
实施例8:裸鼠移植瘤实验
所有动物都饲养在SPF环境中,并且这些方案是在中国科学院昆明动物研究所动物伦理委员会的政策下预先批准和实施的。对于异种移植肿瘤实验形成测定,将10只4周龄的雄性裸鼠分成两组,皮下注射指定的黑色素瘤细胞系(1×106个细胞/点)。注射后每四天用滑动卡尺测量肿瘤,并通过公式长度×(宽度)2/2计算肿瘤体积。实验结束时处死所有小鼠,收集肿瘤并称重。每个肿瘤组织用福尔马林溶液固定并包埋在石蜡中用于后续检测。对于人永生化表皮细胞(HaCaT)稳转细胞株进行的异种移植瘤实验应用2×107个细胞/点注射。注射后,记录无瘤小鼠百分比。对于体内药物治疗测定,皮下注射指示细胞,当异种移植肿瘤大小达到100mm3的体积时,通过腹膜给小鼠注射AMG 706(7.5mg/kg,每天一次)或DDP(7mg/kg,每周一次)。对于肺转移试验,将B16细胞(1×105细胞/小鼠)注射到6~8周龄C57BL/6小鼠的尾静脉中。18天后,处死小鼠,对转移性肺肿瘤进行计数、拍照并统计;对于AMG 706治疗组,腹膜内注射AMG 706(7.5mg/kg,每天一次)。
结果如图38所示,过表达MRGPRF的裸鼠移植瘤形成能力较对照组相比显著降低;图39结果显示,在人永生化表皮细胞HaCaT中敲低MRGPRF的裸鼠移植瘤形成能力较对照组相比显著增强;图40结果显示,裸鼠移植瘤用AMG706处理或与DDP联合用药检测对肿瘤生长的影响,发现AMG706处理或与DDP联合用药可以显著抑制肿瘤的生长。
实施例9:H&E染色、免疫组化和免疫荧光
H&E染色,取长、宽1-1.5cm,厚0.2-0.5cm的组织块,固定于10%中性甲醛中8-12h。脱水程序为:75%乙醇,1h;85%乙醇,1h;90%乙醇,1h;95%乙醇,1h;95%乙醇,1h;100%乙醇,1h;100%乙醇,1h;二甲苯,35min;二甲苯,35min;石蜡,1h;石蜡,1h。包埋后切成5μm厚度,50℃捞片和烘片。HE染色程序为:二甲苯,5min;二甲苯,5min;100%乙醇,2min;95%乙醇,2min;85%乙醇,2min;75%乙醇,2min;自来水,2min;蒸馏水,2min;苏木精,5-10min;自来水,1min;含1%HCl的75%乙醇,几秒;自来水,1min;50℃水返蓝,5-15min;95%乙醇,1min;伊红,几秒;75%乙醇,20s;85%乙醇,20s;95%乙醇,30s;95%乙醇,30s;100%乙醇,30s;100%乙醇,30s二甲苯,2min;二甲苯,2min。封片,拍照。
对于免疫组化,切片成4μm厚度的组织切片并摊片。IHC程序为:二甲苯,10min;二甲苯,10min;二甲苯,10min;100%乙醇,2min;100%乙醇,2min;95%乙醇,2min;85%乙醇,2min;75%乙醇,2min;自来水,2min;蒸馏水,2min;高压锅抗原修复;蒸馏水,1min;含3%H2O2的PBS,20min;PBS,3min;室温封闭(PBS+1%Tween 20+10%NGS),20min;一抗,过夜;PBS,3min;PBS,3min;PBST(PBS+1%Tween20),3min;HRP标记的二抗(Santa CruzBiotechnology,Santa cruz,CA,USA);PBS,3min;PBS,3min;PBST(PBS+1%Tween 20),3min;DAB显色,5min;蒸馏水,1min;苏木精,2-3min(淡染);自来水,1min;含1%HCl的75%乙醇,几秒;蒸馏水(50℃)蓝化,5min;75%乙醇,20s;85%乙醇,20s;95%乙醇,30s;100%乙醇,30s;100%乙醇,30s;二甲苯,2min;二甲苯,2min。封片,拍照(Olympus显微镜)。
结果如图41所示,免疫组化检测MRGPRF在黑色素瘤肿瘤组织和正常皮肤中的表达情况,发现MRGPRF在黑色素瘤肿瘤组织中表达较对照组显著降低。图42结果显示,过表达MRGPRF的裸鼠移植瘤免疫组化染色结果表明当MRGPRF过表达时,Ki67阳性细胞数量显著降低,而活化的cleaved caspase 3阳性信号显著增加。图43结果显示,从不同药物处理(AMG706及与顺铂联用)的裸鼠移植瘤免疫组化染色可知,药物处理后,组织中Ki67阳性细胞显著下降,而MrgprF和CC3阳性率显著增加,同时p-AKT阳性率显著降低,且AMG 706与顺铂联用的表型较AMG 706单独处理更显著。
