CN113151275B - 一种抑制hsa_circ_0001610表达的shRNA及其表达载体 - Google Patents
一种抑制hsa_circ_0001610表达的shRNA及其表达载体 Download PDFInfo
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Abstract
本发明公开了一种抑制hsa_circ_0001610表达的shRNA及其表达载体，属于分子生物学领域。本发明的shRNA具有如SEQ ID NO.4所示的靶点序列。本发明的表达载体包含序列如SEQ ID NO.2～3所示DNA，可靶向hsa_circ_0001610，高效地抑制其表达。
Description
技术领域
本发明属于分子生物学领域。
背景技术
环状RNA(circular RNA，circRNA)是一类具有闭合环状结构的RNA，主要由mRNA前体经过“反向剪接”环化形成。由于circRNA不具有mRNA的5`端帽结构和3`端多聚腺苷酸结构，耐受核糖核酸外切酶的降解、稳定性较好，故具有作为肿瘤标志物的巨大潜力。研究表明，circRNA参与肿瘤的发生发展、肿瘤干性、药物抵抗等，是肿瘤治疗的新方法和新靶点。
研究报道，circRNA主要通过以下机制参与发挥生物学功能：1)作为miRNA的分子海绵，影响miRNA调节的靶基因表达；2)与蛋白相互作用，实现对蛋白功能的调控；3)编码具有功能的多肽。circRNA在肿瘤细胞周期、细胞凋亡、血管形成、侵袭等方面发挥作用。circRNAs的异常表达可能是肿瘤启动的早期关键性事件。其可作为肿瘤的分子标志物或潜在治疗靶点，为肿瘤的早期诊断、疗效评价、预后预测和肿瘤基因治疗提供新靶点。
短发夹RNA(shRNA)是一种常用的敲低(降低靶基因表达)工具，通过载体将其表达序列转入细胞内，并可进一步整合至基因组中，实现shRNA的稳定表达，并可加工成siRNA，从而持续抑制基因表达；其也可通过与目标mRNA互补结合序列特异性地实现靶mRNA降解。但是针对circRNA设计shRNA的难度较大，其靶点序列必须跨越环化位点，可供选择的靶点序列十分有限，往往得到的shRNA敲低效率(敲低效率＝敲低前后靶基因的表达量之差/敲低前靶基因表达量，下同)不够。Guarnerio等设计了一种敲低CircPOK的shRNA，其敲低效率约为60％(Guarnerio J，et al..Intragenic antagonistic roles of protein andcircRNA in tumorigenesis.Cell Res.2019 Aug；29(8)：628-640.)；Chen等设计了一种敲低circRNA_0000285的shRNA，其敲低效率在50％～60％之间(Chen RX，et al.CircularRNA circRNA_0000285 promotes cervical cancer development by regulatingFUS.Eur Rev Med Pharmacol Sci.2019 Oct；23(20)：8771-8778.)。
hsa_circ_0001610是一种由TNFRSF21基因表达的circRNA，其被发现在子宫内膜癌组织中高表达，是子宫内膜癌潜在的治疗靶点。目前尚未见有关使用shRNA敲低hsa_circ_0001610的报道。
发明内容
本发明要解决的问题是：提供一种抑制环状RNA hsa_circ_0001610表达的shRNA及其表达载体。
本发明的技术方案如下：
一种抑制hsa_circ_0001610表达的shRNA，其靶点序列如SEQ ID NO.4所示。
本发明中“靶点序列”指靶基因与shRNA的基因所共有的序列；因为存在该“靶点序列”，shRNA或其加工得到的siRNA产物得以特异性识别靶基因的mRNA。
进一步地，表达该shRNA的DNA的正义链序列如SEQ ID NO.2所示。
一种双链寡核苷酸，其正义链序列如SEQ ID NO.2所示，反义链序列如SEQ IDNO.3所示。
一种抑制hsa_circ_0001610表达的shRNA的表达载体，所述载体包括表达前述的shRNA的DNA序列。
进一步地，所述载体是将表达前述的shRNA的DNA序列插入PLKO.1-TRC载体得到的重组载体。
进一步地，所述表达前述的shRNA的DNA为前述的双链寡核苷酸。
前述载体的构建方法，包括如下步骤：
1)合成寡核苷酸：将序列如SEQ ID NO.2和SEQ ID NO.3的引物先进行变性，再进行退火，得双链寡核苷酸；
2)对PLKO.1-TRC进行Age1和EcoR1双酶切，得2个片段，回收较大片段；
3)将步骤1)所得双链寡核苷酸和步骤2)所得较大片段使用T4连接酶连接。
进一步地，步骤1)的变性程序为：98℃5min。
