CN114606323A - 一种识别胃癌干细胞的标志物lgsn作为胃癌诊断和治疗靶标的应用 - Google Patents
一种识别胃癌干细胞的标志物lgsn作为胃癌诊断和治疗靶标的应用 Download PDFInfo
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- CN114606323A CN114606323A CN202210435019.3A CN202210435019A CN114606323A CN 114606323 A CN114606323 A CN 114606323A CN 202210435019 A CN202210435019 A CN 202210435019A CN 114606323 A CN114606323 A CN 114606323A
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Abstract
本发明公开了一种识别胃癌干细胞的标志物LGSN作为胃癌诊断和治疗靶标的应用,涉及生物医药技术领域。发明人发现了一种新的胃癌干细胞标志物LGSN,其在胃癌组织和胃癌干细胞中均显著高表达,并且与胃癌病人不良预后生存时间有关。一方面,本发明提供一种检测LGSN基因和/或其编码的蛋白表达水平的试剂在制备胃癌的诊断试剂或诊断试剂盒中的应用。另一方面,本发明提供一种降低或抑制LGSN基因和/或其编码的蛋白表达的试剂在制备治疗胃癌的药物组合物中的应用。抑制LGSN表达能够触发焦亡,并导致胃癌干细胞生长抑制及体内成瘤,进而增加化疗药物敏感性,本发明提供了一个全新胃癌干细胞分子靶点治疗药物和化疗药物联用的胃癌治疗策略。
Description
技术领域
本发明涉及生物医药技术领域,尤其涉及一种识别胃癌干细胞的标志物LGSN作为胃癌诊断和治疗靶标的应用。
背景技术
胃癌是全球第五大常见癌症,是癌症相关死亡的第四大常见原因。大量研究指出异常组织驻留胃干细胞是胃癌的主要细胞来源。在胃癌发生过程中,长寿胃干细胞受幽门螺杆菌等造成的慢性炎症诱导所致的基因突变和表观遗传改变,胃干细胞壁龛的扩张,随后多能胃干细胞大量克隆扩增具有基因突变的细胞,最终导致侵袭性癌症。同时,非癌胃干细胞衍生成肿瘤中的侵袭性胃癌干细胞,且这种去分化可能在肿瘤发展过程中持续发生。因此对胃癌发生的深入了解需要详细阐明胃干细胞和胃癌干细胞的作用。
胃癌干细胞继续负责维持着肿瘤异质性,积极构建肿瘤微环境,促进肿瘤生长和治疗耐药性。胃癌干细胞可塑性极大程度造成了临床上胃癌标志物的选择限制,测量放化疗后患者残留胃癌干细胞的定位和自我更新状态并不理想。因此,补充更加特异准确的胃癌干细胞标志物,全面了解胃癌干细胞壁龛是开辟胃癌诊疗新道路的关键。
目前临床上主要使用氟尿嘧啶和铂类等细胞毒性化疗药物治疗中晚期胃癌,副作用大,没有明确的靶点,治疗后胃癌干细胞的持续存在,肿瘤常常出现复发、转移,严重影响了癌症患者的生存质量。最近的研究表明,在化疗后的肿瘤细胞亦可能通过响应来自肿瘤微环境的信号获得的干细胞特性。于是推断出,靶向诱导致肿瘤干细胞获得的胃癌干细胞信号可能比只靶向已存在的癌干细胞更有效。因此,探索胃癌干细胞克隆最脆弱时刻,顺势将其精准根除的分子靶向药物迫在眉睫。这对于填补我国完全自主知识产权的胃癌分子靶向药物的空白,减轻病患经济负担,具有重大民生意义。
LGSN(lens glutamine synthetase-like,基因名LGSN,Uniprot登录号Q5TDP6)为脊椎动物眼睛晶状体特异性表达蛋白,含谷氨酰胺合成酶(Glutamine Synthetase,GS)结构域,为谷氨酰胺合酶家族成员。LGSN是一种参与晶状体终末分化的连接酶。2009年来首次证实,LGSN的异常高表达与癌症有关。LGSN的一个新的剪切形式(splicing variant 4)在肺癌细胞系和肺癌组织中高表达,可能成为潜在新的肺癌特异性抗原,提示LGSN可能在肿瘤的发生发展中发挥作用。但目前LGSN在肿瘤中的作用和分子机制尚未清楚阐明。更重要的是,LGSN在胃癌发生发展过程中的表达模式和生物学功能尚不清楚。
发明内容
基于背景技术中提到的缺陷,本发明公开了LGSN用作识别胃癌干细胞的标志物,在胃癌诊断和治疗靶标上的应用。
本发明公开了一种新的胃癌干细胞干性维持基因LGSN。
在92例胃癌病人组织样本中发现,LGSN在胃癌干细 胞富集区的胃粘膜腺体峡部、颈部和基底胃上皮细胞中高表达,随着胃癌原发灶发展、淋巴结受累程度、远处转移进展和低分化程度,逐渐高表达,且与胃癌病人不良生存率显著正相关,因此可以作为胃癌干细胞标志物及胃癌诊断标记。
随后发现,LGSN在人胃癌干细胞中的表达随着胃癌干细胞的成球生长其表达逐渐增高,与维持胃癌干细胞自我更新能力和抑制其分化紧密相关。
