CN107190006A - 一种靶向IGF‑IR基因的sgRNA及其应用 - Google Patents

一种靶向IGF‑IR基因的sgRNA及其应用 Download PDF

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CN107190006A
CN107190006A CN201710552226.6A CN201710552226A CN107190006A CN 107190006 A CN107190006 A CN 107190006A CN 201710552226 A CN201710552226 A CN 201710552226A CN 107190006 A CN107190006 A CN 107190006A
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董志珍
姚敏
王理
姚登福
郑文杰
蔡胤
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Affiliated Hospital of Nantong University
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Abstract

本发明涉及基因工程技术领域，公开了一种靶向IGF‑IR基因的sgRNA，所述sgRNA的核苷酸序列如SEQ ID NO:2所示。本发明应用Crispr/cas9‑sgRNA慢病毒载体系统，在细胞水平上成功对人肝癌细胞IGF‑IR基因进行敲除或修饰，使肝癌细胞IGF‑IR基因转录水平明显下降，使肝癌细胞的增殖、侵袭、迁移能力下降，为肝癌治疗提供有效新方法，具有临床应用前景。

Description

一种靶向IGF-IR基因的sgRNA及其应用
技术领域
本发明涉及基因工程技术领域，特别涉及一种靶向IGF-IR基因的sgRNA及其应用。
背景技术
新近研究发现IGF信号通路中关键信号分子IGF-IR，具有癌胚性，与肝癌进展间存在密切关系，但其基因转录或活化干预是否影响生物学功能与治疗价值尚不清楚。IGF家族(IGFs)由IGF-I、IGF-II、IGF-IR、IGF-IIR以及6种结合蛋白组成。IGFs与胰岛素有相似的分子结构，它的功能包括促进细胞增殖、分化和各种类型细胞的生存以及保持细胞的各种功能，其在体内生长调控中发挥重要作用。IGF-II的主要功能是产生生长因子，它在许多生长发育过程中起关键作用，并且在生长发育过程中以及成年后都广泛表达。愈来愈多的证据表明IGF-I和II及其酪氨酸激酶受体，参与HCC发生与发展。联合靶向IGF-IR和mTOR通路作为一种新型治疗方法，将最大限度的发挥抗肿瘤作用，且防止耐药机制早期发展。
介于IGF-IR在肝癌形成、恶性转化及侵袭转移过程中都扮演着重要角色，阻断该信号通路，有望使癌细胞生长受抑、诱导癌细胞发生凋亡或增加对放、化疗敏感性，表明酪氨酸激酶受体IGF-IR已成为小分子酪氨酸激酶抑制剂、抗体药物设计及核酸干预的重要靶点。
新近一项CRISPR/Cas9基因敲除新技术，突破物种限制可敲除任何基因。CRISPRRNA是原核生物中的调控RNA，用以抵御病毒和质粒的入侵，在II型CRISPR系统中，其形成的复合物首先特异识别基因组序列，然后Cas9核酸内切酶切断目的基因双链。Cas9以序列特异性方式绑定和切割DNA能力非常强大，近年被广泛应用于各种基因组的研究包括HBV基因。尚未见定向剪接肝癌IGF-IR基因的报道。
发明内容
本发明的目的在于提供一种特异靶向IGF-IR基因的sgRNA导向序列。该sgRNA导向序列可以用于敲除肝癌细胞中IGF-IR基因，使其转录水平明显下降，使肝癌HepG2细胞的生物学特性发生明显改变，表现为癌细胞的增殖、侵袭、迁移能力下降。
为了解决上述技术问题，本发明的技术方案如下：
本发明提供了一种靶向IGF-IR基因的sgRNA，所述sgRNA的核苷酸序列如SEQ IDNO:2所示，能识别人肝癌细胞染色体上IGF-IR基因，和与Cas9核酸酶结合的骨架RNA片段。
本发明另一个目的在于提供由编码所述靶向IGF-IR基因的sgRNA的DNA分子。
