CN115927618A - Snora14a在肝母细胞瘤诊断和治疗中的应用 - Google Patents
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Abstract
本发明涉及SNORA14A在肝母细胞瘤诊断和治疗中的应用。本发明通过在4对肝母细胞瘤组织和癌旁正常肝组织中进行snoRNA测序分析,发现SNORA14A在肝母细胞瘤中显著低表达,并进一步在患者血浆中得到验证,表明其可作为肝母细胞瘤的诊断标志物。同时发现过表达SNORA14A后明显抑制肝母细胞瘤细胞的增殖和克隆形成能力,促进细胞凋亡,表明SNORA14A可作为潜在的抗癌药物靶点加以应用。本发明为肝母细胞瘤的诊断和治疗提供了新的临床手段。
Description
技术领域
本发明涉及生物医药技术领域,具体地说,涉及SNORA14A在肝母细胞瘤诊断和治疗中的应用。
背景技术
肝母细胞瘤(Hepatoblastoma,HB)是儿童最常见的肝脏恶性肿瘤,起源于产生肝脏上皮成分的肝脏干细胞。目前,肝切除术、新辅助化疗和肝移植是HB的主要治疗手段。尽管近年来早期患者的5年生存率提高到70%以上,但由于HB起病隐匿、肿瘤生长迅速,并且缺乏特异的诊断标志物,部分患者就诊时已处于进展期,无法切除肿瘤。广谱化疗药物对这类患者效果很差,严重影响了患者的预后。甲胎蛋白(α-fetoproteinprotein,AFP)是目前临床唯一可用的HB血清诊断标志物,在近90%的患者血清中可检测到AFP水平显著升高。但是,婴幼儿血清AFP基线水平较高,并且在儿童肝细胞癌以及良性肝肿瘤中也明显增加,对于HB的鉴别诊断缺乏特异性。此外,β-catenin、整合酶相互作用分子1和磷脂酰肌醇蛋白聚糖3等免疫组化标志物特异性同样不佳。目前,HB的血清诊断标志物和治疗靶点都非常有限。因此,迫切需要深入研究调控HB发生发展的分子机理,开发兼具敏感性和特异性的血清诊断标志物,挖掘治疗干预的新策略,从而在疾病的早期对其进行干预治疗,提高HB患者的治愈率和生存质量。
SNORA14A是来源于细胞色素P450氧化还原酶基因第1个内含子的box H/ACA核仁小RNA(small nucleolar RNA,snoRNA)。SNORA14A的相关研究鲜少,仅有一篇报道(Kiss,A.M.,et al.,Humanbox H/ACApseudouridylation guide RNAmachinery.Mol Cell Biol,2004.24(13):p.5797-807)证实其能够指导18S rRNAU966位点发生假尿嘧啶修饰。尽管目前尚未有文献报道过snoRNAs与HB发生发展的相关性,但很多研究表明有相当数目的snoRNAs与肿瘤发生发展密切相关。例如,文献(Wang,H.,et al.,Small nucleolar RNAU2_19promotes hepatocellular carcinoma progression by regulating Wnt/β-catenin signaling.Biochem Biophys Res Commun,2018.500(2):p.351-356)和(Wu,L.,et al.,Clinical significance ofC/D box small nucleolar RNA U76 as an oncogeneand aprognostic biomarker in hepatocellular carcinoma.Clin Res HepatolGastroenterol,2018.42(1):p.82-91)报道了snoU2_19和SNORD76通过活化Wnt/β-catenin信号通路促进肝癌细胞增殖和迁移。Cui等证实在荷瘤裸鼠体内注射反义寡核苷酸干扰SNORA23表达,能够显著抑制胰腺导管腺癌的生长、播散与肝转移,提示SNORA23有望成为胰腺癌的治疗新靶点(Cui,L.,et al.,Small Nucleolar Noncoding RNA SNORA23,Up-Regulated in Human Pancreatic Ductal Adenocarcinoma,Regulates Expression ofSpectrin Repeat-Containing Nuclear Envelope 2to Promote Growth and Metastasisof Xenograft Tumors in Mice.Gastroenterology,2017.153(1):p.292-306.e2)。