CN114015686A - 新的与衰老相关的circRNA、筛选方法及应用 - Google Patents

新的与衰老相关的circRNA、筛选方法及应用 Download PDF

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CN114015686A
CN114015686A CN202111081145.5A CN202111081145A CN114015686A CN 114015686 A CN114015686 A CN 114015686A CN 202111081145 A CN202111081145 A CN 202111081145A CN 114015686 A CN114015686 A CN 114015686A
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circrna
atxn2
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宋兴辉
王琳琳
高子颖
邢月婷
李艳伟
刘双双
郭春
王佳佳
黄莹莹
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Zhejiang University ZJU
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Abstract

本发明公开了新的与衰老相关的circRNA、筛选方法及应用，首选筛选得到了新的与衰老相关的circRNA，并对circRNA‑1783(circRNA‑ATXN2)进行了研究，构建了circRNA‑ATXN2过表达载体，并包装成慢病毒，对大鼠皮下脂肪来源的间充质干细胞进行感染，利用流式分选技术，获取空载体(vector‑GFP)和过表达circRNA‑ATXN2(circRNA‑ATXN2‑GFP)的细胞，进行功能检测实验。circRNA‑ATXN2对ADSCs起到抑制增殖，促进凋亡和脂肪分化的作用。circ‑ATXN2(circRNA‑1783)将是提高ADSCs的干细胞治疗能力和ADSCs的内源性修复过程能力的新的潜在靶点。

Description

新的与衰老相关的circRNA、筛选方法及应用
技术领域
本发明具体涉及新的与衰老相关的circRNA、筛选方法及应用。
背景技术
脂肪组织由多种类型的细胞组成，其中脂肪组织来源的间充质干细胞(ADSCs)是脂肪组织中含量较多的细胞，被认为是一种有前途的用于基于干细胞的治疗的再生医学的细胞来源。然而ADSCs的数量和功能会随着年龄的增长而下降，降低干细胞内源性修复过程的能力和影响以干细胞为基础的治疗。
circRNA是一种新的内源性非编码RNA。然而，环状RNA在ADSCs中的功能尚不清楚。
发明内容
针对上述情况，为克服现有技术的缺陷，本发明提供了新的与衰老相关的circRNA、筛选方法及应用。
为了实现上述目的，本发明提供以下技术方案：
新的与衰老相关的circRNA，包括circRNA-1783。
一种新的与衰老相关的circRNA的筛选方法，所述筛选方法能够用于筛选所述的circRNA，包括以下步骤：
(1)采集年轻组和年老组大鼠的皮下脂肪组织；
(2)分别提取年轻组和年老组大鼠的总RNA；
(3)纯化总RNA样本，其中RNA完整性号>7.0；
(4)去除核糖体RNA；
(5)筛选circRNA的表达谱；
(6)通过R package-edgeR筛选两组间差异表达的环状RNA，其倍变幅≥2，p值≤0.05，
(7)进行GO和KEGG富集分析；设定筛选条件进行筛选，得到circRNA-1783。
进一步地，步骤(7)中的筛选条件为：(1)每一个样本均有表达量；(2)exons≥3；(3)有miRNA潜在靶点。
新的与衰老相关的circRNA在调控大鼠脂肪来源干细胞ADSCs增殖、凋亡及脂肪分化中的应用，所述circRNA为以上所述的circRNA。
新的与衰老相关的circRNA在提高ADSCs的干细胞治疗能力和ADSCs的内源性修复能力中的应用，所述circRNA为以上所述的circRNA。
本发明的有益效果是：
本发明筛选得到了新的与衰老相关的circRNA，并对circRNA-1783(circRNA-ATXN2)进行了研究，构建了circRNA-ATXN2过表达载体，并包装成慢病毒，对大鼠皮下脂肪来源的间充质干细胞进行感染，利用流式分选技术，获取空载体(vector-GFP)和过表达circRNA-ATXN2(circRNA-ATXN2-GFP)的细胞，进行功能检测实验。circRNA-ATXN2对ADSCs起到抑制增殖，促进凋亡和脂肪分化的作用。环状RNA，比如circ-ATXN2(circRNA-1783)，将是提高ADSCs的干细胞治疗能力和ADSCs的内源性修复过程能力的新的潜在靶点。
