CN112063718A - Usp14在肝细胞肝癌诊断、预后判断以及治疗中的应用 - Google Patents
Usp14在肝细胞肝癌诊断、预后判断以及治疗中的应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域，尤其是涉及USP14在肝细胞肝癌分子诊断、预后判断及药物与基因治疗的应用。本发明提供了一种与肝癌发生发展相关的分子标志物USP14和一种肝癌癌的诊断产品和手段，通过检测分子标志物的表达水平来判断患者是否患有肝癌的风险。并且提供一种治疗肝癌的药物组合或方法，通过特异性的下调分子标志物来治疗肝癌。实验表明采用基因手段下调USP14表达，能抑制裸鼠体内肝癌细胞的增殖。USP14抑制剂能达到与基因技术一样的肝癌抑制作用，且二者联合应用具有协同抗癌效果。
Description
技术领域
本发明属于生物医药技术领域，尤其是涉及USP14在肝细胞肝癌分子诊断、预后判断及药物与基因治疗的应用。
背景技术
我国是原发性肝癌(Hepatocellular carcinoma，HCC)大国，新发病例和死亡病例占全世界一半以上。由于HCC起病隐匿，早期诊断困难，大部分患者就诊时已进展至晚期，即使手术治疗，术后5年内复发率仍高达40％-60％。因此，深入研究HCC发生发展的分子机制并积极探索有效的治疗措施已成为HCC诊断和治疗亟待解决的问题。缺氧是包括HCC在内实体肿瘤微环境最重要的特征，可介导肿瘤增殖、转移、血管生成等恶性生物学行为并导致患者预后不良。研究发现应用Sorafenib(FDA批准HCC一线用药)能进一步加剧肿瘤内部缺氧，可能是HCC产生Sorafenib治疗抵抗的重要原因。HIF1-α作为重要的氧调节因子，与HCC病人预后及耐药关系密切。因此，发现新的影响HCC低氧环境下生物学行为及调控HIF1-1a功能的新的分子意义重大。
去泛素化酶USP14(ubiquitin specific processing enzyme 14，USP14)属于泛素特异性蛋白酶家族(USPs)，是该家族中唯一一个与26S蛋白酶体复合物相互作用的蛋白酶，它通过与蛋白酶体19S调节颗粒中的Rpn1可逆性结合，极大地增强了其去泛素化作用。近些年来研究表明USP14在实体瘤中可能是重要的促癌因子，抑制USP14的功能能延长荷瘤动物模型的生存期。然而，USP14在肝细胞肝癌中的生物学功能及作用机制尚不明确。
发明内容
鉴于现有技术存在的问题，本发明的目的在于提供USP14在肝细胞肝癌诊断、预后判断以及治疗中的应用。本发明提供了一种与肝癌发生发展相关的分子标志物USP14和一种肝癌的诊断产品和手段，通过检测分子标志物的表达水平来判断患者是否患有肝癌的风险。并且提供一种治疗肝癌的药物组合或方法，通过特异性的下调分子标志物来治疗肝癌。
为了实现上述目的，本发明采用以下技术方案。
检测USP14表达的试剂在制备诊断和/或预后判断肝癌的产品中的应用。
进一步地，所述试剂选自：特异性识别USP14的探针；或特异性扩增USP14的引物。
进一步地，所述的特异性扩增USP14的引物序列如SEQ ID NO.1-SEQ IDNO.2所示。
进一步地，所述产品所述产品包括通过测序技术、核酸杂交技术、核酸扩增技术检测样本中USP14的表达水平的试剂，所述产品包括但不限于试剂、试剂盒、芯片、试纸、高通量测序平台。
USP14抑制剂在制备预防或治疗肝癌的药物及药物组合物中的应用。
进一步地，所述药物组合物包括USP14抑制剂。
进一步地，所述抑制剂选自：以USP14或其转录本为靶序列、且能够抑制USP14表达或转录的干扰分子，包括：shRNA、小干扰RNA、dsRNA、微小RNA、反义核酸，或能表达或形成所述shRNA、小干扰RNA、dsRNA、微小RNA、反义核酸的构建物。
优选的，所述shRNA序列如SEQID NO.1-SEQ ID NO.2所示。
进一步，所述药物组合物还包括与所述抑制剂配伍的其他药类以及药学上可接受的载体和/或辅料。
USP14在筛选预防或治疗肝癌的潜在物质中的应用。
本发明的抑制剂可以通过本领域任何已知的方式配制药物组合物来使用。这种组合物包含活性成分，加上一种或多种药物可接受的载体、稀释剂、填充剂、结合剂及其它赋形剂，这依赖于给药方式及所设计的剂量形式。本领域枝术人员已知的治疗惰性的无机或有机的载体包括但不限于乳糖、玉米淀粉或其衍生物、滑石、植物油、蜡、脂肪、多羟基化合物例如聚乙二醇、水、蔗糖、乙醇、甘油，诸如此类，各种防腐剂、润滑剂、分散剂、矫味剂。保湿剐、抗氧化剂、甜味剂、着色剂、稳定剂、盐、缓冲液诸如此类也可加入其中，这些物质根据需要用于帮助配方的稳定性或有助于提高活性或它的生物有效性或在口服的情况下产生可接受的口感或气味，在这种组合物中可以使用的制剂可是其原始化合物本身的形式，或任选地使用其药物学可接受的盐的形式，本发明的抑制剂可以单独给药，或以各种组合给药，以及与其它治疗药剂一起结合形式给药。如此配制的组合物根据需要可选择本领域技术人员已知的任何适当的方式把抑制剂或激活剂进行给药。使用药物组合物时，是将安全有效量的本发明的抑制剂施用于人，其中口服安全有效量通常至少约100微克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的药物可根据需要制备成各种剂型。包括但不限于，经皮、粘膜、鼻、口颊、舌下或经口使用的片剂、溶液剂、颗粒剂、贴剂、膏剂、胶囊剂、气雾剂或栓剂。
