CN117947156A - Ptbp2在神经母细胞瘤预后评估中的应用 - Google Patents
Ptbp2在神经母细胞瘤预后评估中的应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域,具体涉及PTBP2在神经母细胞瘤预后评估中的应用。本发明首次公开了PTBP2、CCL5、IFNA2、IFNB1中的至少一种在神经母细胞瘤预后评估中的应用,通过PTBP2、CCL5、IFNA2、IFNB1中的至少一种的ROC曲线图可知:PTBP2、CC L5、IFNA2、IFNB1中的至少一种可作为神经母细胞瘤预后评估的标志物。
Description
技术领域
本发明属于生物医药技术领域,具体涉及PTBP2在神经母细胞瘤预后评估中的应用。
背景技术
神经母细胞瘤是儿童最常见的颅外实体肿瘤,起源于交感神经系统的神经嵴细胞,主要位于肾上腺、椎旁腹膜后间隙,其次是后纵隔、颈部、骶前区,其在儿童肿瘤死亡人数中占比超过15%。尽管接受传统治疗如手术、化疗、放疗,能够改善神经母细胞瘤患儿的预后,但不定期的扩散和复发导致总体存活率仍然很低。因此,临床中难治性或复发性神经母细胞瘤患儿急需寻找新的有效的治疗方案。
对于低风险神经母细胞瘤患者,由于进展过程中受体内神经营养因子剥夺、体液或细胞免疫、端粒酶活性丧失和表观遗传调控改变等影响,导致部分神经母细胞瘤出现自发消退现象,主要表现在没有任何治疗干预作用下,原发灶或转移灶的肿瘤组织逐渐变小以致消失。目前,肾癌,恶性黑色素瘤、绒毛膜癌、淋巴恶性肿瘤和神经母细胞瘤均被认为是可以发生自我消退现象的瘤种,其中神经母细胞瘤发生自我消退的概率比临床上能够检测到的疾病高200倍。研究发现在自发流产胎儿的肾上腺中存在类似的神经母细胞病灶,主要发生在妊娠第16~20周之间,之后这些病灶逐渐消失,推测神经母细胞残余病灶可能来源于正常交感肾上腺发育的残留成分,而并非原位恶性神经母细胞瘤的结节。
研究神经母细胞瘤的自我消退机制可能有助于筛选此类患儿的最佳治疗方法,目前针对神经母细胞瘤自我消退的药物作用机制主要是后期延迟激活的原肌球蛋白受体激酶A调节的程序性细胞死亡途径。因此,针对肿瘤抗原的免疫耐受或激活神经营养因子受体途径诱导神经元分化的肿瘤患儿,抑制神经营养因子受体的靶向治疗可用于代替传统化疗或放疗法。近年来,大量研究表明神经母细胞瘤的自发消退与患者的急性感染有一定关系,肿瘤微环境中免疫因子的改变可能在神经母细胞瘤的自我消退中发挥关键作用;此外,早期在神经母细胞瘤合并眼痉挛-肌肉痉挛-共济失调综合症(OMS)患儿中发现患儿自身免疫监控是导致其预后良好的因素。因此,自发消退可能是机体介导的免疫反应的结果。体内实验发现在神经母细胞瘤组织中可观察到肿瘤浸润的淋巴细胞亚群和中和抗体,调控免疫调节的细胞因子可以诱导肿瘤转移的消退,此外诱导肿瘤自我消退的免疫调节疗法还可以减少常规细胞毒性治疗引起的后续炎性效应,并避免对预后良好的患者进行损伤性的外科手术。因此,探索神经母细胞瘤自我消退的机制有可能为难治性神经母细胞瘤甚至其他肿瘤的治疗提供新思路、新策略。
机制上来说,高危神经母细胞瘤细胞通过下调人类白细胞抗原(HLA)I类分子诱导免疫耐受,体外实验可通过干扰素-γ刺激神经母细胞瘤细胞来上调I类抗原,以增强靶向肿瘤抗原的免疫反应,最终引起肿瘤消退。然而,尽管干扰素-γ上调I类抗原分子可增强细胞毒性T细胞对神经母细胞瘤细胞的识别,但同时也降低其对自然杀伤细胞效应的敏感性。研究发现,神经母细胞瘤患者的原发性肿瘤中存在包括I类抗原分子重链的表达同时减少对神经母细胞瘤细胞的杀伤。因此,深入研究这些分子在肿瘤消退中的作用机制是必需的。一项旨在评估不同阶段MYCN非扩增性神经母细胞瘤患者中肿瘤相关巨噬细胞浸润的研究表明,4期患儿组织中抗炎性巨噬细胞标志物CD163明显增加,此外,在<18个月大的转移性神经母细胞瘤组织中巨噬细胞相关标志物(CD33、CD16、IL6R、IL10、FCGR3)高于18个月大的患儿。这表明炎症反应和肿瘤微环境可能对特定患者组中神经母细胞瘤的进展和预后产生重要影响。
临床上针对不同风险级别的神经母细胞瘤的治疗方案不同,低风险患儿在手术或不完全切除的情况下可获得90%以上的长期治愈。中风险患儿优先接受化疗后进行手术,若对化疗敏感性的患者则不需要手术治疗,该治疗方案可使患儿3年无进展生存期达80%,总生存期为95%。而对于高危儿童的治疗很复杂,包含多个连续阶段。第一阶段诱导期接受多药联合化疗,使肿瘤缩减至适合手术切除大小同时减少转移;第二阶段为自体串联骨髓移植的巩固治疗以实现局部治疗控制;第三阶段可通过辐射进行保守疗法;最后一阶段为治疗的维持阶段,将与维甲酸或免疫治疗相结合。进入这一阶段的患者已达到最小残留疾病状态,也是测试单克隆抗体治疗和细胞因子最适合的临床方案。
目前,靶向神经母细胞瘤的免疫疗中,以PD-1/PDL-1抗体为代表的免疫检查点抑制剂疗法、以CAR-T为代表的过继细胞免疫疗法等在一些血液及部分实体肿瘤中已经取得了革命性的进展,但在神经母细胞瘤的免疫治疗中仍缺乏响应。且针对神经母细胞瘤免疫疗法的长期毒性知之甚少,特别是在年幼的婴儿中也是未知。已有的临床试验发现,较高剂量白细胞介素-2存在着严重毒性及较低剂量的未确定疗效,伴随着一些毛细血管渗漏、急性炎症反应和心肺疾病等副作用;低剂量白细胞介素-2耐受性更好,但同时促进了免疫抑制性调节性T细胞的增殖。而接受GD2抗体的儿童也会出现剂量依赖性疼痛、发热、心动过速、毛细血管渗漏,偶尔还会出现过敏反应、低血压、电解质失衡和胃肠道症状。自体骨髓移植期或GD2抗体和过继回输NK细胞也会引起致命的噬血细胞性淋巴组织细胞增多症(HLH)等罕见的副作用。目前的免疫疗法也同时存在着疫苗或抗体的安全性和持续有效性的可靠论证。影响神经母细胞瘤免疫治疗疗效及耐药性的因素一方面是归因于神经母细胞瘤本身免疫原性低,另一方面,肿瘤本身遗传和表观遗传缺陷、及肿瘤微环境的改变都是影响免疫治疗疗效的重要因素。
发明内容
本发明的第一方面的目的,在于提供检测物质在制备神经母细胞瘤预后评估的产品中的应用。
本发明的第二方面的目的,在于提供物质在制备预防和/或治疗神经母细胞瘤的药物中的应用。
本发明的第三方面的目的,在于提供PTBP2和/或CCL5和/或IFNA2和/或IFNB1作为靶点在开发神经母细胞瘤相关产品中的应用。
本发明的第四方面的目的,在于提供一种药物。
本发明的第五方面的目的,在于提供一种标志物组合。
本发明的第六方面的目的,在于提供一种产品。
为了实现上述目的,本发明所采取的技术方案是:
本发明的第一个方面,提供(a1)~(a4)中任意一种、任意两种、任意三种或任意四种在制备神经母细胞瘤预后评估的产品中的应用;
(a1)检测PTBP2的物质;
(a2)检测CCL5的物质;
(a3)检测IFNA2的物质;
(a4)检测IFNB1的物质。
所述检测PTBP2的物质为定量检测PTBP2的物质。
优选地,所述检测PTBP2的物质为在基因水平和/或蛋白质水平上检测PTBP2的物质。
优选地,所述物质是用于选自下组的一种或多种检测技术或方法中的物质:免疫组织化学法、Western印迹法、Northern印迹法、PCR、生物芯片法。
优选地,所述免疫组织化学法选自:免疫荧光法、免疫酶标法(ELISA)和免疫胶体金法。
优选地,所述检测PTBP2的物质选自:对PTBP2具有特异性的物质、PTBP2特异性的探针、基因芯片、PCR引物等。
优选地,所述对PTBP2具有特异性的物质为(b1)~(b3)中的任一种:
(b1)特异性结合PTBP2的抗体;
(b2)特异性结合PTBP2的配体蛋白或多肽;
(b3)特异性识别PTBP2的非蛋白类化合物。
优选地,所述的抗体包括多克隆抗体、单克隆抗体、单链抗体、功能性抗体片段、抗体Fab区,纳米抗体、嵌合抗体、多特异性抗体中的至少一种。
优选地,所述检测PTBP2的物质为PCR引物,所述PCR引物的序列如SEQ ID NO.8和SEQ ID NO.9所示。
