CN111979328A - 一种预测肝癌肺转移的生物标志物及其应用 - Google Patents
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Abstract
本发明属于基因工程领域,涉及基因Sgk1在诊断肝癌肺转移及开发靶向药物中的应用。基因Sgk1的缺失可以使肝癌细胞获得抵抗T细胞免疫进而实施肺转移的能力,Sgk1缺失造成的肺转移瘤可以被免疫检查点(PD‑1,LAG‑3)阻断剂所抑制,Sgk1在原发瘤中低表达的病人更容易发生手术后肝外转移,因此,本发明提供了一种预测肝癌转移和预后的生物标志物Sgk1,并揭示了提高癌症细胞Sgk1的表达水平会抑制肝癌的肝外转移。
Description
技术领域
本发明属于基因工程领域,特别涉及一种预测肝癌肺转移的生物标志物及其应用。
背景技术
在中国,原发性肝癌的死亡率位居所有癌种的第4位。随着早诊早治理念的普及和手术技术的提高,越来越多的病人能够接受根治性肝癌治疗。即便如此,肝癌易于发生肝内复发和肝外转移的特性,仍然是其死亡率居高不下的主要原因。对于肝内复发,病人还有机会通过再次手术切除、肝移植、射频消融、微波消融和放射治疗等方式控制病情进展。然而一旦发生肝外转移,绝大多数病人已丧失手术机会,只能接受姑息治疗和对症支持治疗,中位生存时间仅剩4.9-7.0个月左右。晚期肝癌推荐的靶向药物,给肝外转移病人带来的生存获益也非常有限。
根治性肝癌切除术后,肝外转移的发生率为13.5-36.7%。在所有肝外转移中,肺最常见的转移靶器官器官(38.4%-60%)。有零星研究发现,少数特定的肝癌肺转移病人(无肝内复发或者肝内复发控制良好,能够完整切除转移灶,无瘤间隔时间大于12月,肺转移瘤局限于单个肺段),仍可以通过及时的干预性治疗获益。这些研究提示,对于根治性肝癌切除后肺转移风险较高的病人,实施积极的辅助性治疗控制肿瘤进展以及密切随访早期发现,有望为延长生存创造机会。因此,研发预测肝癌术后肺转移和预后的标志物有着十分重要的临床意义。
目前,在临床实践中,可以通过影像学检查(X线,CT,PET/CT)以及肿瘤血清学指标(AFP,PIVKA)早期诊断肺转移。有报道称可以通过胰岛素样生长因子(insulin-likegrowth factor,IGF),促分裂原激活蛋白激酶(mitogen-activated protein kinase,MAPK),磷脂酰肌醇-3激酶(phosphatidylinositol-3 kinase,PI3K)/蛋白激酶B(ProteinKinase B,AKT)/雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR),和无翅(Wingless/Integrated,WNT)/β-连环蛋白(β-catenin)等信号通路的相关生物标记物,识别处于转移状态的肝癌患者。但是,上述方法只适用于发现已经发生的肺转移,很难在转移开始之前对肺转移进行预警。
肝癌肺转移是一个动态过程,涉及肝癌细胞的迁移,侵入血管,在循环血液中生存,从血管外渗如肺组织,以及在肺组织中定植生长。越来越多的证据表明,在肿瘤转移过程中,离不开与免疫微环境的相互作用。目前文献所报道预测肝癌肺转移风险的标志物,往往只是肿瘤细胞自身特征的体现,而忽视了与免疫系统的相作关系。能同时兼顾肿瘤特性和免疫交互作用的新型预测指标,是开发预测肝癌肺转移生物标志物的方向。
发明内容
本发明为了克服上述技术问题,本发明提供了一种预测肝癌肺转移的生物标志物以及检测方法,通过检测肝癌患者肿瘤细胞中生物标志物Sgk1的基因表达水平,可以有效预测所述肝癌患者肝癌肺转移发生的风险。
