CN117568347B - Ppef1作为神经母细胞瘤药物靶点的应用 - Google Patents
Ppef1作为神经母细胞瘤药物靶点的应用 Download PDFInfo
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Abstract
本发明属于基因诊断和治疗领域，具体涉及PPEF1作为神经母细胞瘤药物靶点的应用。本发明提供了一种磷酸酶PPEF1基因及其编码产物在MYCN扩增神经母细胞瘤中的应用，可以作为MYCN扩增神经母细胞瘤的治疗及药物筛选靶点，对MYCN扩增神经母细胞瘤治疗及药物筛选提供了新的思路。
Description
技术领域
本发明属于基因诊断和治疗领域，具体涉及PPEF1作为神经母细胞瘤药物靶点的应用。
背景技术
神经母细胞瘤（Neuroblastoma，NB）起源于早期神经嵴前体细胞，是儿童最常见的颅外恶性实体肿瘤，常见发病部位是肾上腺。该病发病隐秘，恶性程度高，预后差。根据神经母细胞瘤国际委员会危险度分组可将神经母细胞瘤分为高危，中危，低危和极低危四种。目前NB肿瘤的主要治疗方式是手术切除和放化疗，高危组NB 患儿还可接受自体外周血造血干细胞移植等其他治疗，然而高危NB患儿五年生存率依然不足50%。
MYCN基因属于MYC家族成员，编码转录因子N-Myc，通过转录下游系列靶基因，调控细胞增殖、分化及凋亡等重要生物学过程。研究发现MYCN基因在多种肿瘤存在扩增，如神经母细胞瘤、视网膜母细胞瘤、肺癌、肾癌等。动物实验表明，MYCN转基因小鼠容易自发生成神经母细胞瘤，表明MYCN是NB的致癌基因。研究表明MYCN基因（转录因子，编码癌蛋白N-MYC）多扩增于高危NB患儿，与这些患儿的预后不良密切相关。然而研究发现由于MYCN基因编码蛋白N-Myc缺乏结合药物的“口袋”结构，N-MYC蛋白质很难被靶向，是公认的难以成药靶点，目前尚缺乏直接靶向N-Myc蛋白质活性的抑制剂。因此，越来越多的科学家聚焦N-Myc蛋白质的间接靶向策略，以期实现对MYCN扩增型高危NB的治疗。有研究发现LIN28B和Aurora A激酶可调控N-Myc蛋白质稳定性，且Aurora A抑制剂可在小鼠水平抑制MYCN扩增型高危NB的发生，但由于高剂量抑制剂毒性较大导致其在临床试验中疗效受限。另外，新型反义寡核苷酸可通过靶向N-Myc蛋白质降解，从而抑制MYCN扩增型高危神经母细胞瘤的生长，但其应用前景还需临床试验验证。
因此，针对N-Myc蛋白质直接靶向策略缺乏、现有间接靶向策略存在弊端等现状，亟需探索MYCN扩增型高危NB治疗的新策略、新靶标。
发明内容
本发明发现磷酸酶PPEF1(GENEID:5475)在MYCN扩增NB细胞系高表达，而在MYCN非扩增NB细胞中几乎不表达，与MYCN的表达一致。深入功能研究，发现过表达PPEF1促进MYCN非扩增NB细胞的增殖，而敲低PPEF1明显抑制MYCN扩增NB细胞增殖和MYCN扩增NB肿瘤的发生。以上结果表明，磷酸酶PPEF1促进MYCN扩增高危NB增殖和肿瘤发生，是高危NB的潜在治疗新靶标。
本发明提供了一种磷酸酶PPEF1基因及其编码产物在MYCN扩增神经母细胞瘤中的应用，可以作为MYCN扩增神经母细胞瘤的治疗及药物筛选靶点，对MYCN扩增神经母细胞瘤治疗及药物筛选提供了新的思路。
本发明提供抑制PPEF1基因表达的试剂在制备治疗MYCN扩增神经母细胞瘤的药物中的应用。
根据本发明具体实施方式抑制PPEF1基因表达的试剂的应用，所述抑制PPEF1基因表达的试剂包括特异靶向PPEF1基因的shRNA或siRNA，siRNA的核苷酸序列如SEQ ID NO.1、SEQ ID NO.2所示，
SEQ ID NO.1:5’-GAAACTCTGTACAGATACA-3’；
SEQ ID NO.2:5’-GTACGGATATTGATTTACT-3’。
本发明还提供PPEF1磷酸酶的抑制剂在制备治疗MYCN扩增神经母细胞瘤的药物中的应用。
根据本发明具体实施方式的PPEF1磷酸酶的抑制剂的应用，所述抑制PPEF1基因表达的试剂包括特异靶向PPEF1基因shRNA或siRNA。
siRNA的核苷酸序列如SEQ ID NO.1、SEQ ID NO.2所示，
