CN116875603B - 一种靶向线粒体解偶联蛋白mRNA的miRNA分子及其应用 - Google Patents
一种靶向线粒体解偶联蛋白mRNA的miRNA分子及其应用 Download PDFInfo
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Abstract
本发明公开了一种靶向线粒体解偶联蛋白mRNA的miRNA分子及其应用。本发明所提供的靶向线粒体解偶联蛋白(Uncoupling protein 1,UCP1)mRNA的miRNA分子为SEQ ID No.1所示单链RNA。本发明miRNA可靶向负调控UCP1使得产热能力受损,针对该miRNA设计的抑制剂能够上调棕色脂肪细胞中UCP1蛋白的表达,提升氧气消耗速率,增强线粒体活力,具备促进产热潜能。因此,针对该miRNA的拮抗药物预期可在一定程度上恢复机体代谢平衡,提升人体环境适应能力。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种靶向线粒体解偶联蛋白mRNA的miRNA分子及其应用。
背景技术
产热是包括人类在内的哺乳动物在应对外界冷刺激时发生的重要的代谢功能。主要分为颤抖性产热(shivering thermogenesis, ST)和非颤抖性产热(non-shiveringthermogenesis, NST)。当机体突然处于低温环境时,首先出现的是颤抖性产热,此时肌肉收缩不做外功,能量全部转化为热量。寒冷也会刺激机体产生非颤抖性产热,即不通过肌肉收缩而是通过提高脂肪组织代谢率来增加产热的方式。人类在婴幼儿时期,由于体表面积大,非颤抖性产热是温度调节的主要方式。而对于小型哺乳动物来说,非颤抖性产热是其终身存在的抵御寒冷维持体温的重要机制。例如青藏高原鼠兔依靠非颤抖性产热提高自身代谢抵御严寒,成为高原适居动物。
在生理条件下,产热功能发生紊乱可打破机体能量平衡。例如肥胖是由于能量摄入大于能量输出所引起的。当机体适应性产热水平过低,自身能量代谢水平也相应变低,利用葡萄糖的能力受损,易导致肥胖和糖尿病。而在非正常生理条件下,如人体处于寒冷环境,机体热量大量散失将会对人体的运动系统、心血管系统、免疫系统以及神经系统产生损害,甚至威胁生命。因此,无论是应对外界环境刺激还是正常的生理条件下,机体的产热都需要被严格调控才能维持正常有效的机体稳态平衡。
非颤抖性产热主要由产热脂肪组织介导发挥其重要功能。哺乳动物的脂肪组织可以分为白色脂肪(white adipose tissue, WAT),棕色脂肪(brown adipose tissue, BAT)和米色脂肪(beige adipose tissue)三种类型。白色脂肪组织是机体最丰富的脂肪组织类型。白色脂肪细胞内含单一较大的脂滴,线粒体含量少且活性较低。主要分布在附睾、腹股沟及内脏周围,是重要的能量储库,具有储存能量、缓冲支撑、维持体温及参与能量代谢等重要作用。白色脂肪受到冷刺激或者药物刺激(如肾上腺素能受体激动剂)时,可产生棕色或类棕色脂肪细胞,即米色脂肪。这种白色脂肪组织中出现新生棕色脂肪细胞的现象被称作“米色化”。棕色脂肪是机体最重要的非颤抖性产热组织。棕色脂肪细胞的脂滴数量多且体积小,细胞内有大量线粒体等细胞器,并且高表达线粒体解偶联蛋白(uncouplingprotein 1, UCP1),在冷应激下利用UCP1的线粒体呼吸链解偶联作用产热。UCP1蛋白最早于1976年被分离,随后其编码序列被进一步鉴定。UCP1基因敲除小鼠在急性冷应激时会表现出严重的失温症。而敲除同源蛋白UCP2或者UCP3却无法重现该现象。这些研究指向UCP1是机体适应性产热重要的调节因子。