第一方面,本发明提供了特异性检测生物标志物Sgk1基因表达水平的试剂在制备预测肝癌肺转移的试剂盒或装置中的应用。其中SGK1在NCBI中的信息为Gene ID:6446。
其中“表达水平”是指,来自受试者的样品中由目的基因产生的基因产物的可测量的量,其中基因产物可以是转录产物或翻译产物。因此,表述“测定目的基因的表达水平”可以指,测定所述基因的mRNA或所述mRNA的片段的水平、或所述基因的cDNA或所述cDNA的片段的水平、或由所述基因编码的蛋白或其多肽片段的水平。
在某些优选的实施方案中,所述试剂为能够检测所述标志物的mRNA水平的试剂。这类试剂是本领域公知的,包括但不限于特异性结合目标序列的核酸探针、扩增目标序列的引物、非特异性荧光染料(例如,SYBR Green I)或其组合。在某些实施方式中,所述核酸探针可以为单标记的核酸探针,例如放射性核素(如32P、3H、35S等)标记探针、生物素标记探针、辣根过氧化物酶标记探针、地高辛标记探针或荧光基团(如FITC、FAM、TET、HEX、TAMRA、Cy3、Cy5等)标记探针;所述核酸探针也可以为双标记的核酸探针,例如Taqman探针、分子信标、置换探针、蝎子引物探针、QUAL探针、FRET探针等。在某些实施方式中,所述试剂包括Taqman探针。
采用上述技术方案,本发明的有益效果是检测肝癌患者肿瘤中生物标志物的表达量,可以准确预测其肝癌发生肺转移的风险。
在上述技术方案的基础上,本发明还可以做如下改进。
进一步地,所述的预测包括以下步骤,
步骤a)评估肝癌患者的肿瘤组织中的Sgk1基因的表达;和
步骤b)基于a)的评估结果预测所述肝癌患者是否发生肝癌肺转移的风险。
进一步地,所述评估是指比较肿瘤组织与对照组织中所述Sgk1基因的检测数据,评估所述Sgk1基因的表达水平。
进一步地,所述Sgk1基因的表达水平下调,则表示所述肝癌患者存在肝癌肺转移的风险。
采用上述技术方案,本发明的有益效果是,本发明发现Sgk1基因在人肝癌肺转移瘤中的表达较原发瘤显著下调,同时,Sgk1原发瘤中低表达的病人更容易发生手术后肝外转移,生存时间也更短,并且PD-1治疗有效。
第二方面,本发明还提供了一种包含所述的特异性检测生物标志物Sgk1基因表达水平的试剂的试剂盒。
第三方面,本发明还提供了一种特异性检测生物标志物Sgk1基因表达水平的试剂在制备预测肿瘤细胞抵抗T细胞杀伤力的试剂盒或装置中的应用。
进一步地,所述Sgk1基因的表达水平下调,则表示所述肝癌细胞存在抵抗T细胞攻击的能力,并且这种抵抗可被PD-1单抗中和。
第四方面,本发明还提供了一种特异性检测生物标志物Sgk1基因表达水平的试剂在制备预测肝癌患者对免疫疗法的敏感性的试剂盒或装置中的用途。
进一步地,所述的免疫疗法为使用免疫抑制剂。
进一步地,所述的免疫抑制剂为抗PD-1/PD-L1或LAG-3抑制剂。
第五方面,本发明还提供了生物标志物Sgk1基因的激活剂在制备抑制肝癌肺转移的药物中的应用。所述激活剂通过激活生物标志物Sgk1基因的表达,从而实现靶向抑制肝癌肺转移。
除了针对肝癌肺转移风险的预测,本发明的试剂/试剂盒还有希望用于肝癌肿瘤筛选、风险评估、预后诊断、疾病识别、病症阶段的诊断和治疗性靶标的选择。
相对于现有技术,本发明具有的技术效果:
1)本发明发现Sgk1基因在人肝癌肺转移瘤中的表达较原发瘤显著下调,Sgk1原发瘤中低表达的病人更容易发生手术后肝外转移,生存时间也更短。因此,提供了一个新的能够预测肝癌肺转移的生物标志物Sgk1,丰富了针对肝癌肺转移风险预测的检测靶点。
