CN114574486B - 作用于OPLAH的siRNA、DNA及构建物和应用 - Google Patents
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Abstract
本发明涉及作用于人OPLAH基因的shRNA构建及其用途。本发明针对OPLAH设计了合适的siRNA和表达shRNA的DNA双链序列,并构建了包含上述DNA双链序列的慢病毒载体。通过效果试验证实本发明的siRNA以及表达shRNA的构建物能够显著降低OPLAH mRNA和蛋白水平,敲降作用十分明显。鉴于OPLAH为一种潜在的前列腺癌治疗靶点,因此本发明的siRNA和表达shRNA的构建物可用于制备治疗前列腺癌的有效药物。
Description
技术领域
本发明属于分子生物学领域,具体涉及作用于人OPLAH基因的shRNA构建及其用途。
背景技术
前列腺癌是男性最常见的恶性肿瘤之一,发病率和死亡率均很高,对男性的健康造成严重威胁。在我国,前列腺癌的年发病率呈逐年上升趋势。虽然早期诊断和治疗措施得以改善,但晚期前列腺癌患者预后仍较差。因此对前列腺癌发生发展机制的探索一直是科研工作者研究的焦点,对于提高肿瘤的诊断和治疗水平具有重要意义。
5-羟脯氨酸酶(5-oxoprolinase,ATP hydrolysing,OPLAH)是一种参与γ-谷氨酰基循环的酶,通过ATP水解,催化谷胱甘肽降解产物5-羟脯氨酸转化为谷氨酸。OPLAH基因突变会导致OPLAH缺陷(OPLAHD),以草酸钙/碳酸钙尿石症和尿中过量的5-羟脯氨酸为特征,同时伴有患者反复出现的呕吐、腹泻和腹痛。
OPLAH定位在细胞质中,含有一个羟脯氨酸酶结构域。以往报道OPLAH与心衰、氧化应激等有关,但近期报道提示OPLAH在多种肿瘤中呈高表达,如胃癌、肺癌以及结直肠癌等。目前还未见OPLAH在前列腺癌中的相关研究,也未见抑制OPLAH表达可以抑制肿瘤细胞增殖的相关报道。如果OPLAH能成为潜在的前列腺癌治疗靶点,采用OPLAH的siRNA或抑制剂等方法敲降OPLAH的表达能够在前列腺癌治疗中发挥重要作用。
发明内容
本发明的目的是针对现有技术中的不足,提供一种作用于OPLAH基因的shRNA及其用途。
本发明提供了一种siRNA,所述的siRNA包含以下序列:
正义链:5’-GCGACGUGCUACUGAGCAA-3’,
反义链:5’-UUGCUCAGUAGCACGUCGC-3’。
本发明还提供了一种用于敲降OPLAH基因的DNA双链序列,所述的DNA双链序列包含以下序列:
上游链:5’-CCGGGCGACGTGCTACTGAGCAACTCGAGTTGCTCAGTAGCACGTCGCTTTTT-3’
下游链:5’-AATTAAAAAGCGACGTGCTACTGAGCAACTCGAGTTGCTCAGTAGCACGTCGC-3’
本发明还提供了一种构建物,所述的构建物包含以下DNA双链序列:
上游链:5’-CCGGGCGACGTGCTACTGAGCAACTCGAGTTGCTCAGTAGCACGTCGCTTTTT-3’
下游链:5’-AATTAAAAAGCGACGTGCTACTGAGCAACTCGAGTTGCTCAGTAGCACGTCGC-3’
所述的构建物为慢病毒载体。
本发明还提供了所述的siRNA、所述的DNA双链序列或所述的构建物在制备治疗前列腺癌的药物中的应用。
本发明还提供了所述的siRNA、所述的DNA双链序列或所述的构建物在制备抑制前列腺癌细胞增殖试剂中的应用。
作为本发明的一种具体实施方式,所述的前列腺癌细胞来自于前列腺癌细胞株LNCaP。
本发明还提供了所述的siRNA、所述的DNA双链序列或所述的构建物在非治疗目的的抑制前列腺癌细胞增殖的应用。
本发明优点在于:(1)本发明针对OPLAH设计了合适的siRNA和表达shRNA的DNA双链序列,并构建了包含上述DNA双链序列的慢病毒载体。通过效果试验证实本发明的siRNA以及表达shRNA的构建物能够显著降低OPLAH mRNA和蛋白表达量,敲降作用十分明显。鉴于OPLAH为一种潜在的前列腺癌治疗靶点,因此本发明的siRNA和表达shRNA的构建物可用于制备治疗前列腺癌的药物。(2)本发明首次证实敲降OPLAH可抑制前列腺癌细胞的增殖,提示OPLAH是一种潜在的治疗前列腺癌的新型靶点,可针对前列腺癌OPLAH高表达的病人给予降低OPLAH的表达以达到治疗目的,同时有利于进一步研究前列腺癌的发生发展机制。
