CN109172595A - 微小RNA let-7g的新用途 - Google Patents
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Abstract
本发明公开了一种微小RNA let‑7g的新用途，即其在制备调控哺乳动物乳腺发育及泌乳药物中的应用，所述let‑7g的序列如SEQ ID NO:5所示，本发明通过双荧光素酶报告基因系统证实let‑7g可以通过直接与PRKCA基因的3’UTR结合进而对PRKCA表达进行负向调控，在小鼠乳腺上皮细胞中可发挥调控作用；本发明证实let‑7g可以通过靶向结合PRKCA基因的3’UTR，并对基因表达进行负向调控；且证明抑制let‑7g可增加小鼠乳腺上皮细胞活力，因此微小RNA let‑7g抑制物有应用在制备调控哺乳动物乳腺发育及泌乳药物中的前景。
Description
技术领域
本发明属于分子生物学技术领域，涉及一种微小RNA let-7g的新用途，即在制备调控哺乳动物乳腺发育及泌乳药物中的应用。
背景技术
微小RNA（microRNAs, miRNAs, miRs）是一类长度为18～25 nt 的非编码小RNA，参与调节生长、发育、死亡、疾病等一系列生物进程；它们通过碱基互补配对原则与mRNA的3’UTRs区域结合，与靶基因3’UTR区域互补性结合抑制mRNA，在蛋白表达的转录后阶段起调节作用，从而调控生物学进程。miRNAs的表达具有时间和组织特异性，在处于不同发育阶段的组织细胞和同一发育阶段的不同组织细胞中表达的miRNAs的种类和丰度都不相同，这直接反映了miRNAs对组织细胞分化发育的调控状态；let-7g作为时序性miRNA的代表可从时间和空间上精确控制生物各阶段的发育，在miRNAs的早期研究中早已被人们所关注，但在哺乳动物乳腺发育循环中的作用还未见报道。
蛋白激酶C（PKC）是一系列丝／苏氨酸蛋白激酶家族，存在于许多不同类型的组织和细胞中，并广泛影响多种细胞过程，如细胞的生长和分化、细胞骨架重塑和对刺激产生的多种基因应答等，目前PRKCA是否为let-7g靶基因尚未证实。
发明内容
本发明的目的在于提供一种微小RNA let-7g的新用途，即其在制备调控哺乳动物乳腺发育及泌乳药物中的应用，所述let-7g的序列如SEQ ID NO:5所示（ugagguaguaguuuguacaguu）。
本发明通过双荧光素酶报告基因系统，所述双荧光素酶报告基因系统是指pMIR-REPORT-PRKCA-3’UTR双荧光素酶报告基因载体，检测let-7g可以通过靶基因的3’UTR负向调控PRKCA（蛋白激酶Cα）的表达，let-7g可直接与PRKCA mRNA 3’UTR区直接结合；
本发明通过基因工程技术构建的pMIR-REPORT-PRKCA-3’UTR双荧光素酶报告基因载体，该载体包括双荧光素酶报告基因载体以及SEQ ID NO:1所示核苷酸序列的基因片段，所述核苷酸序列位于PRKCA基因的3’UTR，其中选取的双荧光素酶报告基因载体为pMIR-REPORT Reporter Vector，有美国ABI公司提供。
具体而言，本发明的pMIR-REPORT-PRKCA-3’UTR双荧光素酶报告基因载体是通过系数方法构架而成的：
根据生物信息学预测网站Targetscan（http://www.targetscan.org/）、mirdb（http://mirdb.org/）、microrna.org（http://www.microrna.org/）预测PRKCA的3'UTR可能存在let-7g的结合位点，PRKCA靶基因可能是let-7g的靶基因；通过GenBank及Targetscan查询序列发现，PRKCA 3’UTR在人、小鼠、大鼠、牛等哺乳动物中保守性较高。以小鼠PRKCA mRNA（NM_011101.3）为模板，选取与let-7g相匹配结合的区域向前后延伸50bp，设计短链，上游单链加入酶切位点HindIII，下游单链加入酶切位点SpeI，设计如SEQ IDNO:1和SEQ ID NO:2序列：
PRKCA 3’UTR sense（SEQ ID NO:1）:
5’-CTAGTCGCTCTAGAGCCCTAACCCTCCTACCTCTGTGGCCTAACTACCCA-3’,
PRKCA 3’UTR antisense（SEQ ID NO:2）:
5’-AGCTTGGGTAGTTAGGCCACAGAGGTAGGAGGGTTAGGGCTCTAGAGCGA-3’；
同时设计短链，上游单链加入酶切位点HindIII，下游单链加入酶切位点SpeI，但在PRKCA与let-7g结合位点进行各位剪辑突变，设计如SEQ ID NO: 3和SEQ ID NO: 4序列：
PRKCA-mut-3’UTR sense（SEQ ID NO: 3）:
5’-CTAGTCGCTCTAGAGCCCTAACCCTCGATGCATGTGGCCTAACTACCCA-3’,
PRKCA-mut-3’UTR antisense（SEQ ID NO: 4）:
5’-AGCTTGGGTAGTTAGGCCACAGAGCTACCGAGGTTAGGGCTCTAGAGCGA-3’；
合成的两条单链退火合成两段含有酶切位点的双链；采用SpeI和HindIII限制性内切酶双酶切pMIR-REPORT Reporter Vector载体，将合成两单带有酶切位点的双链PRKCA 3’UTR片段插入同样带有粘性SpeI和HindIII酶切位点的pMIR-REPORT Reporter Vector荧光素酶报告基因载体，构建pMIR-REPORT-PRKCA-3’UTR载体；同样操作构建了pMIR-REPORT-PRKCA-mut-3’UTR。