2)本发明通过敲除Sgk1基因的表达,可使小鼠Hepa1-6肝癌细胞获得抵抗T细胞杀伤的能力,恢复基因Sgk1的表达,可以恢复T细胞对Hepa1-6细胞的杀伤,从而表明生物标志物Sgk1可以成为治疗肝癌肺转移的新靶标。
3)本发明通过将非特异性基因敲除的Hepa1-6细胞分别通过尾静脉注射入免疫健全C57BL/6小鼠和T细胞去除的C57BL/6小鼠循环系统,发现非特异性基因敲除的Hepa1-6细胞无法在免疫健全C57BL/6小鼠形成肺转移瘤,但是去除小鼠T细胞以后可以形成大量肺转移瘤,因此证明T细胞免疫在抑制肝癌细胞肺转移中的重要作用。将Sgk1基因敲除的Hepa1-6细胞和非特异性基因敲除的Hepa1-6细胞分别通过尾静脉注射到免疫健全的C57/BL6J小鼠循环系统后,发现非特异性基因敲除的Hepa1-6细胞无法在小鼠肺部形成转移瘤,而Sgk1基因敲除的Hepa1-6细胞可以在小鼠肺部形成大量肺转移瘤。因此,证明Sgk1基因敲除可以使Hepa1-6细胞获得抵抗体内T细胞免疫进而形成肺转移的能力。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式描述中所需要使用的附图作简单地介绍。
图1实施例1中Hepa1-6尾静脉注射肺转移实验结果;
图3实施例2中体外细胞增殖实验;
图4实施例2中体外肺转移实验,其中,图A是C57BL/6对照小鼠,图B是T细胞去除的C57BL/6小鼠;
图5实施例3中Sgk1基因在原发肿瘤及肺癌转移肿瘤的表达水平;
图6实施例3中预测肝癌肺转移的比例;
图7实施例3中预测肝癌肺转移的总体生存率;
图8实施例3中预测手术后肝内复发的肝外转移比例;
图9实施例4中免疫检查点抑制剂抑制Sgk1敲除Hepa1-6细胞形成肺转移,其中图9A为Hepa1-6细胞形成肺转移结节数量,图9B为小鼠死亡率;
图10实施例5中Sgk1敲除后恢复和激酶沉默实验。
具体实施方式
以下通过实施例对本发明作进一步的阐述,但不限制本发明。
本发明的材料:本申请中提及的细胞株以及培养基均有商品供应或以别的途径能为公众所得,它们仅作举例,对本发明不是唯一的,可分别用其它适合的工具和生物材料来代替。
实施例1
体外培养小鼠肝癌细胞Hepa1-6,收获细胞后计数,分别制备2百万个,4百万个和6百万个细胞的Hepa1-6细胞悬液若干份,以备后续小鼠实验使用。实验小鼠分为五组。第1到第6组使用免疫健全C57BL/6小鼠,每组6只小鼠,第7和8组使用免疫缺陷的裸鼠,每组5只小鼠。在第1到第4组中每只小鼠尾静脉注射2百万个Hepa1-6细胞;第5组每只小鼠尾静脉连续两次分别注射2百万个Hepa1-6细胞,每次注射间隔时间为1天。第6组每只小鼠尾静脉连续三次分别注射2百万个Hepa1-6细胞,每次注射间隔时间为1天。第7和第8组分别尾静脉注射2百万和6百万个Hepa1-6细胞。注射后第五周对小鼠肺部进行CT扫描,结果显示1-4百万Hepa1-6细胞单次注射无法在免疫健全的小鼠体内形成肺转移,2百万Hepa1-6连续注射2次和3次仍然无法在C57BL/6小鼠体内形成肺转移。而将2百万个Hepa1-6尾静脉注射到免疫缺陷的裸鼠体内后,100%形成肺转移,结果如图1所示。裸鼠T细胞缺乏,因此,该实施例提示T细胞是抑制肝癌肺转移的关键因素之一。
实施例2
