CN114085832B - 用于抑制PRR14基因的siRNA分子 - Google Patents
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- CN114085832B CN114085832B CN202111239566.6A CN202111239566A CN114085832B CN 114085832 B CN114085832 B CN 114085832B CN 202111239566 A CN202111239566 A CN 202111239566A CN 114085832 B CN114085832 B CN 114085832B
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Abstract
本发明公开了一种用于抑制PRR14基因的siRNA分子,由正义链SEQ ID NO:1和反义链SEQ ID NO:2组成。本发明的siRNA分子可用于制备治疗皮肤鳞状细胞癌的药物。
Description
技术领域
本发明属于生物医药领域,具体涉及一种用于抑制PRR14基因的siRNA分子及其在制备抗肿瘤药物中的用途。
背景技术
人皮肤鳞状细胞癌(cutaneous squamous cell carcinoma,cSCC)起源于表皮或附属器角质形成细胞,是具有侵袭性和潜在恶性的皮肤肿瘤,占恶性上皮性肿瘤的15%,占非黑色素皮肤肿瘤的20%,仅此于基底细胞癌。cSCC发病因素复杂,可能与紫外线照射、某些化学品的接触、癌前期皮肤病、外伤、瘢痕和免疫抑制剂的应用有关,但其具体发病机制尚不清楚。在大多数情况下,cSCC很容易通过单纯切除或放射治疗达到治愈,然而,局部晚期肿瘤也可出现局部复发,淋巴结或远处转移。常规采用手术、激光、冷冻、放疗等治疗方法,但存在组织结构破坏性大、易复发、复发后再次治疗困难等问题。因此,研究皮肤癌发生机制、治疗手段和预防复发及转移是目前皮肤科领域亟需解决的关键。
富含脯氨酸的蛋白质14(Proline-rich protein 14,PRR14)是富含脯氨酸蛋白质家族的一个成员,该家族包含一个富含脯氨酸的区域,两侧有N端和C端的核定位信号,富含脯氨酸的区域通常通过与各种结构域(尤其是SH3结构域)的结合而参与到多种信号通路。PRR14被认为是核纤层的一个新组成部分,在细胞周期中将异染色质拴在核纤层上,在细胞周期过程中具有特异性和定位异染色质的能力。PRR14很可能是肿瘤发生过程中介导核形态改变及其功能改变的共同机制中的一个关键分子。有报道指出,PRR14在非小细胞肺癌(Non-small cell lung cancer,NSCLC)患者的肿瘤组织中异常过表达,并与预后不良相关,通过激活PI3K/AKT/mTOR信号通路促进肺癌细胞增殖。PRR14作为一种富含脯氨酸的蛋白质,与GRB2的Src同源3(Src homology 3,SH3)结构域结合,导致PI3K活化,可能是一种新的PI3K通路激活剂,促进了肺癌的发生。结果提示,PRR14可作为一个独立的预后指标,在NSCLC的治疗中可能成为一个潜在的治疗靶点。还有研究指出,PRR14在结肠癌组织细胞中也呈高表达,其高表达水平与肿瘤大小、远处转移及淋巴结转移分期有关。功能研究显示抑制PRR14的表达可以抑制结肠癌细胞的生长、迁移和侵袭。此外,PRR14的敲除抑制了上皮-间充质转化(EMT)过程、细胞周期相关蛋白的表达和p-AKT水平。最新研究发现,PRR14在乳腺癌中过度表达,参与乳腺癌的发生,PRR14表达增加促进乳腺癌细胞增殖和肿瘤形成。PRR14在乳腺癌中通过激活PI3K/Akt/mTOR途径和抑制CHEK2途径来保护细胞免受凋亡和刺激增殖。