线粒体最经典的功能是通过氧化磷酸化产生三磷酸腺苷(adenosinetriphosphate,ATP)。与此不同的是,棕色脂肪组织的线粒体内膜上分布着UCP1蛋白,它可以使 H+从线粒体内膜渗漏到线粒体基质中,阻止二磷酸腺苷(adenosine diphosphate,ADP)磷酸化形成ATP,将能量以热能的形式散失,同时缓解因ATP/ADP比值升高而导致的呼吸抑制。嘌呤核苷酸(ATP,ADP,GTP,GDP)通过直接结合UCP1可以有效抑制其介导的线粒体解偶联。受到寒冷刺激后由白色脂肪水解产生的带负电的长链脂肪酸通过直接结合UCP1可以解除这种抑制效应,并促使UCP1作为脂肪酸阴离子/H+的同向转运体把氢离子转运至线粒体基质当中,完成解偶联作用。寒冷暴露会显著诱导棕色脂肪UCP1的表达,预示着棕色脂肪产热通路被激活:寒冷刺激中枢神经系统,致使交感神经末梢释放去甲肾上腺素作用于棕色脂肪细胞,随后通过肾上腺素能受体—Gs蛋白—腺苷环化酶—cAMP—蛋白激酶A(PKA)途径诱导p38促丝裂原活化蛋白激酶(MAPK)磷酸化PGC1-α,使其活化并共激活PPARα、PPARγ等因子形成转录复合物,结合到UCP1启动子区域,开启下游转录。最终,棕色脂肪中UCP1的诱导表达促进了线粒体产热以维持机体体温的稳定。
miRNA(microRNA,微小RNA)是一类长度约为22-24nt的内源单链非编码小分子RNA,在生物体中广泛存在。它们通过与靶基因的3’未翻译区域(UTR)中的互补序列结合来调节mRNA翻译和稳定性。近年来大量研究表明,miRNA能够调控生物体中许多基因的表达,在调节生长发育、细胞增殖、凋亡、抵御环境胁迫等诸多方面发挥重要作用。作为重要的转录后调节方式,目前已报道的可以直接靶向UCP1 mRNA的miRNA还十分有限,针对靶向UCP1的miRNA开展核苷酸药物研发在未来具备广阔的应用前景。
发明内容
本发明的目的是提供一种靶向线粒体解偶联蛋白(UCP1)mRNA的miRNA分子及其应用。本发明所提供的miRNA为来源于脂肪组织的外泌体miRNA,命名为miRNA PC-3p-27388_77。
第一方面,本发明要求保护一种miRNA分子(即miRNA PC-3p-27388_77的成熟体)。
本发明所要求保护的miRNA分子为SEQ ID No.1所示单链RNA。
第二方面,本发明要求保护一种pre-miRNA分子(即miRNA PC-3p-27388_77的前体)。
本发明所要求保护的pre-miRNA分子为SEQ ID No.2所示单链RNA。
前体pre-miRNA能在宿主内加工成前文第一方面中的成熟单链miRNA。
第三方面,本发明要求保护一种miRNA模拟物(miRNA mimic)。
本发明所要求保护的miRNA模拟物(miRNA mimic)为miRNA PC-3p-27388_7模拟物,是双链RNA,一条链如SEQ ID No.1所示,另一条链如SEQ ID No.1的反向互补序列所示。
第四方面,本发明要求保护一种DNA分子。
本发明所要求保护的DNA分子能够转录成前文第二方面中所述的pre-miRNA分子(即miRNA PC-3p-27388_77的前体),并且能够进一步加工形成前文第一方面中所述的miRNA分子(即miRNA PC-3p-27388_77的成熟体)。
第五方面,本发明要求保护含有前文第四方面中所述DNA分子的表达盒、重组载体或重组微生物。
第六方面,本发明要求保护如下任一应用:
(A1)前文第一方面中所述的miRNA分子或前文第二方面中所述的pre-miRNA分子或前文第三方面中所述的miRNA模拟物在下调棕色脂肪组织或棕色脂肪细胞中线粒体解偶联蛋白(UCP1)表达中的应用;
(A2)前文第一方面中所述的miRNA分子或前文第二方面中所述的pre-miRNA分子或前文第三方面中所述的miRNA模拟物或前文第四方面中所述的DNA分子或前文第五方面中所述的表达盒、重组载体或重组微生物在制备用于下调棕色脂肪组织或棕色脂肪细胞中线粒体解偶联蛋白(UCP1)表达的产品中的应用;
(A3)前文第一方面中所述的miRNA分子或前文第二方面中所述的pre-miRNA分子或前文第三方面中所述的miRNA模拟物在降低棕色脂肪组织或棕色脂肪细胞中氧气消耗速率(OCR)中的应用;
(A4)前文第一方面中所述的miRNA分子或前文第二方面中所述的pre-miRNA分子或前文第三方面中所述的miRNA模拟物或前文第四方面中所述的DNA分子或前文第五方面中所述的表达盒、重组载体或重组微生物在制备用于降低棕色脂肪组织或棕色脂肪细胞中氧气消耗速率(OCR)的产品中的应用。
第七方面,本发明要求保护一种miRNA抑制物(miRNA inhibitor)。