图44结果显示,过表达MRGPRF的裸鼠移植瘤免疫组化染色结果中,MRGPRF过表达后,其Ki67和N-cadherin的阳性率显著降低,而E-cadherin蛋白的表达显著增加。图45结果显示,AMG706处理的裸鼠移植瘤的免疫组化染色结果表明,AMG706处理后,组织中MrgprF的表达显著升高,同时其Ki67和N-cadherin的阳性率显著降低,而E-cadherin蛋白的表达显著增加。
实施例10:细胞凋亡检测实验
在流式细胞术检测细胞凋亡实验中,将相应细胞生长到80%覆盖度时,去掉培养基上清,PBS清洗一次,0.25%胰酶1mL消化3min,然后加入5mL培养基终止,吹打成单细胞悬液,用Countstar进行计数,按照每孔2mL培养基体积,细胞总量4×105-6×105个,种于6孔板中,每个样品设置三个重复孔,轻轻混匀以保证细胞分布均匀,然后置于37℃、5%二氧化碳培养箱中过夜。24h后,收集培养基上清,PBS清洗一次,0.25%胰酶1mL消化3min,然后加入2mL培养基终止,吹打成单细胞悬液,注意轻柔吹打,防止因机械力带来的细胞损伤凋亡。收集细胞到离心管中,2000rpm离心5min,以收集漂浮在上清里的凋亡的细胞。去掉上清液,加入5mL PBS洗一次,1500rpm离心5min,重复一次。按照Annexin V-FITC/PI细胞凋亡检测试剂盒说明书,先加入1×binding buffer将细胞重悬,取出三个对照所需细胞(阴性对照,FITC单标对照,PI单标对照),然后每管样品加入FITC和PI各5μL到细胞悬液中(对照样品为:不加染料,加5μLFITC,加5μLPI),轻柔混匀。37℃避光孵育15-30min。利用流式细胞仪检测细胞凋亡比例的变化。数据进行整理分析,绘制细胞凋亡分布图和统计图。
结果如图46所示,过表达MRGPRF的稳转细胞株在有无DDP处理时细胞的凋亡情况显示,在DDP未处理时,MRGPRF过表达与对照相比并无显著差异,而在DDP处理后,MRGPRF过表达与对照相比,细胞凋亡显著增加。图47不同的黑色素瘤细胞系在AMG 706、DDP以及AMG706和DDP共处理时细胞的凋亡检测结果表明,在AMG 706、DDP分别处理后,细胞的凋亡显著增加,而当AMG 706和DDP联用后,细胞的凋亡进一步增加。
实施例11:细胞迁移实验(划痕实验)
划痕实验中,取生长状态良好的细胞,种1-2×106个细胞于6孔板,24h后划线,挑选宽度一致的位置标记并拍照,拍照后置于细胞培养箱;根据不同种类细胞的愈合速度,在愈合24-48h后拍照,整理数据分析并进行统计分析。
结果如图48所示,在不同过表达MRGPRF细胞系(A375和A875)稳转细胞株划痕后,过表达MRGPRF细胞株其迁移能力显著降低。
实施例12:细胞迁移实验(Trans-well迁移实验)
Trans-well迁移实验中,准备Transwell小室:取一个24孔板中首先加入600μL含血清完全培养基,将Transwell小室置于加好含血清培养基的孔中。取对数生长期细胞,去掉培养基,PBS洗除血清,0.25%胰酶消化,然后加培养基终止消化,重悬细胞后离心,用新鲜培养基吹打成单细胞悬液,用Countstar进行计数。取相应数量的细胞离心后,用1mL无血清培养基重悬,然后取出100μL细胞悬液,悬空逐滴加入到第一步准备好的Transwell小室中。孵育24h后,取出Transwell小室,用PBS润湿过的棉签轻轻擦去小室上面未发生迁移的细胞,然后置于预先加入了600μL 4%PFA的孔中固定20min。固定结束后,将小室置于预先加入600μL含0.5%结晶紫的乙醇溶液(100%ETOH配制),使膜浸没在染色液中,室温染色1-2h,取出,蒸馏水清洗约5次,并自然风干。随机取5个以上的视野显微镜拍照并计数。拍照完毕,每孔加入500μL 33%的醋酸,摇床摇10min,充分振荡后在570nm处测定光吸收度。
结果如图49所示,过表达MRGPRF的不同稳转细胞株小室迁移的能力较对照组显著降低;结果如图50所示,在不同细胞系中敲低MRGPRF的表达后敲低表达的稳转细胞株的小室迁移能力较对照组细胞显著增加。图51所示,在不同过表达MRGPRF细胞系稳转细胞株中用SC79处理的小室迁移能力与对照相比显著增强,可以逆转由MRGPRF导致的小室迁移能力的降低。图52所示,在不同细胞系中用AMG 706处理后小室迁移能力受到显著抑制。
