CN110141578A - 环状RNA circ-Ankib1在制备促进神经再生和修复神经损伤药物中的应用 - Google Patents
环状RNA circ-Ankib1在制备促进神经再生和修复神经损伤药物中的应用 Download PDFInfo
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Abstract
本发明公开了环状RNA circ‑Ankib1在制备促进神经再生和修复神经损伤药物中的应用。研究结果表明通过下调或者抑制机体circ‑Ankib1的表达，能够促进Schwann细胞的增殖，进而有利于周围神经损伤修复。
Description
技术领域
本发明属于神经再生和修复技术领域，具体涉及一种环状RNA circ-Ankib1在制备促进神经再生和修复神经损伤药物中的应用。
背景技术
周围神经损伤通常由拉伸性、缺血性、穿透性、挤压性等创伤性损伤引起。与中枢神经系统不同，周围神经系统损伤后轴突具有一定的内在再生能力。Schwann细胞是周围神经系统中重要的胶质细胞，在神经系统中发挥重要作用。周围神经损伤后，Schwann细胞增殖迁移形成细胞索带，同时会通过合成分泌神经营养因子，激活免疫反应等为损伤后的神经再生提供适宜的微环境。从分子的角度研究神经再生过程中的关键因素和途径，有利于发现新的治疗靶点。
环状RNA circular RNAs(circRNAs)是一类不具有5'末端帽子和3'末端poly(A)尾巴、并以共价键形成环形结构的RNA.。circRNAs能通过作为microRNA的分子海绵、剪接和转录调节因子等方式调节基因表达，参与多种生物学过程。
目前关于circRNAs的研究主要集中在癌症方面，是一种非常有前景的生物标记物甚至是治疗靶点。在神经系统发育中circRNAs也发挥着重要的作用，可以影响神经元的迁移和轴突生长。但到目前为止circRNAs在周围神经系统损伤修复中的作用鲜有报道，尤其是对Schwann细胞的表型调节。
发明内容
本发明目的在于提供一种新的大鼠源的环状RNA circ-Ankib1及其用途，可用于制备促进神经再生和修复神经损伤药物。
本发明具体技术方案如下：
一种环状RNA circ-Ankib1，所述circRNA的cDNA核苷酸序列如SEQ ID NO:1所示，首尾连接成环状结构。
本发明另一目的在于提供上述环状RNA circ-Ankib1在制备促进神经再生和修复神经损伤药物中的应用，
所述神经损伤为周围神经系统坐骨神经损伤。
可根据circ-Ankib1核苷酸序列设计干预circ-Ankib1表达的物质，选自化合物、蛋白、多肽、多糖、糖蛋白、糖肽、核酸中的一种或几种。
优选的，所述干预circRNA表达的物质，选自与circ-Ankib1环化位点处的序列互补的siRNA。
本发明另一目的在于提供一种干预上述circ-Ankib1表达的siRNA，核苷酸序列如下：
circ-Ankib1 siRNA1
正义链:5’-CACGAAUGUGAAACAUGUU dTdT-3’(SEQ ID NO:7)
反义链:5’-AACAUGUUUCACAUUCGUG dTdT-3’(SEQ ID NO:8)
circ-Ankib1 siRNA2
正义链:5’-GCUCACGAAUGUGAAACAU dTdT-3’(SEQ ID NO:9)
反义链:5’-AUGUUUCACAUUCGUGAGC dTdT-3’(SEQ ID NO:10)。
本发明研究结果表明，所述siRNA能够干扰circ-Ankib1的表达，促进Schwann细胞增殖。
本发明另一目的在于提供上述干预circ-Ankib1表达的siRNA在制备促进神经再生和修复神经损伤药物中的应用。
本发明构建大鼠坐骨夹伤模型，取损伤不同时间点的坐骨神经组织，通过RNA-seq及生物信息学分析，挑选损伤后表达变化明显的circRNAs。针对筛选到的circRNAs设计特异性的背向引物，再通过PCR验证以及测序鉴定，获得一种新的环状RNA circ-Ankib1。在原代Schwann细胞中，设计siRNA干扰circ-Ankib1的表达，可以促进Schwann细胞增殖。
附图说明
图1为实施例1所述circ-Ankib1和circ-Setd5在大鼠坐骨神经损伤后坐骨神经组织中不同时间点的表达变化(以GAPDH为内参)。
图2为实施例2所述circ-Ankib1和circ-Setd5的siRNA对Schwann细胞中circ-Ankib1和circ-Setd5表达的影响(以GAPDH为内参)。