本发明第三个目的是提供上述sgRNA在特异识别和靶向修饰人肝癌细胞IGF-IR基因中的应用。所述人肝癌细胞IGF-IR基因，其基因组序列是NCBI NM000875中的区域。
本发明第四个目的是提供上述sgRNA在构建人肝癌细胞IGF-IR基因突变库中的应用。所述人肝癌细胞IGF-IR基因，其基因组序列是NCBI NM000875中的区域。
现有技术相比，本发明具有以下优点：
本发明提供了一种特异靶向识别IGF-IR基因的sgRNA，并提供了该sgRNA的编码DNA片段，应用Crispr/cas9-sgRNA慢病毒载体系统，在细胞水平上成功对人肝癌细胞IGF-IR基因进行敲除或修饰，使肝癌细胞IGF-IR基因转录水平明显下降，使肝癌细胞的增殖、侵袭、迁移能力下降，为肝癌治疗提供有效新方法，具有临床应用前景。
附图说明
图1为嘌呤霉素筛选经cas9病毒感染的细胞。
图2为二次感染带荧光的sgRNA病毒，其中，A：普通光镜图；B：同一视野荧光显微镜图；A&B1-3为sgRNA(+)病毒感染组，A&B4为sgRNA(-)病毒感染组。
图3为二次感染带荧光的sgRNA病毒感染效率直方图；sgRNA1-3为sgRNA(+)病毒感染组。
图4为蛋白水平验证IGF-IR基因敲除效率Western blotting图。
图5为蛋白水平验证IGF-IR基因敲除效率Quantity One(Bio-Rad)软件定量条带灰度强度，*：P<0.05。
图6为敲除IGF-IR基因抑制肝癌细胞增殖活性影响的线性图，其中，*P<0.01，**P>0.05。
图7为敲除IGF-IR基因抑制肝癌细胞增殖活性影响的直方图，其中，*P<0.01，**P>0.05。
图8为肝癌HepG2细胞Transwell小室侵袭试验显微镜图，其中：A空白组；B阴性对照组；C sgRNA2感染组穿膜细胞数明显减少,*P<0.01。
图9为肝癌HepG2细胞Transwell小室侵袭试验直方图，sgRNA2感染组穿膜细胞数明显减少,*P<0.01。
图10为划痕试验法检测IGF-IR在肝癌细胞迁移中的作用显微镜图；其中，A1&A2:空白组，B1&B2:阴性对照，C1&C2:sgRNA2干预组。
图11为肝癌HepG2细胞Transwell小室纵向迁移试验显微镜图，其中：A空白组；B阴性对照组；C sgRNA2感染组显示抑制纵向迁移能力,*P<0.01。
图12为肝癌HepG2细胞Transwell小室纵向迁移试验直方图，sgRNA2感染组穿膜细胞数明显减少,*P<0.01。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，下面结合实施例对本发明进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
如无特殊说明，本发明以下实施例中选自以下材料，但并非是对本发明技术方案的限定。
1)人肝癌细胞株
人肝癌HepG2细胞株购自中科院上海细胞所，设三组：空白组(untreated组)、阴性组(LV-neg组)和感染组(LV-sgRNA-IGF-IR组,干扰-1，干扰-2和干扰-3)进行研究。
2)细胞培养相关试剂
DMEM培养基购自南京凯基生物科技有限公司；胎牛血清购自以色列BI公司；0.25％胰蛋白酶溶液购自美国Invitrogen公司；磷酸盐缓冲盐水(Phosphate bufferedsaline,PBS)购自coring有限公司；二甲基亚砜(Dimethyl suLfoxide,DMSO)购自美国Sigma公司。
3)慢病毒感染相关试剂
LV-sgRNA-IGF-IR(PCA00469，PCA00470，PCA00471)，阴性对照病毒Sg-RNA-CON244，LV-cas9-Puro(7768-1)，polybrene感染增强剂，Enis培养液等购自上海吉凯公司；嘌呤霉素购自北京索莱宝公司。
4)蛋白分析相关试剂
RIPA裂解液(强)，苯甲基磺酰氟(PMSF)，BCA蛋白浓度测定试剂盒，十二烷基磺酸钠-聚丙烯酰胺凝胶(SDS-PAGE)蛋白上样缓冲液等购自北京索莱宝公司；鼠抗人IGF-IR抗体、β-actin鼠抗人抗体及辣根过氧化物酶标记的羊抗鼠IgG抗体均购自美国Abcam公司；预染蛋白Marker购自美国Thermo公司旗下Fermentas公司；PVDF膜，Immobilon ECL发光液购自美国Millipore公司。
