CN116637192A - Lztr1联合kras在制备治疗逆转肝癌细胞对仑伐替尼耐药药物中的应用及其验证方法 - Google Patents
Lztr1联合kras在制备治疗逆转肝癌细胞对仑伐替尼耐药药物中的应用及其验证方法 Download PDFInfo
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Abstract
本发明公开了LZTR1联合KRAS在制备治疗逆转肝癌细胞对仑伐替尼耐药药物中的应用,属于生物医学和分子生物学领域,本发明首次公开了可用于检测HCC相关的蛋白质LZTR1及其联合下游蛋白质KRAS在制备HCC诊断、预后标志物和耐药检测相关试剂盒中的应用。本发明创新性发现在肝癌细胞中LZTR1通过降解下游KRAS蛋白,抑制肝癌发展并参与拮抗仑伐替尼耐药的相关机制。同时预示LZTR1在HCC中具有成为新型生物标志物及有效治疗靶点的潜力。
Description
技术领域
本发明涉及生物医学和分子生物学领域,具体而言,涉及LZTR1诱导下游KRAS蛋白降解,联合KRAS蛋白在制备治疗逆转肝癌对仑伐替尼耐药药物中的应用。
背景技术
原发性肝癌(primary liver cancer,PLC)是全球第六大最常见的癌症,也是2020年全球第三大癌症死亡原因。其中肝细胞癌(hepatocellular carcinoma,HCC)是最常见的原发性肝癌,约占75%-85%,它是一种预后较差的侵袭性肿瘤,预计其发病率未来还会持续增加。HCC最常见的病因是乙型肝炎病毒(HBV)或丙型肝炎病毒(HCV)感染,占发展中国家HCC病例的90%以上,其他危险因素包括黄曲霉毒素、酒精性肝病、非酒精性脂肪性肝、自身免疫性肝炎、肥胖和糖尿病等等。
截至目前,HCC的治疗一直以手术切除为主,辅以放化疗、介入、射频消融等。然而,由于肝癌发病隐匿,无典型临床特征,早期诊断困难,首次被诊断时大多已是中晚期,手术切除率不足30%。针对这类患者,治疗选择有限,通常以广谱常规细胞毒性药物化疗为主,然而HCC对常规化疗往往存在高度耐药性,患者5年生存率不足15%。近年来随着靶向药物索拉非尼、仑伐替尼;免疫相关药物纳武利尤单抗、帕博利珠单抗等药物的问世,一定程度上打破了中晚期患者治疗的局限性,给HCC的治疗带来了新的曙光。然而其精准治疗过程中仍存在许多问题。
仑伐替尼是一种口服多靶点受体酪氨酸激酶(RTKs)抑制剂,它的作用是阻断一种可发出癌细胞繁殖信号的异常蛋白质,从而阻止癌细胞的扩散。但近年来的研究发现,人体癌细胞对仑伐替尼极易产生耐药性,仑伐替尼的主要适应症有分化型甲状腺癌、肾癌、肝细胞癌三种癌症,对于不同癌症,仑伐替尼产生耐药的时间也不一样,针对肝细胞癌,单药仑伐替尼治疗肝癌全球无进展生存期仅为7.4个月,总生存期仅13.6个月。
综上所述,研究开发一种逆转肝癌细胞对仑伐替尼耐药的药物,将为肿瘤靶向治疗药物研发提供思路和解决方法,同时具有巨大的社会效益和经济效益。
发明内容
本发明所要解决的问题是针对肝癌治疗药物仑伐替尼在治疗过程中易产生耐药性进而导致治疗失败甚至疾病复发的问题,提供一种逆转肝癌细胞仑伐替尼耐药性的方法。
为解决上述问题,本发明第一方面提供LZTR1表达增强剂逆转肝癌细胞对仑伐替尼的耐药性。
亮氨酸拉链样转录调节因子1(LZTR1)起初被认为是一种转录调节因子,与Noonan综合征(NS),慢性髓系白血病(CML)和神经鞘瘤病的发生密切相关,然而其中的具体机制并不是很清楚。近年来,研究发现LZTR1作为E3泛素连接酶复合物的一部分,介导泛素分子偶联到RAS蛋白上,从而诱导RAS蛋白的泛素化降解,抑制相关通路激活和下游信号转导。因此,LZTR1发生的致病性突变影响对RAS蛋白的调节,这也解释了其在人类神经系统相关疾病中的作用。此外,LZTR1介导的RAS调节同样可能影响肿瘤发生发展,与肿瘤预后相关。本发明利用免疫组织化学染色技术探究LZTR1在HCC中的表达水平,探讨LZTR1作为肝癌标志物的可能性,同时利用克隆形成、划痕实验以及CCK8细胞增殖实验探寻LZTR1对其增殖迁移的影响,此外本发明还发现其可通过诱导下游KRAS蛋白降解抑制肿瘤发展,拮抗仑伐替尼耐药。
进一步地,所述LZTR1表达增强剂为提高肝癌细胞中LZTR1表达水平的分子或制剂。
优选地,所述提高肝癌细胞中LZTR1表达水平的分子或制剂包括过表达质粒,所述过表达质粒为pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro为载体的LZTR1过表达质粒。
进一步地,所述述LZTR1过表达质粒所插入的序列如SEQ ID No.1所示。
本发明第二方面提供一种验证LZTR1过表达质粒逆转肝癌细胞对仑伐替尼耐药的方法,包括以下步骤:
(1):采用生物信息学和免疫组化分析肝癌标本及癌旁组织中LZTR1的表达,并分析LZTR1及预后关系;
