CN106119248A - 针对sox4基因靶点的小干扰rna、短发卡rna及重组载体和应用 - Google Patents
针对sox4基因靶点的小干扰rna、短发卡rna及重组载体和应用 Download PDFInfo
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Abstract
本发明涉及分子生物学、基因工程技术以及生物医药领域,具体涉及一种针对SOX4基因靶点的小干扰RNA、短发卡RNA及重组载体和应用。小干扰RNA包括SEQ ID NO.1所示的正义链和SEQ ID NO.2所示的反义链。短发卡RNA包括SEQ ID NO.3所示的正义链和SEQ ID NO.4所示的反义链。包含短发卡RNA的重组载体能在宿主细胞内转录得到SOX4‑shRNA,经宿主细胞加工后可生成小分子干扰RNA,并靶向SOX4的信使RNA,降低SOX4的表达,导致基因沉默效应,有效抑制食管鳞癌细胞的增殖并促进化疗药物阿霉素诱导的食管鳞癌细胞的衰老,具有抗肿瘤应用价值,为进一步研究SOX4在肿瘤细胞衰老逃逸中的功能及靶向SOX4的药物研发提供了基础。
Description
技术领域
本发明涉及分子生物学、基因工程技术以及生物医药领域,具体涉及一种针对SOX4基因靶点的小干扰RNA、短发卡RNA及重组载体和应用。
背景技术
我国是食管癌高发国家,2015年国家癌症中心发布的数据显示,在全国肿瘤登记地区食管癌的发病率居癌症发病顺位的第6位(21.6/10万人),死亡率居第4位。我国食管癌发病区具有明显的地域特征,主要集中在河南、河北、山西、川北、苏北以及广东汕头和梅县等地区。病理分型上以食管鳞状细胞癌较食管腺癌更为常见。目前食管癌的治疗以手术和放化疗为主,5年存活率低于20%。探寻食管癌发生过程中的病理特点、信号通路和关键分子是肿瘤防治和药物研发的重要基础和前提。
细胞衰老是指细胞丧失增殖能力后进入一种不可逆的相对稳定状态。细胞衰老的主要特征包括:(1)细胞停止分裂,进入类似终末分化的状态;(2)表达特异性的衰老相关半乳糖苷酶;(3)细胞核内染色质凝集,形成衰老相关异染色质斑,端粒缩短,组蛋白H3K9高度甲基化;(4)大量合成一些基质重构蛋白、趋化因子和炎性因子如PAI1/SERPINE1、MMPs、CXCL1、IL1、IL6、IL8等,也被称为衰老相关分泌表型。这些因子不仅是衰老过程的参与者,还能激活免疫应答、参与组织形态重构。衰老是机体抵抗自身细胞恶性转化的一道天然屏障。这是由于肿瘤发生过程中的一些关键性事件如DNA损伤、癌基因过度活化等均能诱发细胞衰老从而抑制肿瘤的发生。其次,一些衰老抑制因素则能促进肿瘤的发生。例如CD11b+Gr-1+骨髓来源抑制性细胞能通过分泌IL1R的拮抗分子IL1RA来增强肿瘤细胞的衰老抵抗并促进其增殖。此外,多数化疗药物能诱发肿瘤细胞的衰老并导致最终死亡。因此细胞衰老/抗衰老的失衡不仅与肿瘤的发生发展密切相关,也有望成为药物治疗的新靶点。
SOX4与SOX11、SOX12同属于SOX C家族,在胚胎期心肌、免疫系统和神经系统发育过程中起着重要作用。SOX4的表达上调在乳腺癌、肝细胞癌、结肠癌中均有报道,并且受到EGFR,TGF-β,Wnt/β-catenin等信号通路的调节。SOX4还是上皮-间质转化的诱导因子,参与肿瘤细胞的转移过程。此外,在条件性敲除SOX4的小鼠中出现了多器官的异常衰老。SOX4是否参与食管鳞癌及其它类型肿瘤细胞的衰老逃逸目前并不清楚。
RNA干扰(RNAi)是利用序列特异性的、与靶基因同源的双链RNA(dsRNA)对靶基因转录后的信使RNA(mRNA)的分解,从而抑制靶基因表达的一种转录后基因沉默技术。其作用机制是:dsRNA被Dicer酶识别,并被切割为小干扰RNA(siRNA)。siRNA与RNA介导沉默复合体(RISC)结合后,识别并降解同源的mRNA,特异性抑制目的基因的表达。由于RNA干扰抑制靶基因表达具有特异性强、快速、高效等优点,尤其适合特异靶向性的基因治疗。
最初RNA干扰采样体外合成siRNA的方法,但存在转染效率低、转移到细胞内的siRNA不能持久性表达、对靶基因表达的抑制作用短暂等缺点,从而限制了其应用。将siRNA合成为短发卡RNA(shRNA)并由载体导入细胞,能在细胞内稳定的转录生成shRNA,并进一步加工生成靶基因特异性的siRNA,可以发挥长期抑制靶基因表达的作用。
发明内容
本发明的目的是以SOX4基因为靶点,提供一种特异性抑制SOX4表达的小干扰RNA。
本发明的另一目的提供稳定表达小干扰RNA的短发卡RNA。
本发明的另一目的提供包含该短发卡RNA的重组载体。