5)细胞增殖相关试剂
Cell Counting Kit-8assay(CCK-8)试剂盒购自日本同仁化学研究所。
6)其他试剂
免疫组化试剂盒，一抗稀释液购自丹麦Dako公司。甘氨酸、Tris购自美国Bio-Rad公司；甲醇、三氯醋酸和醋酸等均为国产分析纯以上级产品。
实施例1
(1)HepG2细胞株复苏
采用迅速融化的方式，将人肝癌HepG2细胞株的冻存管从-80℃冰箱取出后，立即放入37℃恒温水浴锅中快速震荡解冻，持续1min。在超净台先用乙醇消毒，后开启。用吸管将HepG2细胞悬液吸至放有九倍体积的完全培养液(简称完培，含有10％胎牛血清的DMEM)的离心管内，混匀后用离心机低速离心(1000rpm×5min)，弃去上清液，然后加入5mL完全培养液，并吹打混匀后吸入培养瓶中，置于37℃、5％CO2且饱和湿度的培养箱中培养，第二天观察细胞生长情况，并且更换培养液。
(2)HepG2细胞株培养
当细胞融合度大于80％时，弃去原培养液，用PBS洗涤两次，再加入2ml的胰蛋白酶消化液，将培养瓶放置显微镜下观察细胞形态变化，观察到细胞形态变圆、胞质回缩以及细胞间隙增大时弃去胰酶，加入完全培养基吹打重悬细胞，然后将吹打后的细胞悬液分装到三个培养瓶中，继续培养。
(3)HepG2细胞株冻存
遵循“慢冻速融”的原则，取生长状态良好的对数生长期细胞，于冻存前一天进行换液。常规消化收集细胞，加入冻存液(10％DMSO+20％胎牛血清+70％dulbecco'smodified eagle medium，DMEM)，并调整，最终冻存液中细胞浓度为5～10×106/mL，再分装到无菌的冻存管内。管壁标签上注明细胞的名称、代数和日期，后用封口膜封口。将封好的冻存管先置于4℃冰箱30min，再置于-20℃冰箱90min，最后置于-80℃冰箱长期保存。
(4)细胞计数和细胞活力检测
先用乙醇消毒计数板，然后常规消化收集细胞，并制备单细胞悬液，用枪头取吹打好的细胞悬液10μL滴入计数板上盖玻片一侧，待细胞悬液完全铺开后，在显微镜下计数。细胞计数的方法：用10×物镜观察计数板四个角的大方格中的细胞数，细胞数＝(4大格细胞数之和/4)×104/mL时，台盼蓝可穿透发生损伤或死亡的细胞的变性细胞膜，并且可以与解体的DNA结合使其着色，但是活细胞却能阻止台盼蓝进入细胞内，从而鉴别死细胞和活细胞。计算细胞活力＝活细胞总数/(活细胞总数+死细胞总数)×100％。当细胞活力达95％以上可以进行慢病毒感染实验。
实施例2
Crispr/cas9-sgRNA双载体慢病毒构建、感染及筛选
(1)针对IGF-IR基因的慢病毒构建
根据人IGF-IR序列(NCBI：NM000875)，以Crispr/cas9-sgRNA技术原理，设计3条sgRNA序列：
SgRNA-1：5’-TCAGTACGCCGTTTACGTCA-3；(干扰-1)
SgRNA-2：5’-TGTTTCCGAAATTTACCGCA-3’；(干扰-2)
SgRNA-3：5’-GGCTCTCTCCCCGTTGTTCC-3’；(干扰-3)
并构建质粒载体Lenti-CAS9-puro和Lenti-sgRNA-EGFP。
(2)目的细胞慢病毒感染与筛选
将未感染慢病毒的肝癌HepG2细胞接种于6孔板中达到70～80％融合度，在细胞贴壁后，在6孔板中分别加入1μg/mL、2μg/mL、2.5μg/mL、3μg/mL的嘌呤霉素(puromycin)药物，48h后观察细胞形态，来进行致死最低浓度的筛选，从而得出药物处理48h后HepG2细胞全部死亡的最低药物浓度。
