CN117487914A - 靶向zc3h18/pd-l1信号轴在肿瘤免疫逃逸检测、治疗、预后中的应用 - Google Patents
靶向zc3h18/pd-l1信号轴在肿瘤免疫逃逸检测、治疗、预后中的应用 Download PDFInfo
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- CN117487914A CN117487914A CN202311403230.8A CN202311403230A CN117487914A CN 117487914 A CN117487914 A CN 117487914A CN 202311403230 A CN202311403230 A CN 202311403230A CN 117487914 A CN117487914 A CN 117487914A
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Abstract
本发明属于生物技术领域，具体涉及一种靶向ZC3H18/PD‑L1信号轴在肿瘤免疫逃逸检测、治疗、预后中的应用，本发明发现了在ZC3H18过表达的情况下，机体通过招募结合CSNK2A1、PRMT5使PD‑L1转录活性增强的途径显著促进了肿瘤(如胰腺癌)的免疫治疗逃避；本发明联合应用抑制CSNK2A1、PRMT5表达的试剂使ZC3H18过表达的胰腺癌对免疫治疗更敏感；这些结果说明ZC3H18在肿瘤免疫治疗逃避中的重要作用，可作为ZC3H18过表达型肿瘤的新型治疗策略。
Description
技术领域
本发明属于生物技术领域，具体涉及一种靶向ZC3H18/PD-L1信号轴在肿瘤免疫逃逸检测、治疗、预后中的应用。
背景技术
胰腺癌是一种侵袭性极强的恶性肿瘤，是全球癌症相关死亡的第四大常见原因，其中欧洲、北美和澳大利亚/新西兰的发病率最高。尽管近几十年来胰腺癌的诊断和治疗取得了进展，但总体预后极差，中位生存期为6个月，5年生存期约为9％。据统计，2025年胰腺癌的发病率将成为癌症死亡的第三大原因。这种复杂疾病的顽固性源于几个因素，诊断较晚，缺乏敏感和特异性的生物标志物，转移灶早期扩散，特别是对化疗、放疗和目前可用的靶向治疗的耐药性。因此揭示胰腺癌恶性发展的分子机制，寻找诱导胰腺癌靶向药物耐受的关键分子，将对胰腺癌的预防和治疗提供重要的科学根据，对改善肿瘤患者的生存预后具有重要的临床意义。
锌指CCCH结构域蛋白18(ZC3H18)是新发现的定位于16号染色体上的基因，是参与mRNA生物发生和转录调控的多结构域蛋白。是具备增强下游基因转录的反式作用因子。先前的研究表明，ZC3H18在舌蝇血液型锥虫向前环型的转化中起着至关重要的作用。全基因组分析表明，ZC3H18有助于特定蛋白编码基因的转录，这依赖于其cbc相互作用域以及其NEXT-和/或组蛋白相互作用域。此外，ZC3H18已被证明能够特异性结合并激活BRCA1启动子，促进高级别浆液性卵巢癌(HGSOCs)中的同源重组。
免疫治疗已经成为一种非常有前途的癌症治疗方法，对传统疗法(如手术、放疗和化疗)进行了补充。值得注意的是目前免疫治疗策略包括针对程序性细胞死亡1(PD-1)/程序性细胞死亡1配体1(PD-L1)轴信号通路的免疫检查点封锁。临床前和临床研究已经证明了对PD-1/PD-L1进行阻断在各种癌症类型中的疗效，包括黑色素瘤、肺癌和肾癌，在相当大比例的病例中获得了持久的抗肿瘤反应。然而，单独使用PD-L1抑制剂在改善胰腺癌预后方面的效果有限。例如，Amita Patnaik等人的一项研究报告称，在接受PD-L1抑制剂和美雷司替尼联合治疗的胰腺癌患者中，部分缓解率仅为25.0％，且缓解持续时间相对较短(9.8个月)。此外，有证据表明，虽然PD-L1检查点抑制和化疗的联合治疗已经显示出了希望，但PD-L1抑制剂单药治疗在胰腺癌患者中未能产生显著的疗效。因此，全面了解PD-1/PD-L1轴的详细机制，对识别基于PD-L1的组合治疗和开发新的胰腺癌治疗策略至关重要。