本发明的另一目的提供小干扰RNA、短发卡RNA、重组载体的应用。
为达到上述目的之一,本发明采用以下技术方案:
一种针对SOX4基因靶点的小干扰RNA,所述SOX4基因靶点的序列为:5’-GCGACAAGATCCCTTTCAT-3’。
进一步地,所述小干扰RNA包括正义链和反义链,正义链序列为5’-GCGACAAGAUCCCUUUCAU-3’,如SEQ ID NO.1所示;反义链序列为5’-AUGAAAGGGAUCUUGUCGC-3’,如SEQ ID NO.2所示。
一种针对SOX4基因靶点的短发卡RNA,所述SOX4基因靶点的序列为:5’-GCGACAAGATCCCTTTCAT-3’。
进一步地,所述短发卡RNA包含正义链和反义链,正义链序列为5’-GATCCGCGACAAGATCCCTTTCATTTCAAGAGAATGAAAGGGATCTTGTCGCTGA-3’,如SEQ ID NO.3所示;正义链序列为5’-AGCTTCAGCGACAAGATCCCTTTCATTCTCTTGAA
ATGAAAGGGATCTTGTCGCG-3’,如SEQ ID NO.4所示。
上述短发卡RNA在制备抑制SOX4基因表达的生物制剂中的应用。
一种针对SOX4基因靶点的重组载体,所述重组载体包含上述的短发卡RNA。
所述重组载体为质粒载体。
进一步地,所述重组载体为慢病毒载体或腺病毒载体。
进一步地,所述重组载体为在pSilencer 4.1-CMV neo质粒的多克隆位点插入上述短发卡RNA得到的重组载体。
上述重组载体在制备用于治疗食管鳞癌药物中的应用。
本发明具有以下有益效果:
本发明针对SOX4设计了小干扰RNA序列和短发卡RNA序列,并构建了相应的重组载体。本发明的小分子干扰RNA具有与SOX4的信使RNA互补的核苷酸序列,能有效降低食管鳞癌细胞中SOX4基因的表达水平。本发明的重组载体能在宿主细胞内转录得到SOX4-shRNA,经宿主细胞加工后可生成小分子干扰RNA,并靶向SOX4的信使RNA,降低SOX4的表达,导致基因沉默效应,有效抑制食管鳞癌细胞的增殖并促进化疗药物阿霉素诱导的食管鳞癌细胞的衰老,具有抗肿瘤应用价值,为进一步研究SOX4在肿瘤细胞衰老逃逸中的功能及靶向SOX4的药物研发提供了基础。
附图说明
图1为实施例2采用的pSilencer质粒图谱;
图2是实施例3食管鳞癌组织中SOX4的表达结果;
图3为实施例4食管鳞癌细胞KYSE410和KYSE510的SOX4表达的检测结果;
图4为实施例5食管鳞癌细胞KYSE410和KYSE510的增殖实验结果;
图5为实施例6食管鳞癌细胞KYSE410和KYSE510的衰老实验结果。
具体实施方式
下面结合具体实施例对本发明做进一步的说明:
下述实施中所用的方法如无特别说明均为常规方法,具体步骤可参见《MolecularCloning: A Laboratory Manual》(Sambrook, J., Russell, David W., 3rd edition,2001, NY, Cold Spring Harbor)。所用DNA引物均由苏州金唯智生物科技有限公司合成;所用pSilencer载体购自invitrogen公司;各种限制性内切酶和Taq酶均购自TaKaRa公司;T4 DNA连接酶购自NEB公司;胶回收试剂盒和质粒抽提试剂盒购自OMEGA公司;DMEM培养基、胎牛血清均购自HyClone公司;转染试剂Lipofectamine购自Invitrogen公司;KYSE510和KYSE410细胞培养基为含10%胎牛血清的DMEM培养基。如无特别说明,本发明采用的其它试剂均为市售商品。
实施例1
首先针对SOX4基因,根据领域内通常采用的原则设计小干扰RNA序列,并合成相应的短发卡RNA序列。
小干扰RNA正义链序列为5’-GCGACAAGAUCCCUUUCAU-3’,反义链序列为5’-AUGAAAGGGAUCUUGUCGC-3’。
短发卡RNA正义链序列为5’-GATCCGCGACAAGATCCCTTTCATTTCAAGAGAAT
GAAAGGGATCTTGTCGCTGA-3’;
正义链序列为5’-AGCTTCAGCGACAAGATCCCTTTCATTCTCTTGAAATGAAAGGGA
TCTTGTCGCG-3’。
靶向SOX4的区域为:5’-GCGACAAGATCCCTTTCAT-3’。
正义链中,GCGACAAGATCCCTTTCATTTCAAGAGAATGAAAGGGATCTTGTCGC构成转录后所形成RNA的茎环结构,TTCAAGAGA形成环。
