CN110540990A - 沉默小眼畸形转录因子mRNA表达的siRNA分子 - Google Patents
沉默小眼畸形转录因子mRNA表达的siRNA分子 Download PDFInfo
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Abstract
本发明公开了一组沉默小眼畸形转录因子靶基因mRNA表达的小干扰RNA分子。本发明提供的siRNA的反义链由19‑29个核苷酸组成,正义链由15‑21个核苷酸组成;正义链与反义链之间有15‑19个核苷酸互补组成双链结构。本发明提供的siRNA中的所有核苷酸或部分核苷酸等2’‑核糖上可以进行修饰,防止核苷酶降解。本发明提供的siRNA分子或同源性大于60%的分子结构,可单独或联合使用,对靶基因MITF mRNA表达的沉默效率达到40%以上,可治疗MITF突变或过表达引起的疾病或皮肤色素沉着。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种沉默基因或蛋白质表达的siRNA序列及其应用。
背景技术
小眼畸形相关转录因子(Microphthalmia-Associated Transcription Factor,MITF)具有典型的螺旋-环-螺旋-亮氨酸拉链结构(basic helix-loop-helix leucinezipper, bHLXZp),属于MiT转录因子超家族成员(Tachibana M, et al. MITF: a streamflowing for pigment cells. Pigment Cell Res. 2000;13(4):230-240.)。研究发现MITF主要在色素细胞中表达,包括成黑色素细胞(melenoblast)和视网膜色素上皮细胞(retinal pigment epithelium, RPE),其它如肥大细胞和破骨细胞等中也有表达(Carreira S,et al.MITF regulation of Dial controls melanoma proliferation andinvasiveness. Genes Dev.2006;20(24):3426-3439.)。
皮肤黑色素细胞来源于神经嵴,人类皮肤分布着大量成熟黑色素细胞,这些表皮色素细胞具有分裂共那个和杀菌作用,能够抵抗紫外线和其它外界刺激,MITF深入参与其分化(Slominski A. Neuroendocrine activity of the melanocyte. ExperimentalDermatology. 2009;18:(9)760-763.)。
研究发现,MITF参与色素沉着过程,直接作用色素生成相关基因的转录调节。MITF可激活黑色素细胞中的酪氨酸酶(tyrosinase,TYR)、酪氨酸酶相关蛋白1(tyrosinaserelated protein 2, TYRP2)及酪氨酸酶相关蛋白1(tyrosinase related protein 2,TYRP2)等基因的表达。
黑色素是一组天然色素,来源于表皮下的黑色素细胞。黑色素保护人类皮肤免受有害的紫外线辐射(UVR)和不同来源的环境污染物、有毒药物和化学品的压力。皮肤黑素细胞产生异常高的黑色素或黑色素生成会导致色素沉着障碍,如黄褐斑、老年性斑、雀斑、及色素痤疮疤痕。黑色素生成是一个多步骤的生理过程,导致合成一种复杂的深色生物聚合物,称为“黑色素”,是黑色素细胞中与溶酶体相关的细胞器,有助于保护皮肤免受阳光、有毒药物和化学物质的有害影响。
黑色素有两种类型:真黑素(eumelanin)及褐黑素(pheomelanin)。在黑色素生成过程中,有3种主要酶参与:酪氨酸酶(TYR)、酪氨酸酶相关蛋白1(Tyrp 1)和酪氨酸酶相关蛋白2(Tyrp 2)。所有黑色素生成信号通路都与黑素生成的主要调控因子MITF(microphthalmia-associated transcription factor,小眼畸形相关转录因子)有关,MITF调控黑色素生成基因TYR、Tyrp1和Tyrp2的表达。黑色素生成过程及信号传导途径种的关键限速细胞因子为MITF,抑制MITF的表达,具有抑制皮肤色素沉着使皮肤增白的功效。
