CN114854747B - Kiaa1467基因的用途 - Google Patents
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- CN114854747B CN114854747B CN202210440857.XA CN202210440857A CN114854747B CN 114854747 B CN114854747 B CN 114854747B CN 202210440857 A CN202210440857 A CN 202210440857A CN 114854747 B CN114854747 B CN 114854747B
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Abstract
本发明公开了一种KIAA1467基因的用途,属于生物医药技术领域。本发明应用KIAA1467基因的特异性shRNA序列高效抑制KIAA1467基因在人乳腺癌细胞株的表达,shRNA的核苷酸序列如SEQ ID NO.1所示,经CCK8法、EDU实验、克隆形成和Transwell实验,证实KIAA1467基因表达下调能抑制乳腺癌细胞增殖、迁移以及侵袭能力。可见,KIAA1467基因在制备用于治疗乳腺癌药物、用于癌症诊断或预后判断的试剂盒中的具有广泛的应用。
Description
技术领域
本发明属于生物医药技术领域,涉及癌症精准医疗药物技术领域,更具体地说,涉及KIAA1467基因在制备防治乳腺癌药物、诊断及预后评估试剂中的应用。
背景技术
乳腺癌是国内外女性最常见的恶性肿瘤之一。在2020年,新发病例数为226万例,成为全球新发病例数最多的癌症。近年来,虽然在乳腺癌的临床诊断和治疗方面取得了较大的成就,但晚期乳腺癌患者的总体生存率仍然不理想。据目前统计,每年仍有60多万人死于乳腺癌。作为一种具有复杂遗传和多样生化背景的疾病,乳腺癌发生的确切机制至今还未研究清楚,但是,目前的研究证明,乳腺癌细胞具有高异质性及其较强的转移能力是改善预后的主要障碍。因此探索乳腺癌的发生发展机制、寻找新的敏感且特异的分子治疗靶点对乳腺癌诊断及治疗至关重要。
KIAA1467属于KIAA家族,KIAA基因由Kazusa cDNA测序项目鉴定。该项目对直接合成大蛋白(>50kDa)的人类大cDNA序列(>4kb)进行了鉴定,并保存在HUGE数据库(人类未鉴定基因编码大蛋白数据库),这些基因用KIAA加4个阿拉伯数字来命名。由于KIAA基因的筛选和鉴定主要是基于编码大蛋白(>1000个氨基酸残基)的mRNA(>4kb),因此KIAA蛋白具有多种生物学功能,并参与了重要的生命活动。一些KIAA蛋白被认为在多种生物过程中起着关键作用,如DNA损伤、中心粒形成、细胞迁移和侵袭。近年来,KIAA基因在癌症中的潜在价值开始被认识和研究,它作为肿瘤发生过程中各种生物学事件的靶点引起了人们的广泛关注。越来越多的证据表明,KIAA基因的异常表达与多种癌症的发生和预后有关,如胃癌、食管鳞状细胞癌、结肠直肠癌、肺癌和乳腺癌。有研究显示KIAA1324抑制胃癌细胞的侵袭性、生长和致瘤性,促进细胞凋亡,同时KIAA1324低表达与胃癌患者不良预后相关。KIAA1377在伴有淋巴结转移的食管鳞癌中明显扩增。KIAA1199已被确定为包括乳腺癌在内的许多癌症的致癌基因,并与肿瘤侵袭深度、TNM分期和预后不良相关。KIAA1467(也被称为FAM234B)作为KIAA家族的一部分,其具体功能目前尚未得知。有研究表明KIAA1467主要在脑组织中表达,它在儿童白血病复发中也有过报道,是儿童高二倍体急性淋巴细胞白血病下一代测序中的异位基因之一。最新生物信息学分析表明KIAA在乳腺癌中高表达且很大程度上可能与腔面型乳腺癌的预后有关联,不过当前阶段KIAA与与乳腺癌的关系还不明确,其差异对乳腺癌细胞生物学行为的影响及其背后机制尚不清楚。
发明内容
针对现有技术存在的上述问题,本发明所要解决的技术问题在于提供KIAA1467基因在制备防治乳腺癌药物、诊断及预后评估试剂中的应用。
