CN116059238B - circ0000195在肝癌治疗、检测和预后中的应用 - Google Patents
circ0000195在肝癌治疗、检测和预后中的应用 Download PDFInfo
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Abstract
本发明提供了circ0000195在肝癌治疗、检测和预后中的应用。本发明经过研究首次证实，与癌旁组织相比，肝癌组织中circ0000195的表达量明显升高，临床样本资料分析发现circ0000195表达量高的肝癌患者术后易复发，无瘤生存率显著降低。因此，检测circ0000195表达水平的试剂可用于检测肝癌和/或评估肝癌患者的术后易复发及无瘤生存率。进一步地，抑制circ0000195的表达可以促进肝癌细胞的铁死亡，抑制肝癌细胞的增殖。因此，抑制circ0000195表达的试剂可用于制备治疗肝癌的药物。本发明为肝癌的治疗和预后评估提供了新的靶点、新的方案和思路。
Description
技术领域
本发明属于中医药技术领域，具体涉及circ0000195在肝癌治疗、检测和预后中的应用。
背景技术
肝癌即发生于肝脏的恶性肿瘤，可分为原发性肝癌和继发性肝癌两大类。其中，原发性肝癌是指肝细胞或肝内胆管上皮细胞发生的恶性肿瘤；继发性肝癌又称为转移性肝癌，是指全身多个器官起源的恶性肿瘤侵犯至肝脏。一般多见于胃、胆道、胰腺、结直肠、卵巢、子宫、肺、乳腺等器官恶性肿瘤的肝转移。
肝癌的治疗包括手术、放化疗、介入、靶向药物、免疫治疗等多种手段。根据肝癌的不同阶段酌情进行个体化综合治疗，是提高疗效的关键。治疗方法包括手术、肝动脉结扎、肝动脉化疗栓塞、射频、冷冻、激光、微波以及化疗和放射治疗等方法。生物治疗，中医中药治疗肝癌也多有应用。
目前原发性肝癌的病因及确切分子机制尚不完全清楚，认为其发病是多因素、多步骤的复杂过程，受环境和饮食双重因素影响。因此，完善肝癌的发病机制，寻找新的肝癌治疗和预后靶点，对于肝癌的个体化治疗以及解决耐药问题至关重要。
发明内容
基于此，本发明的目的在于提供circ0000195在肝癌治疗、检测和预后中的应用；circ0000195在肝癌组织中表达显著升高，且与肝癌患者术后复发和无瘤生存率明显相关。抑制circ0000195的表达可以促进肝癌细胞的铁死亡，抑制肝癌细胞的增殖。
为达到上述目的，本发明采用如下技术方案。
抑制circ0000195表达的试剂在制备治疗肝癌的药物中的应用，其特征在于，所述circ0000195的编码基因的核苷酸序列如SEQ ID NO.1所示。
在一些实施例中，所述抑制circ0000195表达的试剂为siRNA。
在一些实施例中，所述siRNA选自siRNA1、siRNA2和siRNA3中的至少一种；所述siRNA1包括如SEQ ID NO.2所示的正义链和如SEQ ID NO.3所示的反义链；所述siRNA2包括如SEQ ID NO.4所示的正义链和如SEQ ID NO.5所示的反义链；所述siRNA3包括如SEQ IDNO.6所示的正义链和如SEQ ID NO.7所示的反义链；所述SEQ ID NO.2～SEQ ID NO.7的3’端修饰有TT。
在一些实施例中，所述药物可以促进肝癌细胞铁死亡。
本发明还提供了一种治疗肝癌的药物，所述药物包含抑制circ0000195表达的试剂和药物学上可接受的辅料。
在一些实施例中，所述抑制circ0000195表达的试剂为siRNA。
在一些实施例中，所述siRNA1包括如SEQ ID NO.2所示的正义链和如SEQ ID NO.3所示的反义链；所述siRNA2包括如SEQ ID NO.4所示的正义链和如SEQ ID NO.5所示的反义链；所述siRNA3包括如SEQ ID NO.6所示的正义链和如SEQ ID NO.7所示的反义链；所述SEQID NO.2～SEQ ID NO.7的3’端修饰有TT。
本发明还提供了检测circ0000195表达水平的试剂在制备检测肝癌和/或评估肝癌患者预后的产品中的应用，其特征在于，所述circ0000195的编码基因的核苷酸序列如SEQ ID NO.1所示。
在一些实施例中，所述检测circ0000195表达水平的试剂包括如SEQ ID NO.8所示的上游引物和SEQ ID NO.9所示的下游引物。