所述检测CCL5的物质为定量检测CCL5的物质。
优选地,所述检测CCL5的物质为在基因水平和/或蛋白质水平上检测CCL5的物质。
优选地,所述物质是用于选自下组的一种或多种检测技术或方法中的物质:免疫组织化学法、Western印迹法、Northern印迹法、PCR、生物芯片法。
优选地,所述免疫组织化学法选自:免疫荧光法、免疫酶标法(ELISA)和免疫胶体金法。
优选地,所述检测CCL5的物质选自:对CCL5具有特异性的物质、CCL5特异性的探针、基因芯片、PCR引物等。
优选地,所述对CCL5具有特异性的物质为(c1)~(c3)中的任一种:
(c1)特异性结合CCL5的抗体;
(c2)特异性结合CCL5的配体蛋白或多肽;
(c3)特异性识别CCL5的非蛋白类化合物。
优选地,所述的抗体包括多克隆抗体、单克隆抗体、单链抗体、功能性抗体片段、抗体Fab区,纳米抗体、嵌合抗体、多特异性抗体中的至少一种。
优选地,所述检测CCL5的物质为PCR引物,所述PCR引物的序列如SEQ ID NO.10和SEQ ID NO.11所示。
所述检测IFNA2的物质为定量检测IFNA2的物质。
优选地,所述检测IFNA2的物质为在基因水平和/或蛋白质水平上检测IFNA2的物质。
优选地,所述物质是用于选自下组的一种或多种检测技术或方法中的物质:免疫组织化学法、Western印迹法、Northern印迹法、PCR、生物芯片法。
优选地,所述免疫组织化学法选自:免疫荧光法、免疫酶标法(ELISA)和免疫胶体金法。
优选地,所述检测IFNA2的物质选自:对IFNA2具有特异性的物质、IFNA2特异性的探针、基因芯片、PCR引物等。
优选地,所述对IFNA2具有特异性的物质为(d1)~(d3)中的任一种:
(d1)特异性结合IFNA2的抗体;
(d2)特异性结合IFNA2的配体蛋白或多肽;
(d3)特异性识别IFNA2的非蛋白类化合物。
优选地,所述的抗体包括多克隆抗体、单克隆抗体、单链抗体、功能性抗体片段、抗体Fab区,纳米抗体、嵌合抗体、多特异性抗体中的至少一种。
优选地,所述检测IFNA的物质为PCR引物,所述PCR引物的序列如SEQ ID NO.12和SEQ ID NO.13所示。
所述检测IFNB1的物质为定量检测IFNB1的物质。
优选地,所述检测IFNB1的物质为在基因水平和/或蛋白质水平上检测IFNB1的物质。
优选地,所述物质是用于选自下组的一种或多种检测技术或方法中的物质:免疫组织化学法、Western印迹法、Northern印迹法、PCR、生物芯片法。
优选地,所述免疫组织化学法选自:免疫荧光法、免疫酶标法(ELISA)和免疫胶体金法。
优选地,所述检测IFNB的物质选自:对IFNB1具有特异性的物质、IFNB1特异性的探针、基因芯片、PCR引物等。
优选地,所述对IFNB1具有特异性的物质为(e1)~(e3)中的任一种:
(e1)特异性结合IFNB1的抗体;
(e2)特异性结合IFNB1的配体蛋白或多肽;
(e3)特异性识别IFNB1的非蛋白类化合物。
优选地,所述的抗体包括多克隆抗体、单克隆抗体、单链抗体、功能性抗体片段、抗体Fab区,纳米抗体、嵌合抗体、多特异性抗体中的至少一种。
优选地,所述检测IFNB1的物质为PCR引物,所述PCR引物的序列如SEQ ID NO.14和SEQ ID NO.15所示。
优选地,所述产品为试剂、试剂盒、试纸或芯片。
优选地,所述产品的受试样品选自血液、组织、细胞样品、尿液、粪便中至少一种。
优选地,所述血液选自血浆或血清。
优选地,所述组织选自纵隔区域神经母细胞瘤。
优选地,所检测的物质的水平与预后呈正相关。
本发明的第二个方面,提供(f1)~(f6)中任意一种、任意两种、任意三种、任意四种、任意五种或任意六种在(1)~(19)中任一项中的应用;
(f1)PTBP2蛋白;
(f2)编码PTBP2蛋白的核酸分子;
(f3)含有(f2)中所述核酸分子的表达盒、重组载体或转基因细胞系;
(f4)促进PTBP2蛋白或编码PTBP2蛋白的核酸分子表达的物质;
(f5)促进PTBP2蛋白活性提高的物质;
(f6)单核细胞;
(1)制备预防和/或治疗神经母细胞瘤的药物;
(2)体外非治疗目的地促进CCL表达;
(3)制备促进CCL表达的试剂;
(4)体外非治疗目的地促进促炎性因子表达;
(5)制备促进促炎性因子表达的试剂;
(6)体外非治疗目的地抑制CD209表达;
(7)制备抑制CD209表达的试剂;
(8)体外非治疗目的地促进CD80表达;
(9)制备促进CD80表达的试剂;
(10)体外非治疗目的地诱导单核细胞向炎性巨噬细胞(CD11b+CD80+)分化;
(11)制备诱导单核细胞向炎性巨噬细胞(CD11b+CD80+)分化的试剂;
(12)体外非治疗目的地促进ISGF3复合物形成;
(13)制备促进ISGF3复合物形成的试剂;
(14)体外非治疗目的地促进STAT1表达;
(15)制备促进STAT1表达的试剂;
(16)体外非治疗目的地抑制神经母细胞瘤细胞增殖;
(17)制备制神经母细胞瘤细胞增殖;
(18)体外非治疗目的地抑制神经母细胞瘤细胞迁移;
(19)制备抑制神经母细胞瘤细胞迁移的试剂。
优选地,所述促炎性因子包含:TNF、NOS2、IL1B、IFNA2、IFNB1中的至少一种。
优选地,(f1)~(f5)中任意一种、任意两种、任意三种、任意四种或任意五种和(f6)在(1)~(19)中任一项中的应用。
优选地,所述单核细胞通过常规技术手段制备得到,具体如下:通过抗CD14阳性选择从单个核细胞中分离得到单核细胞。
优选地,所述单个核细胞为外周血单个核细胞或脐带血单个核细胞。
优选地,所述药物还包含药学上可接受的辅料。
优选地,所述药物的剂型为儿童适用的剂型。
优选地,所述儿童为出生28天内的新生儿、1岁以内的婴儿、1~6岁的幼儿、6~18岁的儿童中的至少一种。
优选地,所述药物的剂型为胶囊剂、片剂、微囊制剂、注射剂、栓剂、喷雾剂、散剂、软胶囊剂、滴丸、蜜丸、丸剂、颗粒剂、蜜炼膏剂、缓控释制剂、口服液体制剂、注射剂、咀嚼片、口腔片、透皮贴剂、泡腾片中的至少一种。
本发明的第三个方面,提供(g1)~(g4)中任意一种、任意两种、任意三种或任意四种作为靶点在开发神经母细胞瘤相关产品中的应用;
(g1)PTBP2;
(g2)CCL5;
(g3)IFNA2;
(g4)IFNB1。
优选地,所述产品为:
神经母细胞瘤预后评估的试剂、试剂盒、试纸或芯片;或
预防和/或治疗神经母细胞瘤的药物。
本发明的第四个方面,提供一种药物,包含:(f1)~(f5)中任意一种、任意两种、任意三种、任意四种或任意五种;
和(f6);
(f1)PTBP2蛋白;
(f2)编码PTBP2蛋白的核酸分子;
(f3)含有(f2)中所述核酸分子的表达盒、重组载体或转基因细胞系;
(f4)促进PTBP2蛋白或编码PTBP2蛋白的核酸分子表达的物质;
(f5)促进PTBP2蛋白活性提高的物质;
(f6)单核细胞。
优选地,所述单核细胞通过常规技术手段制备得到,具体如下:通过抗CD14阳性选择从单个核细胞中分离得到单核细胞。
优选地,所述单个核细胞为外周血单个核细胞或脐带血单个核细胞。
优选地,所述药物还包含药学上可接受的辅料。
优选地,所述药物用于预防和/或治疗神经母细胞瘤。
优选地,所述药物的剂型为儿童适用的剂型。
优选地,所述儿童为出生28天内的新生儿、1岁以内的婴儿、1~6岁的幼儿、6~18岁的儿童中的至少一种。
优选地,所述药物的剂型为胶囊剂、片剂、微囊制剂、注射剂、栓剂、喷雾剂、散剂、软胶囊剂、滴丸、蜜丸、丸剂、颗粒剂、蜜炼膏剂、缓控释制剂、口服液体制剂、注射剂、咀嚼片、口腔片、透皮贴剂、泡腾片中的至少一种。
本发明的第五个方面,提供一种标志物组合,包含:(g1)~(g4)中任意两种、任意三种或任意四种:
(g1)PTBP2;
(g2)CCL5;