PD-L1的异常表达，是一种在各种恶性肿瘤中常见的现象，导致免疫逃避和不良的临床结果。癌症中PD-L1表达的失调可由外源性和内在的致癌变化引起。例如，Qin等人已经表明，转录激活子NPM1在TNBC细胞中特异性与PD-L1启动子特异性结合，从而激活PD-L1转录。此外，β-连环蛋白通过将β-连环蛋白/TCF/LEF复合物与PD-L1基因启动子区域结合，促进胶质母细胞瘤中PD-L1的表达，进而促进胶质母细胞瘤的免疫逃避，并促进癌症干细胞的扩增。在胰腺癌中，PD-L1表达的升高与较差的总生存率、***受累阳性、肿瘤晚期和低分化相关。其他研究表明，NEK2可以抑制胰腺癌中PD-L1的降解，而HDAC5可以通过p65去乙酰化调节PD-L1的表达。此外，C-FOXP3直接与PD-L1的启动子区域结合，并诱导PD-L1的表达。然而，PD-L1在胰腺癌中的精确转录调控仍在很大程度上是未知，迫切需要阐明PD-L1的关键调控因子。
发明内容
本发明的目的针对现有技术存在的问题，本发明提供靶向ZC3H18/PD-L1信号轴在肿瘤免疫逃逸检测、治疗、预后中的应用。
为实现上述发明目的，经研究，本发明提供如下技术方案：
ZC3H18作为肿瘤治疗免疫逃避中作为预警靶点分子和预测肿瘤免疫治疗中发生免疫逃避的检测靶点的应用。
一种定量检测ZC3H18的试剂在制备肿瘤治疗免疫逃避中作为预警靶点分子和预测肿瘤免疫治疗中发生免疫逃避的检测靶点的应用，所述定量检测ZC3H18的试剂选自检测ZC3H18基因是否过表达的试剂、定量检测ZC3H18的RNA转录水平的试剂、定量检测ZC3H18的蛋白表达水平的试剂中的至少一种。
一种抑制ZC3H18蛋白表达的物质在制备提高肿瘤对PD-L1单抗疗效的药物中的应用。
一种抑制CSNK2A1、PRMT5表达的试剂在制备治疗ZC3H18过表达型肿瘤药物中的应用。
一种抑制CSNK2A1、PRMT5表达的试剂在制备提高ZC3H18过表达型肿瘤免疫治疗效果的药物中的应用，所述抑制CSNK2A1、PRMT5表达的试剂为化学药物GSK3326595和CX-4945。
一种检测CSNK2A1、PRMT5、PD-L1表达量的试剂在制备肿瘤免疫治疗中作为治疗靶点分子的检测试剂盒或在预测肿瘤免疫治疗效果的检测试剂盒中的应用。
一种肿瘤免疫治疗、肿瘤预后、或/和预测肿瘤免疫治疗效果的检测试剂盒，所述试剂盒中含有检测ZC3H18的试剂，或/和检测CSNK2A1、PRMT5、PD-L1表达量的试剂。
优选的，所述试剂盒中的试剂选自检测ZC3H18基因是否过表达的试剂、定量检测ZC3H18的RNA转录水平的试剂、定量检测ZC3H18的蛋白表达水平的试剂中的至少一种。
一种提高肿瘤对免疫治疗疗效的药剂，所述药剂中含有抑制ZC3H18蛋白表达的物质、抑制CSNK2A1、PRMT5表达的试剂中的至少一种。
一种抑制ZC3H18过表达型肿瘤对免疫治疗疗效的药剂，所述药剂中含有抑制ZC3H18蛋白表达的物质、抑制CSNK2A1、PRMT5表达的试剂中的至少一种。
本发明的有益效果至少包括：
1)本发明发现了在ZC3H18过表达的情况下，机体通过招募结合CSNK2A1、PRMT5使PD-L1转录活性增强的途径显著促进了肿瘤(如胰腺癌)的免疫治疗逃避。本发明联合应用抑制CSNK2A1、PRMT5表达的试剂使ZC3H18过表达的胰腺癌对免疫治疗更敏感。这些结果说明ZC3H18在肿瘤免疫治疗逃避中的重要作用，可作为ZC3H18过表达型肿瘤的新型治疗策略；