进一步进行体内和体外的功能性研究分析证实,LGSN增加正常胃上皮细胞干性后,可异常增加其转移和对化疗药物耐药性,并呈现出与Vimentin互作的新的分子机制。惊喜的是,有效干扰胃癌干细胞中LGSN的表达,能够触发细胞焦亡并导致胃癌干细胞生长抑制及体内成瘤受阻,并极大促进了化疗药物敏感性,因此抑制LGSN表达为胃癌治疗的提供了一个新的有效策略。
综上所述,本研究发现LGSN是调控胃癌干细胞干性和生存的重要分子靶点,可以作为胃癌干细胞标志物及胃癌诊断标记的同时,更能用于制备抗胃癌干细胞及抗胃癌精准治疗的药物。
本发明的第一个目的在于提供LGSN基因和/或其编码的蛋白作为胃癌的患癌风险预警、辅助诊断和/或预后判断的敏感性检测新靶点的应用。
在一个实施方案中,本发明提供了检测LGSN基因和/或其编码的蛋白表达水平的试剂,其用于诊断胃癌和癌前风险。
优选的,所述LGSN与胃癌恶性程度、胃癌发生、发展、转移和预后密切相关。
优选的,所述LGSN在胃癌干细胞成球生长中持续表达,且随着胃癌干细胞的自我更新,其表达逐渐增高。
优选的,所述LGSN是调控肿瘤干细胞生存和生长的关键分子靶点。
优选的,所述LGSN的Ensembl登录号为ENSG00000146166。
优选的,所述LGSN的UniProtKB/Swiss- Prot登录号为Q5TDP6。
本发明的第二个目的在于提供LGSN基因和/或其编码的蛋白在制备杀伤胃癌干细胞的癌症治疗药物中的应用。
在一个实施方案中,本发明提供降低或抑制LGSN基因和/或其编码的蛋白表达的试剂,用作治疗胃癌的药物。降低或抑制LGSN表达的试剂的本质对于本发明来说并不重要,只要它降低或抑制LGSN的表达即可。
优选的,所述降低或抑制LGSN表达的试剂可选择以下试剂中的至少一种:gapmer、反义RNA、siRNA、esiRNA、shRNA、miRNA、RNA适体、TALEN、CRISPR和锌指核酸酶和锌指核酸酶以及LGSN蛋白的抑制剂。
优选的,所述shRNA序列包括SEQ ID NO.1。
优选的,所述药物组合物含有:降低或抑制LGSN基因和/或其编码的蛋白表达的试剂,以及医学上可接受的药用辅料。
所述药物还含有一种或多种药物赋形剂和/或药学上可接受的载体。
优选的,所述药物可以制成包括但不限于显微注射剂、适于转染的剂型、注射液、片剂、粉剂、粒剂、胶囊剂。上述各种剂型的药物均可以按照药学领域的常规方法制备。
优选的,所述药物可用于治疗胃干细胞驱动发生的胃癌。
优选的,所述药物具有减少肿瘤体积,抑制胃癌干细胞成球生长,促进肿瘤干细胞焦亡的作用。
优选的,所述LGSN调控Vimentin功能来调控肿瘤干细胞的干性和生存。
优选的,通过敲低LGSN和Vimentin处理肿瘤干细胞能减少细胞内EMT现象。
优选的,所述Vimentin的Ensembl登录号为ENSG00000026025。
优选的,所述Vimentin的UniProtKB/Swiss-Prot登录号为P08670。
本发明的第三个目的在于提供一种靶向LGSN的胃癌联合治疗的药物组合物。
优选的,所述胃癌联合治疗药物组合物包括LGSN抑制剂和另外的抗癌剂如化疗药,例如用于降低或抑制LGSN表达的试剂和5-氟脲嘧啶(5-FU)和奥沙利铂(L-OHP)联用疗法。
优选的,所述胃癌联合治疗药物组合物能够杀伤胃癌干细胞。
优选的,即使LGSN的抑制足以实现治疗胃癌的效果,但是预期将降低或抑制LGSN表达的试剂与其他抗癌药物如化疗剂组合时,可以获得更强甚至协同的抗癌效果。
本发明的有益效果在于:本发明首次公开了LGSN可以作为制备胃癌干细胞分子靶向治疗药物的靶点,有助于开发针对胃癌干细胞的靶向药物,且可以增加化疗药物5-FU和L-OHP联合治疗敏感性,从而为胃癌的精准诊断和治疗提供新的思路,同时也为降低LGSN驱动型胃癌的复发率奠定坚实的基础。
附图说明
图1显示临床胃癌组织LGSN表达量与恶性程度和不良预后正相关。(A)LGSN在晚期TNM分期胃癌肿瘤和癌旁胃黏膜腺体中高表达的典型免疫组化染色案例。图中,↑(箭头)表示胃黏膜腺体峡部;*(星形)表示胃黏膜腺体颈部和基底部。(B)不同TNM分期胃癌中LGSN阳性染色分数对比。(比例尺:100μm)(C)II-IV期患者LGSN表达上调的百分比比较(每个柱形图区间上的数字表示LGSN上调病例数百分比)(*P<0.05,与I阶段相比)。(D)LGSN的表达与低分化程度显着相关。(E)LGSN的表达与淋巴结转移显着相关。(F)LGSN表达情况与胃癌患者生存预后相关性分析。
图2显示LGSN表达与人胃癌干细胞干性维持正相关。(A)人胃癌干细胞随培养时间(第0、2、4和6天)成球逐渐增大的细胞形态图和(B)细胞大小统计结果。(比例尺:25μm)(C)免疫印迹检测显示,LGSN及干性相关蛋白SOX2、OCT4A和NANOG随着人胃癌干细胞的成球生长表达情况。(D)免疫印迹检测显示,用atRA(反式维甲酸)诱导分化后,LGSN及干性相关蛋白SOX2、OCT4A和NANOG的表达量随人胃癌干细胞分化程度相关性。
