CN114045335B - circSpred1基因作为标志物在纤维化肝脏、肝癌的诊断和药物制备中的应用 - Google Patents
circSpred1基因作为标志物在纤维化肝脏、肝癌的诊断和药物制备中的应用 Download PDFInfo
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Abstract
本发明公开了一种circSpred1基因作为标志物在纤维化肝脏、肝癌的诊断和药物制备中的应用。该circSpred1基因的核苷酸序列如SEQ ID NO.1所示，其编码的蛋白质序列如SEQ ID NO.2所示。本发明提供了一种新的环状RNA分子circSpred1，通过敲低circSpred1的表达量可有效的促进肝细胞再生，表明其可作为治疗纤维化肝脏，及其所引起的相应肝癌病症的新的治疗靶点。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种circSpred1基因作为标志物在纤维化肝脏、肝癌的诊断和药物制备中的应用。
背景技术
肝细胞癌是全球范围内常见的恶性肿瘤，每年有大约90万新发病例和80万死亡病例。其放化疗治疗效果差，肝部分切除术(partial hepatectomy,PH)是其主要临床治疗措施，早期肿瘤切除术后5年生存率高达70％。正常肝脏具有惊人的再生能力，正常肝脏部分切除术后残肝组织通过快速的肝再生，可以有效代偿肝切除丢失的肝组织，从而维持机体正常的肝功能。然而，85％以上肝细胞癌发生于肝纤维化/肝硬化基础上，此慢性肝损伤导致肝脏再生能力显著减弱。既往研究已经证实，纤维化肝脏在接受安全范围内的PH术后，残肝再生能力及再生速率明显弱于正常肝脏。在此情况下按照“根治性切除”的理想切除范围来实施手术，极易因肝切除术后肝功能代偿不足发生肝功能衰竭(post hepatectomyliver failure,PHLF)，发生后患者死亡率高。肝纤维化患者PH术后残肝再生能力不足，是术后发生肝衰竭与死亡的重要原因之一，严重限制了PH的临床应用。如何促进合并有肝纤维化乃至肝硬化的肝细胞癌PH术后的肝再生，从而使更多肝细胞癌患者受惠于肝切除术治疗，成为临床亟待解决的难题。
发明内容
针对现有技术中的上述不足，本发明提供一种circSpred1基因作为标志物在纤维化肝脏、肝癌的诊断和药物制备中的应用，本发明提供了新的环状RNA分子circSpred1，通过敲低circSpred1的表达量可有效的促进肝细胞再生。
为实现上述目的，本发明解决其技术问题所采用的技术方案是：
circSpred1基因作为标志物在制备治疗纤维化肝脏药物中的应用，该circSpred1基因的核苷酸序列如SEQ ID NO.1所示。
进一步地，在制备治疗纤维化肝脏药物时，还可使用与circSpred1基因同源性大于80％，且表达相同功能蛋白的基因。
进一步地，circSpred1基因编码的蛋白质序列如SEQ ID NO.2所示。
一种检测circSpred1基因的试剂在纤维化肝脏诊断或预后中的应用。
一种circSpred1基因表达抑制剂或敲除试剂在制备治疗纤维化肝脏药物中的应用，该药物能够促进肝细胞的增殖。
进一步地，circSpred1基因表达抑制剂或敲除试剂为能够敲低circSpred1基因表达量的siRNA，或包含有该siRNA的腺相关病毒载体。
一种用于纤维化肝脏诊断或预后检测的试剂盒，该试剂盒包括检测circSpred1基因表达量的试剂。
一种治疗肝癌的药物，该药物包括circSpred1基因表达抑制剂或敲除试剂。
进一步地，肝癌为肝纤维化引起的肝癌。
一种用于肝癌诊断或预后检测的试剂盒，试剂盒包括检测circSpred1基因表达量的试剂。
本发明的有益效果：
本发明提供了新的环状RNA分子circSpred1，通过敲低circSpred1的表达量可有效的促进肝细胞再生，表明其可作为治疗纤维化肝脏，及其所引起的相应肝癌病症的新的治疗靶点。
附图说明
图1为纤维化肝脏PH术后再生能力检测结果；其中，A为正常肝脏和纤维化肝脏肝切除术后Ki67、pH3S10的表达；B为肝切除术后不同时相点BrDU的表达；
图2为circRNAs高通量测序结果；
图3为circSpred1基因在纤维化肝脏再生组织中表达量的检测结果；其中，A为qRT-PCR验证circSpred1在两组小鼠肝切除术后36h的表达水平；B为人正常肝组织及肝纤维化组织中circSpred1的表达量；C为肝纤维化小鼠与正常小鼠30％肝切除术后不同时间点circSpred1的表达量；D为各时相点两组归一化后残肝湿重与体重比；
图4为敲低circSpred1的表达量对肝细胞增殖和细胞周期的影响；A为AML-12细胞使用siRNA敲低circSpred1表达的效果；B为CCK-8细胞增殖实验显示敲低circSpred1可促进AML-12细胞增殖；C为EdU细胞增殖实验显示敲低circSpred1后可促进细胞DNA合成；D为免疫印迹显示敲低circSpred1可促进多种细胞周期蛋白的表达；
图5体内验证过表达circSpred1抑制纤维化肝脏的再生；A为验证过表达circSpred1的转染效率；B为过表达circSpred1的纤维化肝脏PH后36h残肝体重比显著低于对照组；C为过表达circSpred1组和对照组PH后36h残肝再生情况的代表性图片。
