CN113943812A - 一种肺腺癌铁死亡敏感性三联标志物及其应用 - Google Patents
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Abstract
本发明涉及生物医学技术领域，具体是一种肺腺癌铁死亡敏感性三联标志物及其应用，所述三联标志物是ACSL4、YAP和LPCAT3。该三联标志物可作为铁死亡敏感性标志物，且实验结果表明该标志物组合相较于单一使用以及其他组合的准确率都有显著性的提升。本发明还提供了该三联标志物在制备用于肺腺癌铁死亡检测试剂盒中的应用。其优点表现在：检测准确率高、成本低、操作简单，可很好的应用于临床上，有很强的实用性。
Description
技术领域
本发明涉及生物医学技术领域，具体地说，是一种肺腺癌铁死亡敏感性三联标志物及其应用。
背景技术
2012年，哥伦比亚大学Stockwell实验室报道erastin等一类小分子抗肿瘤化合物能够诱导大量的细胞死亡，该类型细胞死亡依赖于细胞内的铁离子和脂质过氧化物，并将之命名为ferroptosis，即铁死亡，其在形态学、生物学及基因水平上均明显不同于凋亡、坏死、自噬等其他形式的细胞死亡。除了抑制肿瘤的功能，铁死亡的其余正常生理功能还未探明。但是铁死亡与许多人类疾病和治疗策略相关。铁死亡与癌症(如弥漫性大B细胞淋巴瘤、肾嫌色细胞癌、肝细胞癌等)、神经退行性疾病(如帕金森病，亨廷顿病和阿尔茨海默病)和组织缺血再灌注损伤(如急性肾衰竭和脑室周围白质软化)等疾病息息相关[1,2,3,4]。在多种实验肿瘤模型中可以观察到铁死亡诱导剂抑制肿瘤的作用，这意味着诱导铁死亡可以作为一种新的抗肿瘤治疗方案。
根据2020年全球最新癌症负担数据[5]，由于新发病例数的快速增长，乳腺癌取代肺癌成为全球第一大癌症。然而，肺癌死亡病例仍居所有癌症之首。在我国，肺癌的发病率和死亡率均位于癌症之首。肺腺癌是最常见的亚型，约占所有肺癌病例的40％[6]。虽然手术切除是早期肺腺癌最有效的治疗方法，但晚期患者可能受益于辅助细胞毒治疗[7,8]。然而，化疗耐药往往是肺腺癌患者复发的主要原因[6]。最近，有研究表明顺铂耐药的肺腺癌细胞对铁死亡敏感，诱导铁死亡的疗法可以为顺铂治疗失败的肺腺癌患者提供新的治疗方案[9]。与其他类型的癌症相似，肺腺癌对铁死亡的敏感性可能有所不同，是否存在某种标志物可预示肺腺癌细胞或患者样本对铁死亡的敏感程度尚不清楚。
中国专利申请：201510954097.4公开了一种用于预测肺腺癌患者预后及判断辅助化疗获益的系统，包括检测c-Src、CyclinE1、TTF1、p65、CHK1和JNK1六种蛋白质表达量的系统和蛋白质表达量数据处理系统。检测上述六种蛋白质表达量的系统通过免疫组织化学染色方法测定蛋白质的表达量；蛋白质表达量数据处理系统将来自待预测肺腺癌患者的分离的肺腺癌组织中所述六种蛋白质表达量转换为预后分值，根据预后分值预测待肺腺癌患者的预后和/或待预测肺腺癌患者是否受益于辅助化疗。但该专利中涉及的标志物高达6种，检测方法较为繁杂，且并未涉及到铁死亡内容。
可见是否存在某种标志物可预示肺腺癌细胞或患者样本对铁死亡的敏感程度目前尚不清楚。而本发明首次创造性的提出将肺腺癌铁死亡敏感性的一种三联标志物，为肺腺癌患者提供了一种新的治疗方法，关于本发明一种肺腺癌铁死亡敏感性三联标志物及其应用目前还未见报道。
发明内容
本发明的目的是针对现有技术的不足，提供一种肺腺癌铁死亡敏感性三联标志物及其应用。
为实现上述目的，本发明采取的技术方案是：
第一方面，本发明提供了一种预测肺腺癌铁死亡敏感性的试剂盒，含有检测ACSL4、YAP和LPCAT3表达量的试剂。