图3显示过表达LGSN促进正常胃上皮细胞GES-1干细胞样转化和上皮间质转化(EMT)。(A)免疫印迹检测显示,过表达LGSN后干性蛋白SOX2和NANOG,EMT转录因子Slug和钙粘蛋白N-cadherin表达情况。(B)流式细胞术检测细胞周期分布情况。CCK8检测GES-1过表达LGSN后细胞增殖情况(C),5-FU和L-OHP(D)的IC50。镜下观察成球能力(E)和迁移能力(F)变化情况。(比例尺:200μm)
图4显示LGSN表达被抑制触发焦亡,进而影响人胃癌干细胞干性与增殖。(A)免疫印迹检测显示,敲低LGSN表达后干性蛋白OCT4A表达情况。(B)敲低LGSN后,人胃癌干细胞随培养时间(第0、2、4和6天)成球能力逐渐丧失的细胞形态图和细胞球直径大小统计结果。(比例尺:50μm)(C)流式分析显示,敲低LGSN后,人胃癌干细胞的细胞膜联蛋白V/PI染色双阳细胞数变化情况。(D)镜下细胞形态学观察,冒泡的细胞明显增多。(比例尺:25μm)(E)免疫印迹检测显示焦亡标志物Caspase-1/P20和GSDMD-N表达情况。(F)裸鼠皮下成瘤结果显示敲低LGSN影响人胃癌干细胞体内致瘤能力。(G)人胃癌干细胞GCSC1的皮下移植瘤的肿瘤生长曲线。(比例尺:1cm)
图5显示敲低LGSN表达增加人胃癌干细胞的化疗敏感性。(A)人胃癌干细胞中沉默LGSN后的5-FU和L-OHP的IC50变化情况。(B-D)敲低LGSN表达与联合使用5-FU和L-OHP,流式分析显示导致人胃癌干细胞的细胞膜联蛋白V/PI染色双阳细胞数明显增多。(E)CCK8检测显示敲低LGSN表达后人胃癌干细胞的增殖速率变化情况。(F)免疫印迹检测显示焦亡标志物Caspase-1/P20和GSDMD-N表达情况。(G-H)裸鼠皮下移植瘤显示敲低LGSN与化疗药物联用的治疗效果。(比例尺:1cm)
图6显示LGSN与Vimentin结合维持人胃癌干细胞生存和发展。(A)通过免疫沉淀和质谱法从FLAG-LGSN过表达GES-1细胞中鉴定出Vimentin为LGSN相互作用蛋白。(B)用FLAG进行免疫共沉淀,结合免疫印迹检测证实LGSN和Vimentin存在相互作用。(C-D)免疫荧光染色观察到人胃癌干细胞中LGSN和Vimentin的存在共表达,FLAG-LGSN过表达GES-1细胞用抗LGSN(红色)/抗Vimentin(绿色)免疫标记抗体,细胞核用DAPI(蓝色)复染。(比例尺:50μm)(E)免疫印迹检测显示人胃癌干细胞中沉默LGSN后的Vimentin随之表达降低。(F-G)Transwell实验观察,分化后到人胃癌干细胞细胞再次过表达LGSN对沉默VIM后迁移能力的挽救作用。(J)CCK8检测显示过表达LGSN对沉默VIM后增殖能力的挽救作用。(H-I)Transwell实验观察,分化后到人胃癌干细胞细胞再次过表达VIM对沉默LGSN后迁移能力的挽救作用。(K)CCK8检测显示过表达VIM对沉默LGSN后增殖能力的挽救作用。(比例尺:200μm)
具体实施方式
下面将结合本发明实施例中的附图,对实施例中的技术方案进行清楚、完整地描述。显然,以下将描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
下列实施例中未注明具体条件的实验方法,通常按照本领域常规条件,如《分子克隆实验指南》(第三版)中所述的条件,或按照试剂产品说明书指导条件。所用的试剂均可通过商业购买得到。
以下实施例中所用到的原始试剂和材料均可商购得到。其中:
实验用到以下动物:免疫缺陷BALB/C 4-5周龄裸鼠(购自北京维通利华实验动物技术有限公司)。小鼠根据中国深圳大学教学和研究活体动物使用委员会批准的方案进行饲养和处理。
实验用到以下载体:pLKO.1-puro载体(NTC)(艾基公司);
pCDH-CMV-3xFLAG-EF1-puro载体(VEC-FLAG)(艾基公司);
实验涉及的核苷酸序列如下表:
实验步骤
细胞培养
GES-1细胞和293T细胞购自中科院生化细胞所细胞库,人胃癌干细胞(GCSCs)由四川大学华西医学院莫显明教授慷慨赠与。
GES-1细胞在添加有10%胎牛血清 (FBS)的DMEM(Gibco)中于37℃、5%CO2的条件下培养。使用的所有细胞系均不含支原体。
GCSCs培养按照其文献报道方法(Cell Research,2012,22(1):248-258)进行。
免疫组织化学染色
参照制造商说明书,采用Dako公司EnVision+detection system试剂盒,将组织芯片与识别LGSN(Sigma,HPA039983)的一抗孵育,检测免疫反应信号,最后使用Aperio扫描仪对载玻片成像,全片扫描结果交由两名未知病理数据的病理学专家独立在光镜下,完成染色强度和染色阳性率判读,判定标准参照文献(J.Clin.Pathol.,1995,48:876-878)。
成球实验