SEQ ID NO.1:5’-GAAACTCTGTACAGATACA-3’；
SEQ ID NO.2:5’-GTACGGATATTGATTTACT-3’。
本发明还提供检测PPEF1基因的试剂在制备检测MYCN扩增神经母细胞瘤的试剂中的应用。
根据本发明具体实施方式的治疗MYCN扩增神经母细胞瘤的药物，其含有以下任一或多种：
(1)含有PPEF1基因或含有PPEF1基因的生物材料，所述生物材料为表达盒、表达载体、质粒、宿主菌、宿主细胞；
(2)含有使PPEF1基因在机体内表达量下调的试剂；
(3)抑制或阻断PPEF1基因在机体内表达的试剂。
根据本发明具体实施方式的治疗MYCN扩增神经母细胞瘤的药物，其特征在于，包括特异靶向PPEF1基因的shRNA或siRNA。
本发明中，通过所述抑制或降低待抑制活性蛋白的编码基因表达来实现，具体的，可通过基因敲除或通过基因沉默实现。
所述基因敲除是指通过同源重组使特定靶基因失活的现象。基因敲除是通过DNA序列的改变使特定靶基因失活。
所述基因沉默是指在不损伤原有DNA的前提下使基因不表达或低表达的现象。基因沉默可发生在两种水平上，一种是由于DNA甲基化、异染色质化以及位置效应等引起的转录水平的基因沉默，另一种是转录后基因沉默， 即在基因转录后的水平上通过对靶标RNA进行特异性抑制而使基因失活，包括反义RNA、共抑制、基因压抑、RNA干扰（RNAi）和微小RNA（miRNA）介导的翻译抑制等。
本发明的有益效果：
本发明证明磷酸酶PPEF1在MYCN扩增NB细胞中特异性高表达，而在MYCN非扩增NB中表达很低，从而，磷酸酶PPEF1与MYCN表达一致；在MYCN扩增NB细胞中，敲低PPEF1减弱MYCN扩增NB细胞的增殖和克隆形成能力；在MYCN非扩增NB细胞中，过表达PPEF1显著促进MYCN非扩增NB细胞的增殖能力，从而，磷酸酶PPEF1对MYCN扩增NB细胞恶性增殖是需要的，是NB细胞恶性增殖的新驱动基因，由于PPEF1是个酶分子，提示其可能成为MYCN扩增高危NB的新治疗靶标。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1显示MYCN和PPEF1在7种细胞中的RNA水平；
图2为敲低PPEF1对MYCN扩增NB细胞增殖的影响情况；其中,
A为细胞明场以及结晶紫染色的细胞数目；
B为qRT-PCR方法检测的PPEF1的敲低结果；
图3显示敲低PPEF1对MYCN扩增NB细胞克隆形成能力的影响情况；
图4显示过表达PPEF1对MYCN非扩增NB细胞增殖的影响；其中，
A为细胞明场以及结晶紫染色条件下细胞数量情况；
B为Western 印迹法检测PPEF1蛋白表达水平情况。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将对本发明的技术方案进行详细的描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式，都属于本发明所保护的范围。
实施例 1
选取4种MYCN 扩增NB细胞系MYCN Amp（SK-N-BE(2)、BE2C、IMR32、BE2-M17）、3种MYCN 非扩增NB细胞系MYCN Non（SK-N-AS、SH-SY5Y、SK-N-SH）细胞系，培养2天后，使用TRIzol试剂裂解细胞，并分别提取7种细胞的总RNA，用PrimeScript RT Master Mix反转录试剂盒合成cDNA，合成体系为RNA 1 μg，5×PrimeScript Mix 2 μL，RNase-free水补至10μL。
按照SYBR Green PCR Master Mix试剂盒说明书进行实时荧光定量PCR扩增检测。反应条件为95℃ 10 min，95℃ 15 s，60℃ 1 min，40个循环，读取Ct 值。
以 GAPDH 为内参，Q-PCR技术检测其中MYCN和PPEF1的RNA水平。
结果如图1所示，PPEF1在MYCN 扩增NB细胞系高表达，而在MYCN 非扩增NB细胞系中则不表达，PPEF1与MYCN的表达趋势一致。
实施例 2
6孔培养板每孔接种2.4×105个MYCN 扩增NB细胞系SK-N-BE(2)细胞。