用胰酶将对数生长期的HepG2细胞进行消化，加入完培制成细胞悬液。再将HepG2细胞悬液(细胞数约为5×104)接种于6孔板中，置于37℃5％CO2培养箱中培养，当细胞融合度达到30％时，进行换液，然后根据预实验所得的感染条件和细胞感染复数(multiplicityof infectio,MOI＝10)，用枪头吸入适宜量Cas9病毒，加入到6孔板中。12h后在显微镜下观察细胞状态：如果细胞状态良好，继续培养24h后更换培养基；如果细胞状态差，出现细胞毒性作用，则立即更换培养基。在48h后，加入嘌呤霉素药物筛选48h，从而得到表达Cas9稳定的混合克隆。立即观察筛选后的细胞状态，保证细胞状态良好。HepG2细胞感染cas9-puro病毒48h后加入嘌呤霉素进行筛选，嘌呤霉素经空细胞致死最低浓度筛选之后浓度定为2μg/ml。48h后在光学显微镜下观察，结果如图1，A：感染cas9病毒，B：加入嘌呤霉素48h后；C&D：对照组。A&B组感染cas9病毒的细胞经嘌呤霉素筛选后存活，C&D组未感染的细胞经嘌呤霉素筛选后死亡。
继续培养感染成功的细胞，常规消化后，制成细胞悬液，并且铺板，按照吉凯生物公司慢病毒感染细胞步骤感染表达sgRNA的慢病毒。3天后在荧光显微镜下观察慢病毒感染的细胞的荧光效率，荧光效率在80％左右的细胞开始进行后续实验。
收集存活细胞在扩大培养后行二次感染sgRNA-EGFP病毒，感染48h后在倒置荧光显微镜下观察，成功转染的细胞带绿色荧光(图2A&B)，随机选取五个视野细胞计算感染效率(n＝5)，结果分别为91％，90％，88％和86％(图3)。
实施例3
CCK-8法检测细胞增殖
按照CCK-8试剂盒提供的说明书操作步骤，设空白对照组，阴性对照组，实验组，取生长状态良好的对数生长期细胞，加入胰蛋白酶进行消化，制备成细胞悬液，再接种于96孔板(n＝3)，每孔约100μL，将96孔板置于培养箱中进行预培养(37℃，5％CO2的条件下)，于既定时间点取出并换液，每孔加入10μL的CCK-8试剂，继续在培养箱中孵育1-4h后在酶标仪检测A450值。
成功感染慢病毒的细胞收集后提取蛋白进行Western检测分析蛋白水平的基因敲除效率(图4，图5)，sgRNA2靶点对应的细胞IGF-IR蛋白表达量明显降低，与其余四组细胞相比差异具有统计学意义(*P<0.05)；半定量分析HepG2、sgRNA-neg、sgRNA1和sgRNA3的IGF-IR灰度强度比值分别为1.22±0.13，1.14±1.23，1.01±0.94，0.99±0.82，而sgRNA2比值为0.43±0.79。
实施例4
Transwell迁移检测细胞侵袭能力
第一步准备基质胶：将冻存的BD matrigel置于4度过夜，使其变为液态。
第二步取用24孔Bordend小室，孔径为8μm。用DMEM冲洗小室，再用50mg/LMartrigel 1：8稀释液包被Bordend小室底部膜的上室面，室温风干。
第三步分别加入200μL浓度为l×109/mL各组(空白组、阴性组、sgRNA-2组-干扰-2)的细胞悬液至于上室.上室液为含10g/L BSA无血清DMEM培养基。24孔板下室加入每孔500μL含10％胎牛血清完全培养液。
第四步置于培养箱中培养24h后取出小室.用吸管吸入PBS进行淋洗，再用棉签轻轻擦拭去微孔膜内层的细胞，加入95％酒精固定5min后用4g/L结晶紫染色。
第五步在倒置显微镜下计数移至微孔膜下层的细胞，随机计数五个视野/样本(n＝3)。
细胞侵袭试验显示，与阴性组(160±21)相比，sgRNA2感染组(50±12)穿膜细胞数明显减少，差异具有统计学意义(t＝4.682,P<0.01)，空白组(210±16)与阴性组(160±21)相比，未见明显差异(t＝2.082,P＝0.264),说明敲除IGF-IR能明显抑制肝癌细胞的侵袭能力(图8，图9)。
实施例5
Transwell迁移检测细胞横向迁移能力
划痕愈合：将空白对照组、阴性对照组、sgRNA-2组的细胞用胰酶消化后，制成细胞悬液，以每孔8×105个细胞接种于6孔板中；当细胞达到90％融合时，弃去培养基并用PBS洗涤3次，用枪头在培养板底部进行等宽划痕；用PBS冲洗净刮落细胞后，用吸管加入适量无血清培养基继续培养，48h时置于倒置显微镜下，观察细胞的迁移情况并拍照，采用Picpic图像分析软件测量划痕两侧细胞的迁移数目。每组设3个复孔(n＝3)。
采用划痕实验法测试敲除IGF-IR对细胞横向迁移能力的影响，经倒置相差显微镜计数(图10)，划痕24h后，C2细胞迁移数明显减少，P<0.01。显示C2:sgRNA2感染组细胞迁移数(292±28)与阴性组(564士15)间差异显著(t＝14.831,P<0.01),空白对照(580±14)与阴性对照组(564士15)间未见明显差异(t＝1.351,P＝0.102)。
