CN108546717A - 反义lncRNA介导顺式调控抑制靶基因表达的方法 - Google Patents
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Abstract
本发明公开了一种反义lncRNA介导顺式调控抑制靶基因表达的方法，利用lncRNA具有竞争内源靶RNA的作用，通过CRISPR Cas9基因编辑方法以RNA作为基因组定位工具在靶基因上插入强启动子，通过大量合成一条与靶基因互补的反义lncRNA，实现原位顺式竞争抑制内源靶基因的表达，为一种反义lncRNA介导顺式(cis)调控抑制基因表达的方法。首先构建靶向载体和同源模板链供体载体，将构建的载体同时转染入宿主细胞，通过嘌呤霉素和更昔洛韦共筛选目标克隆，最后对靶基因表达进行检测及功能研究。
Description
技术领域
本发明属于分子生物学领域。更具体地，利用反义lncRNA具有竞争内源靶RNA的作用，通过CRISPR Cas9基因编辑方法在靶基因上反向插入强启动子，通过大量合成一条与靶基因互补的反义lncRNA，实现原位顺式竞争抑制内源靶基因的表达，为一种反义lncRNA介导顺式(cis)调控抑制靶基因表达的方法。
背景技术
人类多种肿瘤中存在某些基因的异常高表达刺激细胞不断地生长、增殖或迁移，最后导致细胞癌变或转移，这些高表达的基因可作为治疗肿瘤的靶标。而如何精准靶向抑制这些基因的表达是关键。目前对基因抑制的手段以转录后水平调控RNA干扰(RNAi)技术为主，通过特异性结合互补链从而抑制基因表达/引发转录后沉默。但体外合成siRNA价格昂贵，并且需要鉴定出有效的siRNA，siRNA在细胞中瞬时表达抑制不具有持久性不能用来进行长期的基因抑制，受转染效率和难转细胞系限制，使用具有一定的局限性，所以许多的研究人员仅用RNAi作为一种工具进行基因功能的研究。现在可以采用Pol III启动子在体内表达短发夹RNA(shRNA)，加工后形成类似siRNAs的分子，能够引发RNAi过程，可使用病毒载体进行转染，该方法使不能持久抑制基因表达的问题迎刃而解，也可使用诱导型启动子如DOX诱导Tet-on系统控制shRNA表达，但他们的共同问题是脱靶效应，siRNA可能与非靶基因结合而导致非靶基因的沉默。
基因表达调控中转录水平的调控占主导地位，通过基因的顺式作用成分和反式调控因子之间的相互作用实现，近年来发现lncRNA在基因表达调控中也起着调节分子的作用。通过转录抑制、染色质重塑、核内RNA-RNA相互作用和胞浆RNA-RNA相互作用等机制，在转录及转录后水平调控靶基因的表达。反义lncRNA参与了X染色体沉默、基因组印记、染色质修饰、转录干扰以及一些疾病的发生和发展等重要的调控过程。
申请人前期研究中发现了一条全新的位于IGF1R基因内含子区域的反义长链非编码RNA(lncRNA)，将之命名为IGF受体I反向印记非编码RNA(IGF1Receptor AntisenseImprinted Noncoding RNA，IRAIN)。正常组织中IGF1R/IRAIN的转录处于一种平衡的状态。而在乳腺癌细胞中IGF1R表达上调的同时IRAIN表达下调。这种表达失衡导致IGF1R信号通路过度激活，活化的IGF1R信号通路可刺激下游PI3K/AKT信号级联反应，促进细胞增殖、抗凋亡，并通过自分泌、旁分泌、内分泌等通路引发耐药反应及肿瘤转移。此外，与原位癌相比，在侵袭性乳腺癌病人样本中IRAIN表达下调，提示我们IRAIN可能为新的非编码RNA抑癌基因成员之一。对IRAIN抑癌功能研究，我们采用CRISPR Cas9基因编辑技术在肿瘤细胞lncRNA IRAIN的前面插入强启动子CMV使其表达量增加，逆转IGF1R/IRAIN的转录失衡，观察发现靶向克隆细胞中IRAIN有效地高表达并可与正向表达IGF1R基因启动子重叠处的mRNA形成顺式竞争，提示该方法为一种“反义lncRNA介导顺式(cis)调控抑制基因表达”的方法(antisense lncRNA-mediated intragenic cis competition，ALIC)，如图2所示，在反义lncRNA介导顺式(cis)调控抑制IGF1R基因中，Cas9是CRISPR Cas9；gRNA是Cas9引导RNA；pCMV是CMV启动子；pH1是RNA聚合酶III H1启动子；Cre是Cre重组酶；pA是SV40poly Asignal；loxP是Cre识别X-over P1的重组位点；Arm1-2是重组序列在gRNAs的引导下，在IRAIN位点处Cas9介导基因重组使IRAIN前面插入pCMV-puro。该方法在乳腺癌细胞中精准修正了IGF1R/IRAIN产物正常表达进而抑制过表达的IGF1R信号通路，细胞增殖，肿瘤成球、迁移和侵袭能力都减慢，从而达到治疗的目的。为精准治疗肿瘤的发展提供了分子基础。