(g3)IFNA2;
(g4)IFNB1。
优选地,所述标志物组合用于神经母细胞瘤预后评估。
本发明的第六个方面,在于提供一种产品,包含:(a1)~(a4)中任意两种、任意三种或任意四种;
(a1)检测PTBP2的物质;
(a2)检测CCL5的物质;
(a3)检测IFNA2的物质;
(a4)检测IFNB1的物质。
优选地,所述检测PTBP2的物质、检测CCL5的物质、检测IFNA2的物质、检测IFNB1的物质为本发明的第一个方面的检测PTBP2的物质、检测CCL5的物质、检测IFNA2的物质、检测IFNB1的物质。
优选地,所述产品为试剂、试剂盒、试纸或芯片。
优选地,所述产品用于神经母细胞瘤预后评估。
本发明的有益效果是:
本发明首次公开了PTBP2、CCL5、IFNA2、IFNB1中的至少一种在神经母细胞瘤预后评估中的应用,通过PTBP2、CCL5、IFNA2、IFNB1中的至少一种的ROC曲线图和/或OS、EFS比较分析图可知:PTBP2、CCL5、IFNA2、IFNB1中的至少一种可作为神经母细胞瘤预后评估的标志物。
本发明首次公开了(f1)~(f6)中任意一种、任意两种、任意三种、任意四种、任意五种或任意六种在预防和/或治疗神经母细胞瘤中的应用,f1)PTBP2蛋白;(f2)编码PTBP2蛋白的核酸分子;(f3)含有(f2)中所述核酸分子的表达盒、重组载体或转基因细胞系;(f4)促进PTBP2蛋白或编码PTBP2蛋白的核酸分子表达的物质;(f5)促进PTBP2蛋白活性提高的物质;(f6)单核细胞;PTBP2在体内通过单核细胞发挥抗肿瘤作用。
发明人首次观察到PTBP2刺激的CCL5分泌是导致TME中单核细胞趋化的原因:利用免疫细胞表型分析,检测NB-TME(GSE3446)的免疫相关差异,观察只有单核细胞和Th2辅助细胞在复发的NBs中减少;同时,通过炎性细胞因子阵列检测方法(AAH-CYT-G6-4)比较PTBP2改变的NB细胞的培养上清,发现RANTES(CCL5)有显著变化。进一步证实,PTBP2在NB细胞中的过表达或下调后,CCL5的浓度和mRNA水平分别增加或降低。
发明人在IRF9的mRNA中发现了一个选择性剪接开关,证实了敲除PTBP2可以有效地加速IRF9的外显子6-7-8区域的剪接进入外显子6-8,从而导致其功能降低:发明人使用RNA-seq分析PTBP2调控的分子信号通路;发现PTBP2处理的NB细胞激活了巨噬细胞的1型干扰素信号通路;接着发明人在IRF9质粒中构建6-7-8-外显子区域,并在PTBP2敲除之前将其导入NB细胞,表明PTBP2敲除能有效地加速IRF9的外显子6-7-8拼接到外显子6-8中;最后,进行交联免疫沉淀反应以确定PTBP2是否直接与IRF9的第6-7-8外显子区域结合,证实了PTBP2与IRF9的第6-7-8外显子区域结合的能力;这一发现揭示了PTBP2校正活性在IRF9选择性剪接和可能的下游生物发生中的关键作用。
发明人揭示了STAT1-CCL5轴在PTBP2调节中的炎症作用:发明人观察到在没有干扰素处理的情况下,STAT1和ISGF3复合体的蛋白水平显著增加,并且干扰素-α/β基因在PTBP2和IRF9过表达时被诱导;抑制PTBP诱导的外显子7跳跃不仅调控IRF9转录,而且在CCL5和干扰素-I信号转导以及单核细胞/巨噬细胞复极化中发挥作用;这一转换不仅证明了PTBP2在IRF9选择性剪接和生物发生中的中心作用,而且也突显了IRF9在下游免疫调节转录程序中的全长作用依赖于STAT1在NB进展过程中诱导的ISGF3的形成。
发明人通过免疫共沉淀研究表明,在对照的NB细胞中,发现IRF9、STAT1和STAT2之间存在明显的关联;PTBP2缺失导致ISGF3复合体中的关联较弱,而PTBP2过表达则显示出相反的作用;PTBP2稳定的敲除或过表达也减少或增加了总STAT1,但对总STAT2表达没有影响,IF染色显示PTBP2敲除或过表达减少或增加了STAT1;此外,PTBP2的稳定敲除或过表达导致NB细胞中IRF9、STAT1和STAT2的细胞核水平上升和下降,但对IRF9和STAT1的细胞质水平略有影响,表明PTBP2的变化与IRF9和STAT1的表达以及ISGF3的形成呈正相关;此外,IRF9(外显子6-7-8)的过表达也显著上调了总的STAT1,并挽救了PTBP2敲除引起的STAT1的下调;相反,IRF9敲除不仅下调了总STAT1的表达,而且抑制了PTBP2诱导的STAT1,这表明PTBP2诱导的IRF9的选择性剪接对总STAT1的表达和ISGF3复合体的形成起作用。
进一步应用靶向STAT1和STAT2的siRNA,观察到PTBP2诱导的CCL5浓度、ISGF3复合体以及IFNA2和IFNB1的表达都被STAT1和STAT2 siRNAs抑制。这些结果表明,PTBP2/IRF9/STAT1轴在ISGF3的形成中发挥核心作用,进而诱导下游的免疫调节转录程序。
附图说明
图1是可变剪切与NB预后相关性的结果图:其中,A、B为蛋白组学差异蛋白热图;C、D为差异蛋白的GO分析结果图;E为差异蛋白的KEGG分析结果图;F为RT-PCR检测不同预后的NB组织中可变剪切基因的表达量图。
图2是PTBP2与NB预后相关性的结果图:其中,A为Western Blot检测PTBP1和PTBP2在不同预后NB组织的表达量图;B为免疫组化检测PTBP1和PTBP2在不同预后NB组织的表达量图;C为RT-PCR检测PTBP1和PTBP2在不同分期NB组织的表达量图;D为公共数据库(GSE3446)转录组中PTBP1和PTBP2分别在复发/未复发组中的表达量图;E为公共数据库(GSE49710)转录组中PTBP1和PTBP2分别在死亡/生存组中的表达量图;F为公共数据库(GSE49710)转录组中PTBP1和PTBP2分别在进展/未进展组中的表达量图。
图3是PTBP2和PTBP1在其它儿童实体瘤中的表达量图:A为PTBP1和PTBP2分别在其它类型NB中的表达量图;B为PTBP1和PTBP2分别在肝母细胞瘤转移/不转移组的表达量图;C为PTBP1和PTBP2分别在肝母细胞瘤死亡/存活组的表达量图;D为PTBP1和PTBP2分别在肾母细胞瘤OS(总生存期)的表达差异图。
图4是PTBP2对NB细胞的功能性作用的影响图:其中,A是PTBP2过表达或敲降对NB细胞活力的影响图;B是PTBP2过表达或敲降对NB细胞克隆数的影响图;C是PTBP2过表达或敲降对NB细胞的迁移的影响图。
图5是NB转录组(GSE3446)中免疫细胞群浸润的差异分析图。
图6是NB细胞PTBP2对原代单核细胞及诱导的巨噬细胞的迁移影响图:其中,A是NB细胞PTBP2对原代单核细胞的迁移影响图;B是NB细胞PTBP2对诱导的巨噬细胞的迁移影响图。
图7是PTBP2改变的NB细胞与单核细胞共培养后的条件培养基对NB细胞的增殖和迁移影响图:其中,A是PTBP2改变的NB细胞与单核细胞共培养后的条件培养基对NB细胞活力的影响图;B是PTBP2改变的NB细胞与单核细胞共培养后的条件培养基对NB细胞克隆数的影响图;C是PTBP2改变的NB细胞与单核细胞共培养后的条件培养基对NB细胞迁移的影响图。
图8是PTBP2通过CCL5诱导单核细胞和巨噬细胞的趋化作用的结果图:其中,A是40个细胞因子队列检测结果图;B是40个细胞因子热图分析结果图;C是Elisa检测PTBP2不同处理组细胞上清CCL5的表达水平图;D为Elisa检测不同预后的NB中CCL5的表达水平图;E是免疫组化检测不同预后的NB中CCL5的表达水平图;F为RT-PCR检测不同预后的NB中CCL5的表达水平图;G是流式检测PTBP2不同处理组细胞上清对单核细胞CCR5的表达的影响图;H是Transwell检测PTBP2不同处理组在加入CCL5或中和抗体进行干预后对单核细胞的趋化作用影响图;I是Transwell检测PTBP2不同处理组在加入CCL5或中和抗体进行干预后对巨噬细胞的趋化作用影响的直观图;J是流式检测PTBP2不同处理组在加入CCL5或中和抗体进行干预后对单核细胞CCR5的表达的表达的影响图。