实施例13:免疫荧光染色实验
在免疫荧光实验中,为了评估细胞形态变化,我们将特定的细胞接种到8孔板上并培养24小时(SC 79和AMG 706接着处理24h),室温用4%PFA固定。用0.1%PBS+Triton X-100透化10min,在室温下用10%正常山羊血清中封闭1h后,将细胞与鬼笔环肽(Sigma,P2141)一起孵育;细胞核用DAPI染色。拍照并观察。
结果如图53所示,过表达MRGPRF后细胞形态发生显著变化,过表达MRGPRF,细胞形态变成长梭形,提示细胞发生MET转变。结果如图54所示,在A375细胞系中过表达MRGPRF后phalloidin染色结果显示过表达MRGPRF细胞伪足数量显著降低,表明过表达MRGPRF可以抑制细胞的迁移;结果如图55所示,在HaCaT细胞系中敲低MRGPRF后phalloidin染色结果显示敲低MRGPRF后,细胞伪足数量显著增加,表明敲低MRGPRF可以促进细胞的迁移;图56结果显示,在不同过表达MRGPRF细胞系稳转细胞株中用SC79处理后phalloidin染色结果显示过表达MRGPRF引起的细胞伪足数量的减少可以被SC79逆转;图57结果显示,A375细胞株用AMG706处理后phalloidin染色结果发现细胞伪足数量显著降低,表明AMG 706可以抑制细胞的迁移。
实施例14:小鼠肺转移动物模型实验
所有小鼠都饲养在SPF环境中,经中国科学院昆明动物研究所动物伦理委员会的政策下预先批准,按照实验方案将B16细胞(1×105细胞/小鼠)注射到6~8周龄C57BL/6小鼠的尾静脉中。18天后,处死小鼠,对转移性肺肿瘤进行计数,拍照并统计。对于AMG 706治疗组,腹膜内注射AMG 706(7.5mg/kg,每天一次)。
结果如图58所示,黑色素瘤肺转移侵袭实验检测结果表明当过表达MRGPRF时,黑色素瘤肺转移侵袭的能力较对照组显著降低;结果如图59所示,AMG 706药物处理后,黑色素瘤侵袭转移较对照组相比,其肺转移侵袭的能力显著降低。
实施例15:免疫共沉淀实验
免疫共沉淀实验中,克隆相应的全长和截短体蛋白表达的DNA序列至pcDNA3.1中。将需要检测的细胞弃去上清培养基,用PBS洗涤1次;根据细胞沉淀的量加入相应的细胞裂解液,于冰上反复冻融裂解3次,期间不停吹打;4℃、12000rpm离心10min,取上清弃沉淀用于后续实验。根据BCA试剂盒说明书的步骤测定总的蛋白浓度。取1μg蛋白于新的1.5mL离心管,分为对照组和实验组分别加入目的蛋白抗体和IgG抗体,置于4℃冰箱低速旋转孵育12-16h。根据需要取适量protein A/G beads于1.5mL离心管,4℃离心机10000g离心5min,弃上清,用IP buffer洗三次。将清洗好的beads加入混合液中,每管20-30μL beads,继续旋转孵育2h。4℃进行离心,弃上清,吸取IP buffer洗涤三次,最后一次弃上清后加入适量2×SDS,置于100℃金属浴处理10min,样品冻存-80℃以备后续免疫蛋白印记实验检测蛋白互作。
结果如图60所示,用免疫沉淀的方法,检测p110-γ、p85和MrgprF的相互结合发现,MrgprF与p110-γ之间存在相互作用。结果如图61所示,用免疫共沉淀的方法证实p110-γ、p101和MrgprF三者可以相互结合形成复合物。结果如图62所示,用免疫共沉淀的方法,检测p110-γ、p101和MrgprF的相互结合发现三者之间存在竞争结合关系,当存在MrgprF时,p110-γ与p101的相互结合变弱。
实施例16:PIP3检测
收集相应的等量细胞沉淀,将细胞沉淀重悬于150-200μL PBS中,冻融3次以破碎细胞。将总细胞提取物以2000rpm离心10min,按照人PIP3 ELISA试剂盒(RUIXIN BIOTECH,RX105304H)使用步骤将上清液用于PIP3检测;
结果如图63所示,在A375和A875细胞中过表达MRGPRF以及在A375和HaCaT中敲低MRGPRF表达后,细胞内PIP3浓度检测结果显示,过表达MRGPRF,细胞内PIP3的浓度降低,而敲低MRGPRF,细胞内PIP3的浓度升高。