(2):制备LZTR1过表达质粒和CRISPR-cas9-LZTR1敲除质粒,并将LZTR1过表达质粒通过质粒转染技术导入肝癌细胞中,实现LZTR1在肝癌细胞中的过表达;
(3):通过实时荧光定量PCR、Western Blot、克隆形成、划痕实验、CCK-8实验验证LZTR1过表达细胞;
(4):检测LZTR1下游蛋白丰度;
(5):制备仑伐替尼耐药肝癌细胞。
优选地,所述肝癌细胞为HepG2和SK-Hep1肝癌细胞。
优选地,所述CRISPR-cas9-LZTR1敲除质粒所选择的靶位点如SEQ ID No.2所示,具体为:
5’-AGTCTTTCACATCGAACCGC-3’。
优选地,所述CRISPR-cas9-LZTR1敲除质粒的载体为pCDH-CMV-MC S-EF1-CopGFP-T2A-Puro。
进一步地,所述实时荧光定量PCR中使用的引物序列如SEQ ID No.3~SEQ IDNo.8所示,具体为:
LZTR1 F:5’-GCGGGGAGATGTACAAGGTT-3’;
LZTR1 R:5’-CCCGTAGTCCTCGTGCAG-3’;
KARS F:5’-AGTCATGGTCACTCTCCCCA-3’;
KARS R:5’-GCAGTCTGACACAGGGAGAC-3’;
GAPDH F:5’-CCCTCAGATGCCTGCTTC-3’;
GAPDH R:5’-CATGCCTTCCGTGTTCC-3’。
本发明具备的有益效果:本发明首次公开了可用于检测HCC相关的蛋白质LZTR1及其联合下游蛋白质KRAS在制备HCC诊断、预后标志物和耐药检测相关试剂盒中的应用,所述的检测主要通过免疫组织化学染色技术及蛋白质免疫印迹技术完成,利用对免疫组织化学染色图片进行分析,通过评估打分比较LZTR1蛋白质在HCC患者样本和正常样本中的表达,以用于辅助诊断HCC,此外可通过蛋白质免疫印迹技术检测LZTR1联合KRAS蛋白的检测评估患者预后及其对仑伐替尼治疗耐药性相关。相对传统肝癌检测技术具有灵敏性高、特异性强、周期短等特点,有利于肝癌的早期诊断,预后评估和治疗选择。本发明创新性发现在肝癌细胞中LZTR1通过降解下游KRAS蛋白,抑制肝癌发展并参与拮抗仑伐替尼耐药的相关机制。同时预示LZTR1在HCC中具有成为新型生物标志物及有效治疗靶点的潜力。
附图说明
图1为LZTR1转录组水平在HCC与癌旁的总体表达差异箱式图;
图2为LZTR1蛋白水平在HCC与癌旁的总体表达差异箱式图;
图3为LZTR1蛋白水平在HCC与配对癌旁组织的配对差异图;
图4为LZTR1蛋白表达差异在HCC患者预后分析的生存曲线图;
图5为76对人HCC组织中,LZTR1免疫组织化学染色的三个代表性图像(n=76,比例尺:100μm(上),25μm(下);
图6为76对人HCC组织及配对癌旁组织免疫组化学染色结果的堆积柱状图及染色百分比及染色强度综合得分柱状图(n=76,***P<0.001);
图7为临床病例特征统计相关热图;
图8为微血管侵犯(MVI)在染色强度阴性组及阳性组的差异柱状图;
图9为稳定表达细胞株的LZTR1转录组水平表达情况;
图10为稳定表达细胞株的LZTR1蛋白水平表达情况;
图11为克隆形成实验检测HepG2和SK-Hep1细胞集落形成能力实验结果图;
图12为克隆形成试验结果的量化柱状图;
图13为LZTR1表达水平对HepG2和SK-Hep1细胞的迁移影响结果图;
图14为细胞迁移实验结果量化柱状图;
图15为LZTR1表达水平对HepG2细胞的增殖影响结果图;
图16为LZTR1表达水平对SK-Hep1细胞的增殖影响结果图;
图17为LZTR1表达水平对HepG2、SK-Hep1细胞的增殖影响结果图;
图18为细胞迁移实验结果量化柱状图;
图19为Western blot验证LZTR1稳定表达细胞株HepG2下游RAS,RIT1,ERK,P-ERK蛋白丰度显色图;
图20为蛋白半衰期实验验证LZTR1过表达对KRAS蛋白降解的影响实验结果图;
图21为仑伐替尼诱导下LZTR1及下游KRAS的蛋白水平表达变化图。
具体实施方式
为使本发明的上述目的、特征和优点能够更为明显易懂,下面对本发明的具体实施例做详细的说明。需要说明的是,以下各实施例仅用于说明本发明的实施方法和典型参数,而不用于限定本发明所述的参数范围,由此引申出的合理变化,仍处于本发明权利要求的保护范围内。
需要说明的是,在本文中所披露的范围的端点和任何值都不限于该精确的范围或值,这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说,各个范围的端点值之间、各个范围的端点值和单独的点值之间,以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围,这些数值范围应被视为在本文中具体公开。