本发明要求保护的miRNA抑制物(miRNA inhibitor)为miRNA PC-3p-27388_7抑制物,是SEQ ID No.1的反向互补序列所示单链RNA。
第八方面,本发明要求保护如下任一应用:
(B1)前文第七方面中所述的miRNA抑制物在上调棕色脂肪组织或棕色脂肪细胞中线粒体解偶联蛋白(UCP1)表达中的应用;
(B2)前文第七方面中所述的miRNA抑制物在制备用于上调棕色脂肪组织或棕色脂肪细胞中线粒体解偶联蛋白(UCP1)表达的产品中的应用;
(B3)前文第七方面中所述的miRNA抑制物在提升棕色脂肪组织或棕色脂肪细胞中氧气消耗速率(OCR)中的应用;
(B4)前文第七方面中所述的miRNA抑制物在制备用于提升棕色脂肪组织或棕色脂肪细胞中氧气消耗速率(OCR)的产品中的应用。
第九方面,本发明要求保护如下任一应用:
(C1)前文第七方面中所述的miRNA抑制物在增强棕色脂肪组织或棕色脂肪细胞中线粒体活力中的应用;
(C2)前文第七方面中所述的miRNA抑制物在制备用于增强棕色脂肪组织或棕色脂肪细胞中线粒体活力的产品中的应用;
(C3)前文第七方面中所述的miRNA抑制物在促进线粒体产热以维持机体体温稳定中的应用;
(C4)前文第七方面中所述的miRNA抑制物在制备用于促进线粒体产热以维持机体体温稳定的产品中的应用。
本发明发现低氧低温应激诱导产生全新脂肪外泌体miRNA:PC-3p-27388_77,该miRNA可靶向负调控UCP1使得产热能力受损,其抑制剂能够上调棕色脂肪组织/细胞中UCP1蛋白的表达,提升氧气消耗速率(OCR),增强线粒体活力,促进线粒体产热。因此,针对该miRNA PC-3p-27388_77的拮抗药物预期可在一定程度上恢复机体代谢平衡,提升人体环境适应能力。
附图说明
图1为PC-3p-27388_77是全新可靶向UCP1的miRNA。A为PC-3p-27388_77种子序列与小鼠UCP1 mRNA 3’UTR匹配的位点示意图。B为PC-3p-27388_77种子序列与小鼠UCP1mRNA 3’UTR匹配的位点序列图。C为PC-3p-27388_77信息总结表。
图2为PC-3p-27388_77可靶向到UCP1的外源性验证。A为psiCHECK2质粒图谱示意图。B为UCP1 mRNA 3’UTR野生型和突变型序列示意图。阴影部分是PC-3p-27388_77种子序列靶向位点,以及对应的突变后序列。C为利用双报告基因检测系统研究PC-3p-27388_77与UCP1 mRNA 3’UTR的相互作用。**p<0.01;NS无显著差异。
图3为低氧刺激(37℃;O2含量1%;5h)成熟棕色脂肪细胞后PC-3p-27388_77表达水平检测。N=6;*p<0.05。
图4为PC-3p-27388_77特异调控靶向UCP1蛋白的表达。A为过表达PC-3p-27388_77模拟物后检测UCP1表达水平。B为过表达PC-3p-27388_77抑制剂后检测UCP1表达水平。
图5为PC-3p-27388_77对细胞耗氧量(OCR)的影响。A和C为细胞线粒体压力测试曲线结果展示。B和D为细胞平均耗氧率的柱状图。N=5;**p<0.01;****p<0.0001。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南,并不以任何方式构成对本发明的限制。
下述实施例中的实验方法,如无特殊说明,均为常规方法,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中所用到的原代棕色脂肪细胞是参考如下两篇文献自行提取诱导获得的:
Oeckl J, Bast-Habersbrunner A, Fromme T, Klingenspor M, Li Y.Isolation, Culture, and Functional Analysis of Murine Thermogenic Adipocytes.STAR Protoc. 2020 Sep 22;1(3):100118. doi: 10.1016/j.xpro.2020.100118. PMID:33377014; PMCID: PMC7757011.