RNA干扰(RNA interference,RNAi)是指在进化过程中高度保守、由双链RNA(double-stranded RNA,dsRNA)诱发的、同源信使RNA(message RNA,mRNA)高效特异性降解的基因转录后沉默现象。近年来,RNAi作为一个基因治疗技术,得到了迅速发展,尤其在抗病毒、抗肿瘤和抗炎症等医药领域显示出广阔的应用前景,已经部分RNAi药物已进入临床试验阶段。小干扰核酸(small interfering RNA,siRNA)是RNAi的效应分子,由两条互补的RNA单链构成,长21~23个核苷酸(nt)。由于RNAi技术可以特异性剔除或关闭特定基因的表达,所以技术广泛地应用于功能基因组学研究、传染性疾病以及抗病毒、抗肿瘤治疗的研究中。自从2016年12月23日美国食品和药物管理局FDA批准了首个用于治疗脊髓性肌萎缩的RNAi药物Spinraza(Nusinersen)的上市以来,国际上已有数十种RNAi药物进入临床阶段。
发明内容
为了得到能有效治疗肿瘤比如皮肤鳞状细胞癌的RNAi药物,发明人以PRR14基因为靶点,设计并筛选出数百种siRNA分子,发现其中一些能够特异性地抑制PRR14表达,其中有两组siRNA分子能有效地抑制PRR14基因。因此,本发明的第一个目的在于提供一种用于抑制PRR14基因的siRNA分子,其由如下序列的正义链和反义链组成:
正义链:5’-CAAUCAGGUUGAACAAGAANn-3’(SEQ ID NO:1),
反义链:5’-UUCUUGUUCAACCUGAUUGNn-3’(SEQ ID NO:2),
其中,正义链和反义链中的N相同或者不同,并且各自独立地是胞嘧啶C、尿嘧啶U、鸟嘌呤G、腺嘌呤A、脱氧胞嘧啶dC、脱氧鸟嘌呤dG、脱氧腺嘌呤dA或脱氧胸腺嘧啶dT;n代表N的个数,n是0、1或2。
在一种实施方式中,所述n为0,即
正义链:5’-CAAUCAGGUUGAACAAGAA-3’(SEQ ID NO:3),
反义链:5’-UUCUUGUUCAACCUGAUUG-3’(SEQ ID NO:4)。
该siRNA分子是该组siRNA分子的主干序列。
在另一种优选的实施方式中,所述N为dT,n为2,即
正义链:5’-CAAUCAGGUUGAACAAGAAdTdT-3’(SEQ ID NO:5),
反义链:5’-UUCUUGUUCAACCUGAUUGdTdT-3’(SEQ ID NO:6)。
本发明的第二个方面提供了上述siRNA分子在制备用于抑制PRR14表达的药物中的用途。
可选地,上述药物是抗肿瘤药物。
优选地,所述药物是治疗皮肤鳞状细胞癌药物。
其中siRNA分子可以作为有效成分用于抑制皮肤鳞状细胞癌细胞的生长、转移,或者促进皮肤鳞状细胞癌细胞的凋亡。
本发明的第三个方面提供了一种RNAi药物,其包含上述的siRNA分子作为药物活性成分以及药学上可接受的载体。
上述药物可以是注射剂型或外用药剂型。
在一种实施方式中,当上述RNAi药物是外用药时,其剂型可以选自冻干剂、水剂、涂液、凝胶剂、软膏、乳膏、霜剂。
可选地,上述RNAi药物还可以包含其他抗癌药物化合物作为辅助活性成分。
上述药物还包含必要的佐剂。
体外实验证明,本发明提供的siRNA分子可特异性地下调人皮肤鳞状细胞癌细胞株A431和HSC-1中PRR14基因表达,达到抑制皮肤鳞状细胞癌细胞生长、转移以及促进癌细胞凋亡的技术效果。
附图说明
图1是PRR14基因的mRNA在A431和HSC-1皮肤鳞状细胞癌细胞中的表达水平的柱状图。与正常皮肤细胞HaCaT相比,A431和HSC-1细胞中PRR14基因的mRNA表达水平显著升高,其中*P<0.05。
图2是本发明的siRNA抑制A431和HSC-1皮肤鳞状细胞癌细胞中PRR14靶基因表达水平的柱状图。其中,A为PRR14的mRNA表达水平的柱状图;B为PRR14蛋白的免疫印迹照片,与阴性对照组siNC处理细胞相比,siRNA显著抑制了PRR14基因的mRNA和蛋白表达水平,其中*P<0.05。
图3是本发明的siRNA抑制A431和HSC-1皮肤鳞状细胞癌细胞增殖的生长曲线图。
其中,A为siRNA抑制A431细胞增殖的生长曲线图;B为siRNA抑制HSC-1细胞增殖的生长曲线图;与阴性对照组siNC处理细胞相比,siRNA在48h、72h显著抑制了细胞的生长,其中*P<0.0。