为了解决上述技术问题,本发明所采用的技术方案如下:
KIAA1467基因的抑制剂在制备防治乳腺癌药物中的应用,该抑制剂抑制KIAA1467基因的表达或翻译,可选自但不限于:核酸分子、碳水化合物、脂类、小分子化学药、抗体药、多肽、蛋白或干扰慢病毒。
所述核酸包括但不限于:反义寡核苷酸、双链RNA(dsRNA)、核酶、核糖核酸内切酶III制备的小干扰RNA(esiRNA)或者短发夹RNA(shRNA)。
本申请中抑制剂为KIAA1467基因的shRNA,其核苷酸序列为:5′-GCTCCATTGTTTGGAGTTACC-3′(SEQ ID NO.1)。
KIAA1467基因的shRNA经酶切后可转为siRNA,进而起到特异性沉默乳腺癌细胞中KIAA1467基因表达的作用。
检测KIAA1467基因的试剂在制备用于诊断KIAA1467表达异常的乳腺癌前期产品中的应用,所述试剂为检测KIAA1467的mRNA或蛋白表达水平的试剂。
检测KIAA1467基因的试剂在制备用于乳腺癌预后判断的产品中的应用,所述试剂为检测KIAA1467的mRNA或蛋白表达水平的试剂。
进一步的,所述产品包括芯片、制剂或试剂盒。
进一步的,所述乳腺癌的细胞为MBA-MD-231细胞和HCC-1937细胞。
相比于现有技术,本发明的有益效果为:
本发明应用KIAA1467基因的特异性shRNA序列高效抑制KIAA1467基因在人乳腺癌细胞株的表达,经CCK8法、EDU实验、克隆形成和Transwell实验,证实KIAA1467基因表达下调能抑制乳腺癌细胞增殖、迁移以及侵袭能力。可见,KIAA1467基因在制备用于治疗乳腺癌药物、用于癌症诊断或预后判断的试剂盒中的具有广泛的应用。
附图说明
图1是Western Blot检测KIAA1467的蛋白质在6对新鲜乳腺癌和癌旁组织中的表达水平;图中,T为癌组织,N为癌旁组织;A为6对乳腺癌和癌旁组织,提取蛋白后采用WB法检测;B为重复WB三次,将结果量化成为柱状图;***P<0.001,****P<0.0001;
图2是IHC检测KIAA1467的蛋白质在乳腺癌及良性组织中的表达图;图中,A,a为KIAA1467蛋白在乳腺癌组织中,癌细胞细胞核及胞浆表达为强阳性;B,b为KIAA1467蛋白在乳腺癌中,癌细胞表达为核阳性;C,c为KIAA1467蛋白在乳腺腺病中,少量腺体细胞表达核阳性;D,d为KIAA1467蛋白在乳腺导管上皮增生中,导管上皮细胞表达阴性;A,B,C,D为40倍,bar=100μm,a,b,c,d为400倍,bar=10μm;
图3是KIAA1467的表达和TNM分期与乳腺癌患者预后的关系图;图中,A为具有高TNM分期(III期)的患者的总生存率低于具有低TNM分期的患者;B为KIAA1467高表达组的患者总体存活率低于KIAA1467低表达组的患者;C为在GEPIA数据库中发现KIAA1467的高表达是乳腺癌的不良预后,危险比(HR)=1.5,P=0.023,n=1069,Y轴为乳腺癌患者生存率,KIAA1467高表达组患者预后显著低于低表达组;
图4是KIAA1467在转染和未转染乳腺癌细胞系中的表达情况图;图中,A为在乳腺上皮细胞MCF-10A和乳腺癌细胞MCF-7,HCC-1937,MDA-MB-231中KIAA1467的蛋白表达的WB结果及量化图,*P<0.05;B为在乳腺癌细胞MDA-MB-231中对照组、成功转染KIAA1467-sh-NCRNA组、成功转染KIAA1467-shRNA组中KIAA1467的表达的WB结果及量化图,*P<0.05;C为在乳腺癌细胞HCC-1937中对照组、成功转染KIAA1467-sh-NCRNA组、成功转染KIAA1467-shRNA组中KIAA1467的表达的WB结果及量化图,*P<0.05;
图5是CCK-8实验在相应的时间点检测转染KIAA1467-shRNA和转染KIAA1467-sh-NCRNA的乳腺癌细胞的增殖指数图;
图6是克隆形成分析转染KIAA1467-shRNA和转染KIAA1467-sh-NCRNA两组中乳腺癌细胞的克隆数目图;