2)本发明发现了ZC3H18基因在胰腺癌中发生肿瘤免疫治疗逃避型的诊断、治疗和预后中的应用。本发明成功构建稳定过表达ZC3H18以及干扰ZC3H18表达的胰腺癌细胞模型，成功建立小鼠胰腺癌肿瘤模型，并通过细胞共培养技术，流式细胞术等实验分析发现，抑制ZC3H18可增强体内外抗肿瘤免疫力。本发明利用基因本体论(GO)富集分析、PPI网络分析、Western Blot以及TCGA数据库分析发现，ZC3H18蛋白表达上调与胰腺癌肿瘤免疫治疗逃避密切相关。因此，该研究结果将为ZC3H18调控肿瘤免疫治疗逃避提供新的机制；
3)机制层面，本发明使用ZC3H18抗体并进行Tag-seq检测，通过CHIP-PCR、荧光素酶报告基因实验等，揭示了ZC3H18与PD-L1启动子结合，增加PD-L1的转录活性；通过进行质谱实验、免疫共沉淀技术以及ChIP实验，发现ZC3H18增加了胰腺癌中PD-L1的表达并促进了肿瘤免疫治疗逃避；深入探讨了转录因子ZC3H18招募人酪蛋白激酶CSNK2A1、蛋白质精氨酸酶甲基转移酶PRMT5蛋白对于促进PD-L1表达的作用，分析了ZC3H18/CSNK2A1/PRMT5轴与下游基因PD-L1启动子的具体结合区域，为癌基因ZC3H18调控靶基因提供了新的分子调控机制；最后，本发明利用收集的胰腺癌临床样品，进行免疫组化等实验并进行分析发现，联合应用CSNK2A1、PRMT5抑制剂极大降低了肿瘤中PD-L1蛋白的表达。这提示ZC3H18基因能作为促进患者肿瘤免疫治疗逃避的重要靶分子。综上所述，本研究发现ZC3H18是胰腺癌细胞免疫逃避的重要促进物，在癌症的发展过程中具有重要意义。这将为临床肿瘤免疫治疗提供新的治疗方向。本发明为肿瘤疾病提供了新的诊断、治疗方法和药物筛选平台。
本发明的特征和优点将在随后的具体实施方式部分予以详细说明。
附图说明
图1为筛选并分析特异性靶向PD-L1启动子的候选蛋白因子流程图。
图2为沉默不同基因对PD-L1表达量的影响示意图。
图3为胰腺癌细胞系(Panc02细胞系和MiaPaCa-2细胞系)的western blot的蛋白条带示意图。
图4为胰腺癌细胞系(Panc02细胞系和MiaPaCa-2细胞系)的流式细胞术的PD-L1蛋白表达量示意图。
图5为ZC3H18 Cuttool Peaks的分布情况(a)，得到ZC3H18的分布情况；ZC3H18Cuttag-seq示意图(b)，检测和分析ZC3H18基因的表达情况；基因间距示意图(c)，显示了基因之间相互作用和调控关系；ZC3H18Cuttag-Seq analyses示意图(d)，显示了对照组和ZC3H18基因过表达组的ZC3H18基因与PD-L1基因之间关系。
图6为胰腺癌细胞系(Panc02细胞系和MiaPaCa-2细胞系)中ZC3H18和lgG在PD-L1基因上的富集程度(a)，其中GADPH作为内参蛋白，用于校正实验中的误差；胰腺癌细胞系(Panc02细胞系和MiaPaCa-2细胞系)的ZC3H8和对照组的荧光素酶活性(b)。
图7是不同蛋白质在PD-L1启动子区域上的富集程度，其中，GAPDH是内参蛋白，P1，P2……表示不同的蛋白质。
图8是PRMT5的质谱分析结果(A)，纵坐标为marker是为了校正实验中的误差；胰腺癌细胞系(Panc02细胞系和MiaPaCa-2细胞系)的ZC3H18在PD-L1启动子区域上的富集程度(B)。
图9是胰腺癌细胞系(Panc02细胞系和MiaPaCa-2细胞系)的ZC3H18-Flag融合蛋白与PRMT5之间的相互作用示意图(C)，PD-L1启动子与H3R2me1、H3R2me2s、ZC3H18之间的关系(D)，H3K4me3，poll和lgG在input中的表达水平以及它们之间的关系(E)，胰腺癌细胞系(Panc02细胞系和MiaPaCa-2细胞系)的ZC3H18和PRMT5-si对PD-L1mRNA表达水平的影响(F)；ZC3H18和vector分别在对照和沉默PRMT5时的荧光强度差异(G)。
