CN116855607A - circCHPT1在制备非小细胞肺癌早期诊断或预后检测试剂盒中的应用 - Google Patents
circCHPT1在制备非小细胞肺癌早期诊断或预后检测试剂盒中的应用 Download PDFInfo
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- CN116855607A CN116855607A CN202310772258.2A CN202310772258A CN116855607A CN 116855607 A CN116855607 A CN 116855607A CN 202310772258 A CN202310772258 A CN 202310772258A CN 116855607 A CN116855607 A CN 116855607A
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Abstract
本发明发现非小细胞肺癌I期患者血液中circCHPT1显著增高；在50例肺癌组织中同样检测到其高表达，并与较差预后相关；在肺癌细胞中过表达circCHPT1促进细胞增殖和周期加速；Pull down实验显示其与MARK3结合并导致RAF1磷酸化，从而激活下游MAPK信号通路。本发明将有助于阐明肺癌恶性进展的机制，并将为肺癌提供潜在的无创性早诊标志物、肺癌预后判断标志物和治疗靶点分子，为临床开发新的早期预测试剂和治疗药物提供理论和实验依据。
Description
技术领域
本发明涉及生物技术领域，具体涉及circCHPT1在制备非小细胞肺癌早期诊断或预后检测试剂盒中的应用。
背景技术
肺癌是全球常见恶性肿瘤，是世界范围中恶性肿瘤致死的主要原因之一。肺癌的早期诊断、预后判断和靶向治疗在其诊治中十分重要。
circRNA共价闭环结构缺乏自由5 '和3 '端，circRNA分子对外切酶Rnase R具有很强的抗性，比线性RNA更稳定。CircRNA在血浆中的平均半衰期超过48小时，而mRNA的半衰期平均为10小时，结构相对稳定。此外，环状RNA在癌组织和非癌组织中有差异表达,具有组织特异性和发育阶段特异性，其表达的差异模式可用于区分癌症类型或组织学亚型。环状RNA可在体液中检测到，如唾液或全血，外泌体中也有大量环状RNA环状RNA存在于体液的游离细胞中，如循环血细胞(血小板和红细胞)和CTC。有试验表明，来源于人类癌症的circRNA可以进入循环系统，利用血清中体外环状RNA能够区分健康对照组和结肠癌患者。Maass等人发现，在20个不同的组织中，血小板环状RNA的表达最高。某些环状RNA可以在癌患者液体活检中出现差异表达，如Li L. 等，在SCLC患者血清中纯化的外泌体中检测到了FECR1circRNA，与正常健康献血者相比，SCLC患者血清-exo- FECR1表达异常升高，且与化疗后病情进展相关。肺癌中源于EML4-ALK融合基因的内源性F-circEA存在于具有EML4-ALK变异体3b易位的H2228细胞，F-circEA不仅存在于非小细胞肺癌组织中，也存在于EML-ALK易位患者的血浆中。因此，基于circRNA的结构稳定及存在于体液中的特点，被认为是适合的无创诊断生物标志物。
除了作为诊断标志物之外，CircRNA以多种方式参与肿瘤的发生发展。作为miRNA海绵或竞争性内源性核糖核酸，与蛋白质相互作用，翻译成蛋白质/肽，调节可变剪接或转录。高表达的circRNA_02231与肺癌患者的晚期TNM分期、淋巴结转移和整体存活率差异显著相关，促进肺癌细胞增殖和侵袭能力。机制方面研究表明，CircRNA作为蛋白质诱饵，通过为特定蛋白质提供结合位点来吸附蛋白质，并通过作为竞争元件来调控蛋白质活性。Circ-Foxo3主要位于细胞质中，它可以结合p21和CDK2，并通过circ-Foxo3-p21-CDK2三元复合物的形成诱导细胞周期停滞；在另一研究中，乳腺癌中的circ-Ccnb1通过结合Ccnb1/Cdk1复合物，抑制肿瘤侵袭和发展。circPABPN1结合HuR，减少PABPN1的翻译。circANRIL与PES1结合，抑制PES1介导的rRNA成熟。