本发明还提供了一种肝癌患者检测和/或预后评估试剂盒，所述试剂盒包含检测circ0000195表达水平的试剂；所述检测circ0000195表达水平的试剂包括如SEQ ID NO.8所示的上游引物和SEQ ID NO.9所示的下游引物。
本发明经过研究首次证实，与癌旁组织相比，肝癌组织中circ0000195的表达量明显升高，临床样本资料分析发现circ0000195表达量高的肝癌患者术后易复发，无瘤生存率显著降低。因此，检测circ0000195表达水平的试剂可用于检测肝癌和/或评估肝癌患者的术后复发及无瘤生存率。进一步地，抑制circ0000195的表达可以促进肝癌细胞的铁死亡，抑制肝癌细胞的增殖。因此，抑制circ0000195表达的试剂可用于制备治疗肝癌的药物。本发明为肝癌的治疗和预后评估提供了新的靶点、新的方案和思路。
附图说明
图1为circ0000195在肝癌组织中的表达(A)和无瘤生存率(B)分析图。
图2为siRNA对circ0000195的抑制作用(A)及抑制circ0000195的表达并用RSL3处理后的MTT检测(B)和活/死细胞染色(C)结果图；*：P<0.05，**：P<0.01，***：P<0.001，比例尺：200uM。
图3为质粒对circ0000195的过表达作用(A)及过表达circ0000195并用RSL3处理后的MTT检测(B)和活/死细胞染色(C)结果图；*：P<0.05，**：P<0.01，***：P<0.001，比例尺：200uM。
具体实施方式
本发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，或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂，均为市售产品。
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。
本发明的术语“包括”和“具有”以及它们任何变形，意图在于覆盖不排他的包含。例如包含了一系列步骤的过程、方法、装置、产品或设备没有限定于已列出的步骤或模块，而是可选地还包括没有列出的步骤，或可选地还包括对于这些过程、方法、产品或设备固有的其它步骤。
在本发明中提及的“多个”是指两个或两个以上。“和/或”，描述关联对象的关联关系，表示可以存在三种关系，例如，A和/或B，可以表示：单独存在A，同时存在A和B，单独存在B这三种情况。字符“/”一般表示前后关联对象是一种“或”的关系。
下面结合具体实施例进行说明。
实施例1
收集18对肝癌和癌旁组织，利用qRT-PCR检测circ0000195的表达量。
所述circ0000195的编码基因的核苷酸序列如SEQ ID NO:1所示：
SEQ ID NO.1：5’-AGTCATCCTTTTTGAACTTCCATGACTCAGACTGCGAACCCAAG GGATCATCACCCTGTGACTCCTTGCTTTCCCTCAACACTGAGAAGATTCTGAGCCAGGCCAAGTCTATTGCAGAACAGAAGAGATTCCCGTTTGCCACTGATAATGACAGCACAAATGAAGAGTTAGCTATTGCTTATGTCTTGATTGGCAGTGGTCTGTATGATGAAGCAATACGGCATTTTTCAACAATGCTTCAG-3’。
利用SYBR@Green试剂盒(购于艾科瑞生物，Cat No.AG11718)进行qRT-PCR检测，操作严格按照试剂盒说明书进行。检测引物包括如SEQ ID NO.8所示的上游引物和SEQ IDNO.9所示的下游引物。
SEQ ID NO.8：5’-TGGCAGTGGTCTGTATGATGAAG-3’；
SEQ ID NO.9：5’-CAAGGAGTCACAGGGTGATGATC-3’。
qRT-PCR检测结果如图1所示。由图1A可知，与癌旁组织相比，肝癌组织中circ0000195的表达量显著升高(P<0.01)。
进一步进行无瘤生存率分析发现，circ0000195表达量高的肝癌患者术后易复发，无瘤生存率降低(图1B)(P<0.05)。
实施例2
在肝癌细胞中敲低或过表达circ0000195，研究circ0000195对肝癌细胞铁死亡的影响。
一、实验方法
1.circ0000195敲低
于体外设计合成siRNA1～siRNA3，其中，
siRNA1包括以下正义链和反义链：
正义链：5’-AUGCUUCAGAGUCAUCCUUUU(SEQ ID NO.2)TT-3’；