图3为实施例2所述circ-Ankib1和circ-Setd5的siRNA干扰circ-Ankib1和circ-Setd5对Schwann细胞增殖的影响。
具体实施方式
以下通过实施例说明本发明的具体步骤，但不受实施例限制。
在本发明中所使用的术语，除非另有说明，一般具有本领域普通技术人员通常理解的含义。
下面结合具体实施例并参照数据进一步详细描述本发明。应理解，这些实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。
在以下实施例中，未详细描述的各种过程和方法是本领域中公知的常规方法。
实施例1：circ-Ankib1的筛选与鉴定
对SD(Sprague Dawley)大鼠坐骨神经夹伤后不同时间点(0d、1d、4d、7d、14d)的坐骨神经进行RNA-seq。根据测序结果挑选全部由外显子组成且环>500nt的表达差异明显的circRNAs。测序结果分析筛选出来的circRNA，进一步通过PCR验证circRNA的存在。根据circRNAs引物设计原则对这些circRNAs设计背向引物，每个circRNA使用NCBI设计引物。根据引物信息选择不同的退火温度进行PCR扩增，再通过琼脂糖凝胶电泳选出条带大小与预期相符的circRNAs，送公司测序，并将测序结果与原始序列比对验证其是否跨越环化位点。最终得到与预期条带大小一致且测序鉴定过环化位点的circRNAs，并挑选其中表达量较高的circ-Ankib1和circ-Setd5进行进一步研究，circ-Ankib1的cDNA序列如SEQ ID NO:1所示，circ-Setd5的cDNA序列如SEQ ID NO:2所示。
一、坐骨神经组织RNA提取
取大鼠坐骨神经夹伤后0d、1d、4d、7d、14d近端的组织按Reagent(Invitrogen)说明书提取组织RNA。大鼠坐骨神经夹伤后不同时间点的近端组织放入1.5mlRNase-free EP管，加入1mlReagent(Invitrogen)；将EP管置于冰中并使用电动匀浆器将组织匀浆，冰上静置5min-10min裂解。加入三氯甲烷200μl，涡旋剧烈振荡20s，室温静置10min；13000rpm，4℃离心15min。小心仔细吸取上清，加入异丙醇800μl，上下颠倒轻柔混匀，-20℃静置1h，13000rpm，4℃离心15min，弃除上清。加入1ml 75％乙醇，轻轻洗涤沉淀，4℃离心13000rpm，5min；去除上清，吹干。加入适量RNase-free H2O，65℃促溶10min；检测RNA的OD值及浓度，-80℃保存备用。
二、RNA逆转录合成cDNA
使用逆转录试剂盒(Takara RR037A)，将500ng的RNA逆转录为cDNA。
(1)circRNA的逆转
冰上操作，每个反应体系10μl，如下：
(2)mRNA的逆转
冰上操作，每个反应体系10μl，如下：
反应程序为：37℃45min，85℃5min，4℃∞。
三、qRT-PCR
根据circRNAs引物设计原则设计circRNAs qRT-PCR引物序列。
circ-Ankib1qRT-PCR引物
circ-Ankib1-F 5’-AGACCGCAGACATGCTCC-3’(SEQ ID NO:3)
circ-Ankib1-R 5’-AGTCCCTAATATCCTATTCATTCCA-3’(SEQ ID NO:4)
circ-Setd5qRT-PCR引物
circ-Setd5-F 5’-TACTCGGCGGTCTTCC-3’(SEQ ID NO:5)
circ-Set5-R 5’-CTCCATCTCCAGCTCTTT-3’(SEQ ID NO:6)
将逆转录反应得到的cDNA，按照1∶10稀释，进行如下qRT-PCR反应。
(1)按下列组份配制qRT-PCR反应液：
(2)反应液混匀，Real-time PCR仪反应程序如下：
(3)反应设置3个复孔，内参为GAPDH，程序结束后，查看溶解曲线与扩增曲线，舍去误差较大的实验数据，根据具体实验要求，对数据进行统计分析。
考察上述circRNA在大鼠坐骨神经损伤后坐骨神经组织中不同时间点的表达变化，结果如图1所示。结果表明circ-Setd5和circ-Ankib1在坐骨神经损伤后表达呈现先下调再上调的状态。
实施例2：circRNA的功能验证
一、原代Schwann细胞的培养