GCSCs球消化分散成单细胞后,计数每孔500个细胞,悬浮培养于超低吸附培养6孔板,无血清、添加生长因子的培养基中,约7天后,显微镜下观察细胞成球状态并计数,使用Image Pro Plus计算细胞球粒径。
免疫印迹
将待测细胞用RIPA裂解液处理获得蛋白上清,及时采用BCA法(Thermo FischerScientific)定量蛋白浓度,随后等量蛋白样品通过SDS-PAGE凝胶(分离胶浓度10%)电泳分离蛋白。经适当分离的蛋白转移到PVDF膜(Millipore)上,接着用5%脱脂牛奶于室温封闭1小时后,使用识别LGSN的抗体(Novus Biologicals,NBP2-85207)在摇床上4℃孵育过夜。次日,TBST缓冲液清洗蛋白转印膜3次,每次5分钟,再用相应的HRP标记二抗于室温孵育1小时。最后膜清洗完毕后,用ECL(SuperSignal West chemiluminescent substrates,Thermo Fisher Scientific)显影液,在ChemiDoc(Bio-Rad)系统上获取化学发光信号,并进行灰度半定量分析。本发明实施例所有抗体均列于表1中。
表1
细胞活力检测实验
实验操作按照细胞增殖CCK8试剂盒(同仁化学研究所)说明书进行。取各组对数生长期细胞(1×104个/mL,100μL)接种于96孔板,每组6个复孔,到达观察所需培养时间后,分别加入CCK8试剂,培养4小时后用酶标仪(Biotek)测量波长450nm的吸光值,绘制各组细胞生长曲线。
细胞迁移实验
将3×104个细胞分别接种于含无血清培养基的Transwell上室中(Corning),将上室置于含完全培养基的下室中,常规培养24小时后的所有实验操作严格按照试剂盒(Becton Dickinson Labware)说明书进行,计数进入下室的细胞量,此实验平行重复3次。
构建慢病毒感染稳转株
使用Lipofectamine 3000(Invitrogen,11668)和第三代慢病毒包装载体pSAX2和pMD2.G,目标基因表达(或敲除)载体和相应对照的质粒转入293T细胞,48小时及72小时后收集慢病毒上清,使用Lenti-X按照试剂说明书浓缩病毒(Clontech)。病毒沉淀经培养基重悬后,联合聚凝胺(2.5μg/ml)感染GCSCs,48小时后用嘌呤霉素(Sigma)筛选2周左右,得到稳定表达细胞株。最后通过免疫印迹检测目的蛋白的表达情况。
裸鼠皮下成瘤
将4×105个实验组细胞或对照组GCSCs分别注射到5只4周龄左右无胸腺裸鼠(BALB/c裸鼠,雄性,北京维通利华)的两侧肋皮下,培养于无菌环境。待肿瘤大小肉眼可见时,密切观察皮下肿瘤生长情况,每隔2天用游标卡尺测量肿瘤的长度和宽度,按公式:V=1/2长径×短径2,计算肿瘤体积,并绘制皮下移植瘤生长曲线。安乐死荷瘤小鼠时,取瘤体拍照并称重。
细胞凋亡检测
用Accutase消化GCSCs,制成单细胞悬液,离心弃上清,用预冷PBS清洗两次,分别加入5μL AnnexinV+5μL PI+95μL AnnexinV Bind Buffer混合物(含钙离子的缓冲液)(BDBiosciences)重悬细胞,室温避光避光15min。上机(Beckman)使用CytoFLEX系统检测签向每个管中加入200μL的AnnexinV BindBuffer混悬并过滤。
细胞周期检测
收集单细胞沉淀,加入1毫升冰浴预冷70%乙醇,轻轻吹打混匀,4℃固定一夜。随后使用Cycletest Plus DNA试剂试剂盒(BD Biosciences),将细胞与500μL的碘化丙啶染色溶液,37℃避光温育30分钟,随即通过流式细胞仪检测细胞分布,并进行进行细胞DNA含量分析。
免疫共沉淀
收集GCSCs,加入适量体积预冷的PierceTM IP裂解缓冲液(Thermo FisherScientific)提取细胞全蛋白,BCA法测定总蛋白浓度,并按照每500μL样品20μL,孵育,加入anti-FLAG(Proteintech,66008)抗体或正常IgG(Santa Cruz Biotechnology,sc-2025)工作液,置于翻转混合仪上4℃孵育过夜,缓冲液冲洗,磁力架分离,去除上清,洗脱互作蛋白进行后续的质谱分析鉴定,或进行免疫印迹分析。
免疫荧光检测
细胞接种至多聚赖氨酸预包被的细胞爬片上,4%多聚甲醛固定,0.2%Triton X-100透化,5%BSA封闭,加入anti-LGSN(Sigma,HPA039983),anti-Vimentin(Proteintech)一抗,4℃过夜,PBST清洗,加入分别标记Alexa Fluor 647(Abcam,ab150075)和AlexaFluor488的二抗(Abcam,ab150113),在室温下孵育1小时,含DAPI的抗淬灭剂(ThermoFisher Scientific)封片,置荧光系统(Biotek)下观察拍照。
统计分析