图7是EDU实验检测KIAA1467对乳腺癌细胞增殖能力的影响图;图中,随机选择3个显微镜视野,并对其进行计数定量分析,检测KIAA1467-shRNA组和转染KIAA1467-sh-NCRNA组细胞的增值率,bar=100μm,*P<0.05,**P<0.01:
图8是KIAA1467表达差异对乳腺癌细胞迁移侵袭能力的影响图;图中,A为转染KIAA1467干扰载体后MDA-MB-231的迁移和侵袭能力检测及对应量化图,bar=100μm;B为转染KIAA1467干扰载体后HCC-1937的迁移和侵袭能力检测及对应量化图,bar=100μm,*P<0.05,**P<0.01。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。
以下实施例中使用的主要试剂为:兔抗人KIAA1467单克隆抗体(biorbyt公司);DAB染色增强液试剂盒(福州迈新生物技术开发有限公司0Kit-0015);GAPDH(Proteintech公司60004-1-lg);SDS-PAGE凝胶快速制备试剂盒(白鲨生物科技公司,BL522A);PVDF膜(Amersham Biosciences公司);RIPA、PMSF(中国上海,碧云天生物技术公司);DMEM基础培养基(BI公司,06-1055-57-1ACS);RPMI1640基础培养基(BI公司,01-100-1ACS);血清Certified Foetal Bovine Semm特级胎牛血清(南京福麦斯生物技术有限公司,04-001-1ACS);胰酶(GIBCO公司,25200-56);无血清细胞冻存液(新赛美生物科技有限公司,20200903);MEGM kit基础培养基(CC-3150);cholera toxin(sigma c8052);大豆胰蛋白酶抑制剂(Invitrogen 17075-029);CCK-8试剂盒(东仁化学科技有限公司);6、24、96孔板(美国Corning公司)等。
以下实施例中使用的主要仪器如下:-80℃超低温冰箱:Thermo SCIENTIFIC公司;倒置相差显微镜:Leica公司;纯水仪:Millipore公司;图像分析系统:Alpha innotechcorp CA公司;自动高压蒸汽消毒柜:日本TOMY公司;低温冷冻离心机:Beckman公司;CO2培养箱:Thermo公司;电泳仪:Bio-Rad公司;医用净化工作台YJ-875:苏州净化设备厂;紫外分光光度计:IimPlen公司;制冰机:SANYO公司。
以下实施例中所使用的乳腺癌和人正常乳腺上皮细胞株:MDA-MB-231,MCF-7,HCC-1937,MCF-10A其来源均为上海中科院细胞库。
实施例1
乳腺癌组织芯片由南通大学附属医院生物样本库制作,乳腺癌组织有143例和良性组织有78例。78例良性组织中包含64例癌旁组织和14例良性肿瘤病例。这些组织样本均是由福尔马林固定,石蜡包埋,并根据最新的WHO诊断标准分级。所有的病例均是由两名病理学专家进行病理组织学确定,患者术前没有接受过免疫治疗、化疗或放疗,临床病例资料详细完整。
一、蛋白免疫印迹法检测标本:
6对新鲜乳腺癌组织和癌旁组织取自通大附院甲乳外科,上述组织手术离体后立即取下后迅速置于液氮内,保存在液氮中。
1、组织蛋白提取:
(1)从液氮中取出组织,后续转移到冰上复温,组织剪碎后,称定100mg,将其添加到1.5ml EP管中,然后添加蛋白裂解液(RIPA:PMSF=100:1)1ml;
(2)用组织研磨仪器将组织研磨;
(3)研磨结束后转移到新的EP管中,4℃离心15min,转速为12000rpm;
(4)留取上清400μl,检测完蛋白浓度后加5×上样缓冲液100μl,将EP管插入金属浴锅中95℃煮10min,存入-80℃冰箱。
2、Western Blot
SDS-PAGE凝胶电泳:
(1)将玻璃板洗净按装置要求组装,加满双蒸水验漏约15min,若液面未下降倒掉双蒸水,用纸吸干残留水分;配10%的分离胶,将混和均匀的分离胶快速加入玻璃平板中,到合适的高度后,将异丙醇加入到分离胶上压平,室温条件下静置40min;