图9是CCL5细胞因子或中和抗体对PTBP2低表达或高表达NB细胞介导的单核细胞表型的影响图。
图10是PTBP2不同处理组的单核细胞在加入CCL5或中和抗体进行干预后对NB细胞的功能作用影响图:其中,A是PTBP2不同处理组的单核细胞在加入CCL5或中和抗体进行干预后对NB细胞的迁移的影响图;B是PTBP2不同处理组的单核细胞在加入CCL5或中和抗体进行干预后对NB细胞的增殖的影响图。
图11是过表达PTBP2对单核细胞的影响图:其中,A是过表达PTBP2组单核细胞的转录组及KEGG分析结果图;B是流式检测PTBP2不同处理组细胞上清对单核细胞的表型影响的统计结果图;C是流式检测PTBP2不同处理组细胞上清对单核细胞的表型影响的流式图;D是转录组PTBP2与CD163相关性分析结果图;E是RT-PCR检测PTBP2不同处理组细胞上清对单核细胞分泌的炎性因子表达量的影响图。
图12是PTBP2敲除诱导IRF9外显子6-7-8区域的选择性剪接及其对CCL5和干扰素α/β的表达的影响图:其中,A是转录组的可变剪切分析结果图;B是转录组的可变剪切验证结果图;C是CHIP检测PTBP2与IRF9的结合区域的结果图;D是RT-PCR、WB检测PTBP2不同处理组对IRF9的表达影响图;E是IF检测PTBP2不同处理组对IRF9的表达影响图;F是WB检测IRF9E6-7-8过表达后不同处理组中IRF9的表达量图;G是RT-PCR检测IRF9 E6-7-8过表达后不同处理组中IRF9的表达量图;H是Elisa检测IRF9 E6-7-8过表达后不同处理组中CCL5的表达量图;I是RT-PCR检测IRF9 E6-7-8过表达后不同处理组中IFNA2,IFNB1的表达量图;J是流式检测IRF9 E6-7-8过表达后不同处理组中对单核细胞表型影响图。
图13是低表达IRF9对PTBP2下游调控作用的影响图:其中,A是Elisa检测IRF9低表达后不同处理组中CCL5表达量图;B是RT-PCR检测IRF9低表达后不同处理组中IFNA2、IFNB1的表达量图;C是流式检测IRF9低表达后不同处理组对单核细胞表型影响图。
图14是PTBP2对STAT1及ISGF复合物的影响图:其中,A是免疫共沉淀(CO-IP)检测PTBP2不同处理组对ISGF3复合物形成的影响图;B是IF检测PTBP2不同处理组对STAT1和STAT2总蛋白的表达量图;C是WB检测PTBP2不同处理组细胞核和细胞浆中IRF9,STAT1和STAT2的表达量图;D是WB检测PTBP2不同处理组对STAT1和STAT2总蛋白的表达量图。
图15是PTBP2在体内通过再培养单核细胞抑制肿瘤生长的结果图:其中,A是不同处理对小鼠转移瘤大小影响的体内生物发光成像图;B是不同处理对小鼠转移瘤大小影响的统计结果图;C是不同处理对小鼠肝脏和肾转移的发展的影响图;D是不同处理对小鼠血液和组织中CD11b+CD80+/CD11b+CD209+的单核细胞/巨噬细胞的比率的影响图;E是不同处理对小鼠肿瘤细胞的增殖影响的Ki67染色结果图;F是不同处理对小鼠肿瘤细胞的增殖影响的评分结果图;G是不同处理对CCL5表达量的影响图;H是不同处理对IFNA2和IFNB1表达量的影响图;I是IFNA2和IFNB1分别在不同预后NB组织的表达量图。
图16是PTBP2高表达组和PTBP2低表达组预计3年OS的比较分析图。
图17是PTBP2高表达组和PTBP2低表达组预计3年RFS的比较分析图。
上述图中,*表示p<0.05;**表示p<0.01;***表示p<0.001。
具体实施方式
以下通过具体的实施例对本发明的内容作进一步详细的说明。
下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。
本实施例中所使用的材料、试剂等,如无特别说明,为从商业途径得到的试剂和材料。
本实施例中采用的材料和方法具体如下:
1.神经母细胞瘤(neuroblastoma,NB)组织标本
冰冻NB组织来源于广州妇女儿童医疗中心手术切除,并根据临床病理资料,按分期或预后进行分类(预后不良(UP)或预后良好(FP)各5名,预后不好的标准:术后反复复发,进展;预后好的标准:术后后无复发)。所有患者在采集样本前均填写书面知情同意书,并得到广州市妇女儿童医疗中心机构审查委员会批准(穗妇儿科伦批字【2022】第070A01号)。
2.细胞分离与培养
人种细胞株HUVECs和NB细胞株SK-N-BE、SK-N-AS、Kelly以及SK-N-SH来源于美国标准培养库(ATCC,Manassas,VA,USA)。SK-N-BE由EMEM和F12以1:1比例配制的培养基培养,HUVECs、SK-N-AS和Kelly细胞由RPMI-1640培养基培养。所有类型的培养基均含有10%的胎牛血清(FBS)和1%青霉素/链霉素。细胞均在37℃,5% CO2中培养,检测无支原体污染。所有类型的细胞均通过短串联重复序列分析进行验证。
通过抗CD14阳性选择(#130-050-201,Miltenyi Biotec,Bergisch Gladbach,Germany)从人外周血单个核细胞(PBMCs)中分离出纯化的单核细胞,并在含有10%胎牛血清和20ng/mL M-CSF(#30025,PeproTech,Rocky Hill,USA)的RPMI-1640培养基中培养分化。7~10天后获得成熟的巨噬细胞,为后续实验步骤做准备。
通过T细胞阴性分离试剂盒(#130-096-535,MiltenyiBiotec,BergischGladbach,德国)从PBMCs中获得总T细胞,并在T细胞扩增培养基(ImmunoCult-XF,StemCell,Vancouver,Canada)中培养,同时加入CD2/CD3/CD28磁珠和IL2(10ng/mL)刺激和扩增。
3.基于TMT的蛋白组学分析与鉴定
选取5对预后不良(UP)或预后良好(FP)的NB患者进行蛋白质组学鉴定。所有蛋白都被提取并被胰蛋白酶水解为多肽,所有的多肽用TMT试剂(Thermo Fisher Scientific,Waltham,MA,USA)标记,用Agilent 1260Infinity II高效液相色谱系统分级。色谱柱经样品平衡加载后分离,然后用Easy nLC系统洗脱,用Q Exactive Plus质谱仪(Thermo FisherScientific,Waltham,MA,USA)分析。随后,原始的图集文件被转换为.mgf文件提交给MASCOT 2.6服务器,通过软件的内置工具进行数据库检索。数据库通过ProteomeDiscoverer 2.1(Thermo Fisher Scientific,Waltham,MA,USA)搜索到文件(.dat文件)后发送到软件,并根据错误发现率(FDR)<0.01的标准进行过滤,获得高度可靠的定性结果。用KEGG方法进行通路分析,并通过比较不同表达蛋白的数量和与之相关的总蛋白的数量来确定显著富集的通路(由Fisher’s精确检验完成)。
4.RNA测序(RNA-seq)和生物信息学分析
通过无RNA酶的DNase I(#M6101,Promega,WI,USA)处理提取总RNA。使用2100系统的生物分析仪(Agilent Technologies,CA,USA)的RNA Nano 6000检测试剂盒检测RNA完整指数(RIN)。所有的RNA样本RIN值均>8。通过AMPure XP系统(Beckman Coulter,Beverly,USA)纯化文库片段,并用PCR扩增。使用Qubit 2.0对cDNA文库质量进行量化,在Bioanalyzer 2100(Agilent,USA)上检测,然后使用Illumina NovaSeq 6000平台进行测序。由DESeq2 R软件包(1.20.0)检测转录组数据中的差异表达基因(DEGs),其倍数变化>2。通过clusterProfiler R包(3.8.1)对DEGs进行基因本体(GO)通路富集分析和KEGG通路富集分析。通过rMATS(4.0.2)软件对AS事件进行蛋白变量分析。