优选地，所述试剂盒还可用于预测脂质活性氧含量。
第二方面，本发明提供了一种试剂组合在制备预测肺腺癌铁死亡敏感性的试剂盒中的应用，所述试剂组合含有检测ACSL4、YAP和LPCAT3表达量的试剂。
第三方面，本发明提供了ACSL4、YAP和LPCAT3在制备预测肺腺癌铁死亡敏感性的药物中的应用。
第四方面，本发明提供了ACSL4、YAP和LPCAT3的促进剂在制备提高肺腺癌铁死亡敏感性的药物中的应用。
优选地，所述ACSL4、YAP和LPCAT3的促进剂是促进ACSL4、YAP和LPCAT3表达量的物质。
优选地，所述ACSL4、YAP和LPCAT3的促进剂选自小分子化合物或生物大分子。
本发明优点在于：
1、本发明首次将ACSL4、YAP和LPCAT3联合使用用于预测肺腺癌铁死亡敏感性，相较于现有技术中单一标志物在准确率上有了显著提升，表明ACSL4、YAP和LPCAT3之间协同作用，能指导临床制定个体化化疗方案，提高疗效。
2、方法简便、成本低，提高了患者的接受度以及为肺腺癌的治疗提供了一种新思路和解决方案，实用性强，应用前景广。
附图说明
附图1是肺腺癌细胞系对铁死亡的敏感程度存在差异。
附图2是LPCAT3与肺腺癌细胞系对铁死亡的敏感程度成正比。
附图3是LPCAT3表达水平和铁死亡敏感程度存在相关性。
附图4是LPCAT3敲除降低了肺腺癌细胞系对铁死亡的敏感程度。
附图5是LPCAT3表达量高的原代肺腺癌细胞对铁死亡更敏感。
附图6是判断YAP，LPCAT3，ACSL4联用作为铁死亡敏感性标志物。
具体实施方式
下面结合具体实施方式，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明记载的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1
1方法
1.1细胞、试剂和质粒
已建立的H358，H1650，PC9，H1975，A549和H1299细胞系均购自上海富衡生物科技有限公司。患者来源的原代肺腺癌细胞系来源于肺腺癌组织。将未坏死的小于1.0cm3的组织用冰冷的DPBS冲洗3次，然后再重悬浮于含有胶原酶(2mg/ml，Solarbio,中国上海)的DMEM培养基中，37℃作用4h，再用DMEM洗涤3次后，在常规条件下进行细胞培养。对于正常细胞培养，细胞用DMEM+10％胎牛血清和1％青霉素/链霉素双抗培养。
细胞实验所用的试剂为：Erastin(Sigma,St Louis,MO,USA),ferrostatin-1(Fer-1,Sigma)，SYTOX Green(Invitgen，Carbsland，CA，USA)，C11-BODIPY581/591(Invitrogen)。
利用LentiCRISPRv2质粒(Addgene,Cambridge,MA,USA)构建LPCAT3的敲除载体。表达LPCAT3的质粒购自中国上海上海佐润生物科技有限公司。
1.2免疫印迹
将细胞蛋白裂解物在SDS聚丙烯酰胺凝胶中分离并转移到硝酸纤维素膜上。将膜在封闭缓冲液(含1％吐温-20的PBS缓冲液中的5％牛奶)中封闭，然后用特异性抗体孵育。所使用的一抗是抗-LPCAT3(Abcam,#ab239585)和抗-GAPDH(Cell Signaling Technology(CST),Boston,MA,USA,#5174)。
1.3定量RT-PCR(qPCR)
用Trizol(Ambion，Carlsad，CA，USA)试剂提取总RNA，并用PrimeScriptTMRT试剂盒(中国大连Takara)反转录成cDNA。对于实时qPCR，使用SYBRpremix Ex Taq(Takara)试剂盒检测mRNA含量。
