CN111849992A - 靶向c-Met基因的siRNA分子及其应用 - Google Patents
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Abstract
本发明公开了一组靶向c‑Met基因的siRNA分子及其应用，属于生物医药技术领域。该siRNA分子由正义链和反义链组成，体外实验证明，本发明的siRNA分子的反义链可特异性地与抑制c‑Met基因的mRNA结合，降解mRNA，从而干扰转录后翻译过程，诱导肿瘤细胞凋亡、抑制肿瘤细胞转移和侵袭，达到治疗肿瘤的目的。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一组靶向c-Met基因的siRNA分子及其应用。
背景技术
原发性肝癌特别是肝细胞肝癌(Hepatocellular carcinoma，HCC)，是最常见的肝脏原发性肿瘤，其发病率及病死率有逐步升高的趋势。目前其全球发病率在所有恶性肿瘤中居第五，而癌症死亡率高居第三位，每年约有80万人死于肝细胞癌。全球每年肝癌新发病例有一半左右发生在我国，列我国癌症死因第二位，严重危害国民健康。肝癌的恶性程度较高，发病隐匿，早期诊断困难，进展较快，确诊时大多数患者已达到中晚期伴有广泛浸润或发生远处转移，不能手术切除。导致肝癌预后不佳的一个重要原因就是缺乏有效的治疗手段，因此除强调和重视早期发现、早期治疗外，探索有效的治疗方法对改善肝癌患者的预后具有重要意义。
c-Met基因位于人类7号染色体长臂(7q31)，大小约110kb，包括21个外显子，启动子区域有许多调控序列，如IL-6和HGF等。在不同组织和细胞系中c-Met的转录产物有多种，各种转录产物的功能尚不清楚，但某些转录产物只在特定的癌组织中出现，因此，这些转录产物可能与特定组织的癌变有关。研究显示，c-Met在非小细胞肺癌、胃癌、脑癌、乳腺癌、结直肠癌、肝癌等多种常见恶性肿瘤中都能检测被异常激活。近年来，诺华、默克等药物公司以c-Met为靶点的抑制剂在治疗肺癌上有突破性进展，由于肺癌和胃癌等在我国频发，故c-Met也已经成为热门研究靶点之一。
RNA干扰(RNA interference e，RNAi)是由小干扰RNA(small interference RNA，siRNA)引发其同源信使RNA(messenger RNA，mRNA)降解的一种转录后基因沉默形式(Nature.1998,391:806-811)。现已被证实在多种病毒感染性疾病和肿瘤的治疗中具有极大的潜力，是理想的阻断基因表达的治疗手段。RNAi技术开辟了一个全新的治疗领域，目前国际上已有数十种siRNA药物进入临床阶段。
发明内容
本发明的目的是提供一组靶向c-Met基因的siRNA分子及其应用，该siRNA分子的反义链可特异性地与抑制c-Met基因的mRNA结合，降解mRNA，从而干扰转录后翻译过程，诱导肿瘤细胞凋亡、抑制肿瘤细胞转移和侵袭。
为了实现上述发明目的，本发明采用以下技术方案：
靶向c-Met基因的siRNA分子，由正义链和反义链组成，其序列为：
正义链：5’-GGAAGAAGAUCACGAAGAUdTdT-3’(SEQ ID NO:1)，
反义链：5’-AUCUUCGUGAUCUUCUUCCdTdT-3’(SEQ ID NO:2)。
上述靶向c-Met基因的siRNA分子在制备抑制细胞中c-Met基因功能的药物中的应用。
上述靶向c-Met基因的siRNA分子在制备预防和/或治疗肝癌的药物中的应用。
进一步地，所述靶向c-Met基因的siRNA分子可以抑制肝癌细胞的迁移。
进一步地，所述靶向c-Met基因的siRNA分子可以抑制肝癌细胞的侵袭。
体外实验证明，本发明的siRNA分子的反义链可特异性地与抑制c-Met基因的mRNA结合，降解mRNA，从而干扰转录后翻译过程，诱导肿瘤细胞凋亡、抑制肿瘤细胞转移和侵袭，达到治疗肿瘤的目的。
本发明的siRNA分子可以应用于制备调节细胞中抑制c-Met基因功能的药物中发挥RNA干扰的作用，诱导肿瘤细胞凋亡、抑制肿瘤细胞转移和侵袭，达到治疗肿瘤的目的。
附图说明
图1为实施例1中实时定量PCR检测c-Met在肝癌细胞HepG2和Huh7和正常肝细胞LO2中的mRNA表达水平。
图2为实施例1中Western Blot检测c-Met在肝癌细胞HepG2和Huh7和正常肝细胞LO2中的蛋白表达水平。
图3为实施例1中实时定量PCR检测siRNA下调肝癌细胞HepG2中c-Met的mRNA表达水平。
图4为实施例1中实时定量PCR检测siRNA下调肝癌细胞HepG2中c-Met的蛋白表达水平。
图5为实施例1中Western Blot检测siRNA下调肝癌细胞Huh7中c-Met的mRNA表达水平。
图6为实施例1中实时定量PCR检测siRNA下调肝癌细胞Huh7中c-Met的蛋白表达水平。
图7为实施例1中MTT法检测siRNA下调肝癌细胞HepG2中c-Met的表达水平后对细胞增值能力的影响。
图8为实施例1中MTT法检测siRNA下调肝癌细胞Huh7中c-Met的表达水平后对细胞增值能力的影响。
图9为实施例1中细胞划痕实验检测siRNA下调肝癌细胞HepG2中c-Met的表达水平后对细胞迁移能力的影响。
图10为实施例1中细胞划痕实验检测siRNA下调肝癌细胞Huh7中c-Met的表达水平后对细胞迁移能力的影响。