进一步地，步骤1)的退火程序为：初始温度95℃，持续8s，以后按照每8s下调温度0.1℃，共下调800次。
一种重组细菌，所述细菌包含前述的载体。
本发明的有益效果：
本发明的shRNA及其载体能高效抑制hsa_circ_0001610的表达，可以令其表达量下降到原表达水平的10％以下(对A549细胞)甚至5％以下(对RKO细胞)，敲低效率远高于现有的针对circRNA的shRNA。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1：载体酶切后电泳图。
图2：PCR验证图。
图3：细胞内环状RNA相对表达水平；CircTNFRSF21指hsa_circ_0001610，纵坐标的U6为内参基因。
具体实施方式
实施例1 shRNA表达载体的构建
环状RNA的信息如下：
名称：hsa_circ_0001610
坐标：chr6：47251673-47254331
Genbank编号：NM_014452
长度：1147nt
序列(SEQ ID NO.1)：
根据如下步骤制备shRNA表达载体。
1.寡核苷酸制备
根据环化位点，人工设计引物如下：
正向引物(Primer F，SEQ ID NO.2)：CCGGGCCATGCTTGGATTCCTTACTCGAGTAAGGAATCCAAGCATGGCTTTTT
反向引物(Primer R，SEQ ID NO.3)：AATTAAAAAGCCATGCTTGGATTCCTTACTCGAGTAAGGAATCCAAGCATGGC
划线部分(SEQ ID NO.4)为靶点序列，即与hsa_circ_0001610序列相同的部分，该部分第二个GC(斜体加粗)是环化位点。
将引物按照如下体系配制，进行变性-退火程序。
变性-退火体系如下：
10×Annealing buffer的配方如下：
5M NaCl 200ul
1M Tris HCl(pH＝7.4) 100ul
ddH2O 700ul
变性-退火程序如下：
98℃ 5min(变性程序)；
95℃ 8s -0.1℃/cycle×800(初始温度95℃，持续8s，以后按照每8s下调温度0.1℃，共下调800次)(退火程序)；
4℃保持。
本步骤最终得到双链寡核苷酸(Oligo)，作为shRNA的表达盒(或表达框)，可表达shRNA，其正义链序列(SEQ ID NO.2)为：
CCGGGCCATGCTTGGATTCCTTACTCGAGTAAGGAATCCAAGCATGGCTTTTT
2.载体制备
以PLKO.1-TRC为骨架载体，浓度300ng/ul，按照如下体系进行酶切：
酶切后，电泳得2个片段，胶回收其中较大片段(plKO.1-Age1-EcoR1)，如图1。
3.连接载体
将前述双链寡核苷酸和胶回收所得片段plKO.1-Age1-EcoR1用T4连接酶连接，温度16℃，时间2h，体系如下：
本步骤得到了本发明抑制has_circ_0001610表达的载体，命名为pLKO.1 sh-0001610。
4.转化
将pLKO.1 sh-0001610转化到感受态细胞(细菌)中，抗生素筛选得到阳性单克隆。具体步骤如下：
加入100ul感受态，轻柔混匀——冰上放置30min——42℃热击90s——冰上放置5-10min——加入900μL LB培养基(不含氨苄青霉素)——37℃摇床培养1h——5000-6000rpm离心2min——取上清，留底部200μL涂板(含氨苄青霉素100μg/mL)，37℃培养过夜。
5.验证
先挑取单克隆，进行PCR验证，电泳，出现长度500-600bp长度之间(实际长度569bp)条带，即表明验证质粒成功转入(图2)。PCR验证的引物序列如下：
上游引物tctttcccctgcactgtacc(SEQ ID NO.5)
下游引物ggcagggatattcaccattatcgtttcaga(SEQ ID NO.6)
再取阳性克隆进行测序，表明pLKO.1 sh-0001610构建成功并成功转入了细菌。测序所得序列(SEQ ID NO.7)如下：
实施例2 pLKO.1 sh-0001610敲低效率验证
将pLKO.1 sh-0001610分别转入A549细胞和RKO细胞，以qPCR检测hsa_circ_0001610(即CircTNFRSF21)的表达水平。携带随机序列的载体(sh-scramble)作为对照，也分别转入A549细胞和RKO细胞。
可见转入pLKO.1 sh-0001610的细胞内hsa_circ_0001610表达量远远低于转入sh-scramble的细胞(图3)
结果表明，hsa_circ_0001610被pLKO.1 sh-0001610有效抑制，本发明成功构建得到了抑制hsa_circ_0001610的shRNA及其载体。
综上，本发明的shRNA及其载体能高效抑制hsa_circ_0001610的表达。