取出生1d的SD大鼠的坐骨神经，用显微剪剪碎，加入3mg/ml的胶原酶1ml，37℃消化30min，每10min摇晃一次；1200rpm，室温离心5min，弃除胶原酶，加入胰酶1ml，置于37℃培养箱中消化8min左右，加入完全培养基3ml终止消化，过200目筛网，1200rpm，室温离心5min；再加入完全培养基2ml洗1-2遍；将细胞种在用左旋多聚赖氨酸(PLL)包被好的培养皿中培养(5％CO2，37℃)；第二天，换成含有Ara-c(10μM)的完全培养基，抑制成纤维细胞的快速增殖；第四天，换成含有HRG(50ng/ml)和Forskolin(2μM)的完全培养基刺激细胞的生长。待细胞长到密度为90％以上后，用补体将细胞纯化。先用胰酶将细胞从培养皿中消化下来并转移到离心管中，室温离心1200rpm，5min；弃上清，用含有1μl anti-thy1.1(1∶1000)的1ml完全培养基将细胞沉淀重悬，置冰上孵育2h；1200rpm，室温离心5min，弃掉管中液体，加入Rabbit complement(250ul Rabbit complement+750ul DMEM)，37℃，孵育1h；DMEM洗三遍；将细胞种在用PLL包被好的培养皿中培养；第二天换液培养(含有HRG和Forskolin)，待细胞长满，纯度达95％以上，用于后续实验。二、Schwann细胞转染
转染特异性针对circ-Ankib1和circ-Setd5的siRNA：本实验中，特异性针对circ-Ankib1 siRNA和circ-Setd5 siRNA(由广州市锐博生物科技有限公司制备，终浓度：100nM)及其对照siRNA Negative Control(NC)，用LipofectamineTM RNAiMAX(Invitrogen)转入Schwann细胞，第二天换成完全培养基，根据需要进行后续实验。
circ-Ankib1 siRNA和circ-Setd5 siRNA如下所示：
circ-Ankib1 siRNA1:
正义链:5’-CACGAAUGUGAAACAUGUU dTdT-3’(SEQ ID NO:7)
反义链:5’-AACAUGUUUCACAUUCGUG dTdT-3’(SEQ ID NO:8)
circ-Ankib1 siRNA2:
正义链:5’-GCUCACGAAUGUGAAACAU dTdT-3’(SEQ ID NO:9)
反义链:5’-AUGUUUCACAUUCGUGAGC dTdT-3’(SEQ ID NO:10)。
circ-Ankib1 siRNA3:
正义链:5’-UCGAGCUCACGAAUGUGAA dTdT-3’(SEQ ID NO:11)。
反义链:5’-UUCACAUUCGUGAGCUCGA dTdT-3’(SEQ ID NO:12)。
circ-Setd5 siRNA1:
正义链:5’-CUUCCCAUGCUGGGUAAUU dTdT-3’(SEQ ID NO:13)。
反义链:5’-AAUUACCCAGCAUGGGAAG dTdT-3’(SEQ ID NO:14)。
circ-Setd5 siRNA2:
正义链:5’-CAUGCUGGGUAAUUACAAA dTdT-3’(SEQ ID NO:15)
反义链:5’-UUUGUAAUUACCCAGCAUG dTdT-3’(SEQ ID NO:16)
circ-Setd5 siRNA3:
正义链:5’-UGGGUAAUUACAAAGUGGA dTdT-3’(SEQ ID NO:17)。
反义链:5’-UCCACUUUGUAAUUACCCA dTdT-3’(SEQ ID NO:18)。
三、Schwann细胞RNA提取、逆转录及qRT-PCR
原代Schwann细胞分别转染特异性针对circ-Ankib1 siRNA及其对照siRNANegative Control(NC)，48h后，收细胞，按Reagent(Invitrogen)说明书提取Schwann细胞RNA，逆转录，进行qRT-PCR，方法同实施例1中所述。结果如图2所示，结果表明circ-Ankib1 siRNA1和circ-Ankib1 siRNA2可以显著干扰circ-Ankib1在Schwann细胞中的表达,circ-Setd5 siRNA 1-3均可以显著干扰Schwann细胞中circ-Setd5的表达，且circ-Setd5 siRNA 2和circ-Setd5 siRNA 3干扰效果显著，***P<0.001。
四、Edu细胞增殖实验