中国专利文献CN110229912A,公开日2019.09.13,公开了一种用于肾透明细胞癌诊断的循环snoRNA生物标志物和试剂盒及用途,所述循环snoRNA生物标志物有如下循环snoRNA组成:SNORA2、SNORD12B、SNORA59B、SNORA70B、SNORD93、SNORD116-2,所述的snoRNA生物标志物可以有效用于肾透明细胞癌诊断和预后。ACA11在肝细胞癌组织中显著高表达,具有较好的诊断效能(AUC=0.81);还有研究报道(Wu,L.,et al.,Small nucleolar RNA ACA11promotes proliferation,migration and invasion in hepatocellular carcinoma bytargeting the PI3K/AKT signaling pathway.Biomed Pharmacother,2017.90:p.705-712)其表达的增加与患者生存时间减少密切相关,可作为肝细胞癌的预后标志物。snoRNAs表达非常稳定,在肿瘤患者的血浆、血清、尿液等体液中都能检测到,是一类非常有潜力的新型生物标志物。然而,目前未见SNORA14A在肝母细胞瘤诊断和治疗中的应用。
发明内容
本发明的目的是针对现有技术中的不足,提供SNORA14A在肝母细胞瘤诊断和治疗中的应用。
第一方面,本发明提供了SNORA14A转录本作为诊断标志物在制备肝母细胞瘤诊断试剂或试剂盒中的应用。
第二方面,本发明提供了检测SNORA14A转录本含量的试剂在制备肝母细胞瘤诊断试剂或试剂盒中的应用。
作为本发明的一个优选例,所述检测SNORA14A转录本含量的试剂选自特异性扩增SNORA14A基因的引物。
作为本发明的另一优选例,所述肝母细胞瘤诊断试剂或试剂盒包括:核酸抽提试剂;和/或聚合酶链反应试剂。
作为本发明的另一优选例,所述肝母细胞瘤诊断试剂或试剂盒的检测样品为组织、血浆、血清或尿液。
第三方面,本发明提供了SNORA14A基因或其转录本的补充剂在制备治疗肝母细胞瘤的药物中的应用。
作为本发明的一个优选例,所述补充剂选自生物大分子。
更优选地,所述生物大分子选自能够输送SNORA14A基因或其转录本到靶部位的逆转录病毒载体、腺病毒载体、腺相关病毒载体、阳离子脂质体、阳离子聚合物。
更优选地,所述生物大分子为SNORA14A基因慢病毒表达载体(SNORA14A-pGMLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro),SNORA14A基因序列如SEQ ID NO:3所示。
第四方面,本发明提供了一种筛选治疗肝母细胞瘤的潜在物质的方法,包括:(1)用候选物质处理不表达或低表达SNORA14A的体系;(2)检测所述体系中SNORA14A的表达;若所述候选物质可增加SNORA14A的表达,则表明该候选物质是需要的潜在物质,反之则表明该候选物质是非需要的潜在物质。
需要说明的是,所述“补充剂”是指能够提高SNORA14A的表达量或活性的物质,达到增强SNORA14A功能的目的,所述的补充剂包括能够输送SNORA14A基因或其转录本到靶部位的逆转录病毒载体、腺病毒载体、腺相关病毒载体、阳离子脂质体、阳离子聚合物等生物大分子。
本发明优点在于:
1、本发明通过在4对HB组织和癌旁正常肝组织中进行snoRNA测序分析,发现SNORA14A在HB中表达明显减少,并进一步在患者血浆中得到验证,表明其可作为诊断HB的标志物,改善了HB诊断标志物匮乏的现状。且检测标本可以是组织、血浆、血清或尿液,来源丰富,易于实现。
2、本发明发现过表达SNORA14A明显抑制HB细胞的增殖和克隆形成能力,并促进细胞凋亡,因此SNORA14A可作为HB治疗的新靶点。总的来说,本发明为HB的临床诊断和治疗提供了新的策略和方法。