图11为实施例1中Transwell细胞侵袭实验检测siRNA下调肝癌细胞HepG2中c-Met的表达水平后对细胞侵袭能力的影响。
图12为实施例1中Transwell细胞侵袭实验检测siRNA下调肝癌细胞Huh7中c-Met的表达水平后对细胞侵袭能力的影响。
具体实施方式
为方便起见，在下文中，术语“siRNA”、“siRNA序列”或“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。
本发明的siRNA分子来源于针对c-Met基因开放阅读框的功能保守区而设计。
siRNA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siRNA、PCR合成siRNA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siRNA分子可以作为制备调节细胞中c-Met基因功能药物的有效成分，尤其是抗肿瘤药物的有效成分。
出于应用目的，可将siRNA分子作为药物直接给药于受药者身上特定部位，比如肿瘤组织。
本发明的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siRNA分子的活性。比如，对于注射用给药系统，剂型可以是冻干粉。
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siRNA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。
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l
一、实验方法
步骤1，细胞培养
肝癌细胞株HepG2和Huh7购于上海中科院细胞研究所，用含10％FBS(Thermo公司)的DMEM培养基(Thermo Fisher公司)培养，所述培养基中加入青霉素和链霉素(Thermo公司)，青霉素和链霉素的终浓度分别为100U/mL和100μg/mL，培养于37℃二氧化碳培养箱。
步骤2，siRNA体外转染
设计靶向c-Met(NCBI号：NM_001127500)基因的siRNA序列c-Met siRNA。设计阴性对照序列(NC)作为对照。序列见表1。
表1 siRNA序列及对照序列
取步骤1中培养得到的处于对数生长期的细胞，96孔板的接种密度为5×104/孔、24孔板按1.5×105/孔、6孔板为1×106/孔接种细胞，将其分为c-Met siRNA转染组和不转染siRNA的未处理组，以及NC转染组作为阴性对照。各实验组采用相应的siRNA按操作说明用脂质体
2000(Thermo Fisher公司)转染到细胞内，使siRNA终浓度为50nM，37℃培养4h后，培养液换成含10％FBS的DMEM培养基。
所有的实验均重复3次，结果均用平均值±SD表示，用SPSS19.0进行统计学分析。统计学差异用单因素方差分析和双侧t检验。P<0.05表明差异显著。在所有的图表中，*表明与未处理组相比差异显著。
二、实时定量PCR(RT-qPCR)检测mRNA表达水平
用上述方法进行细胞培养和siRNA体外转染。所述的体外转染采用96孔板。
转染48h后，细胞总RNA用
RNA提取试剂(Thermo Fisher公司)提取，并按操作说明用RT-qPCR试剂盒(Biomics公司)进行检测。引物序列见表2，PCR反应条件为：95℃预变性5min，45个循环为95℃变性20s、58℃退火30s、72℃延伸30s。
表2 RT-QPCR检测用引物
如图1所示，肝癌细胞株HepG2和Huh7中的c-Met的mRNA表达水平显著高于正常肝细胞LO2(P<0.05)；如图3和4所示，c-Met siRNA有效抑制了HepG2和Huh7细胞中的c-Met基因的mRNA表达。与未处理组相比，c-Met siRNA的抑制率达分别达到83％和85％(P<0.05)。
三、Western Blot检测基因的蛋白表达水平
用上述方法进行细胞培养和siRNA体外转染。
将细胞接种于6孔板中，置于37℃/5％CO2培养箱中培养24h，细胞融合度达70～80％时进行转染siRNA；24h后，弃去培养液，用PBS冲洗2遍；操作在冰上进行，每孔加入50μlSDS蛋白裂解液裂解细胞，充分混匀至粘稠状，用刮子刮下，转移至1.5mL的离心管中，沸水浴10min后置于冰上，4℃，12000rpm×15min，提取上清液；经SDS-PAGE(5％积层胶，8％分离胶)电泳后，采用湿转印仪以200mA恒流转印2h，将凝胶中蛋白质转移到PVDF膜上；将PVDF膜浸没于封闭液(5％的脱脂牛奶)在摇床上缓慢摇晃2h；分别加入一抗(1:1000)，37℃孵育2h；用TBST漂洗膜三次，每次10min；分别加入二抗(羊抗鼠IgG-HRP，1:1000)，37℃孵育2h；二抗孵育结束后，用TBST漂洗膜三次，每次5～10min；洗好的膜进行ECL发光显影；结果分析：以内参基因作为内对照，使用Image J软件分析目的条带的灰度值，计算目的基因相对表达量＝目的条带灰度值/同一样本内参灰度值。
如图2所示，肝癌细胞株HepG2和Huh7中的c-Met的蛋白表达水平显著高于正常肝细胞LO2(P<0.05)；如图5和6所示，c-Met siRNA有效抑制了HepG2和Huh7细胞中的c-Met基因的蛋白表达。与未处理组相比，c-Met siRNA的抑制率达分别达到51％和50％(P<0.05)。