反义链：5’-AAAAGGAUGACUCUGAAGCAU(SEQ ID NO.3)TT-3’；
siRNA2包括以下正义链和反义链：
正义链：5’-UUCAGAGUCAUCCUUUUUGAA(SEQ ID NO.4)TT-3’；
反义链：5’-UUCAAAAAGGAUGACUCUGAA(SEQ ID NO.5)TT-3’；
siRNA3包括以下正义链和反义链：
正义链：5’-CAAUGCUUCAGAGUCAUCCUU(SEQ ID NO.6)TT-3’；
反义链：5’-AAGGAUGACUCUGAAGCAUUG(SEQ ID NO.7)TT-3’。
使用脂质体瞬时转染法，在Hep3B肝癌细胞系中敲低circ0000195。siRNA转染：第一天将肝癌细胞Hep3B以每孔2.5×105个细胞的密度接种于6孔板内。第二天，待细胞融合度合适时，使用Lipofectamine 3000试剂盒，首先配置siRNA-脂质复合物，于室温静置15-20min，将原有培养液更换为基础培养基，均匀滴加250ul/孔的siRNA-脂质复合物，轻轻摇晃使其混合均匀。放入培养箱中培养，于48h后收集细胞以分析转染情况。
2.circ0000195过表达
于体外构建过表达质粒，使用脂质体瞬时转染法，在Hep3B肝癌细胞系中过表达circ0000195。其中，所述过表达质粒序列如SEQ ID NO.10所示：
SEQ ID NO.10：5’-TGAAATATGCTATCTTACAGAGTCATCCTTTTTGAACTTCCATGA CTCAGACTGCGAACCCAAGGGATCATCACCCTGTGACTCCTTGCTTTCCCTCAACACTGAGAAGATTCTGAGCCAGGCCAAGTCTATTGCAGAACAGAAGAGATTCCCGTTTGCCACTGATAATGACAGCACAAATGAAGAGTTAGCTATTGCTTATGTCTTGATTGGCAGTGGTCTGTATGATGAAGCAATACGGCATTTTTCAACAATGCTTCAGGTGAATATATTTTTTCTTGA-3’。
使用脂质体瞬时转染法，在Hep3B肝癌细胞系中过表达circ0000195。质粒转染：第一天将肝癌细胞Hep3B以每孔2.5×105个细胞的密度接种于6孔板内。第二天，待细胞融合度合适时，使用Lipofectamine 3000试剂盒，首先配置质粒-脂质复合物，于室温静置15-20min，将原有培养液更换为基础培养基，均匀滴加250ul/孔的质粒-脂质复合物，轻轻摇晃使其混合均匀。放入培养箱中培养，于24h后收集细胞以分析转染情况。
3.MTT检测细胞增殖能力
第一天将肝癌细胞以每孔5×104个细胞的密度接种于96孔板，于细胞融合度合适时转染(敲低组使用siRNA3进行转染)，于转染后24h用RSL3进行干预，分成转染对照组：无添加RSL3、RSL3低剂量组(RSL3-L)、RSL3高剂量组(RSL3-H)；过表达/敲低组：无添加RSL3、RSL3低剂量组(RSL3-L)、RSL3高剂量组(RSL3-H)，共6组。其中，RSL3低剂量组中RSL3的终浓度为1.25uM；RSL3高剂量组中RSL3的终浓度为2.5uM。在RSL3干预后24h弃旧培养基每孔加入90ul空白培养基及10ul MTT，继续培养4h，4h后弃旧培养基每孔加入150ul DMSO置于摇床上轻轻晃动5-10分钟，酶标仪测定其490nm处的OD值，检测细胞存活率。
4.活/死细胞染色测定
进行活/死细胞染色测定，将细胞接种在96孔板中，并进行如前所述步骤3处理后，将差别处理的细胞与染色工作溶液(2uM骨钙黄因乙酰氧基甲酯，8uM碘化丙啶)在黑暗中孵育30min，随后使用荧光显微镜，观察并捕获其图像。
二、实验结果
1.circ0000195敲低
circ0000195及对照siRNA转染Hep3B后，qRT-PCR分析circ0000195的表达量。如图2A所示，与对照序列相比(NC)，转染siRNA1(S1)、siRNA2(S2)和siRNA3(S3)能显著降低circ0000195的表达，说明成功敲低了circ0000195。
siRNA3(si-circ)及对照siRNA(si-NC)转染Hep3B后，MTT检测RSL3处理24小时后的细胞活性。结果显示(图2B)，siRNA3(si-circ)处理能明显促进RSL3诱导的细胞铁死亡，降低细胞活性。