公共数据集的分析中,基于RNA序列的儿童实体瘤和胶质母细胞瘤的基因表达数据,来源于癌症基因组学的基因表达谱交互分析(GEPIA)数据库。对于基因表达,以P值<0.05为临界值。
5.免疫组织化学实验(IHC)
将NB组织样本和小鼠肿瘤样本固定并包埋进行免疫组化染色。封闭后,分别与鼠抗PTBP2(#sc-376316,Santa Cruz,CA,USA)、兔抗PTBP1(#ab133734,Abcam,Cambridge,MA,USA)、兔抗CCL5(#12,000-1-ap,Proteintech,Chicago,IL,USA)、兔抗Ki67(Cat#12202,Cell Signaling Technology,Beverly,USA)、鼠抗CD68(#ab201340;Abcam)和抗体Twist(#25465-1-AP;ProteinTech)在4℃下过夜,与生物素化抗兔/鼠免疫球蛋白在室温下孵育1小时,并滴加二氨基联苯胺(DAB)(GK500705,Dako,Glostrup,Denmark)观察颜色变化。每个剖面选择3~5个具有代表性的区域进行分析。使用Image-Pro Plus 6.0软件(MediaCybernetics,Rockville,MD,USA)分析平均阳性比例。
6.免疫荧光实验(IF)
固定细胞且通透细胞膜后,分别用PTBP2抗体、STAT1抗体(#10144-2-AP,Proteintech,Chicago,IL,USA)和IRF9抗体(#14167-1-AP,Proteintech,Chicago,IL,USA)孵育。进而用鼠或兔Alexa Fluor荧光二抗(#4414或#4409,Cell Signaling Technology,Beverly,USA)孵育。随后用DAPI(#P0131,Beyotime,Nanjing,China)染细胞核。由配备20×物镜的共聚焦显微镜(Leica SP8,Germany)拍摄细胞图像,使用Image-Pro Plus 6.0软件进行分析。
7.细胞转染
CCL5启动子区的人种属cDNA,包括启动子区FL(全长,hg38_knownGene_ENST00000651122.1range=chr17:35880306-35882360)、Pro1(上游142bp)、Pro1+Pro2(上游282bp)和Pro1+Pro2+Pro3(上游961bp),并克隆且连接到pGL4.10载体的XhoI、HindIII之间。将人种属PTBP2(NM_021190.3)、STAT1(NM_007315.4)和STAT2(NM_005419.4)的全长cDNA克隆并连接到pcDNA3.1载体EcoRI、XhoI之间。将靶向人种属PTBP2的shRNA序列(Y11455-F:CcggGCAGCTATTACTATGGTTATTCAAGAGATAACCATAGTAATAGCTGCTTTTTTg(SEQ IDNO.1,sh#1)、Y11455-R:aattcaaaaaaGCAGCTATTACTATGGTTATCTCTTGAATAACCATAGTAATAGCTGC(SEQ ID NO.2,sh#1);Y11456-F:CcggCCCTGTAACACTTGATGTTcTCAAGAGAAACATCAAGTGTTACAGGGTTTTTTg(SEQ ID NO.3,sh#2)、Y11456-R:aattcaaaaaaCCCTGTAACACTTGATGTTTCTCTTGAgAACATCAAGTGTTACAGGG(SEQ ID NO.4,sh#2))插入载体pDKD-CMV-Puro-U6-shRNA的AgeI、EcoR I之间。使用LipofectamineTM 3000
(Invitrogen,Carlsbad,CA,USA)转染细胞。PTBP2敲降的对照为随机的无义shRNA序列(GL401NC-F:CCGGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTTG(SEQID NO.5)、GL401NC-R:AATTCAAAAAATTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAA(SEQ ID NO.6)),而PTBP2过表达的对照为空载体(EV)。所有质粒均由OBiO科技有限公司(Shanghai,China)提供。通过对目标基因mRNA和蛋白水平的检测进一步验证了该基因的敲除或过表达效率。在NB细胞(RiboBio Co.,Ltd,Guangzhou,China)中利用小干扰RNA对IRF9(siRNA-3:CTCAGAAAGTACCATCAAA,SEQ ID NO.7)进行目标基因瞬时敲除,本研究使用打乱的无义序列作为转染对照。
8.流式细胞术分析
通过对单个细胞进行条件培养基(CM)处理,48小时后用PE/Cyanine 7-CD45(#368532),APC-CD209(#330107),FITC-CD80(#305206),PerCP-CD14(#325032),PE-CD11b(#301305),PE/Cyanine7-CCR5(#359108)(BioLegend,San Diego,CA,USA)抗体染色,并进行流式细胞术可以检测单核细胞或巨噬细胞的表型。染色程序遵循试剂制造商的使用说明。
9.细胞增殖检测实验
细胞增殖活性和克隆形成实验按照试剂盒制造商的使用说明进行。NB细胞在96孔板或6孔板上过夜培养,并按指示处理。通过计算各组96孔板或6孔板cck-8处理后孔内450nm的吸光值或克隆数,获得细胞活力指数。
10.细胞迁移和趋化实验
在细胞迁移实验中,将含有1%胎牛血清的细胞(每孔5×104个)接种在上层小室(Corning,NY,USA)中,然后放入含有10%胎牛血清或特定CM的培养基中(小室孔径为8微米)。12小时后,对迁移至小室下表面的细胞计数,使用Image-Pro Plus 6.0软件进行分析。
单核细胞趋化实验中,将纯化过的单核细胞(每孔1×106个)种在24孔的迁移板(Corning,NY,USA)上,随后将其底层浸入加有CM的下层(孔径为0.4微米)12小时。单核细胞的趋化性是通过计算迁移细胞的数量分析得到的。
11.免疫印迹
免疫印迹按照标准实验步骤进行。用含有蛋白酶和磷酸酶抑制剂的冰冻RIPA缓冲液处理细胞并提取总蛋白。使用BCA蛋白测定试剂盒(Thermo Fisher Scientific,Waltham,MA,USA)对所提的蛋白质进行定量。一抗包括PTBP2抗体、PTBP1抗体、IRF9抗体、STAT1抗体、vinculin抗体(#13901,Cell Signaling Technology,Beverly,USA)、STAT1抗体(#16674-1-AP,Cell Signaling Technology,Beverly,USA)、βactin抗体(#4790,CellSignaling Technology,Beverly,USA)和histone H3(#4499,Cell SignalingTechnology,Beverly,USA)。所有条带均通过增强化学发光可视化,至少重复3次,并用ImageJ软件进行分析量化。
12.RT-PCR分析