图10是CSNK2A1的质谱图(A)；胰腺癌细胞系(Panc02细胞系和MiaPaCa-2细胞系)的westernblot条带示意图，显示了ZC3H18和CSNK2A1的相对表达量，其中input和lgG作为内参蛋白来矫正实验结果(B)；蛋白质免疫印迹实验示意图，显示了Flag-ZC3H18和GST-CSNK2A1的相对表达量，其中GST作为内参蛋白来校正实验结果(C)；胰腺癌细胞系(Panc02细胞系和MiaPaCa-2细胞系)的ZC3H8和CSNK2A1在免疫沉淀实验中的富集程度(D)。
图11是不同浓度CX-4945对CSNK2A1、H2A、p-Y57 H2A蛋白表达的影响的蛋白条带示意图(E)；CX-4945、Flag-H2A(Y57F)、Flag-H2A(WT)、ZC3H18和vector对H2A、H3、H3K4me3的蛋白表达水平的影响的蛋白条带示意图(F)。
图12是shRNA、Control、ZC3H18和vector对细胞中ZC3H18蛋白表达的影响的肿瘤细胞大小示意图(A)；Vector，ZC3H18，shRNA和Control对肿瘤体积的影响示意图(B)，Vector，ZC3H18，shRNA和Control对肿瘤重量的影响示意图(C)，Vector，ZC3H18，shRNA和Control对CD8和CD3的影响(D)，Vector，ZC3H18，shRNA和Control对IFN-r、TNF-a、GzmB和serum evel的影响(E)。
图13是Vector，ZC3H18，shRNA和Control对CD8和CD3的影响(A),胰腺癌细胞系(Panc02细胞系和MiaPaCa-2细胞系)对IFN-r、TNF-a、GzmB和serum evel的相对蛋白表达量的示意图(B)。
图14是；不同治疗方法的给药方式示意图(A)；不同治疗方法的肿瘤细胞大小示意图(B)；不同治疗方法对肿瘤体积影响的示意图(C)；不同治疗方法对肿瘤重量影响的示意图(D)；不同治疗方法对肿瘤细胞中PD-L1表达的影响的示意图(E)；其中，治疗方式分别为对照组，PD-L1单药治疗组，CX-4945单药治疗组，GSK3326595单药治疗组和联合治疗组。
具体实施方式
为了使本发明的目的、技术方案和有益效果更加清楚，下面结合附图和具体的实施方式对本发明作进一步详细的说明。所述实施例的示例在附图中示出。应理解，在下述本发明的实施方式中描述的具体的实施例仅作为本发明的具体实施方式的示例性说明，旨在用于解释本发明，而不构成对本发明的限制。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。在本申请的描述中，除非另有说明，“一个/一种”、“多个/多种”等类似用词的含义是两个/种或两个/种以上。
实施例1：ZC3H18能够增加PD-L1在胰腺癌中的表达
(1)筛选并分析特异性靶向PD-L1启动子的候选蛋白因子方法：设计特异性靶向PD-L1启动子的sgRNA，采用生物素偶联的失活Caspase9***进行捕获分析以及LC-MS/MS筛选出特异性靶向PD-L1启动子的候选蛋白因子，通过STRING进行PPI网络筛选出14个蛋白，再通过IP/MS进行鉴定分析。同时进一步把参与PD-L1转录调控的关键基因进行互作分析，挑选互作作用强(以蛋白丰度为筛选依据)的前15个基因，利用小RNA干扰技术分别单独抑制15个基因的表达后，检测PD-L1的mRNA表达水平。(如图1，2)
结果：在筛选出的有效PD-L1启动子相关蛋白中只有单独沉默ZC3H18、CSNK2A1或PRMT5可以降低PD-L1的mRNA水平，而且ZC3H18是降低PD-L1最显著的基因。
结论：ZC3H18是降低PD-L1表达水平最明显的基因。
(2)建立稳定表达ZC3H18 cDNA和ZC3H18 RNAi(s)的Panc02细胞系方法：通过多位点克隆，将人ZC3H18和鼠ZC3H18基因的编码序列克隆到pCDH-CMV-MCS-3Flag-EF1-CopGFP-T2A-Puro载体上。使用PLKO-U6-EGFP-P2A-PURO载体生成ZC3H18-RNAi(s)。使用Lipofectamine3000试剂(Invitrogen,Carlsbad,CA)根据厂家说明书转染ZC3H18质粒或shRNA。后续进行了western blot(如图3)和流式实验(如图4)进一步验证。