原代Schwann细胞分别转染特异性针对circ-Ankib1和circ-Setd5的siRNA(circ-Ankib1 siRNA1、circ-Ankib1 siRNA2、circ-Setd5 siRNA 2、circ-Setd5 siRNA 3)及其对照(NC)，48h后，根据Cell-LightTM EdU567 In Vitro Kit(广州市锐博生物科技有限公司，C10310-1)进行Edu细胞增殖实验。按1000：1的比例用完全培养基稀释EdU A溶液；加入96孔板，37℃孵育24h，弃去培养基；PBS轻柔清洗细胞1-2次。每孔加入4％多聚甲醛约100μl，室温孵育30min，弃去4％多聚甲醛；每孔加入2mg/ml甘氨酸50μl，孵育5min后，弃甘氨酸溶液；每孔加入PBS 100μl，5min后弃去PBS；每孔加入100μl渗透剂(0.5％TritonX-100的PBS)，10min后弃去液体；PBS清洗1次，5min。每孔加入染色反应液约100μl，室温、避光、孵育30min后，弃除反应液；加入渗透剂(0.5％TritonX-100的PBS)100μl清洗2-3次，每次10min，弃除渗透剂；按100：1的比例用ddH2O稀释试剂F，制备1×Hoechst 33342反应液，避光保存；每孔加入1×Hoechst 33342反应液50μl，室温避光孵育30min后，弃反应液；每次每孔加入PBS 100μl清洗2-3次；每孔加入PBS 100μl荧光显微镜拍照。用circ-Ankib1和circ-Setd5的siRNA(circ-Ankib1 siRNA1、circ-Ankib1 siRNA2、circ-Setd5siRNA 2、circ-Setd5 siRNA 3)与siRNA Negative Control(NC)转染原代培养的Schwann细胞，在转染siRNA 48h后，按上述方法进行EdU标记，Edu实验结果如图3所示，柱状图为siRNA干扰circ-Ankib1、circ-Setd5后Schwann细胞的增殖率，**P<0.01。结果表明：与对照组相比，siRNA干扰circ-Ankib1，显著促进Schwann细胞的增殖，而siRNA干扰circ-Setd5，对Schwann细胞增殖没有影响。
序列表
<110> 南通大学
<120> 环状RNA circ-Ankib1在制备促进神经再生和修复神经损伤药物中的应用
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Claims (7)
1.一种环状RNA circ-Ankib1，所述circ-Ankib1的cDNA核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述的环状RNA circ-Ankib1在制备促进神经再生和修复神经损伤药物中的应用。
3.如权利要求2所述的应用，其特征在于所述神经损伤为周围神经系统坐骨神经损伤。
4.如权利要求2所述的应用，其特征在于根据circ-Ankib1核苷酸序列设计干预circ-Ankib1表达的物质，选自化合物、蛋白、多肽、多糖、糖蛋白、糖肽、核酸中的一种或几种。
5.如权利要求4所述的应用，其特征在于所述干预circRNA表达的物质，选自与circ-Ankib1环化位点处的序列互补的siRNA。
6.一种干预权利要求1所述circ-Ankib1表达的siRNA，其特征在于其正义链核苷酸序列如SEQ ID NO:7或9所示。
7.如权利要求6所述的干预circ-Ankib1表达的siRNA在制备促进神经再生和修复神经损伤药物中的应用。
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| CN110760583A (zh) * | 2019-10-21 | 2020-02-07 | 南通大学 | 一种长链非编码rna bc088259及其应用 |
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| CN110819630A (zh) * | 2019-10-25 | 2020-02-21 | 南通大学 | 环状RNA circ-01477及其应用 |
| CN110819630B (zh) * | 2019-10-25 | 2023-04-07 | 南通大学 | 环状RNA circ-01477及其应用 |
| CN113278690A (zh) * | 2021-05-08 | 2021-08-20 | 南通大学 | 环状RNA circ-26782在制备脊髓损伤诊疗药物中的应用 |
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