(2)弃去异丙醇,用双蒸水冲洗一次,倒干,按要求配浓缩胶,将浓缩胶加至有液体溢出,把提前已清洗并烘干的梳子插入浓缩胶中,不能有气泡,常温下静置40min;
(3)配置电泳液(14.4g甘氨酸+1g SDS+3.02g Tris溶解于双蒸水中,总体积为1000m1),将电泳装置组装好放进电泳槽内,向内槽加满新鲜配置的电泳液,剩余电泳液加于外槽,垂直拔出梳子,根据蛋白浓度计算上样量,将Marker和蛋白依次加入上样孔中,注意位置对称;
(4)设置80V电泳1h左右,再设置120V进行电泳,待溴酚蓝跑到接近凝胶的底端时停止电泳;
(5)此过程中配置转膜液,14.4g甘氨酸+3.02g Tris溶解加入双蒸水至总体积800ml,再加200ml无水甲醇混合,放入冰上预冷。
转膜:
(1)剪PVDF膜至适当大小,浸泡于无水甲醇中3min,之后放在预冷的转膜液中备用;
(2)按顺序组装转膜装置,膜与胶之间不能有气泡;
(3)安装转膜夹按正确的电极方向转移到转膜槽中,将预冷的转膜液倒入槽中,放在冰盒内,条件设为300mA转膜80min(根据分子量大小设置时间);
(4)结束后关闭电源,用镊子轻轻取出PVDF膜。
蛋白质免疫检测:
(1)配置封闭液,即用TBST配置5%脱脂牛奶,把转印好的PVDF膜放置其中,摇床上室温封闭2h;
(2)结束后用TBST洗净封闭液,5min×1次;
(3)孵育一抗,4℃冰箱过夜;
(4)第二天回收一抗,TBST洗膜,10min×4次;
(5)配置二抗,均匀覆盖在膜上,在室温条件下展开孵育,总共持续的时间为1.5h;TBST洗膜,10min×4次;
(6)避光配显影液,于凝胶成像系统显影,拍照,保存。
通过WB方法,检测了在6对乳腺癌组织和其癌旁正常组织中KIAA1467的蛋白质表达水平。其中在6对组织中,KIAA1467蛋白在乳腺癌组织中表达明显高于对应的癌旁组织。将KIAA1467的条带灰度与GAPDH的条带灰度对比,算出KIAA1467条带的相对表达量,得出量化图。结果提示,相比于癌旁组织,乳腺癌组织中KIAA1467蛋白表达量均升高,表达量约为癌旁组织中的2-3倍(图1)。
二、乳腺癌组织芯片免疫组化染色:
乳腺癌组织有143例和良性组织有78例。78例良性组织中包含64例癌旁组织和14例良性病例。143例乳腺癌组织中,年龄低于60岁的91例,超过60岁的51例;病理分级:原位癌3例,I级24例,II级70例,III级34例;淋巴结转移:N0:68例;N1-N3:47例;TNM分期:I期27例,II期57例,III期23例,IV期2例。委托生物样本库制作成组织芯片。
1、组织芯片的制作:
(1)根据HE染色切片的镜检结果,在蜡块上有代表性的癌巢区域作标记;
(2)1∶1混合石蜡与蜂蜡,制作空白受体蜡块;在蜡块上设计10×7孔,共250点组织阵列,然后用组织芯片仪制成TMA空白蜡块;
(3)将供体蜡块在标记的点上选取最有代表性的癌巢区域,取直径2mm的组织块,每例各取1个芯;
(4)将取好的组织芯转移到受体蜡块的孔中,并取相应癌旁组织做对照;
(5)组织阵列块在55℃的恒温烤箱中加热融合10分钟,在快融化之前放至室温冷却,使受体蜡块与供体组织融为一体;
(6)将组织芯片置于4℃条件下冷冻4小时左右,随后用全自动组织切片机对组织阵列块进行修正,速度为20mm/转,等修到所有组织芯完全曝露;
(7)用切片机对组织阵列块进行切片,将连续切片分别漂在凉水中,使其自然展开,再将切片转移至45℃温水中展片2分钟左右,待展开后将其贴在经过防脱片处理的载玻片上晾干;
(8)将切片置于60℃的环境下烤片3分钟,58℃继续烤片16h;
(9)将做好的组织芯片保存于切片盒,置于冰箱4℃冷藏室备用。
2、免疫组化染色(EnVision两步法):
(1)将组织芯片放置在烘箱中先70℃烤片1.5h,再60℃烘片1h;