Aune UL, Ruiz L, Kajimura S. Isolation and differentiation of stromalvascular cells to beige/brite cells. J Vis Exp. 2013 Mar 28;(73):50191. doi:10.3791/50191. PMID: 23568137; PMCID: PMC3641667.
具体操作如下:
第1天:准备消化用的溶液和相关试剂。
用不含血清的DMEM配制同时含1mg/ml的I型胶原酶和1mg/ml的II型胶原酶溶液。第二天使用前按照使用说明加入双抗(#P1400,索莱宝生物)和终浓度为5mM的CaCl2溶液。
第2天:进行小鼠解剖和细胞消化提取。
1、通过CO2窒息对8-10只幼鼠(出生1-2周的小鼠)实施安乐死。
2、解剖肩胛间棕色脂肪组织。在解剖过程中尽量不要损坏主要血管,因为这将有助于最大限度地减少红细胞的污染。
3、用1×PBS冲洗脂肪组织,去除残留的结缔组织和肌肉组织。用1×PBS覆盖以防止组织变干;以无菌方式尽快处理。
4、用灭菌刀片将脂肪组织切成小块,并剁成肉泥。大块会对消化过程产生负面影响,导致细胞产量低。
5、用无菌镊子将上述步骤中剁碎的脂肪肉泥转移至15ml离心管中,随后加入5ml提前配制好并加入双抗和氯化钙的消化缓冲液,开始消化反应。
6、在37℃水浴中消化40-50分钟。每2-3分钟倒置离心管。并确保消化顺利,防止消化过度。
7、加入5ml DMEM/F12培养基(含有10% FPS和双抗)停止消化。将离心管上下颠倒混匀。
8、将细胞过滤器(直径50-70μm)放在新的50ml离心管上,并过滤细胞悬液。然后将细胞悬液转移到新的15ml离心管中。
9、以700g转速离心15分钟。
10、具有诱导分化潜能的血管基质组份(SVF)大部分是聚集在管底部的褐色颗粒,需要保留这部分细胞用于进行后续诱导分化,吸出顶部和大部分液体层的油性成熟脂肪细胞层。
11、在剩余含有血管基质组份细胞的离心管中加入2毫升完全培养基,混合均匀剩余的沉淀物。
12、将2ml悬液加入到含有新鲜47ml DMEM/F12培养基的50ml离心管中混匀。
13、将每孔2mL细胞悬液接种到六孔细胞培养板中。8-10只幼鼠来源的血管基质细胞悬液每种接种4个六孔板。
第3天至第6天:进行细胞换液。
在第二天或接种后至少6小时,使用真空泵小心地除去培养基,并用移液管加入2mL新鲜DMEM/F12培养基。之后每隔一天换液一次,直至细胞长满。注意在第二次换液的时候需要用PBS洗一次细胞,将残余的死亡未贴壁的杂细胞清理干净。
第7天至第9天:进行细胞诱导。
每孔加入2ml的诱导培养基(配方:DMEM高糖培养基,含有10%FBS,含有5μg/ml胰岛素,1nM 3,3ʹ,5-三碘-l-甲状腺原氨酸,125μM吲哚美辛,0.5mM异丁基甲基黄嘌呤,0.5μm罗格列酮,1μM地塞米松)诱导2天。
第10天至第15天:进行细胞分化。
诱导结束后将培养基更换为维持培养基(配方:DMEM高糖培养基,含有10%FBS,补充有5μg/ml胰岛素,1nM 3,3ʹ,5三碘-l-甲状腺原氨酸和0.5μm罗格列酮)再培养5天即可获得成熟的棕色脂肪细胞。
实施例1、靶向线粒体解偶联蛋白mRNA的miRNA分子PC-3p-27388_77及其应用
一、低氧诱导脂肪组织产生全新miRNA PC-3p-27388_77靶向UCP1
(1)PC-3p-27388_77是全新的miRNA