附图说明
附图1.Heatmap图表示HB组织和癌旁正常肝组织中snoRNA差异表达情况。
附图2.火山图表示HB组织和癌旁正常肝组织中snoRNA差异表达情况。
附图3.GO富集分析统计差异表达snoRNA宿主基因的功能。
附图4.qRT-PCR验证SNORA14A在4对用于测序的HB组织和癌旁正常肝组织中的表达。
附图5.SNORA14A在HB组织、血浆及细胞系中表达情况。
A.qRT-PCR检测35例HB患者肿瘤组织和癌旁正常肝组织中SNORA14A表达情况;
B.qRT-PCR检测各8例正常儿童和HB患者血浆中SNORA14A表达情况;
C.qRT-PCR检测QSG-7701、HepG2和HuH6细胞中SNORA14A表达情况。
附图6.过表达SNORA14A对HB增殖和凋亡的影响。
A.qRT-PCR检测SNORA14A基因慢病毒表达载体转染后HepG2和HuH6细胞中SNORA14A表达情况;
B.CCK-8实验检测SNORA14A基因慢病毒表达载体转染后HepG2和HuH6细胞的增殖情况;
C.克隆形成实验检测SNORA14A基因慢病毒表达载体转染后HepG2和HuH6细胞的克隆形成数;
D.流式细胞术检测SNORA14A基因慢病毒表达载体转染后对HepG2和HuH6细胞凋亡的影响。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。
实施例1
1、材料与方法
1.1组织与细胞
人正常肝细胞QSG-7701(目录号:C275)和人肝母细胞瘤细胞HepG2(目录号:SCSP-510;标识符:CSTR:19375.09.3101HUMSCSP510)和HuH6(目录号:TCHu181;标识符:CSTR:19375.09.3101HUMTCHu181)购买自中国科学院典型培养物保藏委员会细胞库。QSG-7701、HuH6和HepG2细胞分别用RPMI 1640完全培养基、DMEM高糖完全培养基和α-MEM完全培养基进行培养,放置在37℃、5%CO2培养箱中。HB患者肿瘤组织及配对癌旁正常肝组织标本,患者和正常儿童血浆标本来自于上海儿童医学中心生物样本库,所有样本已取得患者同意,并通过伦理委员会批准。
1.2 snoRNA测序
收集4例术前未接受放化疗的HB手术患者的肿瘤组织(HB)和配对癌旁正常肝组织(NT),提取总RNA,通过琼脂糖凝胶电泳检测组织RNA的完整性,采用NanoDrop ND-1000分光光度计检测组织RNA浓度和纯度。将待测序RNA依次与3’小RNA接头和5’小RNA接头进行连接反应,然后进行cDNA合成和PCR扩增,通过聚丙烯酰胺凝胶电泳纯化~170-370bp PCR扩增片段(对应~50-250nt小RNAs),使用Agilent BioAnalyzer2100仪器进行文库质检和定量。使用0.1M NaOH将上述文库变性为单链DNA分子,使用NovaSeq 6000S4 Reagent Kit进行簇生成,使用Illumina NovaSeq 6000平台进行测序。使用FastQC软件进行测序质量分析,通过Illumina chastityfilter的raw data用于后续分析。使用NovoAlign(2.07.11)软件将trimmed reads比对到Ensembl数据库。差异表达snoRNAs的阈值设定为两组间TPM值log2fold change>0.585或log2 fold change<-0.585,且p<0.05。对差异表达的snoRNAs进行层次聚类,并绘制散点图。
1.3实时荧光定量PCR(qRT-PCR)检测SNORA14A表达
采用实时荧光定量聚合酶链反应检测SNORA14A的表达,提取组织、血浆或细胞总RNA后通过RNA反转录获取cDNA产物,以cDNA为模板进行qRT-PCR分析,SNORA14A引物序列如下:上游引物5’-TTGGTGGCTTCCCTGTAAATG-3’(SEQ ID NO:1),下游引物5’-GAAATAAGACTGAGCCACAGGAG-3’(SEQ ID NO:2)。
1.4构建稳定过表达SNORA14A细胞株