待细胞密度达到20%-30%，按照Lipofectamine RNAi MAX说明书步骤进行siRNA转染。设置靶向荧光素酶报告基因对照siRNA (siCtrl) 组，利用两种不同序列的siRNA（siPPEF1-2#, siPPEF1-3#），其靶序列为GAAACTCTGTACAGATACA和GTACGGATATTGATTTACT敲低PPEF1,siRNA的转染终浓度为60 nmol·L-1。
处理72 h后，显微镜观察细胞明场以及结晶紫染色的细胞数目，用qRT-PCR方法检测PPEF1的敲低后PPEF1的表达结果。
结果如图2所示，图2A中，siRNA转染后，细胞数量明显减少，且siPPEF1-2#的效果优于siPPEF1-3#；图2B中，siRNA转染后,SK-N-BE(2)细胞中PPEF1的表达下调。
从而，PPEF1敲低导致MYCN扩增NB细胞的增殖能力显著减弱。
实施例 3
构建稳定敲低PPEF1的MYCN扩增NB细胞（SK-N-BE2）:
利用与siRNA相同的靶序列，构建靶向敲低 PPEF1的慢病毒shRNA。将携带靶点序列的工具载体质粒GV654（对照shCtrl为空载质粒）、病毒包装辅助质粒Helper 1.0和Helper 2.0共转染293T细胞。转染后48-72h收集细胞上清液，并浓缩纯化得到高滴度慢病毒保存液。
将4.8×105个SK-N-BE(2)细胞接种于6 cm细胞培养皿内，培养24 h，至细胞汇合度为20%-30%，细胞换液成DMEM完全培养基和HitransG P的混合液，病毒以MOI=10的感染复数感染细胞。
克隆形成实验：
病毒感染24 h后将稳定敲低对照（shCtrl）组、实验组shPPEF1-954#和shPPEF1-955#细胞以每孔600个细胞接种到六孔板中，每组设置3个复孔。
每隔3天更换培养基并观察细胞状态。第14天在显微镜下观察到直径＞1 mm的细胞集落，终止培养。
使用4%多聚甲醛室温固定细胞15 min，结晶紫染色15 min，PBS洗涤细胞，晾干并拍照。统计细胞集落个数，检测该细胞的克隆形成能力，用qRT-PCR方法检测PPEF1的表达结果。
结果如图3所示，感染病毒后，SK-N-BE(2)细胞中PPEF1的表达下调，表明靶向PPEF1这两条shRNA均能有效敲低PPEF1。克隆形成实验结果显示，与对照shCtrl组相比，shPPEF1-954#和shPPEF1-955#组细胞集落数明显减少，抑制NB细胞克隆形成能力。
稳定敲低PPEF1后可抑制MYCN扩增NB细胞SK-N-BE(2)细胞生长。
实施例 4
构建稳定过表达PPEF1的MYCN非扩增NB细胞（SY5Y）：
将PPEF1编码区序列（CDS）克隆到病毒载体GV492上制备目的质粒载体（对照oe-Con335为空载质粒），并与病毒包装辅助质粒Helper 1.0和Helper 2.0共转染293T细胞，转染后48-72h收集细胞上清液，浓缩纯化得到高滴度慢病毒保存液。
将2×105个SY5Y细胞接种于12孔板每个孔内，培养24h，至细胞汇合度为60%-70%。细胞换液成DMEM完全培养基和HitransG P的混合液，病毒以MOI=20的感染复数感染细胞。细胞长满后连续传代培养。
病毒感染8天后，显微镜拍摄对照组（oe-Con335）和实验组（oe- PPEF1）细胞明场以及结晶紫染色的细胞数目。常规提取蛋白质样品，用Western 印迹法检测PPEF1蛋白表达水平，β-tubulin为内参蛋白。
结果如图4所示，图4A中，过表达PPEF1后，细胞数量明显增加。图4B中，稳定过表达细胞株PPEF1蛋白水平明显高于对照组。稳定过表达PPEF1可以明显促进MYCN非扩增NB细胞的增殖能力。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。
Claims (1)
1. 抑制PPEF1基因表达的试剂在制备治疗MYCN扩增神经母细胞瘤的药物中的应用，其特征在于：所述抑制PPEF1基因表达的试剂为特异性靶向PPEF1基因的siRNA，siRNA的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
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