CN110141578B - 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
- Publication number
- CN110141578B CN110141578B CN201910496040.2A CN201910496040A CN110141578B CN 110141578 B CN110141578 B CN 110141578B CN 201910496040 A CN201910496040 A CN 201910496040A CN 110141578 B CN110141578 B CN 110141578B
- Authority
- CN
- China
- Prior art keywords
- circ
- ankib1
- artificial sequence
- seq
- rna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000005036 nerve Anatomy 0.000 title claims abstract description 12
- 230000008929 regeneration Effects 0.000 title claims abstract description 12
- 238000011069 regeneration method Methods 0.000 title claims abstract description 12
- 208000028389 Nerve injury Diseases 0.000 title claims abstract description 11
- 230000008764 nerve damage Effects 0.000 title claims abstract description 11
- 230000001737 promoting effect Effects 0.000 title claims abstract description 10
- 239000003814 drug Substances 0.000 title claims abstract description 9
- 238000002360 preparation method Methods 0.000 title claims abstract description 6
- 108091028075 Circular RNA Proteins 0.000 title abstract description 13
- 230000014509 gene expression Effects 0.000 claims abstract description 19
- 108020004459 Small interfering RNA Proteins 0.000 claims description 27
- 108091081021 Sense strand Proteins 0.000 claims description 9
- 239000002299 complementary DNA Substances 0.000 claims description 7
- 206010039670 Sciatic nerve injury Diseases 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 210000001428 peripheral nervous system Anatomy 0.000 claims description 5
- 230000002452 interceptive effect Effects 0.000 claims description 4
- 210000004116 schwann cell Anatomy 0.000 abstract description 22
- 230000035755 proliferation Effects 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract description 4
- 208000010886 Peripheral nerve injury Diseases 0.000 abstract description 3
- 230000008439 repair process Effects 0.000 abstract description 3
- 230000002222 downregulating effect Effects 0.000 abstract 1
- 230000002401 inhibitory effect Effects 0.000 abstract 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 19
- 108020004414 DNA Proteins 0.000 description 18
- 210000004027 cell Anatomy 0.000 description 13
- 241000700159 Rattus Species 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 230000000692 anti-sense effect Effects 0.000 description 8
- 210000003497 sciatic nerve Anatomy 0.000 description 8
- 239000013642 negative control Substances 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 238000011529 RT qPCR Methods 0.000 description 6
- 208000014674 injury Diseases 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 208000027418 Wounds and injury Diseases 0.000 description 5
- 230000004663 cell proliferation Effects 0.000 description 5
- 230000006378 damage Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- OHCQJHSOBUTRHG-KGGHGJDLSA-N FORSKOLIN Chemical compound O=C([C@@]12O)C[C@](C)(C=C)O[C@]1(C)[C@@H](OC(=O)C)[C@@H](O)[C@@H]1[C@]2(C)[C@@H](O)CCC1(C)C OHCQJHSOBUTRHG-KGGHGJDLSA-N 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 238000007363 ring formation reaction Methods 0.000 description 3
- 238000012453 sprague-dawley rat model Methods 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- SUZLHDUTVMZSEV-UHFFFAOYSA-N Deoxycoleonol Natural products C12C(=O)CC(C)(C=C)OC2(C)C(OC(=O)C)C(O)C2C1(C)C(O)CCC2(C)C SUZLHDUTVMZSEV-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 208000001738 Nervous System Trauma Diseases 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- 238000003559 RNA-seq method Methods 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- OHCQJHSOBUTRHG-UHFFFAOYSA-N colforsin Natural products OC12C(=O)CC(C)(C=C)OC1(C)C(OC(=O)C)C(O)C1C2(C)C(O)CCC1(C)C OHCQJHSOBUTRHG-UHFFFAOYSA-N 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 208000028412 nervous system injury Diseases 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytarabine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 108010015899 Glycopeptides Proteins 0.000 description 1
- 102000002068 Glycopeptides Human genes 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108010025020 Nerve Growth Factor Proteins 0.000 description 1
- 102000007072 Nerve Growth Factors Human genes 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108091036407 Polyadenylation Proteins 0.000 description 1
- 230000013564 activation of immune response Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 210000003050 axon Anatomy 0.000 description 1