本发明中实验结果以均值±标准差(x±s)表示,两组均数比较采用t检验,多组均数比较采用单因素方差分析作为显著性统计分析,生存数据的统计比较采用Kaplan-Meier生存分析法和log-rank统计检验进行生存期单因素分析,当P<0.05时,认为具有统计学差异,用“*”表示。*表示有统计学差异(p<0.05);**表示有显著差异(p<0.01);***表示有明显显著差异(p<0.001);****表示有极为显著差异(p<0.0001)。
实施例1LGSN在临床胃癌组织中的表达水平具有临床意义
选取包含92例临床胃腺癌肿瘤组织样本的组织芯片(上海芯超生物科技有限公司)。分析LGSN表达与胃癌患者临床病理学参数(包括性别、年龄、肿瘤大小、肿瘤位置、肿瘤分化、肿瘤分型、肿瘤侵润深度、淋巴结转移状态、远处转移状态、TNM分期等)之间的关系。
结果发现,LGSN在胃癌和癌旁胃黏膜腺体峡部、颈部和基底中胃癌干细胞富集区高表达(图1A),且表达量随着TNM分期进展逐渐增多(图1B)。结合临床病理学参数进一步分析发现,与I期患者相比,II-IV期患者LGSN上调的患者数量显著增加(图1C),LGSN的表达与胃癌的低分化程度(图1D)、淋巴结转移(图1E)显著正相关,与LGSN低表达的患者(n=47)相比,LGSN高表达的患者(n=45)的总体生存率预后更差(图1F)。
上述结果说明,LGSN可能与胃癌恶性程度,胃癌发生、发展、转移和预后密切相关,有望成为胃癌治疗的靶点和临床诊断应用。
实施例2LGSN参与维持胃癌干细胞自然成球过程
我们跟踪观察了GCSCs成球第0天、第2天、第4天和第6天的细胞球形态(图2A),细胞球径大小从第4天起呈对数增长趋势(图2B)。免疫印迹分析结果显示,LGSN以及干性相关蛋白SOX2、OCT4A和NANOG的表达量随着GCSCs的成球生长而逐渐升高(图2C),表明LGSN在GCSCs自然成球过程中持续表达升高,也说明持续升高的LGSN与GCSCs干性维持、自我更新能力呈正相关。利用促分化剂atRA处理GCSCs,免疫印迹分析表明atRA通过增加CK18(分化标志物)和降低SOX2、OCT4A和NANOG的表达,以有效诱导GCSCs的分化后,LGSN也表达降低,说明LGSN与GCSCs分化能力呈负相关(图2D)。
实施例3LGSN异常高表达促正常胃上皮去分化
将LGSN的过表达质粒转染进入人正常胃上皮细胞GES-1中,免疫印迹分析结果显示,干性蛋白SOX2和NANOG表达随LGSN的过表达而明显升高,同时促EMT转录因子Slug和钙粘蛋白N-cadherin表达显著上调(图3A),从而促进细胞周期从G0/G1期快速进展到M期(图3B),增加细胞增殖率(图3C)。同时,异常高表达的LGSN明显提高GES-1对5-FU和L-OHP(图3D)的耐受能力,其成球干细胞样生长能力(图3E)和迁移能力(图3F)明显增强。说明,LGSN过表达一定程度上促进了正常胃上皮去分化,促使其重新异常获得自我更新能力、过度增殖、耐药能力和迁移能力。
实施例4干扰LGSN表达诱发胃癌干细胞焦亡
我们使用表达LGSN shRNA的慢病毒感染GCSCs,有效敲低LGSN表达后,干性蛋白SOX2表达降低(图4A)。细胞表型上来看,相对于对照细胞的自然成球,LGSN敲低导致GCSCs始终呈单细胞,无法生长成球,生存率显著降低(图4B)。流式分析表明,LGSN敲低后AnnexinV-FITC/PI双阳性细胞显著增加(图4C),细胞球解散,细胞膜肿胀,多处频现泡泡形态(图4D),焦亡过程标志物,切割活性蛋白Caspase 1/P20和细胞膜孔开放蛋白标志物GSDMD-N表达显著增加(图4E),证实LGSN的敲低能极为显著诱导GCSCs焦亡。裸鼠皮下移植瘤结果表明,显示LGSN的敲低极大减弱了GCSCs的体内成瘤能力(图4F),移植瘤生长曲线明显减缓(图4G)。
上述结果表明,本发明构建的LGSN shRNA质粒能有效地抑制LGSN表达;且发现LGSN对GCSCs的生存具有至关重要的作用,是调控GCSCs抵抗焦亡的关键分子,靶向LGSN抗癌效果显著。
实施例5靶向LGSN提高胃癌干细胞对于化疗药物的敏感性
由于LGSN的沉默可以降低GCSCs对于化疗药物的抗性,因此靶向LGSN在提高GCSCs对于化疗药物的敏感性的方面有很大的意义。
1、LGSN的沉默增加胃癌干细胞细胞株化疗焦亡率。
细胞活力检测实验结果显示,LGSN的沉默有效降低了两株GCSCs对于化疗药物5-FU和L-OHP的半数抑制浓度(IC50)(图5A)。
流式分析表明,较之于单独敲低LGSN或单独化疗处理组,LGSN敲低后的化疗组,Annexin V-FITC/PI双阳性细胞显著增加(图5B-D),细胞活力实验结果显示,LGSN的沉默与化疗联用极大阻碍了GCSCs的生长(图5E)。将化疗药物作用后的LGSN的沉默GCSCs进行免疫印迹检测发现,较之于单独敲低LGSN或单独化疗处理组,焦亡过程标志物,切割活性蛋白Caspase 1/P20和细胞膜孔开放蛋白标志物GSDMD-N显著增加(图5F),我们推断,靶向LGSN显著提高了GCSCs对于化疗药物的敏感性,从而阻碍细胞生存,与大范围触发的细胞焦亡有关。