如背景技术所述,由于人体肝癌细胞对仑伐替尼极易产生耐药性,因此研究开发一种逆转肝癌细胞对仑伐替尼耐药的药物,将为肿瘤靶向治疗药物研发提供思路和解决方法,同时具有巨大的社会效益和经济效益。
实施例1
生物信息学分析
下载TCGA及GTEx数据库中肝细胞癌相关数据进行进一步分析。其中TCGA数据下载来源https://portal.gdc.cancer.gov/。GTEx数据下载来源https://xena.ucsc.edu/。下载CPTAC蛋白组学数据库中肝细胞癌相关数据进行进一步分析,下载来源:https://proteomic.datacommons.cancer.gov/pdc/browse/。(3)通过R语言包对数据进行分组合并并分析,分析LZTR1在HCC及配对癌旁正常组织mRNA及蛋白水平的差异,绘制差异箱式图,配对差异图,并结合临床信息分析预后,绘制总体生存率预后曲线。
结果如图1~图4所示,图1~图4为TCGA联合GTEx数据库(mRNA水平)及CPTAC数据库(蛋白水平)对LZTR1在HCC及配对癌旁组织表达上的分析及相应的预后分析。图1为LZTR1转录组水平在HCC与癌旁的总体表达差异箱式图;图2为LZTR1蛋白水平在HCC与癌旁的总体表达差异箱式图;图3为LZTR1蛋白水平在HCC与配对癌旁组织的配对差异图;图4为LZTR1蛋白表达差异在HCC患者预后分析的生存曲线图(ns代表无统计学差异,*P<0.05,**P<0.01,***P<0.001)。
实施例2
组织标本收集和肝癌免疫组织化学染色
纳入研究的76对人肝癌组织样本源自2021年01月01日至2022年7月31日宁波市临床病理诊断中心确诊为HCC。44对福尔马林固定的人肝癌组织标本来源于宁波市临床病理诊断中心。32对人新鲜肝癌组织标本来源于宁波市医疗中心李惠利医院。
新鲜人肝癌组织标本的采集、处理与保存:在符合纳入标准、不影响病理诊断取材的前提下,由专人在标本离体30min内,取肝癌组织及癌旁组织(癌组织旁1-3cm以内),切成小块(0.5×0.5×0.4cm),用生理盐水冲洗标本上残留的血液,将标本放入标本瓶中,旋紧瓶盖,备注患者信息及收集日期等信息,将标本瓶立即放入液氮保存,实验室无液氮时保存于-80℃冰箱。
福尔马林固定肝癌组织标本的采集、处理与保存:福尔马林标本为肝切除术经病理诊断后剩余肝脏组织,常温条件下放于足量10%中性福尔马林溶液中固定。收标本前在1.8mL标本管中加入适量4%多聚甲醛固定液,由专业病理医师在每个样本上取肝癌与癌旁组织各一块(0.5×0.5×1cm),分别放入1.8mL标本管,液体浸没标本,旋紧盖子,置于4℃冰箱中保存备用,定期更换4%多聚甲醛固定液。
纳入标准:1.样本病理诊断明确,确诊为原发性肝细胞肝癌;2.患者未接受术前放、化疗治疗;3.患者既往无其他系统的恶性肿瘤。排除标准为:1.合并其他系统恶性肿瘤;2.术前曾接受过射频消融毁损治疗的患者;3.有梅毒艾滋等传染性疾病者。所有患者经肝切除术治疗,标本经患者签署知情同意书后取得。本实验涉及的所有人体组织标本,均经课题组申请,在宁波大学医学院人体伦理委员会同意的条件下,只限实验室研究。
(1)组织固定与包埋
将福尔马林固定肝癌组织样本用镊子夹到摊开的保鲜膜上,用无菌刀片切成0.5×0.5×0.2cm大小的组织块,浸泡于4%多聚甲醛溶液中12小时以上。组织包埋盒分别用长度一致的白色非吸水线串起来,用无菌镊子将固定组织置于组织包埋盒中,盖紧包埋盒,在包埋盒上用铅笔标注样本相关信息,将包埋盒悬吊于大烧杯中,置于流动自来水下冲洗20min,沥干水分。将组织包埋盒依次放入提前备好的装有不同浓度乙醇的免疫组化染色缸中(定期更换缸中乙醇)梯度脱水,包埋盒放入染色缸后立即盖上盖子,非吸水线置于染色缸外以方便换缸。于75%乙醇溶液浸泡1h×1次;80%乙醇溶液浸泡1h×1次;95%乙醇溶液浸泡1.5h×2次;100%乙醇溶液浸泡1.5h×2次,最后沥干包埋盒上的乙醇溶液。将组织包埋盒放入二甲苯溶液中浸泡1h×2次。将装有熔点58-60℃切片石蜡的蜡缸提前放入60℃烘箱熔化,将组织包埋盒依次放入烘箱中的三个蜡缸,每个蜡缸内浸泡至少1h,包埋盒需充分浸没在液体石蜡中。提前预热包埋机使机器中的石蜡熔化,待石蜡全部熔化,将包埋用的铁盒放入包埋机左侧蜡缸,将烘箱蜡缸中的包埋盒取出放入包埋机右侧蜡缸。去掉包埋盒,将组织放入包埋铁盒正中,用镊子固定组织,向包埋铁盒中灌注液态石蜡使之浸没组织块,将塑料包埋盒的盖子取下,轻轻盖在包埋铁盒上,置于4℃冷冻台上冷却成固体蜡块,弃去包埋铁盒,将蜡块置于密封袋中,袋上做好标记,4℃冰箱保存备用。
(2)免疫组织化学染色