本发明构建的PRKCA-3'UTR双荧光素酶报告基因载体可用于检测let-7g对PRKCA基因的调控作用，可用于细胞和动物水平上研究let-7g对候选靶基因PRKCA的生物学功能和调控机制；本发明采用构建的载体进行检测验证，证明let-7g能够直接与PRKCA 3’UTR直接结合，并对其有负调控作用。实验结果证明：let-7g能够直接与PRKCA 3'UTR直接结合，并对其有负调控作用，let-7g作为微小RNA通过与PRKCA RNA的3'UTR区域直接结合对PRKCA进行转录后调节，通过阻碍降低其PRKCA正常翻译减少PRKCA蛋白表达。这种调控方式书转录后水平的调控手段，在不改变基因组的情况下进行表观遗传学干预来改变蛋白表达水平。
根据miRNA网站mirbase提供的mmu-let-7g序列SEQ ID NO: 5设计反向互补序列SEQ ID NO: 6：
Mmu-let-7g（SEQ ID NO: 5）:5’-ugagguaguaguuuguacaguu-3’
Anti-mmu-let-7g（SEQ ID NO: 6）:5’-AACUGUACAAACUACUACCUCA-3’
通过设计let-7g反向序列抑制物（inhibitor）作用于小鼠乳腺上皮细胞，观察到抑制let-7g可增加小鼠乳腺上皮细胞活力。
本发明证实let-7g可以通过靶向结合PRKCA基因的3’UTR，并对基因表达进行负向调控；并通过设计let-7g反向序列抑制物（inhibitor）作用于小鼠乳腺上皮细胞，证明抑制let-7g可增加小鼠乳腺上皮细胞活力，因此，微小RNA let-7g抑制物有应用在制备调控哺乳动物乳腺发育及泌乳药物中的前景。
附图说明
图1是本发明中pMIR-REPORT-PRKCA-3’UTR示意图；
图2酶切检测pMIR-REPORT-PRKCA-3’UTR质粒载体电泳结果示意图；其中A:pMIR-REPORT-PRKCA-3’UTR酶切；B：pMIR-REPORT -PRKCA-mut-3’UTR；C：pMIR-REPORT；
图3 pMIR-REPORT-PRKCA-3’UTR与let-7g mimic共转染细胞后荧光素酶报告基因检测；
图4 pMIR-REPORT-PRKCA-3’UTR质粒载体结构图；
图5 let-7g inhibior增加小鼠乳腺上皮细胞增殖能力及活力。
具体实施方式
以下结合附图和实施例来进一步阐述本发明的具体内容，本实施例是在人293T细胞中进行，利用同样的方法所构建的pMIR-REPORT-PRKCA-3’UTR质粒载体及其相应生物学功能和基因调控机制上的应用也应视为本发明的保护范围；本实施例中方法如无特殊说明，均为常规方法，所使用试剂如无特殊说明的，均为常规市售试剂。
实施例1：pMIR-REPORT-PRKCA-3’UTR载体构建
1、 PRKCA基因3’UTR核苷酸序列的设计与合成
登录Genebank检索PRKCA基因mRNA编码序列全长，基因序列号为NM_011101.3，结合Targetscan数据库寻找let-7g可能结合的靶位点序列（见图1）；选取与let-7g相匹配结合的区域向前后延伸50bp，设计短链，上游单链加入酶切位点HindIII，下游单链加入酶切位点SpeI（见图4，载体结构图），设计序列及突变对照序列，提交上海生工公司进行合成，暂命名为PRKCA-3’UTR和PRKCA-mut-3’UTR序列。
2、 3’UTR寡核苷酸序列的退火
将合成的3’UTR寡核苷酸片段干粉在12000 rpm离心1min，根据合成量加入适量无菌超纯水进行溶解，并用TE溶液进行进一步的稀释，使寡核苷酸溶液浓度达到100μM，用于退火反应溶液；将配制好的退火反应缓冲液进行混合，短暂离心后放置PCR仪上，用以下程序进行退火：90℃ 5 min，70℃ 10 min，55℃ 10 min，40℃ 10 min，25℃ 10 min；退火后的寡核苷酸可进行连接反应。
反应体系如下：
正义核苷酸单链(100μM) 5μL
反义核苷酸单链(100μM) 5μL
加TE溶液补足至 50μL
用水将退火后双链稀释100倍使用。
3、退火后双链与载体的连接
用HindIII和SpeI双酶切2μg pMIR-REPORT Reporter Vector载体质粒，酶切方法和体系参考说明书进行；将退火后双链与双酶切的pMIR-REPORT Reporter Vector载体（见图1、2、4）。按照以下体系进行16˚C连接过夜；连接反应体系如下：
T4 DNA连接酶 5U
双酶切载体 2μL
稀释后寡核苷酸 2μL
10×连接酶Buffer 1μL
加水补足至 10μL。
4、质粒的测序验证
连接产物的转化和挑斑均按常规方法进行，质粒转化菌送上海生工进行测序验证，完全符合设计要求，无碱基突变；编码序列和设计序列完全一致，表明已成功构建pMIR-REPORT-PRKCA-3’UTR载体。
实施例2：pMIR-REPORT-PRKCA-3’UTR载体转染细胞和双荧光素酶检验
1、pMIR-REPORT-PRKCA-3’UTR载体转染细胞