图6为circSpred1基因编码的蛋白；其中，A为circSpred1的预测ORF，图示显示circSpred1的反向拼接位点在ORF中，下方是ORF序列，红色字体代表所拥有的特异性氨基酸序列；B为AML-12细胞总蛋白经考马斯亮蓝染色结果，下方显示液相色谱串联质谱法检测15-25kDa区域的SDS-PAGE胶结果，证实该特异性肽段存在；C为双荧光素质粒设计图谱，预测IRES序列被搭载至具有独立起始和终止密码子的Rluc和Luc之间；D为上述质粒转染293T细胞后经双荧光素酶报告试验检测结果。结果显示含有IRES的质粒转染后较空白对照组有明显的荧光值变化；E为免疫印迹实验检测过表达circSpred1的AML-12细胞circSpred1-197aa的表达；
图7为CircSpred1差异基因的GO分析。
具体实施方式
下面对本发明的具体实施方式进行描述，以便于本技术领域的技术人员理解本发明，但应该清楚，本发明不限于具体实施方式的范围，对本技术领域的普通技术人员来讲，只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内，这些变化是显而易见的，一切利用本发明构思的发明创造均在保护之列。
实施例1 circSpred1基因与纤维化肝脏
采用CCl4构建小鼠肝纤维化模型，并构建得到小鼠纤维化肝脏部分切除和正常肝脏部分切除动物模型。免疫组化检测发现，细胞增殖标志物Ki67、pH3S10的表达在纤维化肝脏部分切除术后显著低于正常肝脏，表明纤维化肝脏肝切除术后再生能力减弱(图1A)。检测肝切除术后肝脏组织BrDU的表达，发现术后36h表达最高，表明肝再生DNA合成高峰期位于术后36h，拟选择术后36h的肝脏组织进行测序(图1B)。
采用高通量circRNA基因测序的方法，检测小鼠纤维化肝脏30％肝切除后0h和36h肝组织circRNAs的表达，获得了多个差异表达circRNAs(图2)，通过相关网站预测所有差异基因的编码蛋白的能力，选择表达差异明显且具有编码能力的circSpred1进行下一步研究。
使用qRT-PCR验证circSpred1在两组小鼠肝切除术后36h的表达水平，每组6只，发现circSpred1在纤维化肝脏再生肝组织的表达显著高于正常肝脏再生肝组织(图3A)。
在获得四川大学华西医院伦理委员会批准，并签署生物样本使用知情同意书前提下，收集良性肝病(如乙肝肝硬化脾功能亢进行脾切除并肝组织活检患者、无肝纤维化的肝血管瘤切除患者)的肝活检样本，手术样本在离体后15分钟内切取部分肝组织，液氮速冻后转运至-80℃冰箱保存。分别用Trizol和Trizol LS提取组织总RNA，采用qRT-PCR的方法检测circSpred1。
首先检测了16例正常人肝组织及22例肝纤维化患者肝组织中circSpred1的表达量，发现在纤维化肝组织中circSpred1明显低表达(图3B)。提示在未进行肝部分切除的纤维化肝脏中，存在有机体代偿性的低水平肝再生的情况，circSpred1表达降低是顺应并有利于机体自我修复。
继续采用qRT-PCR检测两组小鼠术后各时相点肝组织中circSpred1的表达量，结果发现circSpred1的表达从术后12h开始在纤维化肝脏再生组织中显著高于正常肝脏再生组织，表明circSpred1是纤维化肝脏30％PH后再生能力减弱的重要因素之一(图3C，D)。
实施例2 circSpred1基因敲低对肝细胞增殖的影响
1、体外细胞增殖实验(CCK-8、EdU)，检测在小鼠正常肝细胞AML-12中使用siRNA敲低circSpred1后短期增殖能力的变化。结果显示，敲低circSpred1表达可上调多种周期蛋白表达，并促进肝细胞增殖，提示circSpred1在肝再生过程中发挥抑制肝细胞增殖的作用(图4)。
2、构建能稳定过表达circSpred1的腺相关病毒，按照每只小鼠3×1011v.g/ml的剂量，采用尾静脉注射的方法构建过表达circSpred1的动物模型。注射4周后，在气体麻醉下行30％PH，然后在30％PH后36小时处死小鼠，采集小鼠残肝组织样本计算残肝体重比。结果发现过表达circSpred1的纤维化肝脏PH术后36小时再生显著低于对照组(图5)。
实施例3
通过circRNADb网站数据库显示circSpred1具有高度编码多肽的能力，预测circSpred1含有一跨越拼接位点含有197个氨基酸的开放阅读框，其开放阅读框从拼接位点开始计算，至第516位碱基A开始沿circSpred1环状链向下依次翻译多肽。其中跨过一次拼接位点，至终止密码子TAA结束翻译，共594个碱基，对应有197个氨基酸，我们将circSpred1编码多肽命名为circSpred1-197aa，其氨基酸序列如SEQ ID NO.2所示(图6A)。
选取AML-12细胞总蛋白SDS-PAGE胶电泳的15-25kDa区域的凝胶进行液相色谱-串联质谱检测，结果显示成功检测到此开放阅读框对应的氨基酸序列(图6B)。由于circRNA缺少5’帽子结构，因此编码多肽需要在其内部存在有活性的内部核糖体进入位点序列(Internal ribosome entry site,IRES)。我们设计搭载了IRES序列的质粒，使用双荧光素酶报告系统检测circSpred1的此IRES的活性，结果显示circSpred1中的IRES序列能成功启动萤火虫荧光素的翻译，与对照空白质粒相比具有显著统计学意义(图6C，D)。我们通过免疫印迹实验检测过表达circSpred1的AML-12细胞circSpred1-197aa的表达，发现过表达circSpred1上调了circSpred1-197aa蛋白的表达，提示内源性的circSpred1-197aa在肝细胞中存在同时依赖circSpred1的编码(图6E)。