现有技术中并未报到circCHPT1与非小细胞肺癌早期诊断和预后判断以及非小细胞肺癌治疗之间的关系。
发明内容
针对现有技术的不足，本发明的目的是研究circCHPT1，明确circCHPT1作为非小细胞肺癌早期诊断和预后判断标志物，明确circCHPT1与非小细胞肺癌治疗药物的关系，并研究其机理。
本发明的技术方案是：
本发明提供了circCHPT1的检测试剂在制备非小细胞肺癌早期诊断或预后判断检测试剂盒中的应用。
本发明提供了circCHPT1的检测试剂作为非小细胞肺癌的靶向治疗药物预后判断检测试剂盒中的应用。
本发明还提供了circCHPT1的抑制剂在制备非小细胞肺癌治疗药物中的应用。
进一步的，所述circCHPT1的抑制剂通过对MARK3-RAF1-MAPK信号通路的调控从而参与非小细胞肺癌的治疗。
进一步的，circCHPT1的序列为：
GCACCATACTGGACATACCTTTTATGTGCACTGGGACTTTTTATTTACCAGTCACTGGATGCTATTGATGGGAAACAAGCCAGAAGAACAAACTCTTGTTCCCCTTTAGGGGAGCTCTTTGACCATGGCTGTGACTCTCTTTCCACAGTATTTATGGCAGTGGGAGCTTCAATTGCCGCTCGCTTAGGAACTTATCCTGACTGGTTTTTTTTCTGCTCTTTTATTGGGATGTTTGTGTTTTATTGCGCTCATTGGCAGACTTATGTTTCAGGCATGTTGAGATTTGGAAAAGTGGATGTAACTGAAATTCAGATAGCTTTAGTGATTGTCTTTGTGTTGTCTGCATTTGGAGGAGCAACAATGTGGGACTATACG（SEQ ID NO.1）。
进一步的，所述circCHPT1的抑制剂为si-circCHPT1，si-circCHPT1的具体序列为ATACGGCACCATACTGGACAT（SEQ ID NO.2）。
与现有技术相比，本发明的优势是：
（1）本发明鉴定出新的NSCLC早期诊断和预后判断标志物：①肺癌早期诊断标志物：在本课题中，我们将收集30例肺部良性病变的病人和40例早期肺癌及60例中晚期肺癌病人的血样，全血抽提RNA进行RT-PCR检测circCHPT1的表达水平，我们将首次鉴定出circCHPT1作为早期肺癌的无创性诊断标志物。②同样，我们将收集200例肺癌样本随机分为训练组和验证组，抽提细胞总RNA，以RT-PCR检测circCHPT1的表达水平，结合临床和随访资料分析，明确circCHPT1作为肺癌病人预后判断的标志物。也就是，本发明公开了：circCHPT1的检测试剂可以用于制备非小细胞肺癌早期诊断试剂或预后检测试剂，可以用于检测试剂盒。
（2） 本发明首次阐明 circCHPT1在肺癌发展中的作用机制：提出circCHPT1结合MARK3促进RAF1上调及MAPK信号通路的激活。也就是，本发明公开了：circCHPT1的抑制剂可以用于制备非小细胞肺癌治疗药物。
以下结合附图和具体实施方式对本发明的详细结构作进一步描述。
附图说明
图1为筛选全血中的CircRNA 并验证目标 circRNA的图，其中A.非小细胞肺癌I期患者和健康人群血液采集后RNA提取及测序示意图；B. 聚类热图显示在非小细胞肺癌患者(C)与正常对照人群(N)中出现的差异表达circRNAs；C. 对50个非小细胞肺癌患者的癌与癌旁组织进行验证，circCHPT1的表达存在明显差异。（**P<0.01）。
图2 为显示CircCHPT1的临床意义图，其中A.Kaplan-Meier 分析显示，A.CircCHPT1高表达与肺癌患者的总生存差明显相关（P<0.001); B. CircCHPT1高表达也与肺癌患者的无病生存差显著相关（P<0.01）；C. 在TCGA数据集中CircCHPT1的亲本编码基因CHPT1在肺腺癌和肺鳞癌中CHPT1在癌组织中低表达。D-E.K-M plotter和TCGA数据库多数据集提示CHPT1与肺癌的预后无关。因此，circCHPT1在临床样本上存在差异且与预后相关。