图4是本发明的siRNA抑制A431和HSC-1皮肤鳞状细胞癌细胞迁移的结果图。与阴性对照组siNC处理细胞相比,siRNA显著抑制了A431和HSC-1细胞的侵袭,其中*P<0.05。
图5是本发明的siRNA抑制A431和HSC-1细胞侵袭的结果图。与阴性对照组siNC处理细胞相比,siRNA显著抑制了A431和HSC-1细胞的侵袭,其中*P<0.05。
图6是本发明的siRNA促进A431和HSC-1皮肤鳞状细胞癌细胞凋亡的结果图。与阴性对照组siNC处理细胞相比,siRNA显著促进了A431和HSC-1细胞的凋亡,其中*P<0.05。
具体实施方式
发明人将PRR14基因作为RNA干扰靶标,设计出了120多条长短不一的siRNA分子。理论上讲,针对该PRR14基因设计的siRNA分子或多或少都应该有抑制该人体细胞中该基因表达的生物功能。然而,经过实验之后发现,我们设计的siRNA分子中,绝大部分并没有预期抑制/下调PRR14基因的活性,仅有少数对PRR14基因具有抑制/下调表达效果,包括正义链核苷酸序列为SEQ ID NO:1和SEQ ID NOs:7-16的双链siRNA分子。
5’-GACCAAUCAGGUUGAACAAdTdT-3’(SEQ ID NO:7);
5’-UUGUUCAACCUGAUUGGUCdTdT-3’(SEQ ID NO:8);
5’-GGCGAACUCAGGACCACAAdTdT-3’(SEQ ID NO:9);
5’-UUGUGGUCCUGAGUUCGCCdTdT-3’(SEQ ID NO:10);
5’-GCUCCAAGCUGGAGAGCUUUGdTdT-3’(SEQ ID NO:11);
5’-AAGCUCUCCAGCUUGGAGCGGdTdT-3’(SEQ ID NO:12);
5’-CCUGGAGGAAGAAACAGUAGAdTdT-3’(SEQ ID NO:13);
5’-UACUGUUUCUUCCUCCAGGAGdTdT-3’(SEQ ID NO:14);
5’-GUGGUGCUAGAAGAUGUCAUGdTdT-3’(SEQ ID NO:15);
5’-UGACAUCUUCUAGCACCACGGdTdT-3’(SEQ ID NO:16)。
其中,正义链为SEQ ID NO:1比如SEQ ID NO:5的双链siRNA分子的抑制效果最好,可以特异性靶向PRR14基因,阻断PRR14基因的mRNA转录,阻止基因的翻译,从而根本上抑制PRR14的表达,最终达到抑制肿瘤的目的。
在本文中,有时为了描述简便,会将PRR14蛋白质与其编码基因(DNA)名称混用,本领域技术人员应能理解它们在不同描述场合表示不同的物质。例如,PRR14基因指的是编码PRR14蛋白质的基因DNA。
在本文中,术语“小干扰核酸”、“siRNA”、“siRNA序列”、“siRNA分子”、“双链siRNA”、或“双链siRNA分子”可以互换,它们表示的意思和范围相同。其中,siRNA是正义链和反义链退火形成的双链结构。
siRNA分子的制备可采用多种方法,比如:化学合成法、体外转录、酶切长链dsRNA、载体表达RNA、PCR合成RNA表达元件等,这些方法的出现为研究者提供了可选择的空间,可以更好地获得基因沉默效率。
本发明的RNAi药物的剂型可以为多种形式,只要适合于相应疾病的给药、并且恰当地保持RNA分子的活性。比如,对于注射用给药系统,剂型可以是冻干粉。
当上述RNAi药物是用于治疗皮肤癌的外用药时,其剂型可以选自冻干剂、水剂、涂液、凝胶剂、软膏、乳膏、霜剂。这些剂型配制时可选用制药领域常用的辅助剂,并且其制备工艺也是本领域技术人员所熟知的。
可选地,上述RNAi药物还可以包含其他抗癌药物化合物作为辅助活性成分,以便增强siRNA分子的抗癌活性。
任选地,上述药物剂型中可以包含任何药学上可接受的载体及佐剂,只要其适合于相应的给药体系、并且恰当地保持RNA分子的活性。