附图说明
图1为表达差异显著的环状RNA的火山图和热图；其中，A为表达差异火山图；蓝色点为表达下调的环状RNA，红色点为表达上调的环状RNA，灰色点为表达没有显著性差异的环状RNA；B为表达差异热图，表达差异色带从蓝到红分别表示表达量从低到高。
图2(A)为大鼠皮下脂肪组织中circ-ATNX2测序相对表达量，图2(B)为circ-ATNX2的RT-PCR相对表达量示意图。
图3A(1)为空载的细胞转染图，图3A(2)为过表达circ-ATXN2的细胞转染图；绿色为阳性细胞；B为流式检测的过表达效率图，空载体组和过表达组分别为92.3％和49.1％；C为两组阳性细胞中circ-ATNX2的相对表达量示意图。
图4为circ-ATNX2抑制ADSCs增殖的结果示意图；A.Edu检测细胞增殖的示意图；荧光图片中红色荧光为Edu阳性细胞，蓝色为核染料DAPI；流式图中蓝色线条为空载体组，红色线条为过表达组；B.RTCA检测细胞增殖的示意图。
图5为circ-ATNX2促进ADSCs凋亡的示意图；左边为凋亡检测流式图，右边为统计图。
图6为circ-ATNX2促进ADSCs向脂肪分化的示意图；A为油红染色图；B为油红染色光吸收值测定结果；C为脂肪分化特异性基因PPARr和CEBP/a检测结果图。
具体实施方式
以下结合附图对本发明的技术方案做进一步详细说明，应当指出的是，具体实施方式只是对本发明的详细说明，不应视为对本发明的限定。
以下实施例中所用到的细胞、化学物质、试剂、仪器等均能够通过商业途径购得。以下实施例中，PBS的浓度为10mM,PH为7.2-7.4。
1.动物及样本采集
实验程序经浙江大学动物伦理委员会批准，并按照机构指导原则进行。年轻组(n＝3、10周)和年老组(n＝3、27月龄)雄性SD大鼠由浙江大学动物中心提供。CO2窒息处死大鼠后采集皮下脂肪组织，并立即将一部分组织冻存于液氮中，-80℃保存，用于后续分析和RT-PCR。另外，一部分组织用4％甲醛固定，进行H&E和TUNEL染色。
2.脂肪组织特征分析
(1)在皮下脂肪石蜡切片上测定脂肪细胞的直径和数量。切片用苏木精-伊红(H&E)染色。染色后，用NDP软件测定年轻大鼠(n＝100个脂肪细胞)和年老大鼠(n＝100个脂肪细胞)3个切片的脂肪细胞直径。并计数两组大鼠(n＝3)3个切片相同区域的脂肪细胞数量。
(2)每组(n＝3)按照制造商说明书(in situ apoptosis Detection kit,Takara)进行TUNEL检测。所有染色切片在共聚焦显微镜下成像(A1R，尼康)。
3.核糖核酸测序分析
采用Trizol试剂(Invitrogen,Carlsbad,CA,USA)分别提取年轻和年老大鼠的总RNA。通过RNeasy试剂盒(Cat#217004,QIAGEN,GmBH,Germany)和RNase-Free DNase Set(Cat#79254,QIAGEN,GmBH,Germany)进一步纯化合格的总RNA样本，其中RNA完整性号(RIN)为>7.0。
进一步利用Ribo-ZeroTM rRNA Removal Kit(Illumina,San Diego,USA)去除核糖体RNA。然后用Illumina Hiseq 4000(LC Bio,China)筛选出覆盖9812个circRNA的表达谱。RNA_seq的原始数据已存入GEO(Gene Expression Omnibus)数据库(https://www.ncbi.nlm.nih.gov；加入SUB9913766)。
4.功能分析和生物信息学
首先使用FastQC(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/)对序列质量进行验证。通过R package-edgeR筛选两组间差异表达的环状RNA，其倍变幅≥2,p值≤0.05，具有统计学意义。利用Rsutdio的clusterProfiler软件包进行了Gene Ontology(GO)和Kyoto Ency-clopedia of Genes and Genomes(KEGG)富集分析，以探索circRNA的潜在作用。使用targetscan和miRanda软件预测miRNA-circRNA的相互作用，帮助解决circRNA的功能和机制。