图3为CircCHPT1的结构及定位图，其中A.CircCHPT1在基因组上的位置及其成环位点；B.Northern blot 在不同肺癌细胞系中检测circCHPT1的表达。C.RNase R处理总RNA后进行RT-qPCR检测发现,CHPT1被消化而circCHPT1耐受消化；D.在H1299细胞中加入放线菌素D（ActD）处理,RT-qPCR检测circCHPT1和CHPT1转录本表达水平;E.质核分离试验表明circCHPT1和CHPT基本都位于胞浆，以U1作为核内参，GAPDH作为胞浆内参；F. RNA 荧光原位杂交（FISH）试验，circCHPT1以Cy3标记，细胞核以DAPI染色，结果显示circCHPT1在定位于胞浆。
图4为CircCHPT1在肺癌细胞中的功能图，其中A.不同肺癌细胞系中circCHPT1的表达丰度；B.PC9细胞系中转染过表达circCHPT1质粒后，检测过表达circCHPT1效果；C.A549细胞系中转染circCHPT1的siRNA序列后，检测敲降circCHPT1效果；D.CCK8检测circCHPT1高表达条件下可促进肿瘤增殖；E-F.克隆形成试验检测circCHPT1高表达条件下可促进肿瘤增殖,敲降时肺癌细胞增殖减少；G.流式细胞术检测circCHPT1高表达条件下可促进肿瘤细胞周期进程。
图5为CircCHPT1结合MARK3蛋白的结果图，其中A.circCHPT1过表达且质谱标签载体示意图；B. CircCHPT1标签载体过表达效果检测；（circCHPT1为过表达载体，circCHPT1-MS为带标签过表达）C. CircCHPT1标签载体拉下蛋白进行WB检测；D. 质谱检测拉下蛋白提示MARK3与circCHPT1紧密结合；E. qPCR检测过表达circCHPT1条件下，MARK3的转录本表达水平；F. WB检测过表达circCHPT1条件下，MARK3的转录本及蛋白表达水平;F. RIP实验证明,以MARK3抗体拉circCHPT1，过表达circCHPT1组表达量高于对照组。
图6为CircCHPT1结合MARK3并调控MAPK信号通路，促进RAF1激活的结果图，其中A.一组非小细胞肺癌样本中MARK3在癌组织中高表达（N=116），且该组KARS野生型样本中的MARK3表达更高。C. 过表达circCHPT1的条件下对样本的RNA-seq发现，MAPK经典信号通路被激活；D.WB检测MAPK信号通路相关蛋白变化；E.TCGA数据库中肺癌及癌旁样本中MARK3和RAF1的相关性；F. PPI网络分析提示MARK3和RAF1存在紧密相关性；G. 过表达circCHPT1，同时敲降MARK3, p-RAF1(S259)的蛋白水平基本不变(1——NC，2:——过表达circCHPT1，3——过表达circCHPT1同时si-MARK3）。
实施方式
下面结合说明书和附图对本发明做进一步解释和说明
实施例1
实验方法
（1）明确 circCHPT1 作为无创诊断标志物的发现及验证；
A.全血RNA的提取
采用酚氯仿抽提方法全血样本中总RNA。样本经过预处理后利用水饱和酚和氯仿异戊醇混合物进行提取，并用75%乙醇纯化后，得到总RNA。
B. circRNA测序
提取的总RNA经过安捷伦质检总量大于2ug,OD260/230大于2,且RIN(RNAIntegrity Number)值≥7，复合建库要求。采用illumina 转录组测序（GRCh38.92/hg38作为组装比对的参考）然后样本采用 Ribo-Zero™ Magnetic Kit(Epicentre) 去除rRNA和片段，通过依次合成第一cDNA链及其互补链，在通过尾端修复和加A尾与接头，PCR进行富集。测序所得的原始数据(Raw data)通过bcl2fastq软件进行分析转化为原始测序序列。随后采用FastQC软件对碱基质量进行分析，fastp将原始结果进行过滤。再用Tophats软件对序列进行比对，随后利用CIRCexplorer2软件进行circRNA的鉴定。设定差异筛选标准为|log2FC|≥1 且 P-value＜0.05用以寻找NSCLC I期患者和健康体检人群两组中差异显著的circRNAs。