在人类中,MITF控制黑色素细胞中正常黑色素合成所必需的各种基因的表达,MITF的突变可导致黑色素瘤、Waardenburg综合征和Tietz综合征等疾病(Hodgkinson CA,et al. Mutation at the mouse microphthalmia locus are associated with defectsin a gene encoding a novel basic-helix-loop-helix-zipper protein. Cell.1993;74(2):395-404.)。
除此以外,MITF与细胞周期、分化、衰老、凋亡、增殖和迁移等生物学行为相关。MITF高表达于黑色瘤细胞系,促进增殖相关转录因子 T-box 2的表达,从而促进黑色瘤增殖。另外,MITF调控细胞周期依赖性蛋白激酶2(cyclin-dependent kinase, CDK2)和细胞周期蛋白依赖性激酶抑制因子1A/B(cyclin-dependent kinase inhibitor 1A/B, CDKN1A/B),从而调控细胞生长周期。MITF通过上调B细胞淋巴瘤因子-2(B cell lymphoma-2,Bcl-2)和C-MET等抗凋亡蛋白促进肿瘤的发展。缺氧诱导因子-1α(hypoxia induciblefactor-1α)在黑色素瘤中不仅具有抗凋亡作用,还可刺激血管内皮生长因子(vascularendothelial growth factor, VEGF)的生成。研究发现,MITF可转录激活HIF-1α,从而促进肿瘤血管生成。其它细胞因子如蛋白透光形成素(diaphanous related formin 1,Dial)、miR-211和β-catenin等与黑色素瘤浸润密切相关的蛋白编码基因也受到MITF调控(CheliY,et al. Oncogene. 2012; 31 (19):2461-2470.;Arozarena I, et al. In Melanoma,beta-catenin is a suppressor of invasion. Oncogene. 2011; 30(45): 4531-4543.)。因此,抑制MITF的基因表达,从而抑制MITF的功能,具有治疗黑色瘤的应用前景。
MITF是重要的转录因子,它在生物体生命活动过程中扮演着不可或缺的角色,影响着生物体的生长、发育、分化和衰亡的各个时期,特别对动物的毛发形成具有重要的生物学意义。目前认为MITF在黑色素细胞及色素生成调控方面的作用尤其重要。
小干扰RNA(siRNA)可特异性沉默靶标mRNA表达水平(2006年诺贝尔生理医学奖),因此siRNA分子可以通过特异性沉默引起或促进疾病发生或发展的基因,具有治疗基因过表达及基因突变引起的疾病的应用前景(Science,2016,352(6292):1417-1420;Nat ChemBiol,2006,2(12):689-700.)。
在siRNA双链中,能与Argonaute蛋白(AGO蛋白)组成RNA干扰沉默复合物(RISC)的那条单链称为反义链(Antisense strand)或向导链(Guide strand),该链形成的RISC复合物能与靶mRNA结合,切割并沉默靶mRNA。而另外一条无功能的单链称之为正义链(sensestrand)或过客链(Passenger strand)。利用siRNA分子的特征,可以设计多条沉默靶基因的siRNA序列(Nature Reviews Genetics,2015,16(9):543-552)。
发明内容
发明目的:
提供一种能有效沉默黑色素细胞及黑色素瘤细胞表达的MITF mRNA表达水平的sIRNA及其应用。
技术方案:
在本发明中,采用RNAi原理,设计并筛选获得特异性沉默MITF mRNA表达的siRNA序列,用得到的siRNA抑制MITF的功能,从而实现治疗因MITF过表达或突变引起的疾病和祛除皮肤色素沉着等功效。
本发明公开沉默小眼畸形相关转录因子 mRNA表达的siRNA(小干扰RNA分子)序列,所述的小眼畸形相关转录因子(MITF)表达于黑色素瘤细胞及皮肤黑色素细胞,所述的siRNA序列为具有下列靶序列、正义链和反义链的核苷酸序列中任意一种:
siMITF-M-2
靶序列:CTGGAAATGCTAGAATATA,
正义链:5'-CUGGAAAUGCUAGAAUAUAdTdT-3',