为使本发明更明显易懂,兹以优选实施例,并配合附图作详细说明如下。本领域技术人员应当理解,下述实施例仅用于阐明本发明,并非是对本发明进行限制。
实施例
本文中的siRNA和PCR引物由百奥迈科生物技术有限公司合成。
细胞株A431、HSC-1和HaCaT均购于中科院上海细胞中心。
实施例中,如果对于实验操作温度没有做出具体说明,则该温度通常指室温(10-30℃)。
实施例中涉及到多种物质的添加量、含量及浓度,其中所述的百分含量,除特别说明外,皆指质量百分含量。
实施例1
用人皮肤鳞状细胞癌细胞株A431和HSC-1观察PRR14的表达变化对细胞株的生物学行为的影响。
人皮肤鳞状细胞癌细胞株A431和HSC-1,以及正常皮肤细胞HaCaT作为对照,这些细胞均购于中科院上海细胞中心,在含10%胎牛血清的DMEM培养基(ThermoFisherScientific公司)中,于37℃、5% CO2培养箱中培养。
设计并合成靶向PRR14基因的siRNA序列,包括下述双链siRNA分子:
正义链:5’-CAAUCAGGUUGAACAAGAAdTdT-3’(SEQ ID NO:5),
反义链:5’-UUCUUGUUCAACCUGAUUGdTdT-3’(SEQ ID NO:6)。
分别将正义链和对应的反义链退火成siRNA双链,转染前配置成浓度为20μM。
另外,设计并合成了与人基因不同源的siRNA作为阴性对照的siNC,其序列为:
正义链:5’-AUCAGGAACAUGUAAGACAdTdT-3’(SEQ ID NO:17),
反义链:5’-UGUCUUACAUGUUCCUGAUdTdT-3’(SEQ ID NO:18)。
该siNC具有正义链和反义链。分别将正义链和对应的反义链退火成siNC双链,转染前配置成浓度为20μM。
siRNA转染细胞:将步骤1所得细胞铺到6孔、24孔、48孔或96孔细胞培养板(Sigma-Aldrich公司)中,并在含10%胎牛血清的DMEM培养基中于37℃、5% CO2培养箱中培养过夜。用细胞转染试剂3000(ThermoFisher Scientific公司),按其说明书将siRNA和siNC转染分别转染到A431和HSC-1细胞中,并分别设置正常培养未作任何处理的细胞作为正常组。
实施例2
用实时定量PCR的方法检测siRNA抑制PRR14基因表达后对皮肤鳞状细胞癌细胞中PRR14的mRNA表达水平的影响。按实施例1的方法分别将A431和HSC-1细胞按1.5×105个/孔铺到96孔细胞培养板中,细胞生长24h后至约75%的汇合度进行细胞转染。细胞转染48h后,收集细胞,用TrizolRNA提取试剂Trizol(美国Thermo Fisher Scientific公司)按其说明书进行细胞总RNA的提取。
用PRR14特异性引物检测样本中PRR14基因mRNA表达水平,同时扩增看家基因β-actin作为内参对照。每个样本同时扩增PRR14基因和内参基因β-actin,每个反应做3个平行。用SYBR-Green IOne-Step qPCR试剂盒(ThermoFisher Scientific公司)进行定量PCR反应,建立反应体系为:4μL RNA模板,12.5μL的2×SYBR Green OneStep Mix,上游引物(10μM)和下游引物(10μM)各0.5μL,0.5μL的50×RT/Taq Mix,用无RNase的水补足体系至25μL。混合均匀后,置于实时定量PCR仪器中反应,反应条件为:42℃反转录30min,95℃预变性10min,95℃变性20sec,58℃退火30sec,72℃延伸30sec,循环45次。
检测PRR14基因的定量PCR引物:
上游引物为:5’-CAGGTTGAACAAGAAGGA-3’(SEQ ID NO:19);
下游引物为:5’-CAAAGATGGTCTCAAAGGT-3’(SEQ ID NO:20)。
检测β-actin的定量PCR引物:
上游引物为:5’-TGCACCACCAACTGCTTAGC-3’(SEQ ID NO:21);