使用石蜡切片机对组织进行10μm粗切以除去不含组织的石蜡,并将蜡块表面打磨光滑。将组织蜡块切成4μm薄片,放入40℃水浴锅中展片,挑选组织完整、形状规则的薄片,用带正电荷的载玻片将组织切片捞起使之贴附在载玻片上,在载玻片末端用铅笔标注标本相关信息。将附有组织的载玻片置于已预热65℃的烤片机上进行烤片,至少烘烤2h,或放于37℃烘箱过夜。将满载切片的玻片架依次放入二甲苯中浸泡20min;无水乙醇溶液中浸泡5min×2次;95%乙醇溶液中浸泡5min×1次;75%乙醇溶液中浸泡5min×1次;最后将玻片架置于去离子水中洗涤5min。
将50×的EDTA抗原修复液用去离子水稀释到1×并摇晃均匀,将1×EDTA抗原修复液倒进高压锅中,半合上锅盖,待锅内液体沸腾,将装有切片的玻片架置于高压锅中,使抗原修复液完全浸没切片,旋紧锅盖,待高压锅气阀开始均匀冒气后开始计时,10min后关闭电磁炉电源,结束加热。将高压锅置于流动自来水下降温,打开锅盖,使石蜡切片自然冷却至室温。首先使用去离子水充分洗涤切片5min,接着用PBS缓冲液洗涤切片3min,重复洗涤3次。将30%过氧化氢水溶液用去离子水稀释至3%浓度,现配现用,将石蜡切片放于湿盒内,擦干组织周围水分,用免疫组织化学染色专用疏水笔围绕组织画圈,在载玻片上组织所在位置滴加适量3%过氧化氢水溶液,合上湿盒的盖子,37℃封闭20min。用PBS磷酸盐缓冲液洗涤切片3min×3次,甩掉载玻片上的液体。洗涤后的切片摆放于湿盒内,每张切片上滴加适量10%驴血清封闭液,室温下封闭15min后,甩掉切片上的封闭液。擦净组织周围的封闭液,将切片置于湿盒内,滴加适当浓度的LZTR1抗体稀释液,使抗体稀释液浸没组织,合上湿盒盖子,置于4℃冰箱过夜。用PBS磷酸盐缓冲液洗涤切片3min×3次。擦干组织周围的液体,在组织上滴加适当浓度的HRP标记驴二抗,使抗体完全覆盖组织,合上湿盒盖子,37℃孵育1h。用PBS磷酸盐缓冲液洗涤切片3min×3次。将DAB显色液滴加到载玻片上组织所在位置,避光覆盖适宜时长,将载玻片置于倒置显微镜上观察组织染色情况,待染色强度达到最佳时甩掉显色液,使用去离子水洗涤3min×3次。在载玻片上组织所在位置滴加适量改良Lillie-Mayer苏木素染液,染色1min,甩掉染色液,将装有载玻片的玻片架置于流动自来水下洗涤15min以返蓝。将装有切片的玻片架依次浸泡于75%乙醇溶液5min×1次;95%乙醇溶液中浸泡5min×1次;无水乙醇溶液中浸泡5min×3次。将切片置于二甲苯溶液中浸泡5min×2次。在载玻片上组织所在位置滴加适量中性树胶,用镊子夹取一张盖玻片轻轻覆盖在载玻片上,置于通风橱中晾干成片。将切片置于倒置显微镜上,依次在低倍镜、高倍镜下观察组织形态、细胞核染色、目的蛋白质染色情况等。
相关结果如图5~图8所示,图5~图8为免疫组织化学染色验证LZTR1在人HCC组织及癌旁组织中的表达情况。图5为76对人HCC组织中,LZTR1免疫组织化学染色的三个代表性图像(n=76,比例尺:100μm(上),25μm(下)。图6为76对人HCC组织及配对癌旁组织免疫组化学染色结果的堆积柱状图及染色百分比及染色强度综合得分柱状图(n=76,***P<0.001)。图7为临床病例特征统计相关热图。图8为微血管侵犯(MVI)在染色强度阴性组及阳性组的差异柱状图。
实施例3
患者临床信息相关分析
(1)根据标准化评分标准,将76对免疫组化染色结果分为阴性、弱阳性和强阳性,以表1展示。图1:LZTR1在HCC组织和相邻正常组织中表达的代表性免疫组化图像。星号表示差异显著。*p<0.05,**p<0.01,***p<0.001。
(2)将76对肝细胞癌与癌旁的染色结果进行分组,根据表达分为阴性组和阳性组,将患者临床相关信息按该分组整理为表2。并通过卡方检验分析探索表达在各个临床因素的相关性。
分析结果如表1~表2所示,表1为76对肝癌组织的染色结果根据病理中心标准化打分后分为阴性,弱阳性,强阳性。数据采用双侧fisher精确检验、卡方检验。表2为76位肝癌患者对应的临床病理特征,按照阴性,阳性分组,分析LZTR1表达与临床病理特征的相关性。数据采用双侧fisher精确检验、卡方检验。
表1肝细胞癌与相邻配对癌旁正常组织LZTR1表达的比较
表2肝细胞癌中临床病理信息与LZTR1表达的相关性
实施例4
筛选LZTR1过表达和敲低型稳定细胞株
(1)细胞瞬时转染