反义链:5'-UAUAUUCUAGCAUUUCCAGdTdT-3'。
靶序列:CCTAGAATCAAGTTATAAT,
正义链:5'-CCUAGAAUCAAGUUAUAAUdTdT-3',
反义链:5'-AUUAUAACUUGAUUCUAGGdTdT-3'。
靶序列:GAACGAAGAAGAAGATTTA,
正义链:5'-GAACGAAGAAGAAGAUUUAdTdT-3',
反义链:5'-UAAAUCUUCUUCUUCGUUCdTdT-3'。
siMITF-M-9
靶序列:GAAGAAGATTTAACATAAA,
正义链:5'-GAAGAAGAUUUAACAUAAAdTdT-3',
反义链:5'-UUUAUGUUAAAUCUUCUUCdTdT-3'。
靶序列:CCACTTTAGCAAATAAACA,
正义链:5'-CCACUUUAGCAAAUAAACAdTdT-3',
反义链:5'-UGUUUAUUUGCUAAAGUGGdTdT-3'。
其中A为腺嘌呤核糖核苷酸;G为鸟嘌呤核糖核苷酸;C为胞嘧啶核糖核苷酸;U为尿嘧啶核糖核苷酸; dT为胸腺嘧啶脱氧核苷酸。
一种siRNA分子, 其正义链与所述siRNA分子中正义链序列有60%以上同源性,或者双链均有60%以上同源性;或与所述siRNA分子中反义链序列有60%以上的同源性,或者双链均有60%以上同源性;其正义链与其反义链组成双链siRNA结构。上述序列中,正义链的5'端之后的19个核苷酸序列与反义链的5'端之前的19个核苷酸序列互补。能够获得类似或者更好的沉默效率。
比如,siMITF-5X
靶序列:CCTAGAATCAAGTTAT,
正义链:5'-CCUAGAAUCAAGUUAU-3',
反义链:5'-AUUAUAACUUGAUUCUAGGCUdTdT-3'。
所述siRNA中,所有核苷酸或部分核苷酸中核糖或脱氧核糖进行2’-甲氧基修饰,或2’-氟修饰(修饰的基团较小,不会使得分子链占用的空间过大,导致排列混乱;原子团的活性较大,使得沉默效率提高);核苷酸间骨架可进行硫代磷酸酯修饰(该修饰增加了siRNA脂溶性,能够顺利进入细胞,使得沉默效率提高)。
所述siRNA分子单独或联合使用,对靶基因MITF mRNA表达的沉默效率达到40%以上。
单独或联合使用,可治疗MITF突变或过表达引起的疾病如黑色素瘤或皮肤色素沉着。
采用对照序列:siMITF为阳性对照的siRNA序列,siNC为阴性对照的siRNA序列。
设计及合成获得的候选沉默MITF mRNA表达的siRNA序列中,其siRNA靶序列、正义链和反义链中含有的核苷酸序列分别如下,见表1:
表1.候选沉默MITF mRNA表达的siRNA序列
。
本发明中,采用siRNA设计法则,结合计算机辅助设计软件,并通过对比实验筛选得到。
在此基础上,经过RT-qPCR筛选,各候选siRNA分子的基因沉默效率见下图1, 获得基因沉默效率大于40%的siMITF-2, siMITF-5, siMITF-8, siMITF-9,及siMITF-10用作生物学功能研究。
附图说明
图1是采用RT-qPCR检测中用lipofectamine 2000转染siRNA后,人黑色素细胞A375中MITF mRNA表达水平结果。图1中,纵坐标中文含义:MITF mRNA相对表达水平。此处表示转染siRNA后,细胞中还有多少MITF mRNA表达;表达水平越低,表示加入的 siRNA分子沉默MITF mRNA表达的沉默效率越高。 横坐标是14个不同siRNA分子品种。
图2. Western blot检测siMITF的蛋白质水平沉默效率显影图片。
具体实施方式
实施例1:RT-qPCR方法筛选siMITF对靶基因MITF mRNA的沉默效率
委托专业机构合成得到siRNA分子结构后,将人恶性黑素瘤细胞株A375细胞用培养基DMEM稀释传代,培养基含10%牛血清。Lipo2000转染各组终浓度为100nM的siRNA;另外,采用OPTI-MEM培养基给药,设定为空白对照组(未处理组);含转染试剂培养细胞6小时后恢复全培养基培养48h后,收集细胞,提取细胞总RNA、逆转录合成cDNA,进行qPCR检测沉默效率,以人的beta-actin基因为内参基,利用Real-time PCR试剂盒SYBR Premix(2X)(BIO-RAD750000131)进行实时荧光定量PCR反应。每个样品用3个复孔重复实验,其中Ct的误差控制在±0.5 得到如图1所示的各样品细胞中相应mRNA表达水平,siRNA序列见表1,其中siMITF-M-2、siMITF-M-5、siMITF-M-8、siMITF-M-9、及siMITF-M-10具有>40%以上的基因沉默效率,具体结果可见图1。其中,包括空白对照组(Control),阴性对照组(siNC)。