下游引物为:5’-GGCATGGACTGTGGTCATGAG-3’(SEQ ID NO:22)。
反应结束后,导出数据用2-ΔΔCt法分析实验结果并作图。如图1所示,PPR14在A431和HSC-1中的mRNA表达水平均显著高于正常皮肤细胞株HaCaT,差异有统计学意义(P<0.05)。如图2A所示,与阴性对照siNC处理细胞相比,siRNA均显著抑制了皮肤鳞状细胞癌细胞A431和HSC-1中PRR14基因的mRNA表达水平。比如,在A431和HSC-1细胞中siRNA抑制PRR14的mRNA水平分别达到73%和72%,均显著抑制了其mRNA表达水平,差异均有统计学意义(P<0.05)。
实施例3
用Western blot蛋白印迹的方法检测siRNA抑制PRR14基因表达后对皮肤鳞状细胞癌细胞中PRR14的蛋白表达水平的影响。按实施例1的方法分别将A431和HSC-1细胞按1.5×106个/孔铺到6孔板中,细胞生长24h后至约75%的汇合度进行细胞转染。细胞转染48h后,用预冷的RIPA细胞蛋白裂解液(Promega公司)裂解细胞,置于4℃中,10,000rpm离心20min,取上清用BCA的方法定量蛋白。按每泳道40μg的蛋白进行聚丙烯酰胺凝胶电泳,并将蛋白转印到聚偏氟乙烯(PVDF)(Merck-Millipore公司)膜上。用5%脱脂牛奶进行室温封闭2h,然后用兔抗人PRR14单克隆抗体(Merck公司,1:500稀释),鼠抗人β-actin单克隆抗体(Abcam公司,1:1,000稀释)作为内参。TBST洗涤后,再用辣根过氧化物酶偶联的二抗孵育(PRR14用羊抗兔IgG-HRP,1:2,000稀释;β-actin用羊抗鼠IgG-HRP,1:2,000稀释),用TBST洗涤3次(5min/次)。用ECL Substrate试剂盒(ThermoFisher Scientific公司)显影,结果参见图2B。
如图2B所示,与阴性对照siNC处理细胞相比,siRNA显著抑制了皮肤鳞状细胞癌细胞A431和HSC-1中PRR14蛋白表达水平。比如,在A431和HSC-1细胞中siRNA抑制PRR14蛋白质的水平分别达到68%和62%,均显著抑制了其蛋白表达水平,均差异有统计学意义(P<0.05)。
实施例4
用CCK-8的方法检测siRNA抑制PRR14基因表达后对皮肤鳞状细胞癌细胞增殖的影响。按实施例1的方法分别将A431和HSC-1细胞按1.5×103个/孔铺到96孔细胞培养板中,细胞生长24h后至约75%的汇合度进行细胞转染,重复3个孔,取未转染的细胞为转染0h的细胞组。分别取转染24h、48h和72h后的各实验组的细胞进行CCK-8细胞增殖测定,所述测定方法为:将混合有10μL CCK-8工作液(Sigma-Aldrich公司)的100μL磷酸盐缓冲液(PBS)加入到每个孔中,37℃培养箱避光孵育30min,用酶标仪(Bio-Tek公司)在波长490nm处测定每组细胞的OD值,数据导出分析后制作生长曲线。
如图3所示,与阴性对照siNC处理细胞相比,在A431和HSC-1细胞中,siRNA抑制了细胞的增殖,尤其是在48h和72h抑制效果显著,均差异有统计学意义(P<0.05)。
实施例5
用细胞划痕的方法检测siRNA抑制PRR14基因表达后对皮肤鳞状细胞癌细胞迁移的影响。按实施例1的方法分别将A431和HSC-1细胞按1×105个/孔铺到6孔细胞培养板中,细胞生长24h后至约75%的汇合度进行细胞转染。细胞转染4h后用1mL移液吸头在细胞培养板上的单细胞层进行划痕,分别在0h、24h、48h、72h或96h时间点拍照观察细胞迁移情况。最后,根据测得的细胞迁移距离,分析细胞相对迁移率制作柱形图。
如图4所示,与阴性对照siNC处理细胞相比,siRNA显著抑制了A431和HSC-1细胞的迁移,差异有统计学意义(P<0.05)。
实施例6
