CN116970610A - 一种chi-miR-335-5p的新用途及其调控靶基因DKK1表达的方法 - Google Patents
一种chi-miR-335-5p的新用途及其调控靶基因DKK1表达的方法 Download PDFInfo
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Abstract
本发明公开了一种chi‑miR‑335‑5p的新用途及其调控靶基因DKK1表达的方法，涉及基因治疗领域，一种能够促进毛囊干细胞增殖的miRNA，miRNA为能够促进毛囊干细胞增殖的chi‑miR‑335‑5p；本发明发现chi‑miR‑335‑5p能够促进毛囊干细胞增殖，且lncRNA FABP_AS能够竞争性结合chi‑miR‑335‑5p调控靶基因DKK1的表达，进而影响Wnt/β‑catenin信号通路的活性，对与毛囊干细胞有关的药物上有广泛的应用前景，基于本发明能够制作治疗或预防与毛囊有关的疾病的药物或检测试剂盒。
Description
技术领域
本发明涉及生物领域，特别是一种chi-miR-335-5p的新用途及其调控靶基因DKK1表达的方法。
背景技术
成熟miRNA大小通常为18~25 nt，参与转录后基因调控 (Ha and Kim, 2014)。MiRNA的生物发生包括几个步骤，首先是前体RNA分子由RNA聚合酶II从基因组转录长度为大约1000 nt左右长的初级miRNA（pri-miRNA）；然后，含有RNase III型核酸内切酶Drosha将pri-miRNA加工成细胞核内的前体miRNA（pre-miRNA）；pre-miRNA通过一个细胞核转运蛋白exportin-5输出到细胞质中，随后细胞质核酸内切酶Dicer会对pre-miRNA进行下一步切割，形成一个22 nt左右的双链RNA。MiRNA双链中的一条会与RNA诱导的沉默复合物结合到其目标基因，另外一条通常会被降解(Krol et al., 2010; Kim et al., 2016)。
miRNAs通过其“种子”区（5’端第2~8位核苷酸）与靶基因mRNA 3’UTR的特定位点（5~8核苷酸）互补能够将Argonaute蛋白引导过来，形成RISC复合物调节基因表达(Treiberet al., 2012)。MiRNAs主要通过两种方式调控基因表达：翻译抑制和mRNA降解。其机制是由miRNA与其靶mRNA之间的互补程度决定的；高互补性会通过RNA介导的干扰途径触发mRNA降解；否则，翻译就会受到抑制(Yu et al., 2015)。此外，有报道发现miRNAs也可以结合到CDS区以及5’UTR上发挥作用，与CDS区结合的miRNA同样主要以负调控的方式抑制基因表达，不同的是与5’UTR结合的miRNA可能主要起转录激活作用(Ørom et al., 2008; Tay etal., 2008)。
在调控毛囊发育发面，许多miRNA能够通过靶向一个或多个信号通路调控毛囊的发育。例如，miR-214可以直接降低β-catenin的表达(Ahmed et al., 2014)，也可以靶向EZH2发挥同样的作用(Du et al., 2018)。MiR-214的过表达导致异常的毛囊和毛发形成，而这种表型可以被Wnt激活剂挽救。MiR-214通过直接结合β-catenin的3’UTR调控毛囊发生发育 (Ahmed et al., 2014)。与miR-214相似，miR-195-5p通过靶向低密度脂蛋白受体相关蛋白6（LRP6）抑制Wnt/β-catenin激活从而调节毛囊的诱导性(Zhu et al., 2018a)。在羊驼模型中，miRNA let-7b的表达水平与耳毛和背毛EDA的表达水平呈负相关。let-7b过表达导致EDA mRNA和蛋白水平下降，表明let-7b通过靶向EDA基因调控羊驼毛囊的发育，而EDA基因也与少汗性外胚层发育不良相关(Liu et al., 2018a)。