SEQUENCE LISTING
<110> 四川大学华西医院
<120> 一种抑制hsa_circ_0001610表达的shRNA及其表达载体
<130> GYKH1923-2021P0112951CCR4
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 1147
<212> DNA
<213> 人类（Homo Sapiens）
<400> 1
tcccctggca cagccatctt tccacgccct gagcacatgg aaacccatga agtcccttcc 60
tccacttatg ttcccaaagg catgaactca acagaatcca actcttctgc ctctgttaga 120
ccaaaggtac tgagtagcat ccaggaaggg acagtccctg acaacacaag ctcagcaagg 180
gggaaggaag acgtgaacaa gaccctccca aaccttcagg tagtcaacca ccagcaaggc 240
ccccaccaca gacacatcct gaagctgctg ccgtccatgg aggccactgg gggcgagaag 300
tccagcacgc ccatcaaggg ccccaagagg ggacatccta gacagaacct acacaagcat 360
tttgacatca atgagcattt gccctggatg attgtgcttt tcctgctgct ggtgcttgtg 420
gtgattgtgg tgtgcagtat ccggaaaagc tcgaggactc tgaaaaaggg gccccggcag 480
gatcccagtg ccattgtgga aaaggcaggg ctgaagaaat ccatgactcc aacccagaac 540
cgggagaaat ggatctacta ctgcaatggc catgcttgga ttccttagca ccaccacagc 600
tcagccagaa cagaaggcct cgaatctcat tggcacatac cgccatgttg accgtgccac 660
cggccaggtg ctaacctgtg acaagtgtcc agcaggaacc tatgtctctg agcattgtac 720
caacacaagc ctgcgcgtct gcagcagttg ccctgtgggg acctttacca ggcatgagaa 780
tggcatagag aaatgccatg actgtagtca gccatgccca tggccaatga ttgagaaatt 840
accttgtgct gccttgactg accgagaatg cacttgccca cctggcatgt tccagtctaa 900
cgctacctgt gccccccata cggtgtgtcc tgtgggttgg ggtgtgcgga agaaagggac 960
agagactgag gatgtgcggt gtaagcagtg tgctcggggt accttctcag atgtgccttc 1020
tagtgtgatg aaatgcaaag catacacaga ctgtctgagt cagaacctgg tggtgatcaa 1080
gccggggacc aaggagacag acaacgtctg tggcacactc ccgtccttct ccagctccac 1140
ctcacct 1147
<210> 2
<211> 53
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 2
ccgggccatg cttggattcc ttactcgagt aaggaatcca agcatggctt ttt 53
<210> 3
<211> 53
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 3
aattaaaaag ccatgcttgg attccttact cgagtaagga atccaagcat ggc 53
<210> 4
<211> 19
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 4
gccatgcttg gattcctta 19
<210> 5
<211> 20
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 5
tctttcccct gcactgtacc 20
<210> 6
<211> 30