(2)将切片按照顺序浸入两份新鲜二甲苯中,每份时间为5min;梯度乙醇处理组织芯片:无水乙醇中实施处理对应的时间为5min:100%乙醇中实施处理5min-95%乙醇中实施处理5min-70%乙醇中实施处理5min,浸入到双蒸水中;
(3)将乳腺癌组织芯片置于染色缸中,向染色缸中倒入0.01mol/L柠檬酸稀释液(pH6.0)直至没过芯片,把染色缸放在微波炉100%功率2.5分钟,然后调整功率为20%,加热15分钟,取出后自然冷却至室温;
(4)用组化笔沿组织边缘处画圈,将内源性过氧化物酶阻断剂滴加到组织中,室温孵育30分钟;结束后PBS冲洗,2min×3次;
(5)配好5%BSA溶液封闭20min,弃去封闭液;
(6)用封闭液稀释一抗,浓度为抗体∶封闭液=1∶250,滴满组织表面,4℃冰箱过夜;
(7)次日取出芯片于室温复温(放置1小时左右),用PBS洗去一抗,2min,甩干,将反应增强液滴加到其中,室温条件下进行反应,总共反应30min;用PBS进行冲洗,2min×3次;
(8)根据要求用稀释好二抗滴满组织表面,室温孵育约30min,后用PBS溶液冲洗,2min×3次;
(9)显色:将DAB工作液(1ml稀释液+50μl染色液+50μl DAB浓缩液),滴加在组织芯片表面,约5秒,组织变黄后迅速用去离子水洗净;
(10)芯片放入苏木素中45秒,过盐酸酒精终止染色,在流水下冲洗10分钟;
(11)按照与步骤(2)相反的顺序浸泡组织芯片,依次为70%乙醇3min-95%乙醇5min-100%乙醇5min,最后浸没在二甲苯中5min,后将其取出风干;
(12)滴加中性树脂于组织芯片中间部位,盖片,缓慢按压,此时避免产生气泡,最后将芯片放置在通风橱中晾干。
3、免疫组化结果判断:
染色结果均由多光谱病理扫描成像系统(PerkinElmer,美国)扫描,并设置相应的分析程序由仪器自动打分,分数在0-300之间。
KIAA1467主要在乳腺癌细胞细胞核和胞浆内表达,阳性染色标记(呈棕黄色或褐色)表达主要定位在细胞核。采用X-tile软件分析,选取189作为KIAA1467蛋白表达的临界值,即<189为低表达,189-300分为高表达。
免疫组化法检测了乳腺癌患者样本中KIAA1467的蛋白表达情况(143个乳腺癌样本,64例匹配的癌旁样本和14个良性肿瘤样本),KIAA1467在乳腺癌细胞细胞核和胞浆内表达,阳性染色标记(呈棕黄色或褐色)表达主要定位在细胞核。组化结果表明,在乳腺癌中KIAA1467的蛋白表达显著升高,但在癌旁或良性肿瘤中KIAA1467蛋白低表达或呈阴性(图2)。对组织芯片中的患者进行定量分析数据显示,在乳腺癌组织中有44/143(30.8%)高表达,癌旁组织中仅1/64(1.5%)高表达,14个良性肿瘤标本中无一例高表达(x2=27.080,p<0.001)(表1)。
表1 KIAA1467在肿瘤组织和癌旁组织以及良性组织中的表达
类别 | n(221) | 低表达(%) | 高表达(%) | Pearsonχ2 | P value |
27.080 | <0.001 | ||||
癌 | 143 | 99(69.2) | 44(30.8) | ||
癌旁 | 64 | 63(98.4) | 1(1.6) | ||
良性肿瘤 | 14 | 14(100.0) | 0(0.0) |
4、KIAA1467的表达差异与乳腺癌患者的预后关系
在得到的数据中采用K-M生存分析KIAA1467表达和乳腺癌患者预后的关系,结果表明TNM分期晚和KIAA1467高表达是乳癌患者的不良预后因素(图3A,B)。并在GEPIA数据库中发现KIAA1467的高表达是乳腺癌的不良预后(危险比(HR)=1.5,P=0.023,n=1069图3C)。单因素COX回归分析,提示KIAA1467和患者的预后不良相关(P<0.001)。其他指标如:TNM分期(P<0.001)、淋巴结转移(P=0.001)、病理分级(P=0.001)、远处转移(P=0.007)也是不良预后因素。随后将KIAA1467和TNM分期纳入多因素分析提示KIAA1467高表达(P=0.007)和TNM分期晚(P=0.001)是乳腺癌患者预后的独立影响因子(表2)。