- 230000003376 axonal effect Effects 0.000 description 1
- 238000003766 bioinformatics method Methods 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000000944 nerve tissue Anatomy 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 230000008271 nervous system development Effects 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 230000017511 neuron migration Effects 0.000 description 1
- 239000003900 neurotrophic factor Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000008736 traumatic injury Effects 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Neurosurgery (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Neurology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Epidemiology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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1.5ml RNase-free EP 管，加入1ml
Reagent(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1 μl 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 EdU
567 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100 μl，室温、避光、孵育30min后，弃除反应液；加入渗透剂(0.5％TritonX-100的PBS)100 μl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
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 875
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
aatgtgaaac atgttgtaga agtactccag aagtggctga aggaagaaag aaagtgccac 60
tgcctatcag aaaataaaac aaaacatggg aaatacaacc accaaattcc gcaaagcact 120
catcaatggt gatgaaaacc tggcttgcca aatctatgaa aataatcctc agctaaaaga 180
atcccttgat ccaaatgtat cttatggaga gccctatcag cacaacactc cattacacta 240
tgctgctaga catggaatga ataggatatt agggactttt ctttttggta gagatggaaa 300
cccaaataaa cggaatgtgc acaatgaaac atctatgcat ttgttgtgta tgggacctca 360
aatcatgata tctgaaggaa cccttcatcc tcgcttagca cggcctgtgg aagatgattt 420
cagaagggca gattgtctgc agatgatctt acgatggaaa ggagcaaaac ttgaccaggg 480
tgaatatgag agagcggcta ttgatgcagt tgataacaaa aagaacacac cccttcacta 540
tgctgcagcc tcagggatga aagcctgtgt ggagctttta gtaaaacacg gaggagactt 600
gtttgctgaa aatgaaaata aagatactcc ttgtgattgt gcggaaaagc aacaccacaa 660
agacctggcc ctcagtctgg agtctcagat ggtgttctct cgggacccag aggctgaaga 720
aatagaagct gagtatgctg cactggacaa gcgagagcca tatgaaggac taaggcccca 780
ggatcttcgt aggttaaaag atatgcttat tgtggagacc gcagacatgc tccaggcccc 840
tctgtttact gctgaggcac tgcttcgagc tcacg 875
<210> 2
<211> 589
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
taattacaaa gtggactgtg cttgtcacaa gggaaaccgg aattgcccca tacagaaaag 60
aaatcccaat gctgcagaat tgccactccc acctcctagc tttcccacca ttggagcaga 120
gaccagacgt agaaaagcac ggcggaaaga gctggagatg gagcagcaaa atgaggttcc 180
agaagagaat catgacccac aaccacaaga agttccagaa aaagtaactg tatccagtga 240
gcatgaggaa gttgacaatc cagaagaaaa accggaagaa gaaaaagaag aggccacaga 300
cgaccaggag aactcagctc atagcagaag gactcgggaa gataggaagg ttgaagccat 360
catgcatgct tttgaaactt tagagaagag aaagaaacgg cgggatcagc ctgtagagca 420
gagcaactca gacgtagaga ttactactag cagttcagag ataggagttg gagaagagac 480
aaaaactgca gcccccgagt cagaagttaa caaccctgtt acaaacattg ccatcccaag 540
catcccacag agcactggtg tgaatactcg gcggtcttcc catgctggg 589
<210> 3
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
agaccgcaga catgctcc 18
<210> 4
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
agtccctaat atcctattca ttcca 25
<210> 5
<211> 16
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tactcggcgg tcttcc 16
<210> 6
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ctccatctcc agctcttt 18
<210> 7
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 7
cacgaaugug aaacauguut t 21
<210> 8
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 8
aacauguuuc acauucgugt t 21
<210> 9
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 9
gcucacgaau gugaaacaut t 21
<210> 10
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 10
auguuucaca uucgugagct t 21
<210> 11
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 11
ucgagcucac gaaugugaat t 21
<210> 12
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 12
uucacauucg ugagcucgat t 21
<210> 13
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 13
cuucccaugc uggguaauut t 21
<210> 14
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 14
aauuacccag caugggaagt t 21
<210> 15
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 15
caugcugggu aauuacaaat t 21
<210> 16
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 16
uuuguaauua cccagcaugt t 21
<210> 17
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 17
uggguaauua caaaguggat t 21
<210> 18
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 18
uccacuuugu aauuacccat t 21
Claims (3)
1.一种干预circ-Ankib1 表达的siRNA，其特征在于其正义链核苷酸序列如SEQ IDNO:7 或9所示，所述 circ-Ankib1的cDNA 核苷酸序列如 SEQ ID NO:1 所示。