实施例17:药物毒性实验及IC50检测
为检测过表达MRGPRF对黑色素瘤细胞耐药性的影响和AMG 706对黑色素瘤细胞的杀伤作用,我们采用了磺酰罗丹明B(Sulforhodamine B,SRB)染色的方法检测细胞存活率。消化对数期细胞并计数,种1000个细胞每个孔至96-well中,培养24h。加入不同浓度的相应药物(如:顺铂、BRAF抑制剂Vemurafenib和AMG 706)处理24h。PBS清洗一次,加入适量的10%三氯醋酸(TCA)4℃固定1h,PBS清洗一次,加入适量的0.4%SRB室温染色1h,弃多余染色液,1%醋酸洗涤三次后室温风干,加入10mM Tris溶解,于OD515波长下测定吸光值。以对照组吸光值为100%进行标准化,使用GraphPad Prism 5.0进行IC50的测定。
结果如图64所示,AMG 706处理不同细胞A375、A875黑色素瘤细胞和HaCaT(人永生化表皮细胞)后细胞的存活结果显示AMG 706对A375、A875黑色素瘤细胞有明显的杀伤作用,对HaCaT人永生化表皮细胞的杀伤作用较弱,不同细胞对AMG 706药物IC50的值如图所示,A375为29.1μM,A875为7.2μM,HaCaT为76.3μM。
实施例18:GST pull-down
通过BamHⅠ和NotⅠ酶切位点,将含有MrgprF274-343和p110γ1-216的DNA片段克隆到pGEX-4T-1载体中,在大肠杆菌BL21(DE3)中表达、纯化和洗脱GST融合蛋白(洗脱缓冲液:50mM Tris pH8.8、1mM EDTA、5%甘油、0.01%Triton X-100、50mM NaCl、5mM DTT、20mM谷氨酰胺)。洗脱的蛋白质用于竞争结合测定。对于GST pull-down测定,将与谷胱甘肽树脂结合的GST、GST-MrgprF274-343或p110γ1-216肽段与特定的总细胞提取物在4℃下孵育过夜,将树脂结合的蛋白质洗涤、洗脱并加载到SDS-PAGE并通过免疫印迹检测;
结果如图65所示,将GST-MrgprF或p110γ蛋白截断成不同大小的片段,分别与p110γ全长或MrgprF全长蛋白相互作用筛选其互作区域,发现GST-MrgprF274-343或p110γ1-216肽段是两者相互作用的区段,其中MrgprF-M1的氨基酸序列是SEQ ID NO:4所示氨基酸序列的第1-172位,MrgprF-M2的氨基酸序列是SEQ ID NO:4所示氨基酸序列的第173-343位,MrgprF-M3的氨基酸序列是SEQ ID NO:4所示氨基酸序列的第45-343位,MrgprF-M4的氨基酸序列是SEQ ID NO:4所示氨基酸序列的第1-294位,MrgprF-M5的氨基酸序列是SEQ IDNO:4所示氨基酸序列的第295-343位,M的氨基酸序列是SEQ ID NO:3所示氨基酸序列。
图66结果显示,随着GST-MrgprF274-343肽段浓度的逐渐升高,HA-p101和p110γ-Flag的结合逐渐降低;提示GST-MrgprF274-343肽段可以作为HA-p101和p110γ-Flag相互结合的抑制因子。
序列表
<110> 中国科学院昆明动物研究所
<120> 人MRGPRF基因在肿瘤临床诊疗中的应用
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<213> 人工序列(Artificial)
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<213> 人工序列(Artificial)
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Leu Thr Ile Glu Gln Ile Ala Met Leu Pro Pro Pro Ala Val Met Asn
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Tyr Ile Phe Leu Leu Leu Cys Leu Cys Gly Leu Val Gly Asn Gly Leu