2、利用裸鼠皮下移植瘤模型评价靶向LGSN增效化疗抗肿瘤效果。
为了进一步检测靶向LGSN对胃癌的抑制作用,我们通过对裸鼠进行皮下胃癌干细胞接种,更好地模拟体内GCSCs抵抗化疗而复发的过程。
建立皮下移植瘤10天待其成瘤后开始相关处理,其中化疗处理组尾静脉注射5-FU(10mg/kg/mouse)(MedChemexpress)和L-OHP(5mg/kg/mouse)(MedChemexpress)对照组尾静脉注射生理盐水,每3天注射一次,治疗15天,密切观察皮下肿瘤生长情况。结果再次验证了,只敲低LGSN会降低肿瘤的生长速度,而单独化疗肿瘤效果不明显,将化疗药物联合靶向LGSN最终显著了抑制肿瘤的生长(图5G-H)。
实施例6LGSN与Vimentin相互作用维持胃癌干细胞生存及特性
在正常胃上皮细胞中将LGSN过表达,经过LGSN融合标签FLAG免疫沉淀下LGSN和与之结合的天然蛋白复合体(图6A),并质谱分析鉴定出Vimentin(波形纤维蛋白)为LGSN的重要互作蛋白之一,并通过免疫共沉淀和免疫印迹分析(图6B),及免疫荧光检测证实LGSN与Vimentin于细胞内共表达与定位情况,且随着LGSN表达增多,LGSN与Vimentin共定位信号增强(图6C-D),两者存在显著增强的相互作用。
另一方面,Vimentin随着LGSN的敲低而同比降低表达(图6E),且敲低LGSN导致分化后的GCSCs迁移率下降,而Vimentin在其中重新表达能够部分恢复GCSCs的迁移能力(图6F-G),从而有效地挽救了LGSN敲除诱导的生存抑制作用(图6J)。同时,LGSN对Vimentin的缺失对GCSCs迁移和生长能力抑制也有显著恢复作用(图6H-K)。
上述结果表明,LGSN通过调控Vimentin功能来调控GCSCs的干性和生存。因此,LGSN蛋白有望单独或与Vimentin蛋白联合作为制备肿瘤干细胞分子治疗药物靶点,用于相关药物的开发。
在上述实施例中,对各个实施例的描述都各有侧重,某个实施例中没有详细描述的部分,可以参见其他实施例的相关描述。
以上所述,为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到各种等效的修改或替换,这些修改或替换都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以权利要求的保护范围为准。
序列表
<110> 深圳大学
<120> 一种识别胃癌干细胞的标志物LGSN作为胃癌诊断和治疗靶标的应用
<130> 1
<141> 2022-04-24
<160> 4
<170> SIPOSequenceListing 1.0
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aacccttact tggtgctggc tgcaactgtt gctgccggct tagatggact tcatagcagt 1380
aatgaggtct tggctggtcc agatgagagc acagactttt accaagtgga accttctgag 1440
atccctttaa aactagaaga tgcccttgtg gcactggaag aagatcaatg cctgagacag 1500
gctctaggag aaacctttat tcgatatttt gttgccatga agaaatatga gttggagaat 1560
gaagaaatag ctgcagagag aaataaattc ttagagtatt ttatttaggg atcc 1614
<210> 3
<211> 62
<212> DNA
<213> Homo sapiens
<400> 3
ccggggcacg tcttgacctt gaacgctcga gcgttcaagg tcaagacgtg cctttttgaa 60
tt 62
<210> 4
<211> 1485
<212> DNA
<213> Homo sapiens
<400> 4
gaattcgcca ccatggacta caaagaccat gacggtgatt ataaagatca tgacatcgat 60
tacaaggatg acgatgacaa gtccaccagg tccgtgtcct cgtcctccta ccgcaggatg 120
ttcggcggcc cgggcaccgc gagccggccg agctccagcc ggagctacgt gactacgtcc 180
acccgcacct acagcctggg cagcgcgctg cgccccagca ccagccgcag cctctacgcc 240
tcgtccccgg gcggcgtgta tgccacgcgc tcctctgccg tgcgcctgcg gagcagcgtg 300