C.circCHPT1 qPCR引物设计
前期的测序结果提示cCHPT1的差异表达显著，通过编号在circBase查询到它的全序列与测序结果比对相符，并确定其环化位点。引物设计区别于常规基因组外显子方式，3’端包含cCHPT1环化位点（ Splice Junction Overlapping Divergent Primers)，两条5’端相对。
D.circCHPT1在组织样本中的表达分析
提取三十对带随访资料的癌和癌旁样本RNA，利用Random Primer进行逆转后荧光定量PCR检测circCHPT1在组织中的表达，并结合临床相关指标进行相关性分析。
（2）明确 circCHPT1 促进肺癌细胞恶性增殖；
A.circCHPT1过表达载体构建pLC5-ciR
为研究circCHPT1的功能需要对其进行过表达，以商业化载体pLC5-ciR作为骨架，载体携带了侧翼成环框架，酶切后将circCHPT1成熟序列插入载体中，可用于真核生物表达和慢病毒包装。经转染后在293T和肺癌2株细胞中比对照组高几十甚至上百倍。
B.circCHPT1在肺癌细胞中的功能鉴定
细胞增值能力检测——CCK8：制备细胞悬液后，每孔1000个细胞，培养8小时候，用CCK8试剂染色，OD450nm读值，比较对照组和过表达组之间4天的增殖速度。
细胞迁移能力检测——Transwell 肿瘤细胞的迁移可以因为培养基的营养成分不同对细胞的生长运动产生影响。在24孔板的一个孔内，下层加入700ul的完全培养基，上层小室加入300ul无血清培养基。孔内铺设5*104个细胞约12-15小时，取出一个对照组和实验组的小室，经过生理盐水洗涤，甲醇固定10min，结晶紫染色15min，PBS洗涤晾干后镜检，用IMAGE J统计穿过小室的细胞数量。
C.circCHPT1荧光原位杂交亚细胞定位
对cCHPT1的RNA荧光原位杂交(FISH)的亚细胞定位我们采用寡核苷酸修饰的circCHPT1探针序列是由RiboBio公司(中国广州)合成的。将细胞在PBS中洗涤5分钟后，4%多聚甲醛固定 10分钟，再用PBS洗涤。加入预冷通透液4 °C 静置。加入与杂交液处理30分钟后，将20uM的cCHPT1或18S探针避光在37℃孵育箱进行杂交过夜。第二天，通过杂交洗液和PBS处理后进行DAPI染色，并用PBS 清洗细胞，封片剂封片后上镜检测。
（3）探究circRNA的调控机制，验证 circCHPT1结合MARK3蛋白：
为了检测circCHPT1在胞浆与哪些蛋白互作，我们采用circRNA的 pull-down试验来检测。
circCHPT1标签载体的构建
首先构建特异环状RNA，分别在对照和过表达circCHPT1载体的基础上加入RNA标签系统（一段短的核苷酸序列），通过标签与捕获的蛋白结构上的稳定性提高circRNA原来基于结构序列结合蛋白的不稳定或假阳性，可以提高捕获蛋白的效率。
B.circCHPT1的RNA pull down试验
1）基于上一步构建好的质粒，对他们进行T7体外转录( 采用T7 High EfficiencyTranscription Kit )，并利用磁珠法对RNA进行纯化（EasyPure RNA Purification Kit）。
2）将纯化后的RNA（50pM,根据长度进行换算成体积），加入10*RNA链接反应缓冲液3ul，RNAse抑制剂1ul（40U），Biotinylated Cytidine Bisphosphate 1ul(1nM)以及T4RNA连接酶2ul,30%PEG 15ul，最后用无酶水补齐至30ul总体积，在16℃的恒温条件下，孵育过夜。首先将第一步进行全序列体外转录之后的RNA，用生物素biotin进行标记（Pierce RNA3´End Desthiobiotinylation Kit）；