采用X-tile软件分析,选取189作为KIAA1467蛋白表达的临界值,即<189为低表达,189-300分为高表达,计量资料以均数±标准差表示,组间比较采用单因素方差分析,KIAA1467表达与乳腺癌患者的预后关系分析用Kaplan-Meier生存分析(图3),所有检验结果P<0.05为差异有统计学意义。所有数据统计分析和作图均使用GraphPad Prism 6和SPSS 20.0软件。
表2 KIAA1467的表达和TNM分期与乳腺癌患者预后的关系
实施例2
1、shRNA设计:
针对KIAA1467基因序列,委托广州复能基因有限公司制成shRNA、sh-NCRNA:
KIAA1467-shRNA序列为:5′-GCTCCATTGTTTGGAGTTACC-3′;
KIAA1467-sh-NCRNA序列为:5′-GCTTCGCGCCGTAGTCTTA-3′。
2、乳腺癌细胞系和人正常乳腺上皮细胞的培养:
乳腺癌细胞系,包含:MDA-MB-231,MCF-7,HCC-1937;人正常乳腺上皮细胞:MCF-10A。用1640完培(45ml基培+5ml血清+500μl双抗)培养HCC-1937细胞和MCF-7细胞,DMEM完全培养基(45ml基培+5ml血清+500μl双抗)培养MDA-MB-231,MEGM完全培养基(将choleratoxin与MEGM kit充分混匀,调整浓度为100ng/ml)培养MCF-10A细胞,置于37℃、含5%CO2培养箱中。每24h换液一次,每48h传代一次。
3、细胞蛋白质提取及蛋白量的测定:
(1)各种乳腺癌细胞及人正常乳腺上皮细胞均培养于37℃培养箱,CO2饱和湿度保持在5%,用1640完培(45ml基培+5ml血清+500μl双抗)培养HCC-1937细胞和MCF-7细胞,DMEM完全培养基(45ml基培+5ml血清+500μl双抗)培养MDA-MB-231细胞,MEGM完全培养基(将cholera toxin与MEGM kit充分混匀,调整浓度为100ng/ml)培养MCF-10A细胞,每24h换液一次,每48h传代一次,根据相应的密度传代;
(2)按实验的需求将KIAA1467-shRNA和KIAA1467-sh-NCRNA转染至相应的乳腺癌细胞中(MDA-MB-231、HCC-1937),镜下观察细胞密度;加入裂解液静置20分钟,用细胞刮刮净瓶底的细胞,将刮下的细胞转移至无酶EP管中;
(3)把EP管置于4℃离心机离心15min,转速设置为12000rpm;
(4)吸上清测浓度,根据上清液体积加入5×上样缓冲液,于95℃金属浴加热10min使蛋白变性,存于-80℃冰箱。
Western Blot检测MDA-MB-231,MCF-7,HCC-1937,MCF-10A及相应转染细胞株中KIAA1467的蛋白表达情况,结果表明KIAA1467在MDA-MB-231,MCF-7,HCC-1937乳腺癌细胞中表达高于MCF-10A(图4A)。
4、细胞转染:
(1)提前将计数好的HCC-1937和MDA-MB-231细胞种至六孔板中,16-24h后待细胞贴壁且成形时准备转染;
(2)取无菌EP管,在A管内加238μl基培及12μl lipoFiter,在B管中加入质粒4μg,培养基在其中添加的量具体为250μl,室温条件下进行静置,总共持续的时间具体为5min;
(3)轻轻混合管A和管B,静置20min;
(4)用移液器吸去六孔板中的培养基,用无菌PBS洗涤两遍,更换为2ml的无抗完培,再加入步骤(3)的混合试剂,轻轻混合;
(5)置培养箱中培养6h后换成完培,再培养48小时后进行下一步实验。
将KIAA1467干扰质粒和空白对照质粒分别转染至HCC-1937和MDA-MB-231细胞,Western blot结果显示,转染了KIAA1467-shRNA后的HCC-1937和MDA-MB-231细胞的KIAA1467的蛋白表达明显被抑制(图4B、图4C)。
5、克隆形成实验:
(1)MDA-MB-231(KIAA1467-shRNA转染和KIAA1467-sh-NCRNA转染组)、HCC-1937细胞(KIAA-shRNA转染和KIAA1467-sh-NCRNA转染组),共两组细胞,密度稀释为每个培养皿约800个细胞,均匀分布,置培养箱中;
(2)约半月后出现成团细胞,弃去废液,用无菌PBS洗涤,总共进行的次数为两次,对残留液进行吸取;后续转移到结晶紫溶液中来进行染色,具体时间上约半小时,洗净染液,晾干;
(3)镜下计数,并结合软件分析。
6、CCK-8实验:
(1)收集HCC-1937细胞和MDA-MB-231细胞(KIAA1467-shRNA转染和KIAA1467-sh-NCRNA转染的),将其重悬后进行细胞计数,配成浓度大约为1.5×104个/mL的细胞悬液,96孔板消毒后,在每孔中分别加入200μl的体积,对照组及转染组分别做5个副孔,放在培养箱继续培养;
(2)一天后,根据实验设计,分别于不同的时间点对细胞换液,避光条件配制好10%的CCK8,每个孔吸干净完培,加入100μl配好的CCK8,不可有气泡,避光继续培养2h;
(3)在450nm波长处测量每个小孔的吸光度。
7、EDU实验:
细胞培养:
(1)MBA-MD-231细胞(KIAA1467-shRNA转染和KIAA1467-sh-NCRNA转染组的)、HCC-1937细胞(KIAA1467-shRNA转染和KIAA1467-sh-NCRNA转染组的)收集转染48h后的细胞,离心备用;
(2)1640或DMEM完培重悬细胞,调整至一定浓度,按每孔四十万个细胞接种于24孔板中,培养24h。
EDU标记、细胞固化、染色:
(1)1640或DMEM完培按3000∶1的比例稀释EDU溶液(试剂A),配置30μM EDU培养基大约2ml;
(2)对300μl的30μM EDU培养基实施精密的量取,后转移到培养箱中进行孵育,总共持续的时间为2h,结束后弃去培养基;
(3)无菌PBS清洗细胞1-2次,每次5min;
(4)对150μl完成配置的2mg/ml甘氨酸实施精密的量取,此后就需要添加到其中,摇床孵育,总共持续的时间为5min,将甘氨酸溶液完全的抛弃;
(5)PBS清洗5min,弃PBS;
(6)每孔加入300μl渗透剂,此后则需要在摇床中进行孵育,总共持续的时间为10min;PBS清洗5min;
(7)向每孔加入染反应液(配置比例:去离子水在配置中总共需要添加的量为1407μl+Apollo反应缓冲液在配置中总共需要添加的量为75μl(试剂B)+Apollo催化剂溶液(试剂C)在配置中总共需要添加的量为15μl+Apollo荧光染料溶液在配置中总共需要添加的量为4.5μl(试剂D)+Apollo缓冲添加剂(试剂E)在配置中总共需要添加的量为14mg),用摇床缓慢摇晃室温避光孵育30min,弃去反应液;
(8)于每孔中加入渗透剂,总共添加量为300μl,摇床中来进行清洗,总共进行的次数为2-3次,单次操作持续的时间为10min;甲醇溶液在孔中需要精准地添加300μl,摇床中进行清洗,总共洗2次,每次5min;再用PBS洗5min。
DNA染色:
(1)用双蒸水按100∶1稀释试剂F,配1×Hoechst33342染液,避光保存;
(2)加300μl的1×Hoechst33342至每孔,摇床上避光孵育半小时;
(3)完全弃去染液,对PBS溶液实施精密的量取,分别添加到200μl,清洗总共进行的次数为1-3次,单次操作持续的时间为5min。
图像获取及分析:
染色结束后,立即对其进行观测。镜下观察图像,拍照存档,计算相对增殖阳性率。采用X-tile软件分析,选取189作为KIAA1467蛋白表达的临界值,即得分<189属于低表达,得分在189-300期间则属于高表达。计量资料用x±S表示并采用t检验;计数资料采用x2检验;生存分析采用Kaplan-Meier方法和log-rank检验;P<0.05表示差异有统计学意义。所有数据统计分析和作图均使用GraphPad Prism 6和SPSS 20.0软件。
8、Transwell实验:
(1)收集对数生长期的HCC-1937细胞和MDA-MB-231细胞(KIAA1467-shRNA转染组和KIAA1467-sh-NCRNA转染组)共计2组,消化重悬细胞并调整浓度为2×104个/ml;