通过RNA提取试剂盒(#RN001,essscience,Shanghai,China)提取总RNA,并根据逆转录试剂盒(#RR036A-1,TAKARA,Japan)产品说明逆转录为cDNA。通过定量RT-PCR分析(ABIQ6 System,Applied Biosystems)测定所鉴定基因的相对表达(PTBP2-F:GCAACCGAGGAAGCAGCTATT,SEQ ID NO.8;PTBP2-R:GCCTGAGCACGTTGGTTTAATG,SEQ ID NO.9;CCL5-F:AGCAGTCGTCCACAGGTCAA,SEQ ID NO.10;CCL5-R:CTGGGTTGGCACACACTTGG,SEQ IDNO.11;IFNA2-F:CTTGTGCCTGGGAGGTTGTC,SEQ ID NO.12;IFNA2-R:GGTGAGCTGGCATACGAATC,SEQ ID NO.13;IFNB1-F:GGCGACACTGTTCGTGTTGT,SEQ ID NO.14;IFNB1-R:AGCCTCCCATTCAATTGCCAC,SEQ ID NO.15)。将所有靶基因的表达量归一化后与β-actin的表达量进行比较,并通过2-ΔΔCT比较法计算其变化倍值。
13.酶联免疫吸附实验(ELISA)
利用人CCL5 SimpleStep酶联免疫吸附测定试剂盒(#ab174446,Abcam,Cambridge,MA,USA),将收集到的NB细胞或血浆上清液稀释,检测CCL5含量。在样品孔中加入50微升的样品,然后室温孵育1小时后,每孔加入50微升的抗体,350微升的洗涤液洗涤3次后,再加入TMB显影液,室温孵育10分钟后,加入100微升的终止液,450nm测定吸光值。
14.细胞因子检测
根据试剂制造商的使用说明书,用炎症细胞因子检测试剂(AAH-CYT-G5-4,Raybiotech)测量并定量PTBP2敲除诱导的NB细胞和对照细胞分泌的细胞因子。
15.交联免疫沉淀(CLIP)和染色质免疫沉淀(ChIP)实验
用254nm(400mJ/cm2)紫外光照射PTBP2表达不同的NB细胞。细胞被压成小球状并用加有蛋白酶抑制剂(0.1mg/mL)、1mM DTT以及400U/mL RNA酶抑制剂的NP-40裂解液溶解,37℃放置60分钟,加入30微升DNaseI,37℃放置10分钟,最后加入4微升0.5M EDTA终止消化。在离心管中加入微球菌核酸酶(#10011,Cell Signaling Technology,Beverly,USA)消化总RNA,而input RNA用1%的琼脂糖凝胶电泳分离,将大部分RNA消化为1~5个核小体(150到900bp)。然后,将40微升的预平衡珠子(HY-K0202,MedChemExpress,New Jersey,USA)与10微克正常IgG或PTBP2抗体孵育,4℃过夜。洗珠子,加入1毫升细胞裂解液。混合物在4℃孵育2小时,37℃用FAST AP(#EF0654,Thermo Fisher Scientific,Waltham,MA,USA)去磷酸化1小时。将蛋白质-RNA复合物进一步从珠中洗脱并收集,用于RNA提取,之后通过qPCR实验分析。
利用ChIP试剂盒(#66816,Cell Signaling Technology)的实验使用说明检测与STAT1免疫沉淀的DNA。此复合物进一步用Simple CHIP qPCR Master Mix(#88989,CellSignaling Technology)进行实时荧光定量PCR检测。
16.Co-IP实验
IRF9抗体、STAT1抗体和STAT2抗体也用于Co-IP实验。用免疫印迹法检测免疫沉淀物。兔抗与鼠抗的IgG(#3900和#3420,Cell Signaling Technology,Beverly,USA)作为阴性对照。
17.荧光素酶报告实验
在96孔板上种NB细胞,用含有CCL5启动子和诱导荧光素酶(Lucifer)的质粒转染(pGL4.10-CCL5promoter),并按说明书操作(将5ul含有0.15ul Lipofectamine 3000,的Opti-MEM Medium与5ul含有0.1ul p3000,0.1ug的质粒的Opti-MEM Medium混匀后加到细胞培养孔中并摇匀)。荧光素酶活性通过Steady-Glo Reporter Assay(#E2510,Promega,WI,USA)试剂盒说明书检测。
18.体内实验
通过尾静脉注射,将表达诱导荧光素酶报告基因的SK-N-SH细胞(将含有luciferase序列的慢病毒转入SK-N-SH细胞中构建SK-N-SH-Lucifer稳转株)注射到小鼠(B-NDG背景,4~6周)体内(每只小鼠尾静脉注射100W个SK-N-SH细胞;通过腹腔注射luciferase底物,通过活体成像系统检测光强来监测小鼠肿瘤大小和造模成功率),构造转移性NB模型。将免疫缺陷B-NDG小鼠体内分为两组,一组移植人种属的PBMCs共5×106个(PBMC回输组),另一组为无移植细胞组。分别将两组小鼠随机分为NC、Sh PTBP2、PTBP2、NC+PBMC、Sh PTBP2+PBMC、PTBP2+PBMC共6组(每组n=10,注射100W个SK-N-SH细胞)。使用InVivo Bruker FX PRO荧光素酶成像监测肿瘤大小。拍摄并测量荧光素酶强度,计数并分析转移结节。所有小鼠移植3周后处死,并收集肿瘤组织进行病理分析。
19.统计学分析
所有的免疫印迹、免疫荧光和免疫组化图像都至少为两个独立重复的代表性结果。PTBP2与CD163的表达相关性由Pearson相关性分析完成。所有柱状图皆由means±SDs表示。统计计算至少来自三个独立实验,并通过学生t检验(未配对,双尾)或使用GraphPadPrism 8.0.1软件(GraphPad,La Jolla,CA,USA)进行分析。P值<0.05为有统计学意义。在图中,*表示P<0.05;**P<0.01;和***,P<0.001。
实施例1 PTBP2在神经母细胞瘤预后评估中的应用
鉴于临床中1岁内神经母细胞瘤患儿可出现肿瘤自我消退现象,发明人前期针对本院神经母细胞瘤患儿术后预后好(1岁以内NB患儿手术未能完整切除,且术后未化疗或拒绝化疗,随访5年未复发、无肿瘤残留,n=5)和预后差(年龄>1岁NB患儿手术未能完整切除,但术后化疗,出现反复复发、甚至死亡,n=5)组织标本进行蛋白组学研究,结果如图1所示:mRNA可变剪切通路富集显著(图1中A~E),且可变剪切相关基因(如SFPQ,MATR3,PTBP2,NONO,ELF2B4,HMGB3,PRKCB,RFTN1,和NRAS)在预后差的NB患儿中下调,其中PTBP2下调最明显;而PTBP2在FP-NB患者中显著上调(图1中F)。进一步对可变剪切途径差异表达蛋白的mRNA水平分析表明,PTBP2是FP-NB患者中最显著的上调基因(图1F)。
免疫组化和Western Blot分析PTBP2及同源基因PTBP1的表达进一步验证,PTBP2与NB患儿预后显著正相关(图2中A~C,C图中:Early:病理学诊断为1和2期;Advanced:病理学诊断为3和4期);发明人从公共微阵列数据集(GEO)中进一步研究PTBP1和PTBP2分别在混合型节细胞神经母细胞瘤(2)、高分化(3)和中分化(4)神经节细胞瘤、神经节神经母细胞瘤组织(5)中表达(图3A),发现PTBP2在神经节神经瘤-神经母细胞瘤中的表达最高,而PTBP1表达最低。GEO数据集GSE3446分析显示,PTBP2在复发的NBs的表达低于未复发者,将预后良好和预后不良患者的PTBP2含量进行ROC曲线分析,PTBP2可以有效地区分预后良好和预后不良患者,可作为神经母细胞瘤预后评估的标志物;而PTBP1的表达不明显,而GSE4970对疾病死亡(DOD)和进展(PRO)的分析表明,PTBP2在死亡或进展的NB中的表达,低于在存活和非进展的NB中的表达,而在两个簇中都存在相反的表达(图2中D~F)。GEO数据集GSE131329分析显示PTBP2在肝母细胞瘤(Hepatoblastoma)的转移和死亡队列中低水平表达,而PTBP1高表达(图3中B、C)。