小RNA(miRNA)是一类短的非编码RNA(22-24个核苷酸),通过与靶基因的3’未翻译区域(UTR)中的互补序列结合来调节mRNA翻译和稳定性。作为重要的转录后调节方式,目前已报道的可以直接靶向UCP1 mRNA的miRNA还十分有限,因此靶向UCP1的miRNA相关生物制剂在机体代谢产热的研究领域将具有广阔前景。
发明人经过广泛深入的研究,意外发现低氧可以诱导棕色脂肪细胞表达全新的miRNA:PC-3p-27388_77,该miRNA位于小鼠12号染色体,通过和miRbase数据库比对,发现其属于并未收录的全新miRNA(图1)。
miRNA序列:5’-UUGAACUGUCAAGAACCACUGGU-3’(对应SEQ ID No.1);
pre-miRNA序列:5’-gccgccgugcgcgccgggucuagugguccuaaacauuucacaauugugcuacagaacugUUGAACUGUCAAGAACCACUGGUccaggcgcgccugcaca-3’(对应SEQ ID No.2)。
即PC-3p-27388_77的成熟体序列如序列表中SEQ ID No.1所示(单链RNA,SEQ IDNo.1中用T表示U),前体序列如序列表中SEQ ID No.2所示(单链RNA,SEQ ID No.2中用T表示U)。
根据已有研究,miRNA的5’端2-8个被称为种子序列(seed sequence)的核苷酸如果与靶mRNA的3’UTR匹配,则会通过抑制靶mRNA的翻译来沉默靶基因。我们发现PC-3p-27388_77的种子序列可以与小鼠UCP1 mRNA 3’UTR序列完全互补匹配(图1)。
以上结果为后续进行PC-3p-27388_77的表达水平分析和UCP1调控功能分析提供了坚实基础。
(2)PC-3p-27388_77可靶向UCP1的外源性验证
以PC-3p-27388_77为靶标构建PC-3p-27388_77模拟物(mimic)。miRNA mimic(模拟物)为化学合成的成熟miRNA双链,能增强内源性miRNA功能。包括一条与目标miRNA成熟体序列一致的序列,以及一条与miRNA成熟体序列互补的序列。PC-3p-27388_77模拟物为由序列表中SEQ ID No.1和SEQ ID No.1的反向互补序列组成的双链RNA。
同时构建阴性对照模拟物:由序列表中SEQ ID No.3和SEQ ID No.3的反向互补序列组成的双链RNA(用T表示U)。
将野生型小鼠UCP1基因的3’UTR区(SEQ ID No.4)克隆至荧光素酶报告基因载体psiCHECK-2(Promega,Cat.#C8021)的酶切位点XhoI和NotI之间,构建得到包含野生型小鼠UCP1基因3’UTR序列(SEQ ID No.4)的重组载体,经测序验证正确后命名为psiCHECK2-UCP13’UTR WT。
为了实验的严谨性,同时制备了包含PC-3p-27388_77种子序列结合位点的突变型小鼠UCP1 3’UTR 序列(SEQ ID No.5)的重组载体psiCHECK2-UCP1 3’UTR mut。重组载体psiCHECK2-UCP1 3’UTR mut与psiCHECK2-UCP1 3’UTR WT的区别仅在于将SEQ ID No.4替换为SEQ ID No.5。
按照常规细胞培养方法将HEK293T种植到24孔板上。利用PEI分别转染空载体psiCHECK2、psiCHECK2-UCP1 3’UTR WT、psiCHECK2-UCP1 3’UTR mut,各两个孔。
24小时后,转染空载体psiCHECK2、psiCHECK2-UCP1 3’UTR WT、psiCHECK2-UCP13’UTR mut的孔板中,利用Lipofectamine RNA imax(Life Technologies)转染试剂,对其中一孔转染阴性对照模拟物(100nM),另一孔转染PC-3p-27388_77模拟物(100nM)。