实施例2:RT-qPCR方法验证同源性>60%以上siMITF的基因沉默效率
为了增加基因沉默效率及抑制核苷酶降解,本实施例设计合成了实施1中具有基因沉默效率且同源性大于60%的siMITF序列,按实施1的方法,用RT-qPCR的方法,验证了同源性>60%以上siMITF分子的基因沉默效率,具体的siRNA序列及沉默效率见表2,说明与实施例1中筛选得到的siMITF同源性>60%以上siMITF序列同样具有生物学功能,扩大了原有siMITF分子的应用范围。。
实施例3:Western blot方法验证siMITF的蛋白沉默效率
按照实施例1的方法,用脂质体转染siMITF-2、5、8、9序列。
1、蛋白样品的获得。将转染siMITF-2、5、8、9序列72h的A375-luc细胞用pbs洗一遍,加入100微升RIPA(每1000微升中加入10微升pmsf)后刮取蛋白,冷冻5分钟后将粘稠液体收集至ep管,涡旋后置冰上冷却10分钟。4度13500rpm离心15分钟,取上清至小ep管,再分别取80微升至新的ep管中,各加入20微升5乘SDS蛋白上样缓冲液,混匀后煮沸10分钟。剩余蛋白用于bca蛋白定量。
2、BCA蛋白定量。配置0.5mg/ml蛋白标准品溶液,于96孔板中分别加入0、2、4、8、12、16、20微升,于各孔中加入pbs补至20微升。分别取2微升蛋白样品加入96孔板,加入pbs补至20微升,多余蛋白可同法用作复孔。于各孔加入200微升bca工作液。(A液:B液=50:1)将96孔板置于37度培养箱孵育30分钟后用酶标仪测定562nm处吸光度,绘制标准曲线并计算蛋白样品浓度。
3、Western blot法测定蛋白表达。提前配置10%的SDS-PAGE凝胶和所需电泳液,于槽内加入5微升蛋白Marker以及100微克蛋白样品,恒压80V电泳60分钟,增大电压至120V电泳至溴酚蓝接近分离胶底部后停止电泳。取出凝胶,切取40kda(内参蛋白GAPDH蛋白分子量)与65~70kda(目的蛋白分子量)处凝胶,剪去同等大小PVDF膜用甲醇浸泡10秒活化,制作无气泡“三明治”后恒流0.2A转膜75钟,取出PVDF膜用5%BSA封闭2小时。封闭完成后用TBST洗膜三次,每次各10分钟。加入相应一抗室温摇床孵育30分钟后放置于4℃冰箱过夜。次日取出室温摇床复温30分钟。复温完成后用TBST洗膜三次,每次各10分钟。加入相应二抗室温摇床孵育2小时。孵育完成后用TBST洗膜三次,每次各10分钟。最后配置ECL化学发光液,使用化学发光仪曝光PVDF膜显影。
显影结果见图2,由实验结果可看出,siMITF-5、8、9序列使MITF蛋白表达水平明显下降,与siMITF-5、8、9序列对MITF mRNA基因有较高的沉默效率相关。
SEQUENCE LISTING
<110> 武汉泽智生物医药有限公司
<120> 沉默小眼畸形转录因子mRNA表达的siRNA分子
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Claims (10)
1.一种沉默小眼畸形转录因子mRNA表达的siRNA序列,siRNA序列为正义链和反义链组成双链结构分子的核苷酸序列,其特征在于:所述的siRNA序列的靶序列、正义链和反义链分别为下列品种中的一种:
siMITF-m-2:
靶序列:CTGGAAATGCTAGAATATA,
正义链:5'-CUGGAAAUGCUAGAAUAUAdTdT-3',
反义链:5'-UAUAUUCUAGCAUUUCCAGdTdT-3';
siMITF- m-5:
靶序列:CCTAGAATCAAGTTATAAT,
正义链:5'-CCUAGAAUCAAGUUAUAAUdTdT-3',
反义链:5'-AUUAUAACUUGAUUCUAGGdTdT-3';
siMITF- m-8:
靶序列:GAACGAAGAAGAAGATTTA,