反义链中,GCGACAAGATCCCTTTCATTCTCTTGAAATGAAAGGGATCTTGTCGC构成转录后所形成RNA的茎环结构,TCTCTTGAA形成环。
上述DNA序列由商业化公司合成,退火后成为双链DNA分子,与酶切后的pSilencer载体链接,转化大肠杆菌Ecoli扩增,从而得到大量的靶向SOX4的干扰RNA重组载体。
实施例2
构建靶向SOX4的干扰RNA重组载体
合成DNA序列:正义链和反义链分别溶于双蒸水中,浓度为:1mg/mL。各取2μL,加双蒸水至50μL,混合均匀,95℃水浴锅中放置5min,自然冷却至室温。所得退火双链DNA可存于-20℃冰箱备用。
pSilencer 4.1-CMV neo质粒的图谱如图1所示,其中HindIII(463)与BamHI(516)之间为干扰RNA的DNA序列插入区间。
将pSilencer空载体质粒1μg,内切酶HindIII、BamHI各1μL(10 units),10X酶切缓冲液2μL及适当体积的双蒸水混合使总体积达到20μL,37℃反应30min。反应结束后加10XDNA上样缓冲液2μL于1%琼脂糖凝胶电泳,紫外灯下切割5kbp左右DNA条带所在的胶条并回收DNA测定浓度。
将上述退火后的双链DNA用双蒸水稀释至8ng/μL,取1μL与0.1μg酶切回收后的pSilencer混合,加入10X链接缓冲液1μL,T4 DNA连接酶1μL,用双蒸水补足至总体积10μL,16℃反应2h后转化大肠杆菌,涂布于具卡纳霉素抗性的琼脂板中,37℃过夜后挑取单克隆菌落于含卡纳霉素抗性LB培养基中震荡培养过夜。菌液离心收集后抽提质粒并测序验证,将其命名为pSilencer-SOX4-shRNA。
实施例3
食管鳞癌细胞中SOX4的表达
在取自河南省安阳肿瘤医院的14例食管鳞癌组织及配对的癌旁组织中检测SOX4的表达,结果如图2所示,在13个病例中(92%)SOX4在癌组织中的表达水平高于正常组织;在9个病例中(64%)SOX4在癌组织中的表达水平高于正常组织2倍以上。
RT-qPCR引物:SOX4的正义链为5’-GACCTGCTCGACCTGAAC C -3’,反义链为5’-CCGGGCTCGAAG TTAAAATCC-3’;GAPDH的正义链为5’-CTGGGCTACACTGAGCACC-3’,反义链为5’-AAGTGGTCGTTGAGGGCAATG-3’。反应条件:95℃预变性2min,95℃变性20S,60℃退火20S,73℃延伸30S,40个循环。
实施例4
用pSilencer-SOX4-shRNA降低食管鳞癌细胞SOX4的表达
将人食管鳞癌细胞KYSE410和KYSE510分别按5*106/孔的密度接种于6孔板,过夜后更换新鲜培养基。将2μg的pSilencer-SOX4-shRNA质粒或空载体质粒分别与4μLlipofectamine混合于200μL的无血清DMEM培养基中,静置15min,逐滴加入到六孔板中的细胞培养液中。转染后的细胞于37℃二氧化碳孵箱中培养48h,取出细胞吸弃培养基,经PBS洗涤一遍后每孔加200μL RIPA裂解液,冰上裂解15min,将裂解产物于4℃12000 G离心10分钟,取上清,一部分用于测定蛋白浓度,一部分加蛋白上样缓冲液沸水中放置5min。取50μg蛋白SDS-聚丙烯酰胺凝胶电泳分离,WESTERN BLOT检测SOX4蛋白的水平。所用SOX4抗体购自proteintech公司。WESTERN BLOT检测结果如图3所示,对照组为pSilencer空载体,实验组为pSilencer-SOX4-shRNA,pSilencer-SOX4-shRNA可显著降低食管鳞癌KYSE410和KYSE510细胞中SOX4的表达。
实施例5
用pSilencer-SOX4-shRNA抑制食管鳞癌细胞的增殖
按实施例4的方法,用pSilencer-SOX4-shRNA和空载体分别转染6孔板中的KYSE410和KYSE510细胞。于37℃二氧化碳孵箱中培养24h后,消化收集细胞按每孔1000个细胞的密度重新接种到六孔板中,继续培养10d。吸弃培养基,PBS洗涤一遍,加入1mL浓度为0.1%的结晶紫染色液,室温静置10min,吸弃结晶紫染色液,PBS洗涤两遍,拍照记录各孔中的细胞密度。细胞增殖结果如图4所示,对照组为pSilencer空载体,实验组为pSilencer-SOX4-shRNA,可见实验组中的细胞克隆数明显少于对照组,pSilencer-SOX4-shRNA可显著抑制食管鳞癌KYSE410和KYSE510细胞的增殖。
实施例6
用pSilencer-SOX4-shRNA促进食管鳞癌细胞的衰老