因为TCGA数据库没有NB中PTBP1/PTBP2的OS(总生存期)和无复发生存期(RFS)信息,因此发明人进一步从其它儿科肿瘤中获得PTBP1/PTBP2的OS和RFS信息。公共数据集显示,高表达的PTBP2与胶质母细胞瘤的良好预后显著相关(图3中D)。以上结果表明,PTBP2与大部分儿童肿瘤进展呈负相关。特别是在分期和预后相关的NB肿瘤中,提示我们PTBP2在NB的发生发展及预后中起到了抑制肿瘤进展的作用。发明人通过对GEO数据库(GSE49710)进行分析,该数据库收集了498例确诊后的神经母细胞瘤组织进行常规转录组测序,神经母细胞瘤患儿组织将根据International Neuroblastoma Staging System(INSS)标准进行分期(1,2,3,4and 4S),同时根据转录组表达矩阵绘制图,取PTBP2表达中位数,以中位数为分界线将组织分为高表达和低表达组,进而分析PTBP2表达与预后的相关性及绘制生存曲线,结果如图16、17所示:PTBP2高表达患儿的OS和EFS均显著高于比低表达组,可见,PTBP2可以有效地区分预后良好(OS和/或EFS高)和预后不良(OS和/或EFS低)患者,可作为神经母细胞瘤预后评估的标志物。
实施例2 PTBP2通过肿瘤微环境(TME)调节影响NB进展
发明人进一步敲降或过表达NB细胞PTBP2后发现细胞自身的增殖和迁移都没有明显的改变(图4)。发明人对NB转录组(GSE3446)进行免疫细胞浸润的差异分析发现单核细胞在预后好的NB组中浸润明显高于预后差组(图5),通过体外预实验发明人初步发现过表达PTBP2的NB细胞培养基促进体外原代单核细胞及诱导的巨噬细胞的迁移(图6);此外,过表达PTBP2的NB细胞与单核细胞共培养后的培养基明显抑制NB细胞的增殖和迁移(图7)。可见,PTBP2可能通过诱导单核-巨噬细胞从免疫抑制表型向免疫激活表型极化从而抑制肿瘤细胞生长和迁移。
实施例3 PTBP2通过CCL5诱导单核细胞和巨噬细胞的趋化活性
鉴于前期的功能性结果,发明人对敲降PTBP2后细胞上清与对照进行40个细胞因子队列检测发现,与对照组相比,低表达PTBP2明显抑制单核细胞趋化因子CCL5(RANTES)的分泌(图8中A~B);Elisa结果显示PTBP2降低可抑制SK-N-SH和SK-N-AS中CCL5的分泌,高表达PTBP2后CCL5分泌显著增加(图8中C);Elisa和免疫组化结果进一步验证CCL5与NB预后正相关,将预后良好和预后不良患者的CCL5含量进行ROC曲线分析,CCL5可以有效地区分预后良好和预后不良患者,可作为神经母细胞瘤预后评估的标志物(图8中D~F)。同时通过流式分析结果发现,PTBP2降低可抑制单核细胞表面CCR5表达,高表达PTBP2后CCR5表达增加(图8中G)。进而通过分别加入CCL5细胞因子或中和抗体进行干预后发现,PTBP2诱导的单核/巨噬细胞的趋化及CCR5表达在加入中和抗体后明显减少,反之,CCL5有效增加了PTBP2低表达的抑制作用(图8中H~J)。同时发明人加入CCL5细胞因子或中和抗体进行干预后发现都不能改变PTBP2低表达或高表达NB细胞介导的单核细胞表型的影响(图9)。以上结果表明,PTBP2诱导的单核/巨噬细胞的趋化作用主要由CCL5/CCR5介导。但PTBP2诱导的CCL5并不能改变PTBP2介导的单核细胞对NB的影响。以上结果表明PTBP2除了通过增加CCL5诱导单核/巨噬细胞趋化外,还可能通过共同的上游通路激活单核细胞的表型和功能(图10)。
通过对PTBP2处理组共培养的单核细胞进行转录组分析发现,过表达PTBP2组单核细胞中I型干扰素通路(Type I interferon signaling pathway)通路显著富集(图11中A)。发明人进一步检测PTBP2处理后单核细胞的表型,发现过表达PTBP2可促进单核细胞CD11b+CD80+表型,以及促炎性因子TNF,NOS2和IL1B基因水平,抑制CD209表达(图11中B、C、E);而PTBP2低表达诱导单核细胞上调CD209及抗炎性因子Arg1和TGFB1基因水平(图11中B、C、E);同时转录组分析发现PTBP2与抗炎型的单核/巨噬细胞标志物CD163显著负相关(图11中D);且以上结果表明:NB细胞中过表达PTBP2可诱导单核细胞向炎性巨噬细胞(CD11b+CD80+)的分化。
实施例4 PTBP2敲除诱导IRF9外显子6-7-8区域的选择性剪接
通过提取敲降PTBP2组和对照组的总RNA进行转录组分析发现差异基因中没有筛选出富集免疫通路相关基因。基于PTBP2主要参与可变剪切事件的发生,通过可变剪切分析发现PTBP2敲降可引起IFN信号通路的上游调控基因IRF9发生可变剪切,主要在外显子6-8段区域(24164062-24164955)的外显子跳跃(Chr14:24164613-24164955,lncLevelDifference:0.242)(图12中A)。于是构建IRF9外显子6-8段区域质粒并转染至神经母细胞瘤细胞(同lipo3000转染步骤)。结果证实PTBP2敲降显著降低IRF9外显子6-7-8段区域(E6-7-8)的表达,而在IRF9 E6-7-8正常和过表达条件下,其发生的可变剪切事件增加(图12中B),敲除PTBP2后进一步增加IRF9 E6-7-8剪切为IRF9 E6-8片段。通过PTBP2抗体进行CLIP分析,结果进一步证实PTBP2可直接作用于IRF9 E6-7-8区域(图12中C),过表达或敲降PTBP2时分别增强或减弱其结合能力。以上实验表明,PTBP2在IRF9的可变剪切中发挥关键作用。
实施例5 PTBP2诱导的IRF9选择性剪接影响CCL5和干扰素α/β的表达
RT-PCR、WB及IF染色证实敲降或过表达PTBP2同时降低或增加IRF9蛋白和mRNA水平的表达(图12中D~E)。WB进而发现过表达IRF9 E6-7-8区域的质粒可逆转低表达PTBP2对IRF9的抑制作用,此外与对照组比,过表达IRF9 E6-7-8不仅促进IRF9蛋白及mRNA水平,同时增加对照或PTBP2低表达NB细胞中CCL5,IFNA2/IFNB1的水平及CD11b+CD80+单核细胞的极化,同时对CD11b+CD209+单核细胞则表现相反趋势(图11中F~I),表明IRF9 E6-7-8在IRF9的转录及PTBP2调控中发挥重要作用。相反,靶向IRF9的siRNAs降低对照或PTBP2过表达NB细胞中CCL5,IFNA2/IFNB1的水平及CD11b+CD80+单核细胞的极化(图13中A~C)。以上结果表明PTBP2通过抑制IRF9发生可变剪切而失活,促进CCL-5和IFNα/β的分泌发挥下游对单核细胞的调控作用。
实施例6 PTBP2通过上调STAT1激活ISGF3复合体并诱导干扰素α/β
由于IRF9/p48主要参与I型干扰素通路,它与STAT1/STAT2二聚体形成ISGF3复合物(interferon-stimulated gene factor 3)入核调控干扰素诱导基因的表达,通过免疫共沉淀(CO-IP)实验初步发现,过表达PTBP2明显促进ISGF3复合物的形成(图14中A)。分别提取细胞核(Nucleus)与细胞浆(Cytoplasm)蛋白检测IRF9,STAT1及STAT2表达,结果发现过表达PTBP2明显增加细胞核内IRF9,STAT1及STAT2水平,而胞浆中IRF9和STAT2表达减少,PTBP2降低相应抑制IRF9,STAT1及STAT2的核表达,增加IRF9及STAT2的浆表达,而STAT1在细胞浆中没有明显变化(图14中C)。表明PTBP2可能对STAT1总蛋白表达有直接的调控作用。免疫荧光及WB实验进一步证实:过表达或敲低PTBP2明显上调或下调STAT1的总蛋白表达(图14中B、D)。
实施例7 PTBP2在体内通过再培养单核细胞抑制肿瘤生长