正义链:5'-GAACGAAGAAGAAGAUUUAdTdT-3',
反义链:5'-UAAAUCUUCUUCUUCGUUCdTdT-3';
siMITF- m-9:
靶序列:GAAGAAGATTTAACATAAA,
正义链:5'-GAAGAAGAUUUAACAUAAAdTdT-3',
反义链:5'-UUUAUGUUAAAUCUUCUUCdTdT-3';
siMITF- m-10:
靶序列: CCACTTTAGCAAATAAACA,
正义链:5'-CCACUUUAGCAAAUAAACAdTdT-3',
反义链:5'-UGUUUAUUUGCUAAAGUGGdTdT-3'。
2.根据权利1所述的沉默小眼畸形转录因子mRNA表达的siRNA序列,其特征在于:为采用Western blot方法测试所获知的沉默效率较高的下列靶序列、正义链和反义链构成的siRNA序列中的一种:
siMITF- m-5:
靶序列:CCTAGAATCAAGTTATAAT,
正义链:5'-CCUAGAAUCAAGUUAUAAUdTdT-3',
反义链:5'-AUUAUAACUUGAUUCUAGGdTdT-3';
siMITF- m-8:
靶序列:GAACGAAGAAGAAGATTTA,
正义链:5'-GAACGAAGAAGAAGAUUUAdTdT-3',
反义链:5'-UAAAUCUUCUUCUUCGUUCdTdT-3';
siMITF- m-9:
靶序列:GAAGAAGATTTAACATAAA,
正义链:5'-GAAGAAGAUUUAACAUAAAdTdT-3',
反义链:5'-UUUAUGUUAAAUCUUCUUCdTdT-3'。
3.根据权利1或2述的沉默小眼畸形转录因子mRNA表达的siRNA序列,其特征在于:为采用RT-qPCR筛选法以及Western blot方法测试所获知的沉默效率都高的下述靶序列、正义链和反义链构成的siRNA序列:
siMITF-5:
靶序列:CCTAGAATCAAGTTATAAT,
正义链:5'-CCUAGAAUCAAGUUAUAAUdTdT-3',
反义链:5'-AUUAUAACUUGAUUCUAGGdTdT-3'。
4.根据权利1所述的沉默小眼畸形转录因子mRNA表达的siRNA序列,其特征在于:
其正义链与所述siRNA分子中正义链序列有60%以上同源性,或者其反义链与与所述siRNA分子中反义链序列有60%以上的同源性;且正义链与反义链之间有15-19个核苷酸序列为互补的双链结构。
5.根据权利4所述的沉默小眼畸形转录因子mRNA表达的siRNA序列,其特征在于:siRNA序列的靶序列、正义链和反义链如下:
siMITF-5X:
靶序列:CCTAGAATCAAGTTAT,
正义链:5'-CCUAGAAUCAAGUUAU-3',
反义链:5'-AUUAUAACUUGAUUCUAGGCUdTdT-3'。
6.根据权利4所述的沉默小眼畸形转录因子mRNA表达的siRNA序列,其特征在于:3-5个核苷酸中的核糖进行过2’-甲氧基修饰或2’-氟修饰。
7.根据权利6所述的沉默小眼畸形转录因子mRNA表达的siRNA序列,其特征在于:正义链中的前3个核苷酸中的核糖进行过2’-甲氧基修饰;反义链中的第2个核苷酸中的核糖进行过2’-氟修饰;所得siRNA序列的正义链和反义链:
siMITF-10X:
正义链:(mC)(mC)(mA)CUUUAGCAAAUAAA,
反义链:(mU)(nG)(mU)UUAUUUGCUAAAG(mU)(mG)(mG)dTdT。
8.根据权利1、4或6所述的沉默小眼畸形转录因子mRNA表达的siRNA序列,其特征在于:正义链或反义链,或者双链中的相邻两个或三个核苷酸之间用硫代磷酸酯连接修饰过;所得siRNA序列的正义链和反义链:
siMITF-8X:
正义链:GAACGAAGAAGAAGAUUU~A~dTdT,
反义链:U~A~AAUCUUCUUCUUCG~U~U~CdTdT。
9.一种权利要求1-8中任一所述的siRNA序列的用途, 其特征在于:用于治疗MITF突变或过表达引起的疾病。
10.如权利要求7所述的siRNA序列的用途, 其特征在于:用于治疗MITF突变或过表达引起的黑色素瘤或皮肤色素沉着。
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