将人工合成的SNORA14A基因克隆到慢病毒载体pGMLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro上,SNORA14A基因序列如下:TTGCATTCTTAAACCCTCTTGGTGGCTTCCCTGTAAATGCTTCCAAGATATGAGCGAATGCTATAGAAATTGCAGGAAAGTCCAAAGGGCTGCGCGTCTCCTGTGGCTCAGTCTTATTTCATACCTGCAACATCT(SEQ ID NO:3)。接种293T细胞于10cm培养皿中,使转染时细胞密度达到80%。转染时按如下体系配制转染A液和B液,轻轻混匀,室温孵育5min。
转染体系
A液 | 加入量 | B液 | 加入量 |
OPTI-MEM培养基 | 970μl | psPAX2 | 12μg |
<![CDATA[Lipofectamine<sup>TM</sup>2000]]> | 30μl | pMD2.G | 6μg |
目的质粒 | 12μg | ||
OPTI-MEM培养基 | 补足至1ml |
将A液缓缓加入B液中,轻轻混匀,室温孵育20min。将上述转染混合液加入培养皿中,轻轻混匀,放入37℃、5%CO2培养箱培养。转染12h后更换8mL新鲜完全培养基,之后每24h收集一次病毒液,共收集3次,使用0.45μm滤器过滤病毒液。接种HepG2和HuH6细胞于6孔板中,使感染时细胞密度约为30%。每孔中加入2mL不含抗生素的完全培养基、10mL过滤后的病毒液和2μg/mL聚凝胺,轻轻混匀。感染48h后,更换新鲜完全培养基恢复1天,然后用2μg/mL嘌呤霉素进行筛选,对照组细胞(未感染细胞)完全死亡时终止筛选,之后用qRT-PCR检测SNORA14A表达情况。
1.5 CCK-8实验检测细胞增殖
胰酶消化处于对数生长期的细胞,并制成单细胞悬液。以每孔1000个细胞的密度接种于96孔板中,于37℃、5%CO2培养箱过夜培养。细胞贴壁后,弃去原有培养基,每孔加入含有10μL增强型CCK8溶液的100μL完全培养基,37℃孵育2h,用多功能酶标仪测定450nm波长处的吸光度值(OD450值),记为Day 0吸光度值。之后每隔48h测定一次OD450值,连续测定6天后绘制细胞增殖曲线。
1.6克隆形成实验检测细胞增殖
胰酶消化处于对数生长期的细胞,制成单细胞悬液。以每孔1000个细胞的密度接种于12孔板中,放入37℃、5%CO2培养箱中。连续培养7~10天,当形成肉眼可见的克隆时终止培养。弃去孔中培养基,用1X PBS缓冲液洗涤1次,弃去PBS。然后,每孔加入500μL 4%多聚甲醛溶液在室温下固定细胞20min。弃去固定液,每孔加入500μL 0.1%结晶紫染色液在室温下染色30min。弃去染色液,用ddH2O缓慢洗去残留的染色液,置于37℃培养箱中干燥。将12孔板的每个孔单独拍照,使用Image J软件计算克隆数量。
1.7流式细胞术检测细胞凋亡
胰酶消化细胞,室温800rpm离心5min,沉淀细胞,弃上清。加入1mL4℃预冷1X PBS缓冲液清洗细胞。再次800rpm离心5min,沉淀细胞,弃上清。用ddH2O将5X Binding Buffer稀释成1X工作液。每管样品中加入500μL1XBinding Buffer,5μlAnnexinV-APC和10μL 7-AAD,轻轻吹打混匀,室温下避光孵育5min。立即用流式细胞仪检测细胞凋亡,并用FlowJo软件分析结果。
1.8统计分析
SPSS Statistics 24.0(IBM)和GraphPad Prism 7.0(Graphpad Software)软件用于实验数据分析和图表绘制。独立样本t检验或配对样本t检验用于分析两组数据之间是否存在显著性差异。单因素方差分析One-wayANOVA(Bonferroni检验或Dunnett检验)用于分析三组及三组以上数据之间是否存在显著性差异。*p<0.05,**p<0.01或***p<0.001表示差异有统计学意义。
2、结果
2.1 snoRNA测序分析SNORA14A在HB中的表达