Let-7b也通过转录后的不同机制调控TGF-β受体I来调节EDA的表达 (Yan et al., 2016)。其他几个miRNA参与毛囊的发育，包括miR-21（靶向Pten, Pdcd4, Timp3, Tpm1和BMP信号通路），miR-24（靶向Tcf-3和Wnt/β-catenin通路），miR-218-5p（靶向SFRP2和Wnt/β-catenin通路）和miR-125a（Wnt2和Wnt/β-catenin通路） (Ahmed et al., 2011; Amelio et al., 2013; Chen et al.,2018; Zhao et al., 2019)。
高表达的miRNA通常在特定的细胞和组织类型中发挥关键的调控作用(Andl andBotchkareva, 2015)。MiR-205是一种鳞状上皮miRNA，在皮肤祖细胞和干细胞中富集，特别是在bulge的HFSCs中。据报道，miR-205敲除小鼠表现出严重的发育缺陷，导致新生儿死亡。MiR-205表达的缺失导致毛囊相对较短且角度错位，尽管其对终末分化的影响较弱。MiR-205消融也会阻碍HFSCs和祖细胞的增殖，提示miR-205对毛囊的形态形成至关重要。此外，miR-205通过直接靶向PI3K基因调控PI3K通路，表明miR-205对毛囊的影响可能是通过PI3K通路介导的 (Wang et al., 2013a)。另一项研究表明，在HF周期中miR-29a/b1的过表达缩短了毛发，并最终通过抑制HFSC和基质细胞分化而导致脱发。Wnt和BMP通路与HFSC谱系进展密切相关；miR-29a/b1直接靶向LRP6、β-catenin和Bmpr1a，从而抑制Wnt和BMP信号通路(Ge et al., 2019)。体外过表达miR-214抑制HFSC的增殖和分化，通过直接靶向EZH2的增强子改变细胞周期，并干扰Wnt/β-catenin信号通路；然而，这些发现尚未在体内得到验证(Du et al., 2018)。有趣的是，HFSCs的一个阳性miRNA调控因子也被发现；miR-124被证明可以通过靶向Sox9和Ptbp1促进HFSCs的分化 (Mokabber et al., 2019)。
测序技术的进步促进了几种与色素沉着有关的miRNAs的发现。使用Illumina测序技术研究白色和棕色羊驼皮肤的miRNA表达谱，发现了四种差异表达的miRNAs（miR-211、miR-424、miR-202和miR-184）；这些miRNAs可能通过靶向与黑色素生成途径相关的基因来调节黑色素生成(Tian et al., 2012)。在另一项研究中发现miR-202在C57BL/6黑小鼠皮肤组织中的表达高于BALB/c白小鼠皮肤组织，并且Wnt5a、kit和tcf7受miR-202的负调控，表明miR-202在黑色素生成中的作用(Qu et al., 2017)。此外，miR-10b和miR-211的表达水平在黑色小鼠毛囊中显着高于白色小鼠，有证据表明miR-10b调节MAPK和Notch信号通路，而miR-211与MITF相互作用(Mazar et al., 2010; Mokhtari et al., 2021)。MITF也是其他几种miRNAs靶向的黑色素生成的主要调节因子，包括miR-25、miR-137、miR-148、miR-182、miR-218和miR-340 (Bemis et al., 2008; Haflidadóttir et al., 2010; Zhuet al., 2010; Yan et al., 2012; Guo et al., 2014; Zhao et al., 2017)。此外，miR-137可影响体内色素沉着(Bemis et al., 2008)。Dong等构建了一个miR-137转基因小鼠模型，其中小鼠毛色根据miR-137的表达水平而变化，从深黑色到浅色不等(Dong etal., 2012)。