利用在线分析网站在LZTR1基因的编码区确定了一个sg RNA靶位点(5'-AGTCTTTCACATCGAACCGC-3')。设计pCDH-CMV-MCS-EF1-CopGF P-T2A-Puro(产品号:CD513B)为载体过表达质粒和CRISPR-cas9-LZTR1质粒。设计CD513B载体的LZTR1过表达质粒。在细胞转染前一天,取对数生长期细胞(HepG2和SK-Hep1肝癌细胞),当观察到细胞密度达到80-90%时,按照细胞传代的步骤,将贴壁细胞消化成单细胞悬液。稀释单细胞悬液至适宜程度,取洁净的血球计数板,附上盖玻片,重悬细胞后取15ul稀释后悬液沿盖玻片上侧壁缘滴入。于显微镜下观察视野下方格网中边缘4个大方格中的细胞数量,数量宜在100-160个之间,重复计数两遍,取平均值,平均值即对应稀释后0.1ul细胞悬液中的细胞数量。计算方法:单细胞悬液癌细胞个数/1mL=大方格细胞总数平均值×10000×稀释倍数。按照每孔约5×105个细胞数量,向6孔细胞板中接种细胞,约200-300μL细胞悬液。用移液器向每孔细胞中沿壁缓缓加入2mL完全培养基,盖上板盖,摇匀后消毒置于37℃恒温培养箱中培养至合适状态。取1.5mL无菌离心管6个,各管加入100μL Opti-MEM I Reduced Serum Medium。其中3管离心管中分别加入3μg CD513B载体的LZTR1过表达质粒,CD 513B载体空质粒以及CD513B载体CRISPR-cas9-LZTR1敲除质粒,另一半加入4μL HighGene Transfection Reagent转染试剂,充分混匀后静置5分钟。将CD513B载体的LZTR1过表达质粒、CD513B载体空质粒以及CD513B载体CRISPR-cas9-LZTR1质粒混合液与HighGene Transfection Reage nt转染试剂混合液混匀,静置20min。将待转染的6孔细胞板中的细胞按照上述细胞换液的步骤更换新鲜的完全培养基,向孔中加入质粒-脂质体复合物,轻轻摇匀,培养24h后更换培养基,适时进行下一步实验,其中,LZTR1过表达质粒所插入的序列如SEQ ID No.1所示,CRISPR-cas9-LZT R1敲除质粒所插入的序列如SEQ ID No.3所示。
(2)筛选稳定细胞株
按上述传代步骤向6孔细胞板中接种细胞,查询Puromycin药物筛选说明书建议的筛选浓度,设计梯度筛选预实验。取移液器吸弃6孔板中的培养基,每孔加1ml PBS缓冲液,润洗后弃去,重复2-3次,向每孔细胞中沿壁缓缓加入2mL完全培养基。按从低到高的浓度梯度向孔中加入计算好的Puromycin,消毒6孔板面后放入培养箱继续培养。48h后观察各个孔细胞状态,确定能在48h杀死全部野生型细胞的最低Puromycin浓度。按上述转染步骤分别于6孔板转染CD513B载体的LZTR1过表达质粒,CD513B载体空质粒以及CD513B载体CRISPR-cas9-LZTR1质粒,消毒放入培养箱继续培养。转染48h后,取出6孔板,用75%乙醇消毒双手及6孔板,置入超净台,同上述步骤于各个孔加入Puromycin,使各孔浓度维持在上述预实验确定的能够杀死全部野生型细胞的浓度,消毒后放回培养箱继续培养。48h后观察各个孔细胞状态,弃去原有培养基,轻柔加入1mL PBS缓冲液,润洗后弃去。重复2-3次。用移液器向每孔加入新鲜的2ml完全培养基,消毒放入培养箱继续培养。每日观察细胞存活情况,接下来数日每日换液,待细胞生长密度达到30%以上时,继续更换含Puromycin的培养基,维持各孔浓度为第1轮筛选时的2倍(若细胞抱团生长,影响2轮药筛的效率,必要时可先消化重悬再铺板),同上步骤继续每日观察换液。用荧光显微镜观察镜下荧光细胞比例,直至观察荧光细胞比例为100%,停止药筛,即获得三株稳定株。
实施例5
实时荧光定量逆转录PCR鉴定
从细胞中提取RNA,PCR引物均购自杭州擎科生物科技有限公司。PCR引物序列为:LZTR1 forward:5’-GCGGGGAGATGTACAAGGTT-3’(SEQ ID No.4)and reverse:5’-CCCGTAGTCCTCGTGCAG-3’(SEQ ID No.5),KRAS forward:5’-AGTCATGGTCACTCTCCCCA-3’(SEQ ID No.6)and reverse:5’-GCAGTCTGACACAGGGAGAC-3’(SEQ ID No.7),GAPDH forward:5’-CCCTCAGATGCCTGCTTC-3’(SEQ ID No.8)and reverse:5’-CATGCCTTCCGTGTTCC-3’(SEQID No.9)。使用II 1st Strand cDNA 合成试剂盒,根据制造商说明书进行cDNA逆转录。采用SYBR Green PCR Master Mix试剂盒,根据制造商说明书进行PCR扩增。使用Applied Biosystems QuantStudio 3实时定量PCR仪上进行qRT-PCR反应。实验结果根据定量循环数(quantification cycle,Cq)表示。本研究采用管家基因GAPDH作为qRT-PCR内参,使用ΔCq值来检测目标基因的相对表达量,其计算方式为ΔCq=Cq目的-Cq内参,其值越小,目标基因达到规定荧光阈值所经历的循环数越小,相对表达水平越高。