48小时收取细胞进行检测。按照商品化试剂盒说明书的步骤,利用双荧光素酶检测试剂盒(Promega,E1960)检测每孔细胞里的相对海肾荧光强度。
如图2所示,包含野生型UCP1 3’UTR序列的载体由于被PC-3p-27388_77模拟物靶向,荧光素报告基因无法正常表达,故荧光强度下调。而包含突变型UCP1 3’UTR序列的载体由于位点不能与PC-3p-27388_77模拟物结合,故不能被靶向,荧光素报告基因正常表达,荧光强度不变。上述结果证明PC-3p-27388_77可以靶向野生型UCP1的3’UTR区域。
二、PC-3p-27388_77特异调控靶向UCP1蛋白的表达
为了确认低氧诱导棕色脂肪细胞分泌PC-3p-27388_77,我们提取新生小鼠血管基质细胞在体外培养诱导出成熟的棕色脂肪细胞。随后将成熟的棕色脂肪细胞置于低氧细胞培养箱(37℃;O2含量1%)培养5小时,收取细胞检测PC-3p-27388_77的表达水平。具体方法如下:
(1)提取细胞总miRNA。使用试剂盒为miRcute miRNA提取分离试剂盒(DP501,天根生物)。按照说明书步骤提取低氧应激后细胞的总miRNA,并测定总miRNA浓度。
(2)利用miRcute增强型miRNA cDNA第一链合成试剂盒(KR211,天根生物),按照说明书步骤对总miRNA进行反转录,构建cDNA库。
(3)利用miRcute Plus miRNA荧光定量检测试剂盒(FP411,天根生物),按照说明书,对PC-3p-27388_77的表达水平进行荧光定量检测。
正向引物为试剂盒自带,无需设计。
反向引物为检测PC-3p-27388_77和内参基因U6的引物。
引物序列为:
PC-3p-27388_77:5’-CGTTGAACTGTCAAGAACCACTGGT-3’(SEQ ID No.7);
U6:5’-GCTCGCTTCGGCAGCACA-3’(SEQ ID No.8)。
如图3所示,同未刺激对照相比,PC-3p-27388_77在低氧处理的棕色脂肪细胞中被诱导出高表达。
以PC-3p-27388_77为靶标构建PC-3p-27388_77模拟物(由序列表中SEQ ID No.1和SEQ ID No.1的反向互补序列组成的双链RNA)和阴性对照模拟物(由序列表中SEQ IDNo.3和SEQ ID No.3的反向互补序列组成的双链RNA)。获得原代棕色脂肪细胞系后,用Lipofectamine RNA imax(Life Technologies)把阴性对照模拟物(100nM)和PC-3p-27388_77模拟物(100nM)分别转染进原代棕色脂肪细胞。48小时后收集细胞裂解物,Western blot检测棕色脂肪细胞中的UCP1蛋白和线粒体组份蛋白ATP5A1的表达情况。
以PC-3p-27388_77为靶标构建PC-3p-27388_77抑制剂(为化学合成的成熟miRNA反向互补单链,即为SEQ ID No.1的反向互补序列所示单链RNA)和阴性对照抑制剂(为化学合成的SEQ ID No.6的所示单链RNA)。获得原代棕色脂肪细胞系后,用 Lipofectamine RNAimax(Life Technologies)把PC-3p-27388_77抑制剂(100nM)和阴性对照抑制剂(100nM)分别转染进原代棕色脂肪细胞。48小时后收集细胞裂解物,Western blot检测棕色脂肪细胞中的UCP1蛋白和线粒体组份蛋白ATP5A1的表达情况。