通过效果试验证实本发明的siRNA以及表达shRNA的构建物能够显著降低OPLAHmRNA和蛋白水平,敲降作用十分明显。鉴于OPLAH为一种潜在的前列腺癌治疗靶点,因此本发明的siRNA和表达shRNA的构建物可用于制备治疗前列腺癌的有效药物。
附图说明
图1:OPLAH-siRNA敲降效率验证,图1A为qPCR结果,图1B为Western-blot结果;
图2:pLKO.1-puro慢病毒载体图谱;
图3:OPLAH-shRNA敲降效率验证,图3A为qPCR结果,图3B为Western-blot结果;
图4:在前列腺癌细胞株LNCaP中敲降OPLAH对细胞增殖的影响。
具体实施方式
现结合实例,对本发明做进一步说明。实例仅限于说明本发明,而非对本发明的限定。
实施例1 OPLAH-siRNA的合成及对OPLAH敲降效率的验证
(1)设计OPLAH-siRNA
寻找合适的靶序列,设计、合成针对OPLAH mRNA的OPLAH-siRNA。siRNA为自主设计,由GenePharma合成)
正义链:5’-GCGACGUGCUACUGAGCAA-dTdT-3’,
反义链:5’-UUGCUCAGUAGCACGUCGC-dTdT-3’。
(2)OPLAH-siRNA沉默效果验证
前列腺癌细胞系LNCaP以融合度30%密度接种于6孔板,完全贴壁后用Lipofectamine 2000(#11668019,ThermoFisher)进行转染,具体使用转染试剂5μl+OPLAH-siRNA(20μM)5μl转染,对照组NC转染5μl+NC-siRNA(20μM)5μl。转染48小时后按照文献“BRD4 Promotes Gastric Cancer Progression and Metastasis through Acetylation-Dependent Stabilization of Snail”提取RNA和蛋白,采用qPCR及Western-blot验证敲降效率。
NC-siRNA由GenePharma提供,序列如下:
正义链:5’-UUCUCCGAACGUGUCACGUTT-dTdT-3’,
反义链:5’-ACGUGACACGUUCGGAGAATT-dTdT-3’。
qPCR引物由上海生工生物有限公司合成,序列如下:
ACTB-F | TTGCCGACAGGATGCAGAAGGA |
ACTB-R | AGGTGGACAGCGAGGCCAGGAT |
OPLAH-F | ACCAGAAACCTGCACGACAA |
OPLAH-R | GTTCCAAAGGCACGCAACAT |
结果:图1A为qPCR结果,OPLAH-siRNA组OPLAH mRNA表达量较NC组下降;图1B为Western-blot结果,OPLAH-siRNA组较NC组蛋白表达量下降。
实施例2OPLAH-shRNA病毒包装及稳转细胞系构建
(1)所需OPLAH敲降的真核表达载体pLKO.1-puro如图2,OPLAH-shRNA为自主设计,由上海生工生物有限公司合成,序列如下:
上游链:5’-CCGGGCGACGTGCTACTGAGCAACTCGAGTTGCTCAGTAGCACGTCGCTTTTT-3’
下游链:5’-AATTAAAAAGCGACGTGCTACTGAGCAACTCGAGTTGCTCAGTAGCACGTCGC-3’
(2)对shRNA片段进行变性退火处理,将装有shRNA片段的ep管放入沸水中3分钟,并等待缓慢冷却至室温。如表1:退火shRNA组成;
上游链(100μM) | 11.25μl |
下游链(100μM) | 11.25μl |
10X退火缓冲液 | 2.5μl |
然后用399μl 0.5X退火缓冲液稀释1μl退火shRNA。
10X退火缓冲液:1M NaCl,100mM Tris-HCl,pH=7.4,0.5X退火缓冲液用ddH2O稀释。
(3)载体线性化
用AgeI(#FD1464,ThermoFisher)和EcoRI(#FD0274,ThermoFisher)酶切消化pLKO.1-puro载体,凝胶电泳回收线性化载体质粒。
酶切体系如表2:
pLKO.1-puro | 1μg |
AgeI | 1μl |
EcoRI | 1μl |
10X FD buffer | 2μl |
加ddH2O至20μl |
37℃孵育2小时后,使用胶回收试剂盒(#28704,Qiagen)回收线性化载体,并用50μl洗脱液洗脱。
(4)连接与转化
用T4连接试剂盒(C301-01,Vazyme)连接退火稀释后的shRNA片段与酶切回收的线形化载体,体系如表3:
线性化pLKO.1-puro | 1μl |
退火稀释后shRNA | 1μl |