DPCs的外泌体在对HFSCs的分化中起关键作用。为了研究在毛囊周期循环中DPCs外泌体调控的作用，Yan等共培养DPCs和HFSCs，发现在DPCs外泌体中高度富集的miR-22-5p通过直接与lef1结合，抑制HFSC的增殖 (Yan et al., 2019)。miR-140-5p在低传代DPCs外泌体中富集，并靶向BMP2，促进ORS和基质细胞增殖 (Chen et al., 2020)。
本发明惊喜的发现chi-miR-335-5p能够促进毛囊干细胞(Hair follicle stemcells, HFSCs)增殖，且lncRNA FABP_AS能够竞争性结合chi-miR-335-5p调控靶基因DKK1的表达，进而影响Wnt/β-catenin信号通路的活性，对与毛囊干细胞有关的药物的制备具有广大的应用前景。
发明内容
为解决现有技术的不足，本发明的目的在于提供一种能够促进毛囊干细胞增殖的miRNA及其调控靶基因DKK1表达的方法， 本发明发现chi-miR-335-5p能够促进毛囊干细胞(Hair follicle stem cells, HFSCs)增殖，且lncRNA FABP_AS能够竞争性结合chi-miR-335-5p调控靶基因DKK1的表达，进而影响Wnt/β-catenin信号通路的活性，对与毛囊干细胞有关的药物的制备上有广泛的应用前景。
为了实现上述目标，本发明采用如下的技术方案：
一种能够促进毛囊干细胞增殖的miRNA，miRNA为能够促进毛囊干细胞增殖的chi-miR-335-5p。
前述的一种chi-miR-335-5p的新用途 chi-miR-335-5p成熟体（mimic）以及抑制剂序列信息为chi-miR-335-5p mimic/mimic NC/inhibitor/inhibitor NC；序列如下所示：
。
前述的一种chi-miR-335-5p的新用途 chi-miR-335-5p能够靶向结合DKK1 3’UTR区域。
前述的一种chi-miR-335-5p的新用途过表达chi-miR-335-5p能够促进HFSCs的增殖，抑制chi-miR-335-5p能够抑制HFSCs的增殖。
前述的一种chi-miR-335-5p的新用途过表达chi-miR-335-5p能够升高细胞增殖相关基因MKi67和PCNA的表达，抑制chi-miR-335-5p能够降低细胞增殖相关基因MKi67和PCNA的表达。
一种调控靶基因DKK1表达的方法,过表达chi-miR-335-5p能够显著降低DKK1的表达，抑制chi-miR-335-5p能够显著升高DKK1的表达。
前述的一种调控靶基因DKK1表达的方法,过表达LncRNA-FABP_AS显著抑制HFSCs的增殖，而chi-miR-335-5p过表达后能够抵消这一作用，恢复HFSCs原有的活力，LncRNA-FABP_AS竞争性结合chi-miR-335-5p抑制HFSCs增殖。
一种chi-miR-335-5p在制备治疗与毛囊有关疾病的药物中的应用，前述的chi-miR-335-5p能够促进毛囊干细胞增殖，用于制备治疗与毛囊有关疾病治疗或预防的药物。
一种chi-miR-335-5p在制备治疗与毛囊有关疾病的药物中的应用，前述的chi-miR-335-5p能够促进毛囊干细胞增殖，用于制备检测与毛囊有关疾病的试剂盒。
专有名词释义：
。
FABP_AS命名原因是：lncRNA TCONS_00462852在细胞分化期高表达，比对基因组发现其与A-FABP基因反向互补；FABP_AS属于top 15差异表达lncRNAs之一，表明其可能参与调控毛囊形态发生过程。
本发明的有益之处在于：
本发明发现chi-miR-335-5p能够靶向结合DKK1 3’UTR区域，能够促进毛囊干细胞增殖；
过表达chi-miR-335-5p能够升高细胞增殖相关基因MKi67和PCNA的表达，抑制chi-miR-335-5p能够降低细胞增殖相关基因MKi67和PCNA的表达；