用CRIPSR技术来构建Sgk1敲除的细胞株,我们针对靶向Sgk1的kinase domain和AGC-kinase C-terminal设计了3条sgRNA(CGTGTTCCGGCTATAAAACG(SEQ ID NO:1)、,CCCGTTTTATAGCCGGAACA(SEQ ID NO:2)、,GACCGGCTCCTCGGTAAACT(SEQ ID NO:3),最终我们挑选了4个Sgk1-KO的克隆(Sgk1-KO-2和3来源于同一条sgRNA),同时也构建了两个非特异性基因敲除的Hepa1-6细胞系(NTC)作为对照。之后我们利用构建好的Sgk-KO细胞和NTC细胞进行T细胞共培养实验,简单来说就是在day0时从OT-1转基因小鼠脾脏中提取CD8+T细胞,之后用5ng/ml IL7和100ng/ml IL15刺激T细胞。在day2时将Sgk-KO细胞和NTC细胞种到96孔板中,1500个细胞每个孔。在day3时,往Sgk-KO细胞和NTC细胞中加入OVA抗原多肽或者对照PBS,在4小时后,更换培养基,除去培养基中的抗原,并加入T细胞,6000个T细胞每个孔。在day5和day6时,洗去96孔板中的T细胞,检测剩余的肿瘤细胞活性。Hepa1-6和T细胞共培养实验结果如图2所示,T细胞对NTC Hepa1-6细胞存在明显的杀伤能力,而敲除sgk1基因后可显著抑制T细胞对其的杀伤。体外细胞增殖实验结果如图3所示,表明敲除Sgk1基因并不显著影响Hepa1-6的增殖能力。体内实验部分分为六组,每组8只免疫健全的C57BL/6小鼠。第1组和第2组每只小鼠分别尾静脉注射2百万个NTC Hepa1-6细胞;第3-6组分别尾静脉4个Sgk1-KO的克隆,即Sgk1-1、Sgk1-2、Sgk1-3、Sgk1-4,每只小鼠注射2百万个细胞。尾静脉2百万个NTC细胞不能在小鼠体内形成肺转移,而尾静脉注射2百万个Sgk1敲除Hepa1-6细胞后,可抵抗小鼠体内的T细胞免疫进而形成肺转移(图4A)。如果我们在小鼠体内注射T细胞抗体去除T细胞后,2百万个NTC和Sgk1敲除Hepa1-6细胞均能形成肺转移瘤(图4B)。因此,该实施例提示Sgk1敲除后可使肝癌细胞获得T抵抗能力并在肺内形成转移瘤。
实施例3
获取肝癌原发灶和配对肺转移灶12对石蜡标本,提取每个标本中的RNA并建库,建库产物进行RNA测序。通过生物信息分析获得每个标本的全转录组基因的表达水平(TPM)。比较原发肝癌组织和其配对同一病人的肺转移组织中的Sgk1基因表达水平,结果如图5显示,我们发现Sgk1基因在人肝癌肺转移瘤中的表达较原发瘤显著下调。同时,通过对一个包含110例原发性肝癌手术切除病人队列的石蜡标本进行Sgk1的免疫组化染色,结果显示,Sgk1蛋白在原发瘤中低表达的病人更容易发生手术后肝外转移(见图6),生存时间也更短(见图7)。但是,Sgk1基因在原发瘤中的表达水平对手术后肝内复发却没有影响(见图8)。因此,Sgk1基因为一个能够预测肝癌转移和预后的生物标志物。
实施例4
利用CRISPR技术构建Sgk1基因敲除Hepa1-6细胞,体外培养细胞并收获细胞后,制备成含有2百万个细胞的细胞悬液若干份,以备后续试验使用。实验小鼠为C57BL/6小鼠,分为三组,每组8只,第1组为Lag-3抗体治疗组,第2组为PD-1治疗组,第3组为IgG对照组。通过尾静脉将2百万个Sgk1基因敲除的Hepa1-6细胞分别注射到这三组小鼠循环系统内,随后第1组腹腔注射Lag-3抗体100ug/只/周,第2组腹腔注射PD-1抗体10mg/kg/隔天一次,第3组腹腔注射IgG抗体100ug/只/周。在尾静脉注射细胞后第三周和第五周分别对小鼠肺部进行CT扫描。结果显示,PD-1和Lag-3可以显著抑制Sgk1敲除Hepa1-6细胞形成肺转移(见图9A),降低小鼠死亡率(见图9B)。因此该小鼠体内实验表明Sgk1低表达的肝癌可能成为预测肝癌免疫治疗(PD-1或Lag-3抗体)疗效的生物标志物。