通过snoRNA测序分析4对HB组织和配对癌旁正常肝组织中的snoRNA表达情况,结果显示与正常肝组织组相比,在HB组织组中表达显著增加且log2fold change>0.585的snoRNAs有8个,表达显著下降且log2 fold change<-0.585的snoRNAs有64个(图1-2)。由于snoRNAs没有自己的功能数据库,无法对snoRNAs直接进行功能富集分析,因此我们对差异表达snoRNAs的宿主基因进行GO富集分析,从而间接反映这些差异表达snoRNAs在HB中可能发挥的生物学功能,结果显示这些宿主基因参与了细胞内转录后调控、翻译调控、核糖体功能、mRNA代谢过程等(图3)。测序结果显示,SNORA14A在64个表达下调的snoRNAs中下调倍数排名第一,log2 fold change为-2.522,引起我们的注意。随后,我们采用qRT-PCR方法在4对用于测序的HB组织和癌旁组织中验证SNORA14A表达,结果发现,与正常肝组织相比,SNORA14A的表达在4个HB组织中均显著下调(图4)。
2.2 SNORA14A在HB患者组织、血浆以及HB细胞系中表达显著减少
为了进一步验证SNORA14A在HB中的表达,我们在35对HB组织和癌旁正常肝组织中进行扩大样本量验证,结果发现,与正常肝组织相比,SNORA14A的表达在HB组织中明显减少(图5A)。接着,我们在各8例HB患者和正常儿童血浆中进行验证,结果表明SNORA14A在HB患者血浆中表达同样减少(图5B),提示血浆SNORA14A可作为HB诊断标志物。随后,我们检测了SNORA14A在人HB细胞株(HepG2和HuH6)和人正常肝细胞株(QSG-7701)中的表达水平,结果显示,与QSG-7701相比,SNORA14A在HB细胞中的表达水平明显下降(图5C)。
2.3 SNORA14A抑制HB发生发展
为了探索SNORA14A在HB中的生物学功能,我们首先构建了稳定过表达SNORA14A的HepG2和HuH6细胞,并采用qRT-PCR方法验证其过表达效率(图6A)。CCK8和克隆形成实验证实在HB细胞中过表达SNORA14A,能够显著抑制细胞的增殖和克隆形成能力(图6B-C)。流式细胞术结果表明过表达SNORA14A后HB细胞的早期凋亡和晚期凋亡均显著增加(图6D)。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
Claims (10)
1.SNORA14A转录本作为诊断标志物在制备肝母细胞瘤诊断试剂或试剂盒中的应用。
2.检测SNORA14A转录本含量的试剂在制备肝母细胞瘤诊断试剂或试剂盒中的应用。
3.根据权利要求2所述的应用,其特征在于,所述检测SNORA14A转录本含量的试剂选自特异性扩增SNORA14A基因的引物。
4.根据权利要求2所述的应用,其特征在于,所述肝母细胞瘤诊断试剂或试剂盒包括:核酸抽提试剂;和/或聚合酶链反应试剂。
5.根据权利要求1-4任一所述的应用,其特征在于,所述肝母细胞瘤诊断试剂或试剂盒的检测样品为组织、血浆、血清或尿液。
6.SNORA14A基因或其转录本的补充剂在制备治疗肝母细胞瘤的药物中的应用。
7.根据权利要求6所述的应用,其特征在于,所述补充剂选自生物大分子。
8.根据权利要求7所述的应用,其特征在于,所述生物大分子选自能够输送SNORA14A基因或其转录本到靶部位的逆转录病毒载体、腺病毒载体、腺相关病毒载体、阳离子脂质体、阳离子聚合物。
9.根据权利要求8所述的应用,其特征在于,所述生物大分子为SNORA14A基因慢病毒表达载体(SNORA14A-pGMLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro),SNORA14A基因序列如SEQ IDNO:3所示。
10.一种筛选治疗肝母细胞瘤的潜在物质的方法,包括:
(1)用候选物质处理不表达或低表达SNORA14A的体系;
(2)检测所述体系中SNORA14A的表达;若所述候选物质可增加SNORA14A的表达,则表明该候选物质是需要的潜在物质,反之则表明该候选物质是非需要的潜在物质。
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