为了在体内系统中验证体外数据,发明人进一步通过静脉注射稳定的、含有过表达或下调的PTBP2的SK-N-SH细胞系来建立转移性NB模型。小鼠接受人外周血单核细胞移植。体内生物发光成像显示,PTBP2的过表达显著抑制了转移瘤的大小,而PTBP2的下调则扩大暴露于人PBMC移植的转移瘤大小,而在没有人PBMC的组(无移植细胞组)之间没有明显的差异(图15中A、B)。因此,随着人源PBMC回输后,能观察到敲降PTBP2组的小鼠肝和肾转移的发展显著增加,相反,过表达PTBP2组显著减少了肝脏和肾转移(图15中C)。此外,与PBMC回输组的对照组相比,PTBP2过表达组在血液和组织中的人CD11b+CD80+/CD11b+CD209+的单核细胞/巨噬细胞的比率较高,而在PTBP2敲除后较低(图15中D)。肿瘤切片的Ki67染色表明,PBMC回输组中,下调或过表达PTBP2分别促进或抑制了小鼠肿瘤细胞的增殖(图15中E和F)。此外,PTBP2诱导的人CCL5浓度,以及IFNA2和IFNB1的表达,也在PBMC移植后的肿瘤中得到证实(图15中G、H)。这些结果表明,PTBP2在小鼠体内通过单核细胞发挥抗肿瘤作用。
实施例8 IFNA2和IFNB1的高表达与NB的预后正相关
发明人最后检测了IFNA2和IFNB1在预后好和预后差的神经母细胞瘤患儿(每组各10例)中的表达。与预后差组相比,预后好组的IFNA2和IFNB1水平升高(图15中I)。将预后良好和预后不良患者的IFNA2和/或IFNB1含量进行ROC曲线分析,IFNA2和/或IFNB1可以有效地区分预后良好和预后不良患者,即IFNA2和/或IFNB1可以作为神经母细胞瘤预后评估的标志物。将预后良好和预后不良患者的PTBP2、CCL5、IFNA2、IFNB1中的任意一种、两种、三种、四种含量进行ROC曲线分析,PTBP2、CCL5、IFNA2、IFNB1中的任意一种、两种、三种、四种可以有效地区分预后良好和预后不良患者,即PTBP2、CCL5、IFNA2、IFNB1中的任意一种、两种、三种、四种可以作为神经母细胞瘤预后评估的标志物。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.(a1)~(a4)中任意一种、任意两种、任意三种或任意四种在制备神经母细胞瘤预后评估的产品中的应用;
(a1)检测PTBP2的物质;
(a2)检测CCL5的物质;
(a3)检测IFNA2的物质;
(a4)检测IFNB1的物质。
2.根据权利要求1所述的应用,其特征在于:
所述检测PTBP2的物质包含在基因水平和/或蛋白质水平上检测PTBP2的物质;
优选地,所述检测CCL5的物质包含在基因水平和/或蛋白质水平上检测CCL5的物质;
优选地,所述检测IFNA2的物质包含在基因水平和/或蛋白质水平上检测IFNA2的物质;优选地,所述检测IFNB1的物质包含在基因水平和/或蛋白质水平上检测IFNB1的物质。
3.根据权利要求2所述的应用,其特征在于:
所述物质是用于选自下组的一种或多种检测技术或方法中的物质:免疫组织化学法、Western印迹法、Northern印迹法、PCR、生物芯片法;
优选地,所述检测IFNB1的物质选自:对IFNB1具有特异性的物质、IFNB1特异性的探针、基因芯片、PCR引物;
优选地,所述检测PTBP2的物质选自:对PTBP2具有特异性的物质、PTBP2特异性的探针、基因芯片、PCR引物;
优选地,所述检测CCL5的物质选自:对CCL5具有特异性的物质、CCL5特异性的探针、基因芯片、PCR引物;
优选地,所述检测IFNA2的物质选自:对IFNA2具有特异性的物质、IFNA2特异性的探针、基因芯片、PCR引物;
优选地,所述检测PTBP2的物质为PCR引物,所述PCR引物的序列如SEQ ID NO.8和SEQID NO.9所示;
优选地,所述检测CCL5的物质为PCR引物,所述PCR引物的序列如SEQ ID NO.10和SEQID NO.11所示;
优选地,所述检测IFNA2的物质为PCR引物,所述PCR引物的序列如SEQ ID NO.12和SEQID NO.13所示;
优选地,所述检测IFNB1的物质为PCR引物,所述PCR引物的序列如SEQ ID NO.14和SEQID NO.15所示;
优选地,所述产品为试剂、试剂盒、试纸或芯片;
优选地,所述产品的受试样品选自血液、组织、细胞样品、尿液、粪便中至少一种;
优选地,所述血液选自血浆或血清;
优选地,所述组织选自纵隔区域神经母细胞瘤。
4.(f1)~(f6)中任意一种、任意两种、任意三种、任意四种、任意五种或任意六种在(1)~(19)中任一项中的应用;
(f1)PTBP2蛋白;
(f2)编码PTBP2蛋白的核酸分子;
(f3)含有(f2)中所述核酸分子的表达盒、重组载体或转基因细胞系;
(f4)促进PTBP2蛋白或编码PTBP2蛋白的核酸分子表达的物质;
(f5)促进PTBP2蛋白活性提高的物质;
(f6)单核细胞;
(1)制备预防和/或治疗神经母细胞瘤的药物;
(2)体外非治疗目的地促进CCL表达;
(3)制备促进CCL表达的试剂;
(4)体外非治疗目的地促进促炎性因子表达;
(5)制备促进促炎性因子表达的试剂;
(6)体外非治疗目的地抑制CD209表达;
(7)制备抑制CD209表达的试剂;
(8)体外非治疗目的地促进CD80表达;
(9)制备促进CD80表达的试剂;
(10)体外非治疗目的地诱导单核细胞向炎性巨噬细胞(CD11b+CD80+)分化;
(11)制备诱导单核细胞向炎性巨噬细胞(CD11b+CD80+)分化的试剂;
(12)体外非治疗目的地促进ISGF3复合物形成;
(13)制备促进ISGF3复合物形成的试剂;
(14)体外非治疗目的地促进STAT1表达;
(15)制备促进STAT1表达的试剂;
(16)体外非治疗目的地抑制神经母细胞瘤细胞增殖;
(17)制备制神经母细胞瘤细胞增殖;
(18)体外非治疗目的地抑制神经母细胞瘤细胞迁移;
(19)制备抑制神经母细胞瘤细胞迁移的试剂。
5.(g1)~(g4)中任意一种、任意两种、任意三种或任意四种作为靶点在开发神经母细胞瘤相关产品中的应用;
(g1)PTBP2;
(g2)CCL5;
(g3)IFNA2;
(g4)IFNB1。
6.根据权利要求5所述的应用,其特征在于:
所述产品为:
神经母细胞瘤预后评估的试剂、试剂盒、试纸或芯片;或
预防和/或治疗神经母细胞瘤的药物。
7.一种药物,包含:(f1)~(f5)中任意一种、任意两种、任意三种、任意四种或任意五种;
和(f6);
(f1)PTBP2蛋白;
(f2)编码PTBP2蛋白的核酸分子;
(f3)含有(f2)中所述核酸分子的表达盒、重组载体或转基因细胞系;
(f4)促进PTBP2蛋白或编码PTBP2蛋白的核酸分子表达的物质;
(f5)促进PTBP2蛋白活性提高的物质;
(f6)单核细胞。
8.根据权利要求7所述的药物,其特征在于:
所述药物还包含药学上可接受的辅料;
优选地,所述药物用于预防和/或治疗神经母细胞瘤;
优选地,所述药物的剂型为儿童适用的剂型;
优选地,所述儿童为出生28天内的新生儿、1岁以内的婴儿、1~6岁的幼儿、6~18岁的儿童中的至少一种;
优选地,所述药物的剂型为胶囊剂、片剂、微囊制剂、注射剂、栓剂、喷雾剂、散剂、软胶囊剂、滴丸、蜜丸、丸剂、颗粒剂、蜜炼膏剂、缓控释制剂、口服液体制剂、注射剂、咀嚼片、口腔片、透皮贴剂、泡腾片中的至少一种。
9.一种标志物组合,包含:(g1)~(g4)中任意两种、任意三种或任意四种:
(g1)PTBP2;
(g2)CCL5;
(g3)IFNA2;
(g4)IFNB1。
10.一种产品,包含:(a1)~(a4)中任意两种、任意三种或任意四种;
(a1)检测PTBP2的物质;
(a2)检测CCL5的物质;
(a3)检测IFNA2的物质;
(a4)检测IFNB1的物质。
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