综上所述，传统抑制基因表达的方法具有很大的局限性，开发的新方法采用最新发展起来的一种强有力的分子生物学工具CRISPR Cas9基因编辑技术，以RNA作为基因组定位工具，通过20个左右的核苷酸特异识别靶基因，利用反义lncRNA具有竞争内源靶RNA的作用，在靶基因上反向插入强启动子，使其大量合成一条与靶基因互补的反义lncRNA，实现原位顺式竞争抑制内源靶基因的表达，为一种反义lncRNA介导顺式(cis)调控抑制基因表达的方法。
发明内容
本发明克服传统方法的局限性，利用lncRNA具有竞争内源靶RNA的作用，通过CRISPR Cas9基因编辑方法以RNA作为基因组定位工具在靶基因上插入强启动子，通过大量合成一条与靶基因互补的反义lncRNA，实现原位顺式竞争抑制内源靶基因的表达，为一种反义lncRNA介导顺式(cis)调控抑制基因表达的方法。首先构建靶向载体和同源模板链供体载体，将构建的载体同时转染入宿主细胞，通过嘌呤霉素和更昔洛韦共筛选目标克隆，最后对靶基因表达进行检测及功能研究。
一种反义lncRNA介导顺式调控抑制靶基因表达的方法，该方法首先采用CRISPRCas9基因编辑方法在靶基因上反向插入强启动子，通过大量合成一条与靶基因互补的反义lncRNA，实现原位顺式竞争抑制靶基因的表达；所述的基因为编码RNA或编码蛋白的碱基序列，所述的基因包括位点本身带有内源性反义lncRNA，也可为本身无反义lncRNA的基因。
所述的基因为过表达的癌基因，或为具有治疗作用的靶基因。
所述的反义lncRNA包括靶基因位点固有的内源性反义lncRNA和重新诱导合成的反义lncRNA。
所述的强启动子为固定表达的非调控型启动子或为可调控型启动子。所述的固定表达的非调控型启动子为CMV启动子；所述的可调控型启动子为tet-ON启动子或tet-OFF启动子。所述原位顺式竞争抑制靶基因的表达由图1表示。
一种反义lncRNA介导顺式调控抑制靶基因表达的方法能应用到检测试剂盒、转基因细胞、转基因鼠的制备。
附图说明
图1是反义lncRNA介导顺式(cis)调控抑制靶基因表达。
图2是反义lncRNA介导顺式(cis)调控抑制IGF1R基因。
图3是IGF1R ALIC靶向抑制肿瘤特征。
具体实施方式
一、靶位点的选择
根据靶基因序列设计，在靶基因启动子下游序列选择PAM前间区序列临近基序CRISPR Cas9-gRNA复合物特异性识别并结合的靶位点，包含由三个核苷酸NGG组成的PAM并可与gRNA分子互补结合。所述的抑制内源靶基因是利用反义lncRNA具有竞争内源靶RNA的作用，通过CRISPR Cas9基因编辑技术在靶基因上插入强启动子，大量合成一条与靶基因互补的反义lncRNA，实现原位顺式竞争抑制内源靶基因的表达。
二、构建靶向载体pCD Cas9-gRNA1-pU6-gRNA2
克隆两个分别带有pU6和pH1启动子的gRNA到靶向载体中，用Pme I和Not I酶切位点插入在Cas9核定位序列(NLS)后。该靶向载体在宿主细胞内既能表达野生型具有切割DNA双链活性的Cas9核酸酶又能表达特异性gRNA。Cas9结合gRNA形成CRISPR Cas9-gRNA复合物，识别PAM后利用gRNA碱基配对识别靶点序列，揭开DNA双螺旋，Cas9特异切割靶基因双链，产生DNA双链断裂，从而诱发DNA损伤修复机制。其中的NHEJ(Non-homologous EndJoining)修复是一种易错修复，而同源重组修复(homologous repair:HR)，提供同源修复模板供体，实现在基因组特定位置的基因敲入。
三、构建同源模板链供体载体pArm1-loxP-pCMV-Puro-loxP-Arm2-TK