本发明的药物的施用途径不受限制，只要它能发挥期望的治疗效果或预防效果即可，包括但不限于静脉内，腹膜内，动脉内，口服，肌肉内，皮下的。在某些情况下，可以系统地给药。在某些情况下是局部地给药。
本发明的药物的剂量不受限制，只要获得期望的治疗效果或者预防效果即可，可以依据症状、性别、年龄等来恰当的确定。本发明的治疗药物或预防药物的剂量可以使用例如对疾病的治疗效果或者预防效果作为指标来确定。
附图说明
图1是免疫组化法比较USP14在HCC组织中表达显著高于正常肝组织中的表达。
图2是生存分析提示HCC患者中USP14表达高的病人总生存率(OS)低于USP14表达低的病人——即预后较差。
图3是裸鼠皮下注射HCC细胞，慢病毒介导下调USP14表达组荷瘤体积小于对照组。
图4是口服USP14抑制剂IU1能显著抑制裸鼠皮下成瘤体积，且与下调USP14表达的慢病毒协同应用，能进一步抑制肿瘤生长。
具体实施方式
下面结合附图和实施例对本发明作进一步详细的说明。实施例中未注明具体条件的实验方法，通常按照常规条件，例如教科书和实验指南中所述的条件，或按照制造厂商所建议的条件，为本领域普通技术人员熟知或易于获知，以下实施例仅为本发明的优选实施例，并不限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
实施例1。
一、材料方法。
1.USP14在HCC中的表达及其与临床病理特征之间的关。
收集约100例临床标本，应用免疫组化方法检测USP14表达情况，根据标本对应的临床资料及预后情况，分析USP14表达与临床病理特征及病人预后的关系。
2.USP14在小鼠体内对HCC生长的影响。
将带有USP14和shUSP14慢病毒感染HCC-LM3细胞，对4周龄，体重18-22g的雄性裸鼠进行皮下原位注射。2周后，再将所有鼠分为两组，按40mg/kg/d IU1灌胃给药，连续给药2周。分别于皮下注射2周和4周后观察并检测裸鼠体内肿瘤大小，来评估USP14及其抑制剂IU1在小鼠体内对HCC生长影响。
设计针对USP14的引物：USP14 shRAN序列：正义链：TGTGCCTGAACTCAAAGATGC；反义链：ATATACTGCGCTGAAGCCATTT。
USP14慢病毒病毒相关信息见表1。
表1.USP14慢病毒病毒相关信。
二、实验结果。
1.USP14在HCC病灶中表达显著高于正常肝组织，如图1所示。
2.HCC病人中，USP14表达高的人群5年总生存率显著低于USP14表达低的人群，如图2所示。
3.裸鼠皮下注射人来源HCC细胞，基因手段下调USP14表达组的成瘤体积显著小于对照组，如图3所示。
4.在结果3基础上，口服USP14抑制剂IU1(小鼠灌胃)，能显著抑制皮下成瘤大小，其中与基因敲减USP14联合应用组，荷瘤体积进一步得到抑制，如图4所示。
三、实验结论。
1.USP14可能成为HCC早期诊断的新型分子标记物。
2.USP14的蛋白表达水平水平能判断HCC病人的预后，即USP14表达与患者预后呈负相关。
3.采用基因手段下调USP14表达，能抑制裸鼠体内肝癌细胞的增殖。
4.USP14抑制剂IU1能达到与基因技术一样的肝癌抑制作用，且二者联合应用具有协同抗癌效果。
序列表
<110> 中国医科大学
<120>USP14在肝细胞肝癌诊断、预后判断以及治疗中的应用
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> 人工序列
<400> 1
TGTGCCTGAA CTCAAAGATG C 21
<210> 2
<211>
<212> DNA
<213> 人工序列
<400> 2
ATATACTGCG CTGAAGCCAT TT 22
Claims (10)
1.检测USP14表达的试剂在制备诊断和/或预后判断肝癌的产品中的应用。
2.如权利要求1所述的应用，其特征在于，所述试剂选自特异性识别USP14的探针或特异性扩增USP14的引物。
3.如权利要求2所述的应用，其特征在于，所述的特异性扩增USP14的引物序列如SEQIDNO .1-SEQ ID NO .2所示。
4.如权利要求1所述的应用，其特征在于，所述产品包括通过测序技术、核酸杂交技术、核酸扩增技术检测样本中USP14的表达水平的试剂，所述产品包括但不限于试剂、试剂盒、芯片、试纸、高通量测序平台。
5.USP14抑制剂在制备预防或治疗肝癌的药物及药物组合物中的应用。
6.如权利要求5所述的应用，其特征在于，所述药物组合物包括USP14抑制剂。
7.如权利要求5所述的应用，其特征在于，所述抑制剂选自：以USP14或其转录本为靶序列、且能够抑制USP14表达或转录的干扰分子，包括：shRNA、小干扰RNA、dsRNA、微小RNA、反义核酸，或能表达或形成所述shRNA、小干扰RNA、dsRNA、微小RNA、反义核酸的构建物。
8.如权利要求7所述的应用，其特征在于，所述shRNA序列如SEQID NO .1-SEQ ID NO.2所示。
9.如权利要求5所述的应用，其特征在于，所述药物组合物还包括与所述抑制剂配伍的其他药类以及药学上可接受的载体和/或辅料。
10.USP14在筛选预防或治疗肝癌的潜在物质中的应用。
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