实施例5
为了进一步确认是Sgk1的缺失导致了Hepa1-6细胞对T细胞杀伤的抗性,我们在Sgk1-KO-3中过表达SGK1,然后检测这些细胞对T细胞杀伤的反应。我们从Invitrogen的cDNA文库中克隆了SGK1的transcript variant 1,然后将SGK1的cDNA克隆到带有EGFP的载体上,得到了SGK1-IRES-EGFP的载体。此外,我们还在构建好的载体的基础上构建了SGK1激酶活性缺失的突变体(S422A)。之后,我们将构建好的载体包装成慢病毒,并感染Sgk1-KO-3Hepa1-6细胞。最终我们得到了SGK1-rescue和SGK1-kinase-dead-rescue的两株细胞,以及与之对应的表达EGFP空载的细胞。
在Sgk1敲除的Hepa1-6细胞中再次恢复Sgk1表达,其T细胞抵抗能力显著被抑制。进一步,通过基因编辑的方法构建Sgk1敲除且激酶活性沉默的Hepa1-6细胞系,再次恢复它们的Sgk1表达后,Hepa1-6细胞的T细胞抵抗能力并没有被抑制(见图10),因此,Sgk1的免疫抗性是通过其激酶活性来实现的。这一结果表明Sgk1可为治疗肝癌肺转移的新靶点。
除非另外具体说明,否则在这些实施例中阐述的数值并不限制本发明的范围。在这里示出和描述的所有示例中,除非另有规定,任何具体值应被解释为仅仅是示例性的,而不是作为限制,因此,示例性实施例的其他示例可以具有不同的值。
SEQUENCE LISTING
<110> 复旦大学附属中山医院,上海顿慧医疗科技发展有限公司,上海科技大学
<120> 一种预测肝癌肺转移的生物标志物及其应用
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Claims (10)
1.特异性检测生物标志物Sgk1基因表达水平的试剂在制备预测肝癌肺转移的试剂盒或装置中的应用。
2.根据权利要求1所述的应用,其特征在于,所述的预测包括以下步骤,
步骤a)评估肝癌患者的肿瘤组织中的Sgk1基因的表达;和
步骤b)基于a)的评估结果预测所述肝癌患者是否发生肝癌肺转移的风险。
3.根据权利要求1所述的应用,其特征在于,所述评估是指比较肿瘤组织与对照组织中所述Sgk1基因的检测数据,评估所述Sgk1基因的表达水平;优选地,所述Sgk1基因的表达水平下调,则表示所述肝癌患者存在肝癌肺转移的风险。
4.一种包含权利要求1所述的特异性检测生物标志物Sgk1基因表达水平的试剂的试剂盒。
5.特异性检测生物标志物Sgk1基因表达水平的试剂在制备预测肿瘤细胞抵抗T细胞杀伤力的试剂盒或装置中的应用。
6.特异性检测生物标志物Sgk1基因表达水平的试剂在制备预测肝癌患者对免疫疗法的敏感性的试剂盒或装置中的用途。
7.根据权利要求6所述的用途,其特征在于,所述的免疫疗法为使用免疫抑制剂的免疫疗法,所述免疫抑制剂为抗PD-1/PD-L1或LAG-3抑制剂。
8.基因Sgk1作为靶点在开发或筛选或制备用于预防和/或治疗肝癌肺转移疾病的药物中的应用。
9.基因Sgk1的激活剂在制备预防和/或治疗肝癌肺转移的药物中的应用。
10.根据权利要求9所述的应用,其特征在于,所述激活剂通过激活基因Sgk1的表达,从而实现靶向预防和/或治疗肝癌肺转移。
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