上述沉默ZC3H18的RNA序列为:
CCGGCCAGCACGGTTCTCATGTAAACTCGAGTTTACATGAGAACCGTG CTGGTTTTTG(SEQ IDNO:1)
结果：获得ZC3H18过表达、ZC3H18沉默以及空白对照的胰腺癌细胞系，并且发现与对照组相比，过表达ZC3H18会使PD-L1蛋白水平持续上调，沉默ZC3H18的话PD-L1蛋白水平则下调。
结论：建立稳定表达ZC3H18以及沉默ZC3H18的胰腺癌细胞系。与此同时发现ZC3H18增加了胰腺癌细胞中PD-L1的表达。
实施例2：ZC3H18结合PD-L1启动子，增加PD-L1的转录活性。
(1)研究ZC3H8在胰腺癌中上调PD-L1蛋白水平的分子机制。
方法：使用ZC3H18抗体做CUT&Tag-seq(如图5)，再通过CHIP-PCR检测ZC3H18与PD-L1启动子的相关性(如图6-a)。采用荧光素酶报告基因检测PD-L1启动子的荧光酶活性(如图6-b)。
结果：seq分析表明PD-L1可能是ZC3H18的有效靶点。CHIP-PCR结果显示ZC3H18与PD-L1启动子显著相关。荧光素酶报告基因检测显示过表达ZC3H18可以增强PD-L1启动子的荧光素酶活性，敲低ZC3H18则降低PD-L1启动子的荧光素酶活性。
结论：ZC3H18与PD-L1启动子显著相关。过表达ZC3H18可降低PD-L1启动子的荧光素酶活性，敲低ZC3H18则降低PD-L1启动子的荧光素酶活性。(2)研究ZC3H18是否参与PD-L1的转录调控方法：将PD-L1启动子(-2500bp)进行分段处理(如图7)。
结果：发现ZC3H18与PD-L1启动子区域内的3区结合。
结论：ZC3H18与PD-L1启动子区域内的3区结合。
实施例3：PRMT5是ZC3H18诱导PD-L1上调所必需的
(1)探讨ZC3H18转录上调PD-L1的机制方法：采用IP/MS分析ZC3H18的互作蛋白(如图8-A)，并结合CHIP-PCR检测来进行分析(如图8-B)。
结果：IP/MS分析发现PRMT5可能是ZC3H18的一个有效互作蛋白，CHIP-PCR检测发现抑制PRMT5的表达显著降低了ZC3H18过表达细胞中PD-L1的转录活性水平。
结论：PRMT5是ZC3H18的一个有效互作蛋白，并且参与ZC3H18对PD-L1的转录调控。
(2)检测PRMT5是否参与ZC3H18诱导的PD-L1上调
方法：采用共免疫沉淀(co-IP)分析ZC3H18与PRMT5的相互作用(如图9)，同时在胰腺癌细胞中，对富集PD-L1启动子上的组蛋白翻译修饰后进行CHIP-qPCR分析。另外对细胞用流式细胞术分析PD-L1的平均荧光强度(MFI)。
结果：免疫共沉淀(co-IP)分析结果表明，ZC3H18与PRMT5在体外和体内都发生了相互作用。在ZC3H18过表达的胰腺癌细胞中，PD-L1启动子上H3R2me1的富集水平显著升高，而在ZC3H18沉默细胞中H3R2me1的富集水平降低。然而，我们发现使用siRNA沉默PRMT5或使用EPZ015866抑制PRMT5活性显著降低了ZC3H18过表达胰腺癌细胞PD-L1启动子上的H3R2me1和H3R2me2s水平。还发现PRMT5或WDR5的抑制显著降低了胰腺癌细胞PD-L1启动子上H3K4me3和聚合酶Ⅱ的丰度。重要的是，沉默PRMT5可以降低ZC3H18过表达的胰腺癌细胞中的PD-L1的mRNA和蛋白水平。
结论：PRMT5介导H3R2me1和H3R2me2s修饰参与了ZC3H18上调PD-L1的表达。ZC3H18通过PRMT5对H3R进行甲基化修饰，进而使H3K4再PD-L1启动子发生甲基化，从而上ZC3H18的转录水平。