cccggggtgc ggctcctgca ggactcggtg gacttctcgc tggccgacgc catcaacacc 360
gagttcaaga acacccgcac caacgagaag gtggagctgc aggagctgaa tgaccgcttc 420
gccaactaca tcgacaaggt gcgcttcctg gagcagcaga ataagatcct gctggccgag 480
ctcgagcagc tcaagggcca aggcaagtcg cgcctggggg acctctacga ggaggagatg 540
cgggagctgc gccggcaggt ggaccagcta accaacgaca aagcccgcgt cgaggtggag 600
cgcgacaacc tggccgagga catcatgcgc ctccgggaga aattgcagga ggagatgctt 660
cagagagagg aagccgaaaa caccctgcaa tctttcagac aggatgttga caatgcgtct 720
ctggcacgtc ttgaccttga acgcaaagtg gaatctttgc aagaagagat tgcctttttg 780
aagaaactcc acgaagagga aatccaggag ctgcaggctc agattcagga acagcatgtc 840
caaatcgatg tggatgtttc caagcctgac ctcacggctg ccctgcgtga cgtacgtcag 900
caatatgaaa gtgtggctgc caagaacctg caggaggcag aagaatggta caaatccaag 960
tttgctgacc tctctgaggc tgccaaccgg aacaatgacg ccctgcgcca ggcaaagcag 1020
gagtccactg agtaccggag acaggtgcag tccctcacct gtgaagtgga tgcccttaaa 1080
ggaaccaatg agtccctgga acgccagatg cgtgaaatgg aagagaactt tgccgttgaa 1140
gctgctaact accaagacac tattggccgc ctgcaggatg agattcagaa tatgaaggag 1200
gaaatggctc gtcaccttcg tgaataccaa gacctgctca atgttaagat ggcccttgac 1260
attgagattg ccacctacag gaagctgctg gaaggcgagg agagcaggat ttctctgcct 1320
cttccaaact tttcctccct gaacctgagg gaaactaatc tggattcact ccctctggtt 1380
gatacccact caaaaaggac acttctgatt aagacggttg aaactagaga tggacaggtt 1440
atcaacgaaa cttctcagca tcacgatgac cttgaataag gatcc 1485
Claims (10)
1.一种检测LGSN基因和/或其编码的蛋白表达水平的试剂在制备胃癌的诊断试剂或诊断试剂盒中的应用。
2.根据权利1所述的应用,其特征在于,所述试剂为:特异性扩增LGSN基因DNA链和/或其cDNA链的PCR引物或LGSN特异性抗体。
3.根据权利要求1或2所述的应用,其特征在于,所述LGSN的Ensembl登录号为ENSG00000146166。
4.根据权利要求1或2所述的应用,其特征在于,所述LGSN的UniProtKB/Swiss-Prot登录号为Q5TDP6。
5.一种降低或抑制LGSN基因和/或其编码的蛋白表达的试剂在制备治疗胃癌的药物组合物中的应用。
6.根据权利要求5所述的应用,其特征在于,所述药物组合物含有:降低或抑制LGSN基因和/或其编码的蛋白表达的试剂,以及医学上可接受的药用辅料。
7.根据权利要求5所述的应用,其特征在于,所述降低或抑制LGSN和/或其编码的蛋白表达的试剂选自以下试剂中的至少一种:gapmer、反义RNA、siRNA、esiRNA、shRNA、miRNA、RNA适体、TALEN、CRISPR和锌指核酸酶和锌指核酸酶以及LGSN蛋白的抑制剂。
8.根据权利要求5所述的应用,其特征在于,所述降低或抑制LGSN基因表达的shRNA,序列如SEQ ID NO.1。
9.一种靶向LGSN的胃癌联合治疗的药物组合物,其特征在于,包括降低或抑制LGSN表达的试剂或化疗药物、抑制LGSN与Vimentin结合的试剂或化疗药物。
10.根据权利要求9所述的药物组合物,其特征在于,所述药物组合还包括医学上可接受的其他抗癌剂。