3）提取细胞总蛋白，建立circCHPT1过表达，空白对照和总蛋白3组。然后将生物素标记的RNA胞浆蛋白进行功孵育，形成RNA-蛋白质复合物（Pierce™ Magnetic RNA-Protein Pull-Down Kit）。磁珠吸附RNA-蛋白质复合物，缓冲液清洗3次，收集最后蛋白浆。
4）复合物洗脱后，收集到的RNA结合蛋白，可以通过质谱检测再用Western Blot实验验证。
C.circCHPT1结合蛋白质谱检测
1）蛋白酶切：将4 µL 0.05 M TCEP 溶液加入蛋白浆，60℃反应1 h之后，加入2 µL55 mM MMTS溶液，室温避光45 min转移至超滤管，12000g离心20 min；
再加入100 µL UA（8 M尿素pH 8.5）溶液离心后，加入100 µL 0.25 M TEAB，12000g离心20 min，随后加入50 µL 0.5 M TEAB，最后加入2%胰酶（胰酶与蛋白质量比为1:50），37度孵育过夜（12 h）；次日补加1%胰酶（胰酶与蛋白质量比为1: 100），37度孵育4 h；更换新的收集管，离心收集滤液，低温真空抽干。
1）质谱检测：肽段用样品0.1%甲酸、2%乙腈的溶解液溶解，低位高速离心20 min，取上清，上机检测45min,分离后的肽段直接进入Thermo Scientific Q Exactive质谱仪进行在线检测，然后用MASCOT（http://www.matrixscience.com/）检索uniprot数据库，比对检测到的肽段归属的蛋白质。
D.RIP检测MARK3为抗体条件下，拉下circCHPT1的定量检测
RIP（RNA Immunoprecipitation）是研究细胞内RNA和蛋白结合的关键技术，通过本实验用于研究circCHPT1和MARK3的互作结合。
1）裂解细胞去除DNA：裂解10cm大皿107个肺癌细胞后，加入蛋白酶和RNA酶抑制剂。加入 4.5 μL的DNA盐驻物（DNase salt stock）、10 μL的DNase (20 U)去除体系内DNA，37℃ 条件下孵育 10分钟之后，将样品转入冰浴环境，加入4.5 μL 的0.5M EDTA，1.8 μL的0.5M EGTA和9 μL的DTT 并充分混匀；在4℃环境下，离心10分钟速度 16,000 g并转移上清到一个新的无 RNA酶污染的离心管。
2）抗体孵育：1mL的样品可以按照 0.8 mL（IP）、0.1 mL（ Input）分成两份，加入MARK3抗体，对照组加入IgG垂直旋转的立体混匀器 4℃旋转16 小时。
3）磁珠平衡：首先在珠子中加入 0.5 mL的聚合物裂解缓冲液（polysome lysisbuffer）上下颠倒混匀洗涤每组样品中预备使用的20 µL的protein A/G beads磁珠，利用磁力架吸附磁珠去上清；重复一次之后，再加入20 µL 的polysome lysis buffer 恢复体系。
4）免疫沉淀：将前一步处理好的磁珠加入抗体和蛋白混合物，低温环境下0.5m L的polysome washing buffer 和 5 µL的DTT 洗涤三次；在IP 样品中加入 94.5 μL的polysome washing buffer 和0.5 μL的DNase salt stock以及5 μL的DNase (5 U)，在37℃ 温育 10 分钟之后，将EP管放于磁力架收集磁珠并去上清；然后IP 样品加入 0.5 mL的polysome washing buffer 和 5 µL的DTT 洗涤两次，每次在低温4℃条件下，摇动孵育 5分钟再放磁力架收集磁珠并去上清；IP 样品加入 100 µL的polysome elution buffer和1µL的DTT以及2 µL的proteinase K（去除蛋白）重悬珠子， Input 样品从-80℃冰箱取出之后加入 1 µL的DTT 及 2 µL的proteinase K；IP和Input样品同时在55℃ 孵育1小时（注意封口避免液体蒸发）之后洗脱 RNA，再次使用磁力架收集磁珠，保留上清并将其转移至新的无 RNA 酶离心管中。