实施例4：CSNK2A1调控H2A酪氨酸磷酸化，增加了ZC3H18诱导的PD-L1的转录激活
(1)探讨参与胰腺癌中PD-L1的转录激活的其他因素
方法：同样是通过LC-MS/MS筛选出特异性靶向PD-L1启动子的候选蛋白因子，再采用亲和纯化/质谱(IP/MS)鉴定其与ZC3H18之间的关系并通过co-IP和western blotting来进行分析(如图10)。
结果：CSNK2A1也是PD-L1启动子的调控蛋白之一，并且通过IP/MS鉴定发现CSNK2A是与ZC3H18相互作用的蛋白之一。Co-IP和western blotting的结果表明CSNK2A1直接与ZC3H18相互作用。
结论：CSKN2A1与ZC3H18是相互作用的蛋白。
(2)分析其他因素对ZC3H18诱导的PD-L1的转录影响
方法：通过CHIP-PCR检测CSNK2A1的表达对ZC3H18过表达细胞的PD-L1的转录水平的影响。用western blotting检测CSNK2A1的小分子抑制剂CX-4945通过对CSNK2A1活性的影响来影响H2A上的酪氨酸磷酸化水平(如图11)。
结果：CHIP-PCR检测发现抑制CSNK2A1的表达显著降低了ZC3H18过表达细胞中PD-L1的转录水平。通过western blotting发现在过表达ZC3H18的胰腺癌细胞中，使用CX-4945抑制CSNK2A1的活性，显著降低了H2A上酪氨酸磷酸化水平。还通过western blotting发现如果Y57突变或使用CX-4945会导致H3K4me3蛋白的丢失，但是不会导致ZC3H18过表达的胰腺癌细胞中H3K4me2的丢失。
结论：CSNK2A1调控的H2A酪氨酸磷酸化增加了ZC3H18诱导的PD-L1的转录激活。
实施例5：抑制ZC3H18可增强体内抗肿瘤免疫能力
方法：肿瘤细胞皮下种植小鼠模型：C57BL/6小鼠随机分为两组(n＝5/组)。将细胞系(Panc02/Vector、Panc02/ZC3H18、Panc02/对照、Panc02/ZC3H18-shRNA)注射到左背侧C57BL/6小鼠皮下。每周检查两次肿瘤和小鼠的体重。用卡尺测量肿瘤的长度和宽度，肿瘤体积用公式(L*W2)/2计算肿瘤体积。第28天，动物被安乐死，肿瘤被切除，用于流式细胞术检测(如图12)。
结果：小鼠体内实验表明，注射Panc02/ZC3H18细胞的小鼠形成的肿瘤更大，而注射Panc02/ZC3H18-shRNA细胞形成的肿瘤大小和重量均比对照组小。通过流式细胞术分析肿瘤浸润淋巴细胞，发现ZC3H18基因沉默的肿瘤中CD3+CD8+T细胞数量显著增加，而ZC3H18过表达的肿瘤中显著减少。此外，CD8+T细胞产生的各种免疫相关细胞因子包括IFN-γ、TNF-a、GzmB的血清水平在ZC3H18沉默肿瘤中显著升高，而在ZC3H18过表达肿瘤中显著降低。
结论:抑制ZC3H18的表达使胰腺癌肿瘤生长也得到抑制，增强了抗肿瘤免疫能力。
实施例6：抑制ZC3H18可增强体外抗肿瘤免疫力
方法：从C57BL/6小鼠的脾获得CD8+T细胞，培养在含10％胎牛血清、1％谷氨酰胺、1％β巯基乙醇以及1％青霉素链霉素的RPMI-1640培养基中，放置在细胞培养箱中。24h后，将上述CD8+T细胞与Panc02细胞系(Panc02/Vector、Panc02/ZC3H18、Panc02/对照、Panc02/ZC3H18 shRNA)共培养48h，加入刺激剂(BioLegend)处理后，进行流式细胞技术，对细胞进行分析。同时将人jurkat T细胞与Miapaca-2细胞共培养，并进行流式分析。用FlowJo X对结果进行分析(如图13)。