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---|---|---|---|---|
CN117805381A (zh) * | 2024-02-29 | 2024-04-02 | 首都医科大学附属北京积水潭医院 | 检测Gasdermin蛋白家族的物质在制备检测骨肉瘤的产品中的应用 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060275313A1 (en) * | 2002-11-29 | 2006-12-07 | Clark Georgina J | Therapeutic and diagnostic agents |
CN101448938A (zh) * | 2006-03-28 | 2009-06-03 | 佐藤升志 | 新肿瘤抗原肽 |
US20180126003A1 (en) * | 2016-05-04 | 2018-05-10 | Curevac Ag | New targets for rna therapeutics |
CN109529018A (zh) * | 2019-01-10 | 2019-03-29 | 南方医科大学南方医院 | Atxn2l用于制备逆转胃癌奥沙利铂耐药的药物的用途 |
CN111107866A (zh) * | 2017-06-12 | 2020-05-05 | 黑曜石疗法公司 | 用于免疫疗法的pde5组合物和方法 |
CN112410429A (zh) * | 2020-11-26 | 2021-02-26 | 四川大学华西医院 | Fxyd3作为胃癌诊断标志物和治疗靶标的应用 |
-
2022
- 2022-04-24 CN CN202210435019.3A patent/CN114606323B/zh active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060275313A1 (en) * | 2002-11-29 | 2006-12-07 | Clark Georgina J | Therapeutic and diagnostic agents |
CN101448938A (zh) * | 2006-03-28 | 2009-06-03 | 佐藤升志 | 新肿瘤抗原肽 |
US20180126003A1 (en) * | 2016-05-04 | 2018-05-10 | Curevac Ag | New targets for rna therapeutics |
CN111107866A (zh) * | 2017-06-12 | 2020-05-05 | 黑曜石疗法公司 | 用于免疫疗法的pde5组合物和方法 |
CN109529018A (zh) * | 2019-01-10 | 2019-03-29 | 南方医科大学南方医院 | Atxn2l用于制备逆转胃癌奥沙利铂耐药的药物的用途 |
CN112410429A (zh) * | 2020-11-26 | 2021-02-26 | 四川大学华西医院 | Fxyd3作为胃癌诊断标志物和治疗靶标的应用 |
Non-Patent Citations (3)
Title |
---|
GRAEME WISTOW等: "Expressed sequence tag analysis of adult human lens for the NEIBank Project: over 2000 non-redundant transcripts, novel genes and splice variants", pages 171 - 184 * |
MUNEHIDE NAKATSUGAWA等: "SOX2 is overexpressed in stem-like cells of human lung adenocarcinoma and augments the tumorigenicity", vol. 91, pages 1796 - 1804 * |
朱志强等: "shRNA-CXCR4对人胃癌BGC823细胞增殖迁移的影响", no. 11, pages 1899 - 1902 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117805381A (zh) * | 2024-02-29 | 2024-04-02 | 首都医科大学附属北京积水潭医院 | 检测Gasdermin蛋白家族的物质在制备检测骨肉瘤的产品中的应用 |
CN117805381B (zh) * | 2024-02-29 | 2024-05-28 | 首都医科大学附属北京积水潭医院 | 检测Gasdermin蛋白家族的物质在制备检测骨肉瘤的产品中的应用 |
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