5）提取RNA：在收集的IP上清和之前备用同一批-80℃保存的100uL的Input中加入等体积100uL的混合液体（苯酚-氯仿-异戊醇）颠倒混匀15秒后高速离心，加入 1 µL的Glycogen（帮助驻留RNA）, 10 µL的sodium acetate 以及 250 µL 100% ethanol（沉淀RNA） 充分颠倒混匀； 在–20℃ 静置过夜沉淀 RNA 之后再次在 4℃ 预冷条件下，16000 g离心 30 分钟去上清（此时应该在管底看到白色小点状的RNA）；加入预冷的 1 mL 的80%乙醇，在 4℃条件下16100 g 离心 10 分钟后弃上清，室温静置风干 10分钟； 加入 20 µL的RNase-free water 溶解 RNA备用，随即检测浓度和质量及逆转。进行RT-qPCR检测。
(4)证实 circCHPT1-MAPK3—RAF1轴促进肺癌细胞增殖和转移及肺癌的恶性进展；
A.蛋白免疫印迹检测过表达circCHPT1条件下，MARK3蛋白表达水平检测
利用上一步构建好的circCHPT1过表达载体，转染肺癌细胞H1299，并以空载作为对照。24小时候收取六孔板中蛋白，进行定量变性。10%SDS-PAGE胶进行蛋白电泳，转膜孵育MARK3一抗，次日孵育二抗后进行曝光检测。判断circCHPT1过表达条件下对MARK3蛋白表达量的影响。
B.IF检测circCHPT1和MAPK3共定位
RNA荧光原位杂交(FISH)。寡核苷酸修饰的circCHPT1探针序列是由公司合成合成以CY3标记。将细胞固定后在PBS中洗涤，用RNase R在37℃处理15分钟，然后再次固定。利用梯度乙醇70、80和100%乙醇脱水，在37℃的黑暗潮湿的室内进行杂交过夜。在SSC液体中洗涤三次后，377℃中避光孵育30分钟，并用含有DAPI的封片剂封片。
C.组织样本检测circCHPT1和MARK3以及RAF1表达相关性
1）在前期研究中，RT-qPCR检测提示circCHPT1高表达患者预后差，后期拟扩大样本量，在 90例（已有大部分）不同临床分期NSCLC手术后的新鲜组织样本和血液样本，进行RT-qPCR检测 circCHPT1、MARK3和RAF1 的表达情况，同时检测组织中MARK3以及RAF1之间的表达情况。
2）免疫组织化学技术检测200 例NSCLC石蜡样本中 MARK3和RAF1的表达。
在上一步中，明确了NSCLC组织中MARK3和RAF1在小样本中RNA与蛋白相关性， 所以拟进一步扩大样本量，在200例（大部分已有的）手术后病理检测在不同临床分期的NSCLC石蜡标本中，进行 IHC检测 MARK3和RAF1的表达情况。
实验结论
（1）circCHPT1在外周血和肺癌组织中的表达和临床意义
为了筛选出潜在的肺癌早期诊断标志物，我们首先收集了手术前IA期7例非小细胞肺癌患者和3例正常人外周血样本。这7例肺癌病人术前未接受过任何抗癌治疗，术后病理诊断均为NSCLC的I期，同时这些病人排除其他慢性呼吸系统疾病如COPD等，以及其他肿瘤转移到肺部；正常对照血液样本来自本院体检人群，对应肺癌患者年龄和性别比例，确定无其他肿瘤、慢性疾病及呼吸道疾病等的健康人。全血样本提取RNA后送公司进行高通量RNA测序（图1A）。根据P value<0.05和差异倍数|log2FC|≥2的筛选原则，我们在测序结果中发现78个circRNA在肺癌病人外周血中上调，91个下调（图1B）。为了找出来源于肺癌组织的circRNA，我们选取了在全血中上调前十位的circRNA设计PCR引物，在50例肺癌及配对癌旁肺组织样本中用RT-qPCR的方法检测这十个circRNA表达。结果发现，与癌旁肺组织相比，circCHPT1在肺癌中上调最显著，癌组织样本中位值高于癌旁肺组织2.5倍（图1C）。