采用转录组RNA-seq对siRNA敲减circSpred1后差异表达的基因进行分析，共发现719种差异基因，其中核糖体相关的通路的基因在circSpred1下调后显著上调，再次印证circSpred1下调后促进了细胞增殖(图7)。
序列表
<110> 四川大学
<120> circSpred1基因作为标志物在纤维化肝脏、肝癌的诊断和药物制备中的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 553
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
taatagttat gcacgagtgc gagctgtggt gatgaccaga gatgactcaa gtggtggatg 60
gctaccactc ggagggagcg gactgagcag cgtcactgtc ttcagagtcc ctcatcagga 120
agagaatggc tgtgcggact tttttatccg tggagagaga ctcagagaca aaatggtggt 180
tttggaatgt atgcttaaga aagacctcat ctataataag gtcactccca catttcacca 240
ctggaagatc gatgacaaga agtttggcct tacctttcag agtcctgctg atgccagggc 300
ttttgatcga ggcattcgaa gagctataga ggatatatct ctagggtgcc cagcgtcaaa 360
aactgaggct gaaggaggag atgatgattt acaaacgact gaagaagaca cttcccgttc 420
cctagtgaaa gatcactttt tccagcaaga gacagttgtt accagtgaac cttacagaag 480
ctcagacata agaccgttac cctttgaaga tctgaatgcc agaagagtct acttgcaaag 540
ccaagtcagc cag 553
<210> 2
<211> 197
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Pro Glu Glu Ser Thr Cys Lys Ala Lys Ser Ala Ser Asn Ser Tyr
1 5 10 15
Ala Arg Val Arg Ala Val Val Met Thr Arg Asp Asp Ser Ser Gly Gly
20 25 30
Trp Leu Pro Leu Gly Gly Ser Gly Leu Ser Ser Val Thr Val Phe Arg
35 40 45
Val Pro His Gln Glu Glu Asn Gly Cys Ala Asp Phe Phe Ile Arg Gly
50 55 60
Glu Arg Leu Arg Asp Lys Met Val Val Leu Glu Cys Met Leu Lys Lys
65 70 75 80
Asp Leu Ile Tyr Asn Lys Val Thr Pro Thr Phe His His Trp Lys Ile
85 90 95
Asp Asp Lys Lys Phe Gly Leu Thr Phe Gln Ser Pro Ala Asp Ala Arg
100 105 110
Ala Phe Asp Arg Gly Ile Arg Arg Ala Ile Glu Asp Ile Ser Leu Gly
115 120 125
Cys Pro Ala Ser Lys Thr Glu Ala Glu Gly Gly Asp Asp Asp Leu Gln
130 135 140
Thr Thr Glu Glu Asp Thr Ser Arg Ser Leu Val Lys Asp His Phe Phe
145 150 155 160
Gln Gln Glu Thr Val Val Thr Ser Glu Pro Tyr Arg Ser Ser Asp Ile
165 170 175
Arg Pro Leu Pro Phe Glu Asp Leu Asn Ala Arg Arg Val Tyr Leu Gln
180 185 190
Ser Gln Val Ser Gln
195
Claims (4)
1.circSpred1基因作为标志物在制备治疗纤维化肝脏药物中的应用，其特征在于，所述circSpred1基因的核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的应用，其特征在于，所述circSpred1基因编码的蛋白质序列如SEQ ID NO.2所示。
3.一种circSpred1基因表达抑制剂或敲除试剂在制备治疗纤维化肝脏药物中的应用，其特征在于，所述药物能够促进肝细胞的增殖。
4.一种治疗肝纤维化引起的肝癌的药物，其特征在于，所述药物包括circSpred1基因表达抑制剂或敲除试剂。
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