2.如权利要求 1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。
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。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910496040.2A CN110141578B (zh) | 2019-06-10 | 2019-06-10 | 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 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910496040.2A CN110141578B (zh) | 2019-06-10 | 2019-06-10 | 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 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110141578A CN110141578A (zh) | 2019-08-20 |
| CN110141578B true CN110141578B (zh) | 2022-06-14 |
Family
ID=67590895
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910496040.2A Active CN110141578B (zh) | 2019-06-10 | 2019-06-10 | 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 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110141578B (zh) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110760583B (zh) * | 2019-10-21 | 2023-03-28 | 南通大学 | 一种长链非编码rna bc088259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 |
| CN113278690B (zh) * | 2021-05-08 | 2023-02-07 | 南通大学 | 环状RNA circ-26782在制备脊髓损伤诊疗药物中的应用 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710458A (zh) * | 2007-06-08 | 2014-04-09 | 比奥根艾迪克Ma公司 | 预测抗tnf响应性或无响应性的生物标志物 |
| EP2806274A1 (en) * | 2013-05-24 | 2014-11-26 | AIT Austrian Institute of Technology GmbH | Lung cancer diagnostic method and means |
-
2019
- 2019-06-10 CN CN201910496040.2A patent/CN110141578B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710458A (zh) * | 2007-06-08 | 2014-04-09 | 比奥根艾迪克Ma公司 | 预测抗tnf响应性或无响应性的生物标志物 |
| EP2806274A1 (en) * | 2013-05-24 | 2014-11-26 | AIT Austrian Institute of Technology GmbH | Lung cancer diagnostic method and means |
Non-Patent Citations (1)
| Title |
|---|
| Cell-Type Specific Features of Circular RNA Expression;Julia Salzman等;《PLOS Genetics》;20130930;第9卷(第9期);第1-15页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110141578A (zh) | 2019-08-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110141578B (zh) | 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 | |
| US20220251578A1 (en) | Genetic elements driving circular rna translation and methods of use | |
| Melamed et al. | Alternative splicing regulates biogenesis of miRNAs located across exon-intron junctions | |
| Kang et al. | Bone morphogenetic protein 4 promotes vascular smooth muscle contractility by activating microRNA-21 (miR-21), which down-regulates expression of family of dedicator of cytokinesis (DOCK) proteins | |
| CN108546702B (zh) | 靶向长链非编码RNA DDX11-AS1的siRNA及其在肝癌治疗中的应用 | |
| US20130158096A1 (en) | Mirnas involved in the blood brain barrier function | |
| Luo et al. | microRNA133a targets Foxl2 and promotes differentiation of C2C12 into myogenic progenitor cells | |
| Mehi et al. | MicroRNA-339 and microRNA-556 regulate Klotho expression in vitro | |
| Pang et al. | miR-1275 inhibits adipogenesis via ELK1 and its expression decreases in obese subjects | |
| Gallo et al. | A-to-I RNA editing and cancer: from pathology to basic science | |
| KR101299733B1 (ko) | ｍｉＲＮＡ의 발현 억제를 이용한 성체줄기세포의 노화 억제 방법 | |
| CN114432452A (zh) | RNA Hsa_circ_0063865抑制剂在制备抗食管鳞状细胞癌药物中的应用 | |
| CN110819630B (zh) | 环状RNA circ-01477及其应用 | |
| CN107760718B (zh) | 绵羊cnksr2基因双荧光素酶报告基因载体及构建方法和应用 | |
| CN113186306A (zh) | 一种与羊的皮肤毛囊成熟相关的miRNA及应用 | |
| CN110760583B (zh) | 一种长链非编码rna bc088259及其应用 | |
| CN109486816B (zh) | 一种用于肿瘤治疗的多聚核苷酸及其应用 | |
| CN116004630A (zh) | 一种调控tpd52基因表达的试剂在制备影响反刍动物卵巢颗粒细胞增殖的产品中的应用 | |
| CN109679960B (zh) | 一种调节肝细胞增殖的基因RGD1559786的siRNA及其应用 | |
| CN114517204A (zh) | 一种用于肿瘤治疗靶点和诊断生物标志物的circPOLK及其应用 | |
| Brenner et al. | Evaluation of BMP2/miRNA co-expression systems for potent therapeutic efficacy in bone-tissue regeneration | |
| CN109055429B (zh) | 一种靶向RunX2基因的小鼠成骨样细胞慢病毒载体及其构建方法 | |
| CN110540990A (zh) | 沉默小眼畸形转录因子mRNA表达的siRNA分子 | |
| LU501763B1 (en) | SMALL INTERFERING RNAs (siRNAs) FOR INTERFERING WITH ZINC FINGER PROTEIN 24 (ZNF24) GENE EXPRESSION AND USE THEREOF IN INHIBITING CELL PROLIFERATION AND MIGRATION | |
| Huichalaf et al. | Regulation of the promoter of CUG triplet repeat binding protein, Cugbp1, during myogenesis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20190820 Assignee: NANTONG EGENS BIOTECHNOLOGY Co.,Ltd. Assignor: NANTONG University Contract record no.: X2023980046726 Denomination of invention: Application of circular RNA circ-Ankib1 in the preparation of drugs that promote nerve regeneration and repair nerve damage Granted publication date: 20220614 License type: Common License Record date: 20231114 |