进一步，结合NSCLC患者临床资料进行Kaplan-Meier分析发现高表达circCHPT1的患者预后更差，总生存率和无病生存率相较于低表达circCHPT1患者减少（图2A-B）。此外，我们排除了circCHPT1所在基因组的编码基因CHPT1,它在TCGA大数据样本库中，不同数据集的总生存情况，可以看到CHPT1表达差异前提下，总生存率并无差异（图2C-E）。结果提示，当circCHPT1高表达情况下，它是一个危险因子，暗示其具有潜能参与非小细胞肺癌的发生发展过程。因此我们选择circCHPT1作为我们后续研究的目标circRNA。
（2）circCHPT1的起源和细胞内定位
circCHPT1的亲本基因CHPT1编码胆碱磷酸转移酶1，其DAN序列位于12号染色体长臂的2区3带（12q23.2)，而circCHPT1的成环位点见（图3A）。我们通过Northen blot试验检测不同肺癌细胞系中的circCHPT1（图3B）。当用RNase R处理肺癌细胞总RNA后，再进行RT-qPCR检测，结果发现CHPT1被消化而circCHPT1耐受[28]（图3C）。表明扩增产物是cirRNA具有环状RNA的稳定性。接着，我们通过RNA合成抑制剂放线菌素D处理肺癌细胞，qRT-PCR检测RNA，结果发现，相比CHPT1，circCHPT1在细胞内具有非常好的稳定性（图3D）。
生物大分子在细胞内的定位与其生物学功能密切相关。为了要研究circCHPT1的功能，我们首先需要明确其在细胞内的位置。因此，我们采用质核分离实验分别提取了H1650细胞系的细胞质与细胞核，以U1作为核内阳性参照，GAPDH作为胞浆阳性参照，结果发现，circCHPT1及其所在的基因约90%都位于细胞浆（图E）。为进一步确定circCHPT1在细胞内的定位，我们又进行了RNA 荧光原位杂交（FISH）实验，结果同样证明circCHPT1在细胞中的定位于胞浆（图3F），提示其在胞浆中可能通过结合某些RNA或者蛋白来发挥生物学作用。
（3） circCHPT1细胞生物学功能
随后我们在现有肺癌细胞系中检测了circCHPT1的表达丰度（图4A），选取低表达的细胞系PC9，对circCHPT1进行过表达（图4B），以及高表达A549细胞中敲降circCHPT1（siRNA序列特异针对其环化位点区段），可以看到si-1效果较好既能敲降circCHPT1又不影响CHPT1，si-2对circCHPT1无效并且会影响CHPT1基因（图4C）。由于circRNA在体内的本底表达水平并不高,我们先观察它在体外过表达条件下对细胞功能的影响。结果提示circCHPT1高表达条件下可促进肿瘤增殖（图4D），克隆形成增多（图4E-F）以及加速周期进程（图4G）。这些结果表明了circCHPT1促进肿瘤增殖这一表型的发生，提示circCHPT1可能在肿瘤进展过程中发挥重要作用。
（4）明确circCHPT1结合并调控MARK3
前面证实circCHPT1主要定位于胞浆，根据前人的报道提示circRNA可作为蛋白支架或诱饵结合某些蛋白而行使生物学功能。例如，前述circ-Foxo3可以与细胞周期相关蛋白包括p21和p27相互作用，从而阻断这些蛋白在癌细胞周期进程中的作用。circPABPN1，已被证明结合到一个著名的RBP——HuR，减少PABPN1的翻译。circANRIL被证明能与PES1结合，抑制PES1介导的rRNA成熟。为了探索circCHPT1的可能作用机制，我们构建了带有MS标签的circCHPT1过表达载体并检测其过表达效率（图5A-B），该标签有助于将circCHPT1结合的蛋白进行富集并通过Flag抗体进行RNA-pull down试验（图5C）。随后质谱分析对照组和过表达circCHPT1拉下的蛋白，排除内参等干扰，选取了差异显著的肽段证实为一种蛋白激酶MARK3 (Microtubule Affinity Regulating Kinase 3) （图5D），这提示circCHPT1能与MARK3结合。