结果：流式细胞术分析显示，过表达ZC3H18显著降低了CD3+CD8+T细胞数量的水平，而ZC3H18缺失显著增加了CD3+CD8+T细胞数量。人jurkat T细胞与Miapaca-2细胞共培养，发现过表达ZC3H18中D3+CD8+T细胞数量显著减少，而ZC3H18缺失明显增加了CD3+CD8+T细胞数量。重要的是，免疫相关细胞因子如IFN-γ、TNF-a、GzmB在ZC3H18沉默细胞中显著增加，而在ZC3H18过表达细胞中显著减少。
结论：抑制ZC3H18可增强胰腺癌的体外抗肿瘤免疫力。
实施例7：GSK3326595和CX-4945联合抗pd-l1治疗效果较好
方法：首先将Panc02/ZC3H18细胞系注射到左背侧C57BL/6小鼠皮下。对照组IgG和DMSO治疗；GSK3326595单药治疗组采用IgG和GSK3326595；CX-4945单药治疗组分别给予IgG和CX-4945。抗PD-L1单药治疗组每日给予DMSO和抗PD-L1；每4天进行一次。联合治疗组采用GSK3326595和CX-4945和抗PD-L1。肿瘤大小每周检查两次。用卡尺测量肿瘤的长度和宽度，肿瘤体积用公式(L*W2)/2计算肿瘤体积。在治疗结束时，处死小鼠，切除肿瘤，部分用于石蜡包埋，并进行IHC分析；部分用于流式细胞术检测(如图14)。
结果：小鼠体内实验结果显示GSK3326595、CX-4945和α-PD-L1联合治疗最显著地减少了肿瘤的大小和肿瘤重量。免疫组化实验显示，与单独治疗和对照组相比，GSK3326595、CX-4945和α-PD-L1联合治疗显著降低了PD-L1阳性率。
结论：这些数据表明PRMT5和CSNK2A1均参与了ZC3H18诱导PD-L1的表观遗传学上调的过程，而α-PD-L1与GSK3326595和CX-4945对ZC3H18过表达的胰腺癌有更好的治疗效果。
Claims (10)
1.ZC3H18作为肿瘤治疗免疫逃避中作为预警靶点分子和预测肿瘤免疫治疗中发生免疫逃避的检测靶点的应用。
2.一种定量检测ZC3H18的试剂在制备肿瘤治疗免疫逃避中作为预警靶点分子和预测肿瘤免疫治疗中发生免疫逃避的检测靶点的应用，其特征在于，所述定量检测ZC3H18的试剂选自检测ZC3H18基因是否过表达的试剂、定量检测ZC3H18的RNA转录水平的试剂、定量检测ZC3H18的蛋白表达水平的试剂中的至少一种。
3.一种抑制ZC3H18蛋白表达的物质在制备提高肿瘤PD-L1单抗疗效的药物中的应用。
4.一种抑制CSNK2A1、PRMT5表达的试剂在制备治疗ZC3H18过表达型肿瘤药物中的应用。
5.一种抑制CSNK2A1、PRMT5表达的试剂在制备提高ZC3H18过表达型肿瘤免疫治疗效果的药物中的应用，其特征在于，所述抑制CSNK2A1、PRMT5表达的试剂为化学药物GSK3326595和CX-4945。
6.一种检测CSNK2A1、PRMT5、PD-L1表达量的试剂在制备肿瘤免疫治疗中作为治疗靶点分子的检测试剂盒或在预测肿瘤免疫治疗效果的检测试剂盒中的应用。
7.一种肿瘤免疫治疗、肿瘤预后、或/和预测肿瘤免疫治疗效果的检测试剂盒，其特征在于，所述试剂盒中含有检测ZC3H18的试剂，或/和检测CSNK2A1、PRMT5、PD-L1表达量的试剂。
8.根据权利要求7所述的一种肿瘤免疫治疗、肿瘤预后、或/和预测肿瘤免疫治疗效果的检测试剂盒，其特征在于，所述试剂盒中的试剂选自检测ZC3H18基因是否过表达的试剂、定量检测ZC3H18的RNA转录水平的试剂、定量检测ZC3H18的蛋白表达水平的试剂中的至少一种。
9.一种提高肿瘤对免疫治疗疗效的药剂，其特征在于，所述药剂中含有抑制ZC3H18蛋白表达的物质、抑制CSNK2A1、PRMT5表达的试剂中的至少一种。
10.一种抑制ZC3H18过表达型肿瘤对免疫治疗疗效的药剂，其特征在于，所述药剂中含有抑制ZC3H18蛋白表达的物质、抑制CSNK2A1、PRMT5表达的试剂中的至少一种。
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