相关实验结果如图9~图10所示,图9~图10为实时荧光定量逆转录PCR(qRT-PCR)及蛋白质免疫印迹技术(Western blot)验证HepG2和SK-Hep1肝癌细胞经Puromycin筛选后,稳定表达细胞株的LZTR1表达。图9为稳定表达细胞株的LZTR1转录组水平表达情况。图10为稳定表达细胞株的LZTR1蛋白水平表达情况。
实施例6
蛋白提取,蛋白质免疫印迹技术鉴定
从细胞中提取蛋白质,进行电泳,配制10%的分离胶,注入制胶玻璃板后75%乙醇液封,待分离胶凝固后倒去乙醇。配制浓缩胶,注入制胶玻璃板,立即插入加样梳,凝固30min后,待用。于电泳液中拔出梳子,取20ul上样,先用80V恒压电泳至marker开始分离后再用恒压130V进行电泳,直至目的条带分开置合适位置。转膜:将负极板、海绵垫、滤纸、胶、PVDF膜、滤纸、海绵垫、正极板按次序放置,连接电源,恒流220mA转膜2h。封闭及孵育一抗:配置5%脱脂奶粉封闭液,室温摇床封闭1h,4℃孵育适宜稀释比一抗过夜。洗膜及孵育二抗:室温使用TBST洗膜5次,5min/次,室温摇床孵育辣根过氧化物酶标记的二抗1h。再次用TBST洗膜5次,5min/次,滴加ECL发光液,并在暗室内进行胶片曝光。使用的主要抗体和试剂如下:LZTR1兔pAb(A7350,Abclonal,中国武汉)、ERK1/2兔单抗(A4782,Abclonal,中国武汉)、KRAS兔pAb(12063-1-AP,Proteintech,中国武汉)、RIT1兔pAb(A15715,Abclonal,中国武汉)、Phospho-ERK-T202/204+ERK2-T185/Y187兔pAb(AP0472,Abclonal,中国武汉)、GAPDH兔pAb(AC001,Abclonal,中国武汉)、HRP山羊抗兔IgG(AS014,Abclonal,中国武汉)。
实施例7
克隆形成
取上述药物筛选的3株HepG2肝癌稳定株细胞,将贴壁细胞消化成适宜浓度均匀细胞悬液。吸取15μL上述均匀细胞悬液加入细胞计数板中,如上述计数方法测其浓度,按照相应的比例稀释细胞悬液,用移液器吹打均匀。取数个6孔细胞板,每孔加入1.5×103个细胞,定量2mL,每个处理组设置3-4个复孔,利用“8”字法摇匀,置于恒温培养箱,每5天换液一次,培养1-2周。当六孔板内肉眼可见细胞集落时,弃去孔内培养基,用PBS缓冲液轻柔润洗2次,每孔加入适量4%多聚甲醛固定细胞,冰上放置30min。弃去固定液,用PBS缓冲液润洗2次,向每孔加入800μL 0.1%结晶紫染料,放于摇床上染色10min,回收染料,用PBS缓冲液润洗3次,置于37℃烘箱过夜,待六孔板晾干后拍照并统计数据。
相关实验结果如图11~图12所示,图11~图12为克隆形成验证LZTR1表达水平对HepG2和SK-Hep1细胞生长的影响。图11表示克隆形成实验检测HepG2和SK-Hep1细胞集落形成能力。图12表示克隆形成试验结果的量化柱状图(ns代表无统计学差异,*P<0.05,**P<0.01,***P<0.001)。
实施例8
划痕实验
取上述药物筛选的3株HepG2和3株SK-Hep1肝癌稳定株细胞,将贴壁细胞消化成适宜浓度均匀细胞悬液。吸取15μL上述均匀细胞悬液加入细胞计数板中,如上述测量其浓度,按照相应的比例稀释至适宜的细胞悬液,用移液器吹打均匀。取2个6孔细胞板,用马克笔在六孔板背面间隔0.5-1cm划5条横线,每株细胞设2-3个复孔,每孔接种30万个细胞/孔,定量2mL,放入恒温培养箱培养。待细胞密度达80%-90%时,用200μL枪头按照六孔板背面的横线进行垂直划线,用PBS缓冲液洗去漂浮细胞,更换2%血清DMEM培养基,放入培养箱继续培养。显微镜下观察0、24h的划痕愈合情况,拍照存储备用,使用Image J软件测量各组的划痕愈合面积。
相关实验结果如图13~图14所示,图13~图14为划痕实验验证LZTR1表达水平对HepG2和SK-Hep1细胞迁移的影响。图13为LZTR1表达水平对HepG2和SK-Hep1细胞的迁移影响。图14为细胞迁移实验结果量化柱状图(ns代表无统计学差异,*P<0.05,**P<0.01,***P<0.001)。
实施例9
CCK8实验
取上述药物筛选的HepG2和SK-Hep1肝癌稳定株细胞,将贴壁细胞消化成适宜浓度均匀细胞悬液。吸取15μL上述均匀细胞悬液加入细胞计数板中,如上述测量其浓度,按照相应的比例稀释至适宜的细胞悬液,用移液器吹打均匀。取3块96孔细胞培养板,每个处理组定6个复孔,每孔加入100μL细胞悬液,定量1.5×103个细胞,分别培养24、48、72、96、120、144h。培养结束后避光条件下向每孔中加入10μL的CCK-8试剂。用75%酒精消毒96孔细胞培养板,继续培养2h,使细胞裂解。2h后,将酶标仪波长设定为450nm,测定每孔吸光度,记录数据。