用含基质胶(Matrigel)的Transwell法检测siRNA抑制PRR14基因表达后对皮肤鳞状细胞癌细胞侵袭的影响。按实施例1的方法分别将A431和HSC-1细胞按1.5×105个/孔铺到24孔细胞培养板中,细胞生长24h后至约75%的汇合度进行细胞转染。细胞转染48h后用DMEM培养进行重悬,调整浓度至1.5×106个细胞/mL。Transwell板(Sigma-Aldrich公司)内室用DMEM培养基预孵育1h,上室和下室用包被有50μL的Matirgel(0.5mg/mL,BD公司)的8μm的聚碳酸酯膜隔开。将100μL的细胞重悬液与600μL含10% PBS的DMEM培养基或者条件培养基(上述48h处理的细胞培养基上清)加到每个上室中。24h后,留在上室中的细胞用棉签去除,迁移到下室中的细胞用10%甲醛固定30sec。最后,细胞用0.5%结晶紫染色4min,随后用PBS缓冲液洗3遍。细胞在200倍放大视野中计数,每个条件下计数5个视野。算出侵袭细胞数,制作柱形图。
如图5所示,与阴性对照siNC处理细胞相比,siRNA显著抑制了细胞HSC-1和A431细胞的侵袭,差异有统计学意义(P<0.05)。
实施例7
用Annexin V-FITC/PI双染并流式细胞术的方法检测siRNA抑制PRR14基因表达后对皮肤鳞状细胞癌细胞凋亡的影响。按实施例1的方法分别将A431和HSC-1细胞按3×105个/孔铺到6孔细胞培养板中,细胞生长24h后至约75%的汇合度进行细胞转染。用AnnexinV-FITC凋亡测定试剂盒(Sigam-Aldrich公司)进行细胞凋亡检测分析,A431和HSC-1细胞转染48h后,收集细胞并在1,000rpm离心5min后,每组样品用195μL 1×Annexin V-FITC结合缓冲液重悬细胞至浓度为1×106个细胞/mL,每组样品中加入5μL的Annexin V-FITC和10μLPI染液并室温避光孵育10min。每组样品细胞用BD FACSCanto II流式细胞仪(BD公司)分析细胞凋亡情况,导出数据并计算细胞凋亡率,制作柱形图。
如图6所示,与阴性对照siNC处理细胞相比,siRNA显著促进了A431和HSC-1细胞的凋亡,差异有统计学意义(P<0.05)。
由上述实验可见,本发明的siRNA分子SEQ ID NOs:5-6表现出高效抑制PRR14表达的活性,可以抑制A431和HSC-1细胞的增殖、迁移、侵袭,并显著促进皮肤鳞状细胞癌细胞株A431和HSC-1的凋亡,因而具有应用于抗肿瘤、尤其是皮肤鳞状细胞癌治疗的潜力。
序列表
<110> 南通大学附属医院
<120> 用于抑制PRR14基因的siRNA分子
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Claims (7)
1.一种用于抑制PRR14基因、治疗皮肤鳞状细胞癌的siRNA分子,由如下序列的正义链和反义链组成:
正义链:5’-CAAUCAGGUUGAACAAGAA-3’(SEQ ID NO: 3),
反义链:5’-UUCUUGUUCAACCUGAUUG-3’(SEQ ID NO: 4);或者
正义链:5’-CAAUCAGGUUGAACAAGAAdTdT-3’(SEQ ID NO: 5),
反义链:5’-UUCUUGUUCAACCUGAUUGdTdT-3’(SEQ ID NO: 6)。
2.如权利要求1所述的siRNA分子在制备用于抑制PRR14表达的药物中的用途,所述药物是治疗皮肤鳞状细胞癌药物。
3.如权利要求2所述的用途,其特征在于,所述治疗皮肤鳞状细胞癌药物用于抑制皮肤鳞状细胞癌细胞的生长、转移,或者促进皮肤鳞状细胞癌的凋亡。
4.一种RNAi药物,其特征在于,包含如权利要求1所述的siRNA分子作为药物活性成分以及药学上可接受的载体。
5.如权利要求4所述的RNAi药物,其特征在于,所述药物是注射剂型或外用药剂型。
6.如权利要求5所述的RNAi药物,其特征在于,外用药剂型选自冻干剂、水剂、涂液、凝胶剂、软膏、乳膏、霜剂。
7.如权利要求4所述的RNAi药物,其特征在于,还包含其他抗癌药物活性成分。
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