MARK3也称为PAR-1a,C-TAK1蛋白分子量约78KD，具有丝氨酸/苏氨酸蛋白激酶活性；它与KSR1形成复合物于胞浆并且磷酸化KSR1的S392位点形成14-3-3蛋白锚定位点。14-3-蛋白通常结合在RAF1上，遮蔽其磷酸化位点。当MARK3减少，KSR1磷酸化S392程度降低，结合的14-3-3蛋白变少，下游的RAF1蛋白遮蔽并影响磷酸化活性及下游信号传递[30]。下游MEK至ERK信号入核，促进Cyclin D1结合并磷酸化CDK4/6加速细胞G1/S期的转化而促进细胞增殖。 当我们过表达circCHPT1时MARK3的转录本变化不大而蛋白水平升高（图5E-F），而反向用MARK3抗体拉circCHPT1可以发现，相较于对照组，过表达组有一个高的富集（图5G）。前人研究已知MARK3激活KSR-1丝氨酸392位点，形成一个磷酸化，帮助14-3-3蛋白锚定，阻滞下游RAF1的激活，抑制MAPK信号通路。
（5） 探索 circCHPT1通过结合MARK3调控RAF1的机制
在肿瘤细胞的增殖和侵袭转移过程MAPK信号通路的参与调控，当受到胞外信号刺激时激活并将信号从核外向核内传导。肿瘤细胞中，MAPK 通路异常与其上游的K-ras 或B-raf 突变有关，两种突变不并存且单一针对RAS或RAF抑制临床疗效有限[34]。近年来报道了曲美替尼（trametinib）等多个MEK 抑制剂存在耐药问题。同样在胞浆中，RAF1是MAPK/MEK/ERK信号放大的关键部分。circCHPT1可促进MARK3的蛋白表达。根据Oncomine中NSCLC一组116个样本的数据分析MARK3在肺癌组织中高表达，且该样本集中KRAS野生型的MARK3高表达（图6A-B），通常KARS突变型可以用MEK抑制剂，而野生型无合适药物,本研究可能成为潜在的KRAS-wt预测靶点。根据过表达circCHPT1的条件下对样本的RNA-seq发现，MAPK经典信号通路被激活（图6C）。对通路中的相关经典蛋白进行WB验证发现RAF1有升高，下游MEK1和p-MEK1被激活（图6D）。PPI蛋白互作系统提示MARK3与RAF1存在紧密相关性（图6E），TCGA数据库中肺癌及癌旁样本中MARK3与RAF1存在密切相关性R 2=0.74（图6F）。基于此，申请人假设，circCHPT1在帮助MARK3蛋白水平提高前提下，激活KSR1磷酸化，形成14-3-3蛋白铆定，释放其与RAF1结合的基础上，帮助RAF1暴露胞外信号磷酸化位点，从而触发MAPK经典级联信号放大通路。过表达circCHPT1时，可提高p-RAF1，同时敲降MARK3的可阻断这一信号的传导（图6G）。
综上所述，本项目前期的研究结果：（1）肺癌增殖相关circCHPT1在非小细胞肺癌I期患者血液中上调，回顾性研究验证其在NSCLC组织样本中也是高表达并与预后差相关；（2）CircCHPT1定位于细胞浆，过表达circCHPT1能促进肺癌细胞增殖；（3） RNA pull down及蛋白质谱分析证明circCHPT1与MARK3结合并抑制后者功能，导致RAF1被磷酸化，从而激活下游MAPK信号通路。基于上述前期结果，我们得出结论：circCHPT1通过与MARK3结合促进RAF1磷酸化从而激活MAPK经典信号通路导致肺癌细胞增殖。
以上所述为本发明的具体实施方式，但本发明的保护范围不局限于此，任何熟悉技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其构思加以等同替换或改变，都应涵盖在本发明权利要求的保护范围之内。
Claims (6)
1.circCHPT1的检测试剂在制备非小细胞肺癌早期诊断或预后检测试剂盒中的应用。
2.circCHPT1的检测试剂作为非小细胞肺癌的靶向治疗药物预后检测试剂盒中的应用。
3.circCHPT1的抑制剂在制备非小细胞肺癌治疗药物中的应用。
4.根据权利要求3所述应用，其特征是，所述circCHPT1的抑制剂通过对MARK3-RAF1-MAPK信号通路的调控从而参与非小细胞肺癌的治疗。
5.根据权利要求3或4所述应用，其特征是，所述circCHPT1的抑制剂为si-circCHPT1。
6.根据权利要求5所述应用，其特征是，所述si-circCHPT1的具体序列如SEQ ID NO.2所示。
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