实施例6
Transwell迁移检测细胞纵向迁移能力
胰酶消化三组(空白对照组、阴性对照组、sgRNA-2组)的细胞，每孔下室加入500μL10％胎牛血清的培养基，Transwell上室(上室滤膜无Matrigel包被)分别加入200μL浓度为l×109/mL各组(空白对照组、阴性对照组、sgRNA-2组)的细胞悬液.上室液为含10g/L BSA无血清培养基，置于培养箱中培养24h后取出小室。用吸管吸入PBS进行淋洗，再用棉签轻轻擦拭去微孔膜内层的细胞，加入95％酒精固定5min后用4g/L结晶紫染色，在倒置显微镜下计数移至微孔膜下层的细胞，随机计数五个视野/样本，并取平均值(n＝3)。
细胞迁移(图11，图12)显示，sgRNA2感染组的肝癌HepG2细胞接种上室24h后，穿膜细胞数均明显减少，肝癌HepG2细胞空白组、阴性对照组和sgRNA2感染组穿膜细胞数，分别为176±12、155±24和59±19个，感染组与阴性组相比，差异具有统计学意义(t＝5.432,P<0.01),空白组与阴性组相比，未见明显差异(t＝1.355,P＝1.006)，敲除IGF-IR能明显抑制HepG2细胞纵向迁移能力。
本发明首次以CRISPR/Cas9双载体慢病毒构建、成功转染肝癌细胞并筛选出有效序列。肝癌HepG2细胞中IGF-IR基因被CRISPR/Cas9-sgRNA靶向敲除后，癌细胞增殖受抑，以划痕愈合、迁移运动及侵袭试验证明肝癌细胞侵袭、迁移等生物学行为发生变化，提示IGF-IR基因可望成为肝癌基因治疗一个有效靶目标。
综上所述，临床上多数肝癌在确诊时已属中、晚期，已不能进行手术切除，放疗或化疗有其局限性，并且预后较差。HCC组织IGF-IR表达明显高于癌旁，癌组织IGF-IR表达与HCC患者生存期密切相关；肝细胞恶性转化过程中IGF-IR介导的信号转导通路异常激活，以酪氨酸激酶抑制剂、单克隆抗体、microRNA干扰靶向IGF-IR已成为肝癌治疗的热点。然而对IGF-IR通路作用机制认识有待深入研究，敲除IGF-IR可抑制癌细胞增殖、侵袭、迁移，但研发以IGF-IR为靶点既安全又有效的基础和临床试验，与手术、介入、化疗或放疗等相结合，将会为肝癌治疗提供有效新方法，具有临床应用前景。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
SEQUENCE LISTING
<110> 南通大学附属医院;南通大学
<120> 一种靶向IGF-IR基因的sgRNA及其应用
<130> GW2017I1303
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> RNA
<213> 人工序列
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tcagtacgcc gtttacgtca 20
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<212> RNA
<213> 人工序列
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tgtttccgaa atttaccgca 20
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<212> RNA
<213> 人工序列
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ggctctctcc ccgttgttcc 20

Claims (4)

1.一种靶向IGF-IR基因的sgRNA，其特征在于，所述sgRNA的核苷酸序列如SEQ ID NO:2所示。
2.编码权利要求1所述sgRNA的DNA。
3.权利要求1所述的sgRNA在特异识别和靶向修饰人肝癌细胞IGF-IR基因中的应用。
4.权利要求1所述的sgRNA在构建人肝癌细胞IGF-IR基因突变库中的应用。
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