供体载体包含“pArm1-loxP-pCMV-Puro-loxP-Arm2-TK”、5’,3’-lncRNA片段(Arm1、Arm2)、pCMV和嘌呤霉素片段、特异性选择基因(HSV-TK)，并通过重叠PCR连接成完整片段，连接后的完整片段用Cla I和Nhe I酶切位点克隆入pcDNA3.1载体(Invitrogen,CA)。其中5’,3’-lncRNA片段Arm1、Arm2为切割位点上下游1kb左右的同源序列；特异性选择基因HSV-TK为单纯疱疹病毒胸苷激酶基因，该基因编码的酶蛋白，可以使低毒的更昔洛韦转化为强细胞毒性物质，杀死宿主细胞，也是肿瘤基因治疗研究中常用的自杀基因。
四、阳性细胞筛选及鉴定
将靶向载体和同源模板链供体载体同时转染入宿主细胞，通过嘌呤霉素和更昔洛韦共筛选目标克隆，PCR验证和酶切验证同时进行，保证同源重组成功整合至基因组的实现原位顺式竞争抑制内源靶基因的表达。
五、以IGF1R为例表达抑制后细胞功能的检测，如图3所示：
乳腺癌细胞MDA-MB-231中对IGF1R采用“反义lncRNA介导cis式调控抑制基因表达”的方法，筛选出来的两个阳性克隆ALIC1和ALIC2，与对照组细胞CTL相比IRAIN表达增强，逆转录PCR及western检测靶基因mRNA和蛋白表达明显下调(图3中的A、B)；检测细胞增值(图3中的C)，显示两个阳性克隆ALIC1和ALIC2细胞wst-1增值检测，提示增殖减慢；流式细胞分析细胞周期(图3中的D)，显示已敲低IGF1R的MDA-MB-231细胞克隆的细胞周期S期细胞显著增加同时G2期细胞减少，表明IGF1R通路下调导致细胞周期的S期阻滞。接下来我们验证了ALIC理论改变细胞迁移和侵袭能力。采用transwell的方法，检测ALIC方法靶向修饰IGF1R显著降低的两个细胞克隆的迁移能力较对照组野生型减慢(图3中的E)；对于侵袭，将ALIC方法敲低IGF1R的MDA-MB-231细胞铺在基底胶包被的transwell小室内，计算穿过基质胶的细胞数量，做为对照的野生型MDA-MB-231侵袭呈恶性表型而ALIC方法敲低IGF1R组显著降低(图3中的F)。
图3是IGF1R ALIC靶向抑制肿瘤特征，其中：
A.筛选出来的两个细胞克隆ALIC1和ALIC2中IGF1R基因mRNA表达情况，实时定量PCR检测表达。**p<0.01与野生型对照组相比(CTL)。
B.筛选出来的两个阳性克隆ALIC1和ALIC2中IGF1R基因蛋白表达情况，灰度分析定量结果。**p<0.01与野生型对照组相比(CTL)。
C.细胞增殖检测。两个细胞克隆用MTT方法做增殖能力分析。
D.细胞周期定量分析。
E.细胞的迁移能力检测。
F.细胞的侵袭能力检测。
如图1所示，是本发明的反义lncRNA介导顺式(cis)调控抑制靶基因表达。
Claims (7)
1.一种反义lncRNA介导顺式调控抑制靶基因表达的方法，其特征在于：首先采用CRISPR Cas9基因编辑方法在靶基因上反向插入强启动子，通过大量合成一条与靶基因互补的反义lncRNA，实现原位顺式竞争抑制靶基因的表达；所述的基因为编码RNA或编码蛋白的碱基序列，所述的基因包括位点本身带有内源性反义lncRNA，也可为本身无反义lncRNA的基因。
2.根据权利要求1所述的一种反义lncRNA介导顺式调控抑制靶基因表达的方法，其特征在于：所述的基因为过表达的癌基因，或为具有治疗作用的靶基因。
3.根据权利要求1所述的一种反义lncRNA介导顺式调控抑制靶基因表达的方法，其特征在于：所述的反义lncRNA包括靶基因位点固有的内源性反义lncRNA和重新诱导合成的反义lncRNA。
4.根据权利要求1所述的一种反义lncRNA介导顺式调控抑制靶基因表达的方法，其特征在于：所述的强启动子为固定表达的非调控型启动子或为可调控型启动子。
5.根据权利要求4所述的一种反义lncRNA介导顺式调控抑制靶基因表达的方法，其特征在于：所述的固定表达的非调控型启动子为CMV启动子；所述的可调控型启动子为tet-ON启动子或tet-OFF启动子。
6.根据权利要求4所述的一种反义lncRNA介导顺式调控抑制靶基因表达的方法，其特征在于：所述原位顺式竞争抑制靶基因的表达由图1表示。
7.权利要求1所述反义lncRNA介导顺式调控抑制靶基因表达的方法应用到检测试剂盒、转基因细胞、转基因鼠的制备。
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