具体地，利用基因测序的方法获取了年轻大鼠和年老大鼠皮下脂肪组织的环状RNA的表达谱，依据fold change≥2和p value≤0.05获得67个表达差异显著的环状RNA，并基于宿主基因的表达通过GO和KEGG分析其功能；
通过设定的三个筛选条件(1、每一个样本均有表达量；2、exons≥3；3、有miRNA潜在靶点)，最终得到circRNA-1783。
本发明将circRNA-1783命名为circRNA-ATXN2并进行研究。为了研究circRNA-ATXN2的功能，构建了circRNA-ATXN2过表达载体，并包装成慢病毒，对大鼠皮下脂肪来源的间充质干细胞进行感染。利用流式分选技术，获取空载体(vector-GFP)和过表达circRNA-ATXN2(circRNA-ATXN2-GFP)的细胞，用于下面的功能检测实验。
5.细胞培养和处理
雄性SD大鼠(4日龄)由浙江大学动物中心提供。从皮下脂肪中分离并培养ADSCs。具体地，将皮下脂肪用质量分数为1％胶原酶(Gibco Life Technologies Inc.)在37℃的水浴摇瓶中充分消化2小时，800g离心3分钟。离心后，细胞在添加体积分数10％胎牛血清(FBS)(Gibco Life Technologies Inc.)和青霉素(100U/ml)/链霉素(100μg/ml)的低糖Dulbecco's modified Eagle培养基(DMEM)中培养。每3天更换一次培养基。当细胞达到80％汇合时进行传代培养。之前的流式细胞术分析表明，分离的ADSCs在CD90和CD105阳性的基础上具有较高的纯度。
6.慢病毒的构建和感染
为了过表达circ-ATXN2，使用了plc5-circ-gfp(#GS0108)载体(Geneseed，中国广州)。将全长circ-ATXN2(chr12:40329454-40335680)插入到EcoRI和BamHI酶切位点之间的plc5-circ-gfp载体中，包装进慢病毒并感染到大鼠ADSCs中。用流式细胞仪(BeckmanCulter,moflo Astrios EQ)对载体-gfp和circ-ATXN2-GFP细胞进行分选，进行下一步实验。
7.细胞增殖实验
(1)xCELLigence实时细胞分析(RTCA)系统增殖实验
RTCA系统(ACEA Biosciences,DP)是一种可以通过阻抗读数连续实时测量细胞活力的机器。电阻指数(CI)反映电子传感器阻抗的任意单位。将50μL的ADSCs培养基加入16-E-Plates(ACEA Biosciences)(n＝4)孔中，测量背景阻抗并显示为CI指数。空载体-GFP和circ-ATXN2-GFP细胞分别以2000个/孔的细胞密度种在16-E-Plate中。之后，将板置于层流罩内室温保存30分钟，然后转移到细胞培养箱内的RTCA仪上。在整个实验期间，每隔15分钟立即开始数据记录。
(2)5-Ethyny1-2’-denoxyridine(EdU)掺入法检测增殖
使用EdU增殖试剂盒(iFluor647,abcam)检测细胞增殖。将vector-GFP和circ-ATXN2-GFP细胞(1x104)接种于96孔板中过夜。次日，用EdU溶液(50μM)37℃孵育细胞2小时。随后，用质量分数4％多聚甲醛固定细胞30分钟，用甘氨酸(2mg/ml)孵育5分钟。在接下来的步骤中，用体积分数0.5％Triton X-100的PBS渗透细胞10分钟，然后用Hoechst染色在黑暗中30分钟。最后，用共聚焦显微镜(IX81-FV1000,Olympus)对细胞成像。
同时采用Beckman Culter、cytoflex LX流式细胞仪检测EdU-阳性细胞百分率。
(3)流式细胞术检测细胞周期
从培养皿中收集vector-GFP和circ-ATXN2-GFP细胞(1×106)，PBS洗涤，体积分数70％乙醇固定，4℃保存过夜。然后用2.5μg/ml碘化丙啶(PI,Sigma)在12.5μg/ml RNase(LifeTechnologies)存在下染色。采用FC500流式细胞仪(Beckman Culter)测定细胞周期各期细胞数。
(4)流式细胞术检测细胞凋亡
为了观察过表达circ-ATXN2对细胞凋亡的影响，采用CoCl2制作细胞凋亡模型。将Vector-GFP和circ-ATXN2-GFP细胞以每孔1×105个细胞的方式放置在6孔板上，用500μMCoCl2处理24小时。Annexin V-APC/PI凋亡试剂盒(MULTI SCIENCES)检测细胞凋亡情况。收集细胞，在500μl、5μl Annexin V-APC和5μlPI的结合缓冲液中室温黑暗孵育15分钟。用DxFLEX流式细胞仪(Beckman Coulter)检测凋亡细胞。Annexin v-APC阳性细胞为凋亡细胞，Annexin v-APC阴性和PI阳性细胞为坏死细胞，Annexin v-APC和PI双阳性细胞为晚期凋亡细胞。Annexin v–APC阳性和PI阴性细胞为早期凋亡细胞。