过表达chi-miR-335-5p能够显著降低DKK1的表达，抑制chi-miR-335-5p能够显著升高DKK1的表达；
过表达LncRNA-FABP_AS显著抑制HFSCs的增殖，而chi-miR-335-5p过表达后能够抵消这一作用，恢复HFSCs原有的活力，LncRNA-FABP_AS竞争性结合chi-miR-335-5p抑制HFSCs增殖。
本发明提供一种chi-miR-335-5p的新用途，chi-miR-335-5p能够促进毛囊干细胞(Hair follicle stem cells, HFSCs)增殖，用于制备治疗与毛囊有关疾病的药物。
附图说明
图1是本发明的chi-miR-335-5p促进HFSCs增殖的实验结果图；
图2是本发明的chi-miR-335-5p靶向下调DKK1的实验结果图；
图3是本发明的lncRNA FABP_AS可作为ceRNA竞争性结合chi-miR-335-5p的验证实验结果图；
图4是本发明的LncRNA-FABP_AS竞争性结合chi-miR-335-5p抑制HFSCs增殖的实验结果图。
具体实施方式
以下结合附图和具体实施例对本发明作具体的介绍。
一，验证chi-miR-335-5p的功能
根据miRBase数据库得到chi-miR-335-5p的成熟体序列信息，由上海吉玛制药技术有限公司设计合成chi-miR-335-5p mimic/mimic NC/inhibitor/inhibitor NC，序列信息详见表1。
表1 chi-miR-335-5p相关序列信息
。
6孔板培养HFSCs，当密度达到80%以后转染chi-miR-335-5p mimic/mimic NC/inhibitor/inhibitor NC。细胞转染48 h后，提取细胞RNA，RT-qRCR检测chi-miR-335-5p的表达。结果表明，与mimic NC/ inhibitor NC组相比，mimic/inhibitor组显著升高/降低了chi-miR-335-5p的表达（图1A）。随后利用CCK8、结晶紫染色、EdU和MTT验证chi-miR-335-5p在HFSCs中的功能。结果表明，过表达/抑制chi-miR-335-5p能够显著促进/抑制HFSCs的增殖（图1B-E）。进一步通过RT-qRCR检测发现，过表达/抑制chi-miR-335-5p能够显著升高/降低细胞增殖相关基因MKi67和PCNA的表达（图1F）。引物序列信息详见表2。
表2 RT-qPCR引物序列信息
。
二，探究chi-miR-335-5p的分子调控机制
通过TargetScan数据库对chi-miR-335-5p的下游靶基因进行预测，并从筛选出DKK1基因。已有研究表明Wnt/β-catenin信号传导途径对真皮毛乳头细胞的生长和维持至关重要，而Dkk1是Wnt信号通路中重要的拮抗分子之一，能够特异地抑制经典Wnt信号通路。将野生型和突变型的DKK1 3’UTR序列分别构建到psiCHECK2载体上（图2A），双荧光素酶试验结果表明，与对照组相比，过表达/抑制chi-miR-335-5p能够显著降低/升高荧光素酶活性，而位点突变后则无显著差异，说明chi-miR-335-5p能够靶向结合DKK1 3’UTR区域（图2B和2C）。进一步通过RT-qRCR检测发现，过表达/抑制chi-miR-335-5p能够显著降低/升高DKK1的表达（图2D）。此外，通过miRDB数据库发现lncRNA FABP_AS可以作为ceRNA竞争性结合chi-miR-335-5p调控下游靶基因，且通过RT-qRCR检测表明过表达/抑制lncRNA FABP_AS能够显著升高/降低DKK1的表达（图2E）。
将野生型和突变型的chi-miR-335-5p与lncRNA FABP_AS结合的序列分别构建到psiCHECK2载体上（图3A），双荧光素酶试验结果表明，与对照组相比，过表达/抑制chi-miR-335-5p能够显著降低/升高荧光素酶活性，而位点突变后则无显著差异，说明chi-miR-335-5p能够与lncRNA FABP_AS互作（图3B和3C）。进一步通过RT-qRCR检测发现，过表达/抑制lncRNA FABP_AS能够显著降低/升高chi-miR-335-5p的表达（图3D）。载体构建引物信息详见表3。