四、MTT法检测细胞增殖
用上述方法进行细胞培养和siRNA体外转染，所述的体外转染采用96孔板。转染前待细胞汇合度达到约75％时，将对数生长期的细胞铺到96孔细胞培养板中，接种密度为5×104/孔，重复3个孔。
分别测定转染24h、48h、72h、96h时的c-Met siRNA转染组，NC转染组和未处理组样本，以及上述的未转染的样本的OD值，所述测定方法为：每孔加入10μl MTT，37℃培养箱避光放置4h；每孔加150μl DMSO，37℃培养箱放置10min；吹打混匀后取120μl于另一干净96孔板中，并取120μl DMSO作为空白对照调零，酶标仪(Bio-Rad公司)上测OD，波长为490nm；进行数据处理，绘制细胞生长曲线。
如图7和8所示，与未处理组和NC组相比，c-Met siRNA处理HepG2和Huh7细胞48h、72h和96h时具有显著细胞生长抑制效果(P<0.05)。
五、细胞划痕实验检测细胞迁移
用上述方法进行细胞培养和siRNA体外转染。
用密度为1.5×105/孔的HepG2和Huh7细胞铺到24孔板里并转染。48h后，用移液器吸头在汇合的细胞中划痕，然后用pH为7.4的PBS缓冲液洗涤，加入无血清的DMEM培养基(Thermo Fisher公司)。划痕后24h和48h后拍照观察细胞迁移，实验做3组平行，每板拍4个视野。
如图9和图10所示，与未处理组和阴性对照组相比，在c-Met siRNA处理HepG2和Huh7细胞24和和48h时能有效抑制HepG2和Huh7的迁移(P<0.05)。
六、Transwell细胞侵袭实验
用上述方法进行细胞培养和siRNA体外转染。所述的体外转染采用24孔板。
转染后72h用24-well membrane filters(美国Corning Bioscience公司)检测细胞迁移。1.5×105个细胞铺到上室中以向含血清的DMEM培养基(Gibco公司)中迁移24h。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30s。最后，细胞用0.1％结晶紫染色4min，随后用pH为7.4的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。
如图11和12所示，与未处理组和NC组相比，c-Met siRNA能显著抑制HepG2和Huh7细胞的侵袭(P<0.05)。
序列表
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Claims (5)
1.靶向c-Met基因的siRNA分子，其特征在于：由正义链和反义链组成，其序列为：
正义链：5’-GGAAGAAGAUCACGAAGAUdTdT -3’，
反义链：5’- AUCUUCGUGAUCUUCUUCCdTdT -3’。
2.权利要求1所述的siRNA分子在制备抑制细胞中c-Met基因功能的药物中的应用。
3.权利要求1所述的siRNA分子在制备预防和/或治疗肝癌的药物中的应用。
4.根据权利要求3所述的应用，其特征在于：所述靶向c-Met基因的siRNA分子可以诱导肝癌细胞凋亡。
5.根据权利要求3所述的应用，其特征在于：所述靶向c-Met基因的siRNA分子可以抑制肝癌细胞的转移和侵袭。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080113377A1 (en) * | 2002-11-14 | 2008-05-15 | Dharmacon, Inc. | siRNA Targeting proto-oncogene MET |
| US20130023578A1 (en) * | 2009-12-31 | 2013-01-24 | Samyang Biopharmaceuticals Corporation | siRNA for inhibition of c-Met expression and anticancer composition containing the same |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080113377A1 (en) * | 2002-11-14 | 2008-05-15 | Dharmacon, Inc. | siRNA Targeting proto-oncogene MET |
| US20130023578A1 (en) * | 2009-12-31 | 2013-01-24 | Samyang Biopharmaceuticals Corporation | siRNA for inhibition of c-Met expression and anticancer composition containing the same |
Non-Patent Citations (1)
| Title |
|---|
| 张盛周;张宏霞;潘飞燕;李朝军;: "腺病毒介导的siRNA下调c-Met表达抑制肝癌细胞生长" * |
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