(5)Adiopogenesis检测
当细胞融合达到100％时，使用添加10％胎鼠血清、1μmol/L地塞米松、100μg/mL3-异丁基1-1-甲基黄嘌呤和2μg/L胰岛素(sigma，上海，中国)的DMEM(Gibco LifeTechnologies Inc)培养基诱导vecl-gfp和circ-ATNX2-GFP细胞成脂。培养基每2天更换一次。诱导后48h，RT-PCR检测与脂肪形成相关的基因。诱导后第14天进行油红O染色。
8.RT-PCR分析
用Trizol试剂(invitrogen)提取年轻大鼠和老年大鼠SAT总RNA。用RNase R(3U/μg,Epicentre,Madison,WI,USA)处理RNA 20min，用RT-PCR检测circ-ATNX2的表达水平。
用Trizol试剂(Invitrogen)提取Vector-GFP和circ-ATNX2-GFP细胞的总RNA，检测其成脂分化相关基因的表达。每个反应在480II系统上使用SYBR green试剂盒进行三次重复(罗氏，美国)。所有基因的表达归一化为GAPDH。RT-PCR引物序列如表1所示。
表1PCR引物
Genes Forward(5'-3') Reverse(5'-3')
circ-ATXN2 AGTTATGCACGAAGAGCCACCT AGAAATCGTAGGCTGAGGCAG
PPARγ ACCACAGTTGATTTCTCCAG TGTTGTAGAGCTGGGTCTTT
CEBP/α CGACTTCTACGAGGTGGAG ATGTAGGCGCTGATGTCTAT
GAPDH CCACCACCCTGTTGCTGTAG CTTGGGCTACACTGAGGACC
9.油红O染色及定量
为了检测脂滴的产生，去除细胞培养基，在室温下用4％甲醛固定。脂肪细胞的脂质染色，在室温下用过滤过的油红O溶液处理细胞20min，然后用体积分数为60％异丙醇去除残留的染料。在显微镜下观察所得的红色脂滴，并用异丙醇萃取。在540nm处测量吸光度以量化脂肪细胞内的残余脂肪含量。
10.统计分析
所有的实验都至少进行三次后采用GraphPad Prism 8.0对结果进行图形化和统计分析。结果显示为平均值±扫描电镜。*p<0.05被认为有统计学意义，通过学生t检验或双向方差分析。
本发明利用基因测序的方法获取了年轻大鼠和年老大鼠皮下脂肪组织的环状RNA的表达谱，依据fold change≥2和p value≤0.05获得67个表达差异显著的环状RNA，并基于宿主基因的表达通过GO和KEGG分析其功能；设置fold change≥2和p value≤0.05，能够确保表达量有显著性差异。
通过设定的三个筛选条件(1、每一个样本均有表达量；2、exons≥3；3、有miRNA潜在靶点)，得到的circRNAs成为研究功能的候选环状RNA，circRNA-1783。
设置前两个条件能够确保环状RNA有表达，第三个条件能够确保环状RNA有作用。三个条件综合运用，这样有利于精确地得到相应功能的环状RNA。
circRNA-1783的序列为：
CCACCTGCCTCACCGGTTTGGGTTTGATAAAGCGAGTTGTGTTTCGTCTTCCTCTCTGTGGCAGTCTCTCCCTGGCCTGGTAGCACACAGCCACTGAGATTTCCTGTCACACAGGATGGCCCCTTCCACACTCAGCCTGGCCTGTGTTTGAGTAGAATTAGAAGATTATATTTTATGTTCTCTTTAATTTAAATAAAATGAAGATTGAATTTAACAAAACAATATTTATTAATCTTTGCAGAGGTCGGAACAGTAGCAAAGGACTGCCTCAGCCTACGATTTCTTTTGACGGAATCTATGCAAACGTGAGGATGGTTCATATACTTACATCGGTTGTGGGATCGAAATGTGAAGTACAAGTGAAAAACGGAGGTGTATATGAAGGAGTTTTTAAAACATACAGTCCTAAGTGTGATTTGGTACTTGATGCTGCACATGAGAAAAGTACAGAATCCAGTTCGGGGCCAAAACGTGAAGAAATAATGGAGAGTGTTTTGTTCAAATGCTCAGACTTCGTTGTGGTACAATTTAAAGATACAGACTCGAGTTATGCACGAAGAG