表3 载体构建引物序列信息
。
三，LncRNA-FABP_AS竞争性结合chi-miR-335-5p抑制HFSCs增殖
为了进一步确定LncRNA-FABP_AS能够作为chi-miR-335-5p的ceRNA发挥作用，对LncRNA-FABP_AS和chi-miR-335-5p进行过表达处理并进行回补试验。利用CCK8、结晶紫染色、EdU和MTT等进行功能试验。结果表明，过表达LncRNA-FABP_AS显著抑制HFSCs的增殖，而chi-miR-335-5p过表达后能够抵消这一作用，恢复HFSCs原有的活力（图4A-D）。进一步通过RT-qRCR检测发现，DKK1基因以及Wnt/β-catenin信号通路相关基因CTNNB1、Cyclin D1、C-myc的表达变化也和功能试验结果相符合（图4E）。
本发明发现chi-miR-335-5p能够促进毛囊干细胞(Hair follicle stem cells,HFSCs)增殖，且lncRNA FABP_AS能够竞争性结合chi-miR-335-5p调控靶基因DKK1的表达，进而影响Wnt/β-catenin信号通路的活性。
以上显示和描述了本发明的基本原理、主要特征和优点。本行业的技术人员应该了解，上述实施例不以任何形式限制本发明，凡采用等同替换或等效变换的方式所获得的技术方案，均落在本发明的保护范围内。本发明不涉及疾病的诊断和治疗方法，本发明要求保护药物的新用途，这样的药物可以在产业上利用的，且不会妨碍医生的自由。
Claims (8)
1.一种chi-miR-335-5p的新用途，其特征在于，所述chi-miR-335-5p能够促进毛囊干细胞增殖；所述chi-miR-335-5p的成熟体序列信息为chi-miR-335-5p mimic/mimic NC/inhibitor/inhibitor NC；序列如下所示：
。
2.根据权利要求1所述的一种chi-miR-335-5p的新用途，其特征在于，所述chi-miR-335-5p能够靶向结合DKK1 3’UTR区域。
3.根据权利要求1所述的一种chi-miR-335-5p的新用途，其特征在于，过表达所述chi-miR-335-5p能够促进HFSCs的增殖，抑制所述chi-miR-335-5p能够抑制HFSCs的增殖。
4.根据权利要求1所述的一种chi-miR-335-5p的新用途，其特征在于，过表达chi-miR-335-5p能够升高细胞增殖相关基因MKi67和PCNA的表达，抑制chi-miR-335-5p能够降低细胞增殖相关基因MKi67和PCNA的表达。
5.一种调控靶基因DKK1表达的方法,其特征在于，过表达chi-miR-335-5p能够显著降低DKK1的表达，抑制chi-miR-335-5p能够显著升高DKK1的表达。
6.根据权利要求5所述的一种调控靶基因DKK1表达的方法,其特征在于，过表达LncRNA-FABP_AS显著抑制HFSCs的增殖，而chi-miR-335-5p过表达后能够抵消这一作用，恢复HFSCs原有的活力，LncRNA-FABP_AS竞争性结合chi-miR-335-5p抑制HFSCs增殖。
7.一种chi-miR-335-5p在制备治疗与毛囊有关疾病的药物中的应用，其特征在于，如权利要求1所述的chi-miR-335-5p能够促进毛囊干细胞增殖，用于制备治疗或预防与毛囊有关疾病的药物。
8.一种chi-miR-335-5p在制备治疗与毛囊有关疾病的药物中的应用，其特征在于，如权利要求1所述的chi-miR-335-5p能够促进毛囊干细胞增殖，用于制备检测与毛囊有关疾病的试剂盒。
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