1.4代谢物的测定
不稳定铁和MDA检测试剂盒购自Abcam公司，根据制造商的说明测定代谢物。
1.5细胞死亡和脂质活性氧生成的测量
SYTOX Green染色分析细胞死亡，随后采用流式细胞仪测定。在细胞收获前加入终浓度为2μM的荧光探针C11-BODIPY581/591，37℃下孵育30分钟，随后用流式细胞仪检测脂质活性氧阳性的细胞。
1.6免疫组化及染色指数分析
免疫组化可用来分析特定蛋白的组织原位表达情况，首先对切片进行脱蜡，抗原修复，内源性过氧化物酶修复等步骤，封闭后加入合适一抗二抗，DAB显色后苏木素染色，最后梯度酒精二甲苯脱水封片，显微镜拍照。所用的一抗包括抗-YAP(Abcam，#ab52771)，抗-LPCAT3(Abcam,#ab239585)，抗ACSL4(Abcam，#ab155282)，抗-TFRC(Abcam，#ab214039)，抗-PTGS2(Abcam，#ab179800)，抗-NOX1(Abcam，#ab131088)。染色指数(0-12)等于染色强度和染色区域的乘积。染色强度的分数被定为：阴性0分；弱1分；中等2分；强阳性3分。阳性细胞的频率被定义为：少于5％，0分；5％-25％，1分；26％-50％，2分；51％-75％，3分；大于75％，4分。0到7是低表达，8到12是高表达。根据染色指数判断YAP，LPCAT3，ACSL4表达情况。
1.7统计分析
所有实验结果均采用均数±标准差(mean±SD)表示，每个实验三重复。用于统计分析的检验方法有Student’s-t检验、one-wayANOVA和Spearman相关性检验。P<0.05表示有统计学意义，其中*表示P<0.05，**表示P<0.01。
2结果
2.1肺腺癌细胞系铁死亡处理后存在敏感性差异
铁死亡是新发现的一种脂质过氧化物驱动的依赖于铁的调节性细胞死亡方式[10,11]。铁死亡过程有3个显著特点[12]：1)具有氧化还原活性的铁的存在；2)含有多不饱和脂肪酸的磷脂发生过氧化作用；3)脂质过氧化修复网络失衡。通过检测细胞死亡情况、脂质活性氧含量、不稳定铁含量和丙二醛(脂质过氧化物的产物及标志物)的含量，我们发现六株肺腺癌细胞系(A549、H358、H1299、H1650、PC9和H1975细胞)在erastin处理后对铁死亡的敏感程度存在差异(图1A-D)。其中，H1975细胞对铁死亡最为敏感，A549细胞最为抵抗。H1975细胞在加入erastin后细胞死亡数，Lipid ROS阳性细胞，游离铁以及MDA上升程度最大，而A549细胞趋势则相反(图1A-D)。
2.2 LPCAT3与肺腺癌细胞系铁死亡敏感性成正比
通过免疫印迹和qPCR，我们检测了六株肺腺癌细胞系中LPCAT3在翻译水平和转录水平的表达情况(图2A-B)。我们发现LPCAT3蛋白表达量和LPCAT3 mRNA含量存在显著的正相关关系(R＝0.9729，p＝0.0011)(图2C)。此外，LPCAT3蛋白表达量和铁死亡指标细胞死亡情况(R＝0.8342，p＝0.0389)以及脂质活性氧含量(R＝0.8742，p＝0.0227)均存在正相关性(图2D-E)。
2.3过表达LPCAT3，肺腺癌细胞变敏感
我们在A549和H1975细胞系中过表达LPCAT3，使其蛋白表达含量形成梯度递增(图3A)。在A549和H1975细胞系中均可观察到LPCAT3蛋白表达量和细胞死亡情况(R＝0.9821，p＝0.0178；R＝0.9752，p＝0.0248)呈正向相关(图3B)。同时，我们也观察到A549和H1975细胞系中LPCAT3蛋白表达量与脂质活性氧含量(R＝0.9662，p＝0.0338；R＝0.9849，p＝0.0151)存在正相关性(图3C)。