相关实验结果如图15~图16所示,图15~图16为CCK8实验验证LZTR1表达水平对HepG2和SK-Hep1细胞增殖的影响。图15为LZTR1表达水平对HepG2细胞的增殖影响;图16为LZTR1表达水平对SK-Hep1细胞的增殖影响(ns代表无统计学差异,*P<0.05,**P<0.01,***P<0.001)。
实施例10
Transwell迁移实验
取上述药物筛选的3株SK-Hep1肝癌稳定株细胞,将贴壁细胞消化成适宜浓度均匀细胞悬液。将细胞悬液转移至无菌的1.5mL微量离心管中,1000×g离心5min,加入1mlPBS充分重悬。1 000×g离心5min,弃去上清液,用1mL无血清培养基重悬细胞。吸取15μL上述均匀细胞悬液加入细胞计数板中,如上述测量其浓度。取Trans-well小室,在下室中加入750μL完全培养基。在上室内加入200μL用无血清培养基的稀释后的单细胞悬液,使得上室内细胞数量为4×104个。静置30min后将其置于37℃恒温培养箱中培养36h。弃培养基,用PBS缓冲液洗涤2次。向小室内加入800μL细胞固定液,静置20min,使穿过聚碳酸酯小室膜的细胞固定。回收细胞固定液,用PBS缓冲液清洗小室1-2次,加入800μL0.1%结晶紫染料,置于摇床上染色20min。染色完成后,回收结晶紫染料,用去离子水轻柔漂洗小室,将染料洗去并晾干。40倍镜观察视野,100倍镜每组随机挑选3个细胞分布均匀视野,统计细胞数量。
相关实验结果如图17~图18所示,图17~图18为为Transwell迁移实验证实LZTR1表达水平对细胞迁移能力的影响。图17为LZTR1表达水平对HepG2、SK-Hep1细胞的增殖影响。图18为细胞迁移实验结果量化柱状图(ns代表无统计学差异,*P<0.05,**P<0.01,***P<0.001)。
实施例11
LZTR1下游蛋白丰度检测
待稳定株扩大培养后取六孔板中单孔稳定株细胞,弃去完全培养基,用PBS缓冲液洗涤贴壁细胞2次,彻底吸除PBS。加入200μL细胞裂解液,置于4℃恒温摇床上摇晃20min,将细胞裂解液转移至1.5mL离心管中,放入-80℃冰箱中过夜。按照上述蛋白质免疫印迹技术进行后续操作。
相关实验结果如图19所示,图19为Western blot验证LZTR1稳定表达细胞株HepG2下游RAS,RIT1,ERK,P-ERK蛋白丰度显色图。
实施例12
KRAS蛋白半衰期检测
取HepG2过表达及Control细胞按上述种板步骤按照每孔约3×105个细胞数量向12孔细胞板中接种细胞,每组各6个孔。用移液器向每孔细胞中沿壁缓缓加入1mL完全培养基,盖上板盖,摇匀后消毒置于37℃恒温培养箱中培养至合适状态。取放线菌酮(CHX),以DMSO为溶剂配置适宜浓度,待细胞生长到对数生长期时,更换含50μM CHX的新鲜培养基阻断蛋白质合成。在加入CHX后的0、4、8、12、20和28h后的各个节点按上述制样步骤制备细胞裂解液,按照蛋白质免疫印迹技术进行后续操作。
相关实验结果如图20所示,图20为蛋白半衰期实验验证LZTR1过表达对KRAS蛋白降解的影响实验结果图。
实施例13
仑伐替尼药物诱导肝癌细胞耐药实验
取对照组HepG2细胞按上述种板步骤按照每孔约5×105个细胞数量向6孔细胞板中接种细胞,用移液器向每孔细胞中沿壁缓缓加入2mL完全培养基,盖上板盖,摇匀后消毒置于37℃恒温培养箱中培养至合适状态。取仑伐替尼,以二甲基亚砜(DMSO)为溶剂配置适宜浓度,待细胞生长到对数生长期时,6孔细胞分别更换含0μM、4μM、8μM、12μM、16μM、20μM仑伐替尼浓度的新鲜培养基,继续培养48h,期间根据细胞生长及营养消耗情况可选择期间更换一遍上述培养基。48h后按上述制样步骤制备细胞裂解液,按照蛋白质免疫印迹技术进行后续操作。
相关实验结果如图21所示,图21为仑伐替尼诱导下LZTR1及下游KRAS的蛋白水平表达变化图。
结果分析:具体实施方式所有实施例确保了实验数据及收集的原始临床资料进行全面检查、校对、整理,确保资料尽可能的完整、准确、无误。然后利用Excel软件分别建立组织、细胞数据库,对涉及的临床原始资料进行分组、归纳后将其录入表格中。最后利用SPSS26.0统计软件对最终的实验数据进行整理、分析。所有数据统计检验均取双侧概率,按照α=0.05的检验水准进行统计检验,若P<0.05,即认为两组间的差异有统计学意义,反之则认为差异无统计学意义。柱状图、折线图等实验结果图的绘制,利用GraphPad Prism 9.0、SPSS 26.0软件共同完成。
首先我们通过调研TCGA联合GTEx数据库,发现LZTR1转录组mRNA水平表达在HCC和癌旁正常组织统计学无显著性差异。我们继续分析CPTAC蛋白数据库,发现LZTR1蛋白水平在癌旁组织表达显著高于癌组织(***P<0.001),生存分析提示HCC组织中LZTR1蛋白表达高的患者总生存时间优于LZTR1蛋白表达低的患者(*P<0.05)。