(2)把小室在基培里放置5min,使之正反两面湿润;
(3)24孔板下室加入600μl细胞悬液,侵袭实验的小室提前铺好基质胶,待凝固后同样加入细胞悬液600μl在transwell小室中,放入24孔板,常规培养48小时;
(4)取出小室,PBS清洗两遍,放入0.1%结晶紫中染色5分钟,清洗干净后风干;
(5)倒置在镜下观察。
将KIAA1467-shRNA和KIAA1467-sh-NCRNA别转染至对应乳腺癌细胞中,采用CCK-8实验、克隆形成实验、EDU实验、Transwell实验分析KIAA1467在乳腺癌细胞中对其增殖和迁移侵袭能力的调节。CCK-8结果显示,与对照组相比,抑制了KIAA1467表达的HCC-1937和MD-MB-231细胞增殖能力显著下降(图5)。同时,克隆形成实验表明,下调KIAA1467的表达可抑制细胞克隆的形成(图6),此外,通过EDU细胞增殖实验检测了KIAA1467对细胞增殖活性的影响,敲降KIAA1467后,细胞的增殖活性减少(图7)。Transwell实验表面,敲降KIAA1467的表达后,HCC-1937和MDA-MB-231乳腺癌细胞的迁移能力和侵袭能力明显下降(图8)。
序列表
<110> 南通大学附属医院
<120> KIAA1467基因的用途
<130> 1
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> KIAA1467-shRNA(Artificial)
<400> 1
gctccattgt ttggagttac c 21
Claims (1)
1.KIAA1467基因的抑制剂在体外抑制乳腺癌细胞系增殖或迁移或侵袭能力中的应用,其特征在于,所述抑制剂为KIAA1467基因的shRNA,其核苷酸序列如SEQ ID NO .1所示;所述乳腺癌细胞系为MDA-MB-231细胞和HCC-1937细胞。
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---|---|---|---|---|
CN108535480A (zh) * | 2018-03-05 | 2018-09-14 | 南通大学附属医院 | EphA8基因在制备抗乳腺癌药物及其诊断试剂盒中的应用 |
CN108841965A (zh) * | 2018-08-14 | 2018-11-20 | 江门市中心医院 | Tlcd1在乳腺癌转移的诊断、预后及治疗中的应用 |
Non-Patent Citations (4)
Title |
---|
Cyclin B2 (CCNB2) Stimulates the Proliferation of Triple-Negative Breast Cancer (TNBC) Cells In Vitro and In Vivo;Wu S等;《Disease Markers》;20210726;第2021卷;Article 5511041 * |
Homo sapiens KIAA1467 protein(KIAA 1467), mRNA;Strausberg RL等;《GenBank Dtatabase》;ACCESSION NO. NM_020853 * |
LINC00665在乳腺癌细胞增殖和侵袭中的作用及其机制研究;吕民豪;《中国博士学位论文全文数据库(电子期刊) 医药卫生科技辑》(第2022年第04期);第E072-69页 * |
Overexpression of FAM234B Predicts Poor Prognosis in Patients with Luminal Breast Cancer;Lyu L等;《Cancer Management and Research》;第12卷;摘要、第12466页-12467页讨论 * |
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