2.4敲除LPCAT3细胞，肺腺癌细胞系敏感性消失
借助CRISPR/Cas9技术，我们构建了LPCAT3敲除的六株肺腺癌细胞系(图4A)。加入erastin处理后通过检测细胞死亡情况(图4B)和脂质活性氧的含量(图4C)，我们发现LPCAT3敲除后，肺腺癌细胞对铁死亡变得抵抗。综上，可以得出结论：LPCAT3敲除降低了肺腺癌细胞系对铁死亡的敏感程度。
2.5 10株原代细胞培养，LPCAT3高组，低组存在明显铁死亡敏感性差异
通过免疫印迹，我们将肺腺癌患者来源的10株原代细胞根据LPCAT3蛋白表达量分为LPCAT3表达量高组和LPCAT3表达量低组(图5A)。加入erastin处理后，我们可以观察到与LPCAT3表达量低组相比，LPCAT3表达量高组细胞死亡百分比显著升高(图5B)。同时，LPCAT3表达量高组细胞产生的脂质活性氧和不稳定铁的含量明显高于LPCAT3表达量低组(图5C-D)。由此可以得出结论，LPCAT3表达量高的肺腺癌细胞对铁死亡较为敏感。这些结果表明，LPCAT3有潜力成为显示肺腺癌对铁死亡敏感程度差异的标志物。
2.6 ACSL4，YAP，LPCAT3三者联用可更好判断铁死亡敏感性。
已有研究证实ACSL4以及YAP均可作为铁死亡敏感性标志物[13,14]，我们对新鲜组织120例取一部分培养于含erastin(10μM)培养基中，24小时后检测MDA含量，另一部分直接用免疫组化检测ACSL4，YAP，LPCAT3含量(图6A)，染色强度前20％认为高表达(high)而其余80％为低表达(low)。我们发现三者均高表达标本加入erastin后MDA产生量明显高于某一/二指标高表达标本，而且三者均低表达标本MDA产生量更低，该结果提示用ACSL4、LPCAT3、YAP联合检测可判断铁死亡敏感性(图6B)。此外，我们还探究了其他铁死亡相关标志物联用的方式，包括本研究主要关注的LPCAT3与YAP/ACSL4两者之一和其他铁死亡敏感性潜在标志物(包括PTGS2，TFRC以及NOX1)的联用，发现其它的联用方式判断准确率显著低于ACSL4、LPCAT3、YAP的联用(图6C)，说明ACSL4、LPCAT3、YAP联合检测判断铁死亡敏感性的方式具有一定优越性。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明原理的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
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Claims (7)
1.一种预测肺腺癌铁死亡敏感性的试剂盒，其特征在于，含有检测ACSL4、YAP和LPCAT3表达量的试剂。
2.根据权利要求1所述的试剂盒，其特征在于，所述试剂盒还可用于预测脂质活性氧含量。
3.一种试剂组合在制备预测肺腺癌铁死亡敏感性的试剂盒中的应用，其特征在于，所述试剂组合含有检测ACSL4、YAP和LPCAT3表达量的试剂。
4.ACSL4、YAP和LPCAT3在制备预测肺腺癌铁死亡敏感性的药物中的应用。
5.ACSL4、YAP和LPCAT3的促进剂在制备提高肺腺癌铁死亡敏感性的药物中的应用。
6.根据权利要求5所述的应用，其特征在于，所述ACSL4、YAP和LPCAT3的促进剂是促进ACSL4、YAP和LPCAT3表达量的物质。
7.根据权利要求6所述的应用，其特征在于，所述ACSL4、YAP和LPCAT3的促进剂选自小分子化合物或生物大分子。
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