按实施例4的方法,用pSilencer-SOX4-shRNA和空载体分别转染6孔板中的KYSE510和KYSE410细胞。转染24h后,更换新鲜培养基,并加入阿霉素使其终浓度为0.1μg/ml,继续培养48h后吸弃培养基,PBS培养基洗涤一遍,用β-半乳糖苷酶染色试剂盒染色过夜。倒置显微镜下拍照记录,蓝色细胞为衰老细胞。衰老结果如图5所示,对照组为pSilencer空载体,实验组为pSilencer-SOX4-shRNA,箭头代表SA-β-Gal阳性的衰老细胞,转染pSilencer-SOX4-shRNA后能显著促进阿霉素诱导的KYSE410和KYSE510细胞衰老。
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,任何属于本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到的变化或替换,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该以权利要求的保护范围为准。
<110> 深圳大学
<120> 针对SOX4基因靶点的小干扰RNA、短发卡RNA及重组载体和应用
<130>
<160> 4
<170> PatentIn version 3.5
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<213> 人工序列
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gcgacaagau cccuuucau 19
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<213> 人工序列
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agcttcagcg acaagatccc tttcattctc ttgaaatgaa agggatcttg tcgcg 55
Claims (10)
1.一种针对SOX4基因靶点的小干扰RNA,其特征在于,所述SOX4基因靶点的序列为:5’-GCGACAAGATCCCTTTCAT-3’。
2.根据权利要求1所述的小干扰RNA,其特征在于,所述小干扰RNA包括正义链和反义链,正义链序列为5’-GCGACAAGAUCCCUUUCAU-3’,如SEQ ID NO.1所示;反义链序列为5’-AUGAAAGGGAUCUUGUCGC-3’,如SEQ ID NO.2所示。
3.一种针对SOX4基因靶点的短发卡RNA,其特征在于,所述SOX4基因靶点的序列为:5’-GCGACAAGATCCCTTTCAT-3’。
4.根据权利要求3所述的短发卡RNA,其特征在于,所述短发卡RNA包含正义链和反义链,正义链序列为5’-GATCCGCGACAAGATCCCTTTCATTTCAAGAGAATGAAAGGG
ATCTTGTCGCTGA-3’,如SEQ ID NO.3所示;
正义链序列为5’-AGCTTCAGCGACAAGATCCCTTTCATTCTCTTGAAATGAAAGGGA
TCTTGTCGCG-3’,如SEQ ID NO.4所示。
5.权利要求3或4所述的短发卡RNA在制备抑制SOX4基因表达的生物制剂中的应用。
6.一种针对SOX4基因靶点的重组载体,其特征在于,所述重组载体包含权利要求3或4所述的短发卡RNA。
7.根据权利要求6所述的重组载体,其特征在于,所述重组载体为质粒载体。
8.根据权利要求6所述的重组载体,其特征在于,所述重组载体为慢病毒载体或腺病毒载体。
9.根据权利要求6所述的重组载体,其特征在于,所述重组载体为在pSilencer 4.1-CMV neo质粒的多克隆位点插入权利要求3或4所述的短发卡RNA得到的重组载体。
10.权利要求6所述的重组载体在制备用于治疗食管鳞癌药物中的应用。
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CN111321142A (zh) * | 2020-02-05 | 2020-06-23 | 翁炳焕 | 一种新型冠状病毒肺炎dsRNA疫苗的制备方法 |
CN115990270A (zh) * | 2022-07-14 | 2023-04-21 | 郑州大学 | 一种抑制肿瘤干性的纳米载体及其制备方法和应用 |
CN115990270B (zh) * | 2022-07-14 | 2023-08-11 | 郑州大学 | 一种抑制肿瘤干性的纳米载体及其制备方法和应用 |
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