siRNA3(si-circ)及对照siRNA(si-NC)转染Hep3B后，活/死细胞染色法检测RSL3处理24小时后对细胞活力的影响。结果显示，与对照siRNA处理的RSL3低剂量组(si-NCRSL3-L)和RSL3高剂量组(si-NC RSL3-H)相比，siRNA3处理的RSL3低剂量组(si-circRSL3-L)和RSL3高剂量组(si-circ RSL3-H)能明显促进RSL3诱导的细胞铁死亡，减少活细胞数量(图2C)。
2.circ0000195过表达
circ0000195过表达质粒(Ov-circ)及对照质粒(Ov-NC)转染Hep3B后，qRT-PCR分析circ0000195的表达量。如图3A所示，与对照质粒(Ov-NC)相比，过表达质粒(Ov-circ)显著增加了circ0000195的表达，说明过表达成功。
circ0000195过表达质粒(Ov-circ)及对照质粒(Ov-NC)转染Hep3B后，MTT检测RSL3处理24小时后的细胞活性。如图3B所示，过表达circ0000195(Ov-circ)能明显增加RSL3处理后的细胞活性，降低RSL3诱导的细胞铁死亡。
circ0000195过表达质粒(Ov-circ)及对照质粒(Ov-NC)转染Hep3B后，活/死细胞染色法检测RSL3处理24小时后对细胞活力的影响。结果显示(图3C)，未进行RSL3处理时，circ0000195过表达质粒(Ov-circ)及对照质粒(Ov-NC)转染组细胞活性差别不显著；与对照组质粒处理的RSL3低剂量组(Ov-NC RSL3-L)和RSL3高剂量组(Ov-NC RSL3-H)相比，过表达circ0000195质粒处理的RSL3低剂量组(Ov-circ RSL3-L)和RSL3高剂量组(Ov-circRSL3-H)能明显抑制RSL3诱导的细胞铁死亡，增加活细胞数量。
综上所述，检测circ0000195表达水平的试剂可用于检测肝癌和/或评估肝癌患者的术后易复及无瘤生存率。抑制circ0000195表达的试剂可用于制备治疗肝癌的药物。本发明为肝癌的治疗和预后评估提供了新的靶点、新的方案和思路。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对以上实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (5)
1.抑制circ0000195表达的试剂在制备治疗肝癌的药物中的应用，其特征在于，所述circ0000195的编码基因的核苷酸序列如SEQ ID NO. 1所示；所述抑制circ0000195表达的试剂为siRNA；所述siRNA选自siRNA1、siRNA2和siRNA3中的至少一种；所述siRNA1的正义链如SEQ ID NO. 2所示，反义链如SEQ ID NO. 3所示；所述siRNA2的正义链如SEQ ID NO. 4所示，反义链如SEQ ID NO. 5所示；所述siRNA3的正义链如SEQ ID NO. 6所示，反义链如SEQ ID NO. 7所示；所述SEQ ID NO. 2~ SEQ ID NO. 7的3’端修饰有TT。
2.如权利要求1所述的应用，其特征在于，所述药物可以促进肝癌细胞铁死亡。
3.一种治疗肝癌的药物，其特征在于，所述药物包含抑制circ0000195表达的试剂和药物学上可接受的辅料；所述抑制circ0000195表达的试剂为siRNA；所述siRNA选自siRNA1、siRNA2和siRNA3中的至少一种；所述siRNA1的正义链如SEQ ID NO. 2所示，反义链如SEQID NO. 3所示；所述siRNA2的正义链如SEQ ID NO. 4所示，反义链如SEQ ID NO. 5所示；所述siRNA3的正义链如SEQ ID NO. 6所示，反义链如SEQ ID NO. 7所示；所述SEQ ID NO. 2~SEQ ID NO. 7的3’端修饰有TT。
4.检测circ0000195表达水平的试剂在制备检测肝癌和/或评估肝癌患者预后的产品中的应用，其特征在于，所述circ0000195的编码基因的核苷酸序列如SEQ ID NO. 1所示。
5.如权利要求4所述的应用，其特征在于，所述检测circ0000195表达水平的试剂为如SEQ ID NO. 8所示的上游引物和SEQ ID NO. 9所示的下游引物。
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