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Val Leu Trp Phe Phe Gly Phe Ser Ile Lys Arg Asn Pro Phe Ser Ile
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Tyr Phe Leu His Leu Ala Ser Ala Asp Val Gly Tyr Leu Phe Ser Lys
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Ala Val Phe Ser Ile Leu Asn Thr Gly Gly Phe Leu Gly Thr Phe Ala
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Asp Tyr Ile Arg Ser Val Cys Arg Val Leu Gly Leu Cys Met Phe Leu
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Thr Gly Val Ser Leu Leu Pro Ala Val Ser Ala Glu Arg Cys Ala Ser
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Val Ile Phe Pro Ala Trp Tyr Trp Arg Arg Arg Pro Lys Arg Leu Ser
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Ala Val Val Cys Ala Leu Leu Trp Val Leu Ser Leu Leu Val Thr Cys
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Leu His Asn Tyr Phe Cys Val Phe Leu Gly Arg Gly Ala Pro Gly Ala
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Ala Cys Arg His Met Asp Ile Phe Leu Gly Ile Leu Leu Phe Leu Leu
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Cys Cys Pro Leu Met Val Leu Pro Cys Leu Ala Leu Ile Leu His Val
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Glu Cys Arg Ala Arg Arg Arg Gln Arg Ser Ala Lys Leu Asn His Val
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Ile Leu Ala Met Val Ser Val Phe Leu Val Ser Ser Ile Tyr Leu Gly
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Cys Pro Pro Gly Asn Ala Ser
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Claims (3)
1.人MRGPRF基因在筛选黑色素瘤治疗药物中的应用,其特征在于:基于人MRGPRF基因对PI3K/AKT信号通路的抑制作用,筛选能够抑制PI3K/AKT信号通路的人MRGPRF小肽作为黑色素瘤治疗药物,所述人MRGPRF蛋白序列如SEQ ID NO:4所示。
2.根据权利要求1所述的应用,其特征在于:所述小肽为MrgprF274-343,小肽的氨基酸序列如SEQ ID NO:3所示。
3.根据权利要求2所述的应用,其特征在于:小肽MrgprF274-343能与人p110-g蛋白相互作用,阻断或降低p110-g与p101的相互结合,进而降低PI3K/AKT信号通路活性作用。
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