T4 ligase | 1μl |
10X ligase buffer | 1μl |
ddH2O | 6μl |
16℃孵育6小时后,加入50μl DH5α化学感受态细胞(#11802ES80,YEASEN),冰浴30分钟,42℃热激90秒后迅速冰浴2分钟。向离心管中加入LB培养基至1ml,在37℃摇床中220rpm震摇1小时,取100μl已转化的感受态细胞均匀涂布在含100μg/ml氨苄霉素(A8180,Solarbio)的LB琼脂糖培养基上,37℃倒置培养12-16小时。挑选2个单克隆接种于3ml LB培养基中培养,各取1ml菌液送生工生物有限公司测序验证插入序列正确性,测序引物为U6-renyuan,序列如下:ATGGACTATCATATGCTTACCGTA。
(5)病毒包被
将HEK-293T细胞接种于6孔板中,待细胞融合度为70-80%时,将原有培养基弃掉,向其中加入10%FBS DMEM(#C11885500BT,Invitrogen)培养基2ml,然后暂放37℃,体积浓度5%CO2的空气培养箱中。取出四个1.5ml的ep管,先各加入0.125ml OPTI-MEM(#31985070,Invitrogen)培养基,然后混转染体系。其中两管分别加入pLKO.1-OPLAH-puro或pLKO.1-puro 1μg,包装质粒psPAX2(#VT1444,youbio)加0.75μg,包装质粒pMD2.G(#VT1443,youbio)加0.25μg,另两管加转染试剂Lipofectamine2000 5μl,将混合质粒加入Lipofectamine2000中混匀,室温放置20分钟后逐滴加入细胞中。转染6h后,将培养基换成2ml含10%FBS的DMEM培养基。于转染后24小时和48小时收集病毒液,并用0.45μm滤膜过滤,去除杂质及细菌,-80℃贮存备用。
(6)pLKO.1-OPLAH病毒侵染LNCaP细胞并验证敲降效率
提前一天将LNCaP细胞铺到六孔板中,待细胞融合率为30%时,弃掉原培养基并加入1ml 10%FBS 1640培养基(#C11875500BT,Invitrogen)和1ml病毒液。侵染24小时后补加1ml 10%1640培养基,侵染48小时后,更换含嘌呤霉素(ant-pr-1,InvivoGen)培养基筛选,嘌呤霉素浓度为2μg/ml,持续筛选直到野生型细胞全部被杀死后即可获得稳定敲降OPLAH(pLKO.1-OPLAH)和对照组(pLKO.1-puro)的LNCaP细胞系。取适量稳转细胞通过qPCR和Western-blot验证敲降效率。
结果,图3A qPCR和图3B Western-blot结果表明OPLAH敲降组OPLAH mRNA和蛋白水平显著低于对照组。
实施例3敲降OPLAH抑制前列腺癌细胞的增殖
在前列腺癌细胞株LNCaP中敲降OPLAH后可抑制前列腺癌细胞增殖。如实施例2,在稳转细胞系构建成功并验证敲降效率后,用CCK8法检测增殖效率。具体地,将稳定表达pLKO.1-puro和pLKO.1-OPLAH的LNCaP细胞进行计数,以每孔1000个细胞/100μl加入96孔板。在检测前2小时将培养基换成含10%CCK8的培养基,用酶标仪测定24、48、72小时OD450nm的吸光度。
结果,CCK8结果见图4,敲降OPLAH之后,前列腺癌细胞LNCaP的增殖受到抑制。
Claims (4)
1.一种siRNA、DNA双链序列或构建物中的一种或二种以上在制备治疗前列腺癌的药物中的应用,其特征在于,
所述的siRNA序列如下所示:
正义链:5’-GCGACGUGCUACUGAGCAA-3’,
反义链:5’-UUGCUCAGUAGCACGUCGC-3’;
所述的DNA双链序列如下所示:
上游链:5’-CCGGGCGACGTGCTACTGAGCAACTCGAGTTGCTCAGTAGCACGTCGCTTTTT-3’
下游链:5’-AATTAAAAAGCGACGTGCTACTGAGCAACTCGAGTTGCTCAGTAGCACGTCGC-3’;
所述的构建物包含所述DNA双链序列。
2.根据权利要求1所述的应用,其特征在于,所述的构建物为慢病毒载体。
3.权利要求1所述的siRNA、DNA双链序列或构建物中的一种或二种以上在制备抑制前列腺癌细胞增殖试剂中的应用。
4.权利要求1所述的siRNA、DNA双链序列或构建物中的一种或二种以上在非治疗目的的抑制离体的前列腺癌细胞增殖中的应用。
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