文献报道了circRNA-1783的宿主基因ATXN2与糖尿病、高血压、神经萎缩等疾病相关。将circRNA-1783命名为circRNA-ATXN2，研究circRNA-ATXN2的功能，构建了circRNA-ATXN2过表达载体，并包装成慢病毒，对大鼠皮下脂肪来源的间充质干细胞进行感染。利用流式分选技术，获取空载体(vector-GFP)和过表达circRNA-ATXN2(circRNA-ATXN2-GFP)的细胞，用于功能检测实验，分别检测了circRNA-ATXN2对ADSCs的增殖、凋亡以及脂肪分化能力的作用。
图1为表达差异显著的环状RNA的火山图和热图。其中，A为表达差异火山图。蓝色点为表达量下调的环状RNA，红色点为表达量上调的环状RNA，灰色点为表达量没有显著性差异的环状RNA。B为表达差异热图，表达差异色带，从蓝到红分别表示表达量从低到高。由图1可知各种环状RNA在年轻和年老大鼠脂肪组织中表达量的差异。
图2为大鼠皮下脂肪组织中circ-ATNX2测序相对表达量(A)和RT-PCR相对表达量(B)。
图3为circ-ATNX2过表达效率和相对表达量。A.circ-ATNX2的过表达效率荧光图，绿色为阳性细胞。B为流式检测的过表达效率，空载体组和过表达组分别为92.3％和49.1％。C.两组阳性细胞中circ-ATNX2的相对表达量。
图4为circ-ATNX2抑制ADSCs增殖的结果示意图。A.Edu检测细胞增殖的示意图，其中，荧光图片中红色荧光为Edu阳性细胞，蓝色为核染料DAPI；比例标尺(Scale bars)为20μm；流式图中蓝色线条为空载体组，红色线条为过表达组；图中13.8％,7.16％分别表示空载体组、过表达组的Edu阳性细胞的百分比含量。B.RTCA检测细胞增殖的示意图。由图4A、4B可以得到在大鼠脂肪来源的干细胞中过表达环状RNA会抑制细胞增殖。
图5为circ-ATNX2促进ADSCs凋亡的示意图。左边为凋亡检测流式图，右边为统计图。由图5可知，在大鼠脂肪来源的干细胞中过表达环状RNA会促进细胞凋亡。
图6为circ-ATNX2促进ADSCs向脂肪分化的示意图。A为油红染色图。B为油红染色光吸收值测定结果。C为脂肪分化特异性基因PPARr和CEBP/a检测结果图。由图6可知，在大鼠脂肪来源的干细胞中过表达环状RNA会促进细胞向脂肪分化。
综上，实验结果显示，circRNA-ATXN2对ADSCs起到抑制增殖，促进凋亡和脂肪分化的作用。
脂肪组织逐步被证明是一个针对基本衰老机制的有价值的目标组织。更好地了解脂肪组织老化对ADSCs功能的影响可能对再生医学具有广泛的意义。因此，脂肪组织老化对于年龄相关机制的研究具有重要价值，也是对抗衰老和年龄相关疾病新疗法的一个强有力的治疗靶点。
本发明筛选并研究了一种新的与年龄相关的环状RNA—circ-ATXN2，其在调节ADSCs增殖、凋亡和脂肪形成中具有重要作用。环状RNA，比如circ-ATXN2，将是提高ADSCs的干细胞治疗能力和ADSCs的内源性修复过程能力的新的潜在靶点。
显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本发明保护的范围。

Claims (5)

1.新的与衰老相关的circRNA，其特征是，包括circRNA-1783。
2.一种新的与衰老相关的circRNA的筛选方法，其特征是，所述筛选方法能够用于筛选权利要求1所述的circRNA，包括以下步骤：
(1)采集年轻组和年老组大鼠的皮下脂肪组织；
(2)分别提取年轻组和年老组大鼠的总RNA；
(3)纯化总RNA样本，其中RNA完整性号>7.0；
(4)去除核糖体RNA；
(5)筛选circRNA的表达谱；
(6)通过R package-edgeR筛选两组间差异表达的环状RNA，其倍变幅≥2，p值≤0.05；
(7)进行GO和KEGG富集分析；设定筛选条件进行筛选，得到circRNA-1783。
3.根据权利要求1所述的新的与衰老相关的circRNA，其特征是，步骤(7)中的筛选条件为：(1)每一个样本均有表达量；(2)exons≥3；(3)有miRNA潜在靶点。
4.新的与衰老相关的circRNA在调控大鼠脂肪来源干细胞ADSCs增殖、凋亡及脂肪分化中的应用，其特征是，所述circRNA为权利要求1中所述的circRNA。
5.新的与衰老相关的circRNA在提高ADSCs的干细胞治疗能力和ADSCs的内源性修复能力中的应用，其特征是，所述circRNA为权利要求1中所述的circRNA。
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