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 6
ggcagggata ttcaccatta tcgtttcaga 30
<210> 7
<211> 999
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 7
gctcgttccg gacacgccca gagcagccgc gtccctgcgc aaacccaggg ctgccttgga 60
aaaggcgcaa ccccaacccc ctcgagccgc ggccaaagtg gatctctgct gtccctgtaa 120
taaacccgaa aattttgaat ttttgtaatt tgtttttgta attctttagt ttgtatgtct 180
gttgctatta tgtctactat tctttcccct gcactgtacc ccccaatccc cccttttctt 240
ttaaaattgt ggatgaatac tgccatttgt ctcgaggtcg agaattaaaa agccatgctt 300
ggattcctta ctcgagtaag gaatccaagc atggcccggt gtttcgtcct ttccacaaga 360
tatataaagc caagaaatcg aaatactttc aagttacggt aagcatatga tagtccattt 420
taaaacataa ttttaaaact gcaaactacc caagaaatta ttactttcta cgtcacgtat 480
tttgtactaa tatctttgtg tttacagtca aattaattcc aattatctct ctaacagcct 540
tgtatcgtat atgcaaatat gaaggaatca tgggaaatag gccctcggtg aagggggcgg 600
ccgctcgagg ctagtctcgt gatcgatacc gtcgagatcc gttcactaat cgaatggatc 660
tgtctctgtc tctctctcca ccttcttctt ctattccttc gggcctgtcg ggtcccctcg 720
gggttgggag gtgggtctga aacgataatg gtgaatatcc ctgcctaact ctattcacta 780
tagaaagtac agcaaaaact attccttaac cctaccaagc ctccctacta tcatcataga 840
ataactttta tatacccaca gcccaatttg ttaatgttaa acccatttcc acaaaccttg 900
cacatttatc caaattccaa taattccttg ttcattcttt tcttggctgg ttttgcgatt 960
cctccattta aaggagtgta ttaagccttg tgtaattgt 999
Claims (10)
1.一种抑制hsa_circ_0001610表达的shRNA，其特征在于，其靶点序列如SEQ ID NO.4所示。
2.如权利要求1所述的shRNA，其特征在于：表达该shRNA的DNA的正义链序列如SEQ IDNO.2所示。
3.一种双链寡核苷酸，其特征在于：其正义链序列如SEQ ID NO.2所示，反义链序列如SEQ ID NO.3所示。
4.一种抑制hsa_circ_0001610表达的shRNA的表达载体，其特征在于：所述载体包括表达权利要求1或2所述的shRNA的DNA序列。
5.如权利要求4所述的载体，其特征在于：所述载体是将表达权利要求1或2所述的shRNA的DNA序列插入PLKO.1-TRC载体得到的重组载体。
6.如权利要求4或5所述的载体，其特征在于：所述表达权利要求1或2所述的shRNA的DNA为权利要求3所述的双链寡核苷酸。
7.权利要求6所述载体的构建方法，其特征在于：包括如下步骤：
1)合成寡核苷酸：将序列如SEQ ID NO.2和SEQ ID NO.3的引物先进行变性，再进行退火，得双链寡核苷酸；
2)对PLKO.1-TRC进行Age1和EcoR1双酶切，得2个片段，回收较大片段；
3)将步骤1)所得双链寡核苷酸和步骤2)所得较大片段使用T4连接酶连接。
8.如权利要求7所述的方法，其特征在于：步骤1)的变性程序为：98℃5min。
9.如权利要求7所述的方法，其特征在于：步骤1)的退火程序为：初始温度95℃，持续8s，以后按照每8s下调温度0.1℃，共下调800次。
10.一种重组细菌，其特征在于：所述细菌包含权利要求4～6任一所述的载体。
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