UCP1抗体:proteintech,Cat No.23673-1-AP。
ATP5A1作为阴性对照,用于指示PC-3p-27388_77的特异性。PC-3p-27388_77只影响UCP1表达,不影响ATP5A1。ATP5A1抗体:proteintech,Cat No.14676-1-AP。
Vinculin:内参蛋白。Vinculin抗体:CST,#13901。
结果如图4所示,过表达PC-3p-27388_77模拟物后,棕色脂肪中UCP1蛋白表达显著下调。而过表达PC-3p-27388_77抑制剂后可以有效上调UCP1蛋白表达。这两种情况下,线粒体组份蛋白ATP5A1表达并未受影响,表明PC-3p-27388_77确实可以特异调控靶向UCP1蛋白的表达。
三、PC-3p-27388_77对细胞耗氧量(OCR)的影响
在原代棕色脂肪细胞系内验证PC-3p-27388_77对细胞耗氧率(oxygenconsumption rate,OCR)的影响。参照已有的实验流程和实验方法,将上一步骤中转染有不同PC-3p-27388_77模拟物或PC-3p-27388_77抑制剂的棕色脂肪细胞利用 Seahorse XFe24分析仪(Seahorse Bioscience)以24孔板形式测量活细胞的耗氧率(OCR),具体步骤按照“Agilent Seahorse XFe24实验操作手册”进行。用以反映PC-3p-27388_77在细胞内对UCP1的调控是否最终影响脂肪细胞的呼吸代谢。
如图5所示,在线粒体压力测试实验通过依次加入线粒体电子传递链(ETC)的靶向药物测量细胞的氧气消耗速率(OCR)而得到反映线粒体功能的关键参数。这几种药物分别为:寡霉素、异丙肾上腺素、三氟甲氧基羰基氰苯腙和鱼藤酮。
耗氧率是海马细胞能量代谢仪(型号:XF24,安捷伦)中最基本的指标之一,反映的是细胞内线粒体呼吸链的功能状况。当线粒体的功能受损时,耗氧率通常会下降。过表达PC-3p-27388_77模拟物的时候,可以显著降低棕色脂肪细胞的氧气消耗速率,而过表达PC-3p-27388_77抑制剂的时候,则使得棕色脂肪细胞氧气消耗速率提升,指示PC-3p-27388_77抑制剂的应用使得细胞线粒体活力增强。
上述结果表明,PC-3p-27388_77抑制剂能够特异性上调棕色脂肪组织/细胞中UCP1蛋白的诱导表达,进而促进线粒体产热以维持机体体温的稳定。
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。
Claims (7)
1. miRNA分子,其特征在于:所述miRNA分子为SEQ ID No.1所示单链RNA。
2. pre-miRNA分子,其特征在于:所述pre-miRNA分子为SEQ ID No.2所示单链RNA。
3. miRNA模拟物,其特征在于:所述miRNA模拟物为双链RNA,一条链如SEQ ID No.1所示,另一条链如SEQ ID No.1的反向互补序列所示。
4. DNA分子,其特征在于:所述DNA分子能够转录成权利要求2所述的pre-miRNA分子,并进而加工形成权利要求1所述的miRNA分子。
5.含有权利要求4所述DNA分子的表达盒、重组载体或重组微生物。
6. miRNA抑制物,其特征在于:所述miRNA抑制物为SEQ ID No.1的反向互补序列所示单链RNA。
7.权利要求6所述的miRNA抑制物在制备用于促进线粒体产热以维持机体体温稳定的产品中的应用。
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