转染前一天，以不含双抗的DMEM培养基将生长状态良好的293T细胞铺至六孔细胞培养板中，待细胞生长至融合度约80-90%时，参考Invitrogen产品说明，取2μg pMIR-REPORT-PRKCA-3’UTR/ pMIR-REPORT-PRKCA-mut-3’UTR与50nM let-7g mimic /NC，使用脂质体Lipofectamine 2000 Reagent共转染，pMIR-REPORT-PRKCA-mut-3’UTR和NC作为对照；转染6小时后，更换新鲜含1%新生牛血清的DMEM培养基进行维持培养48h。
2、双荧光素酶报告基因检验
将转染后培养48h的细胞，吸去旧的培养基以PBS洗两遍，按Dual-LuciferaseReporter Assay System试剂盒说明书向每孔加入100μlPLB，室温作用20min，再向没空加入20μl LAR-II试剂，放入仪器读取荧光素酶荧光值，然后再加入20μl的Glo试剂，放入仪器上读取β-gal读取β半乳糖苷酶数值；通过Dual-Luciferase报告基因检测系统测定各实验组中萤火虫荧光素酶的荧光值和β半乳糖苷酶相对活性值进行数值统计；let-7g可抑制PRKCA 3'UTR载体的荧光素酶报告基因表达（图3）。结果表明，PRKCA是let-7g的靶基因，let-7g可通过与PRKCA 3'UTR结合靶向抑制PRKCA表达，let-7g对PRKCA起到负调控作用。
实施例3：let-7g inhibitor对小鼠乳腺上皮细胞增殖能力及活力的检验
1、 pMIR-REPORT-PRKCA-3’UTR载体转染细胞
转染前一天，以不含双抗的DMEM培养基将生长状态良好的小鼠乳腺上皮细胞铺至24孔细胞培养板中，待细胞生长至融合度约70%时，参考Invitrogen产品说明，取25nM、50 nM、100nM let-7g inhibitor / 50nM NC，使用脂质体Lipofectamine 2000 Reagent分别转染， NC作为对照；转染6小时后，更换新鲜含1%新生牛血清的DMEM培养基进行维持培养72h。
2、双荧光素酶报告基因检验
将转染后培养24h、48h、72h的细胞，吸去旧的培养基以PBS洗两遍，加入CCK8作用2小时后放入仪器上读取数值；根据不同时间点细胞数目变化数据进行图表绘制；见图5，图中结果表明，let-7g抑制物inhibitor可增加小鼠乳腺上皮细胞增殖能力。
序列表
<110> 昆明理工大学
<120> 微小RNA let-7g的新用途
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 50
<212> DNA
<213> 人工序列(Artificial)
<400> 1
ctagtcgctc tagagcccta accctcctac ctctgtggcc taactaccca 50
<210> 2
<211> 50
<212> DNA
<213> 人工序列(Artificial)
<400> 2
agcttgggta gttaggccac agaggtagga gggttagggc tctagagcga 50
<210> 3
<211> 49
<212> DNA
<213> 人工序列(Artificial)
<400> 3
ctagtcgctc tagagcccta accctcgatg catgtggcct aactaccca 49
<210> 4
<211> 50
<212> DNA
<213> 人工序列(Artificial)
<400> 4
agcttgggta gttaggccac agagctaccg aggttagggc tctagagcga 50
<210> 5
<211> 22
<212> DNA
<213> 人工序列(Artificial)
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ugagguagua guuuguacag uu 22
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial)
<400> 6
aacuguacaa acuacuaccu ca 22
Claims (1)
1.微小RNA let-7g在制备调控哺乳动物乳腺发育及泌乳药物中的应用，所述let-7g的序列如SEQ ID NO:5所示。
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| CN103933582A (zh) * | 2014-04-28 | 2014-07-23 | 南通大学 | Let-7家族miRNA在制备治疗神经再生相关疾病的药物中的应用 |
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| CN103933582A (zh) * | 2014-04-28 | 2014-07-23 | 南通大学 | Let-7家族miRNA在制备治疗神经再生相关疾病的药物中的应用 |
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| Title |
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| 冯丽等: "let-7g对小鼠乳腺发育和泌乳相关功能基因Tgfbr1的作用及其机理", 《中国兽医学报》 * |
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