通过免疫化学组织染色技术,我们发现在肝癌组织中,LZTR1的阳性表达率为64.5%(49/76),而癌旁组织为84.2%(64/76),差异有统计学意义(X2=24.417,***P<0.001)。LZTR1在癌旁组织中的表达水平明显高于相应的肝癌组织(***P<0.001)。临床因素相关分析上,阴性组微血管侵犯水平高于阳性表达组,差异有统计学意义(X2=4.632,*P=0.031)。
克隆形成实验显示,与Control组HCC细胞相比,LZTR1高表达组细胞集落的数量明显减少,而LZTR1敲低组细胞集落的数量明显增加。细胞划痕实验结果提示,LZTR1表达下调促进HCC细胞迁移。CCK8实验显示,LZTR1低表达组较Control组细胞增殖速度加快,LZTR1高表达组较Control组细胞增殖速度减慢。Transwell迁移实验证明LZTR1低表达组较Control组细胞迁移能力强,LZTR1高表达组较Control组细胞迁移能力弱。
我们发现在HCC细胞中,LZTR1通过降解KRAS参与调节RAS-RAF-MEK-ERK信号通路,此外发现LZTR1过表达HepG2细胞中KRAS的降解较Control组加快,半衰期更短。
通过在Control组HepG2细胞中设计不同梯度浓度的仑伐替尼诱导耐药实验,我们发现随着浓度的升高,LZTR1蛋白表达水平下降,与此同时KRAS蛋白水平逐渐升高,揭示LZTR1通过抑制RAS蛋白继而拮抗仑伐替尼耐药的潜在机制。
该发明通过检测LZTR1在HCC组织的表达以及LZTR1高表达以及低表达对肝癌细胞增殖迁移的影响,探索其通过降解KRAS蛋白参与抑制HCC发展及拮抗仑伐替尼耐药的相关机制,结合了相关统计学原理和现代生物学技术,这为HCC的诊断,预后评估,耐药预判等方面提供、灵敏度高、特异性强、周期短和结果稳定的检测方法,为肝癌患者的分子靶向药物选择和治疗提供科学依据,为新型分子靶向治疗的开发提供研发思路,数据支持。
上述说明并非对本发明的限制,本发明也并不限于上述举例。本技术领域的普通技术人员在本发明的实质范围内,做出的变化、改型、添加或替换,也应属于本发明的保护范围。
虽然本公开披露如上,但本公开的保护范围并非仅限于此。本领域技术人员,在不脱离本公开的精神和范围的前提下,可进行各种变更与修改,这些变更与修改均将落入本发明的保护范围。
Claims (9)
1.LZTR1联合KRAS在制备治疗逆转肝癌细胞对仑伐替尼耐药药物中的应用,其特征在于,通过LZTR1表达增强剂逆转肝癌细胞对仑伐替尼的耐药性。
2.如权利要求1所述的LZTR1联合KRAS在制备治疗逆转肝癌细胞对仑伐替尼耐药药物中的应用,其特征在于,所述LZTR1表达增强剂为提高肝癌细胞中LZTR1表达水平的分子或制剂。
3.如权利要求2所述的LZTR1联合KRAS在制备治疗逆转肝癌细胞对仑伐替尼耐药药物中的应用,其特征在于,所述提高肝癌细胞中LZTR1表达水平的分子或制剂包括过表达质粒,所述过表达质粒为pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro为载体的LZTR1过表达质粒。
4.如权利要求3所述的LZTR1联合KRAS在制备治疗逆转肝癌细胞对仑伐替尼耐药药物中的应用,其特征在于,所述LZTR1过表达质粒所插入的序列如SEQ ID No.1所示。
5.一种验证LZTR1过表达质粒逆转肝癌细胞对仑伐替尼耐药的方法,其特征在于,包括以下步骤:
(1):采用生物信息学和免疫组化分析肝癌标本及癌旁组织中LZTR1的表达,并分析LZTR1及预后关系;
(2):制备LZTR1过表达质粒和CRISPR-cas9-LZTR1敲除质粒,并将LZTR1过表达质粒通过质粒转染技术导入肝癌细胞中,实现LZTR1在肝癌细胞中的过表达;
(3):通过实时荧光定量PCR、Western Blot、克隆形成、划痕实验、CCK-8实验验证LZTR1过表达细胞;
(4):检测LZTR1下游蛋白丰度;
(5):制备仑伐替尼耐药肝癌细胞。
6.如权利要求5所述的验证LZTR1过表达质粒逆转肝癌细胞对仑伐替尼耐药的方法,其特征在于,所述肝癌细胞为HepG2和SK-Hep1肝癌细胞。
7.如权利要求5所述的验证LZTR1过表达质粒逆转肝癌细胞对仑伐替尼耐药的方法,其特征在于,所述CRISPR-cas9-LZTR1敲除质粒所选择的靶位点如SEQ ID No.2所示,具体为:
5’-AGTCTTTCACATCGAACCGC-3’。
8.如权利要求7所述的验证LZTR1过表达质粒逆转肝癌细胞对仑伐替尼耐药的方法,其特征在于,所述CRISPR-cas9-LZTR1敲除质粒的载体为pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro。
9.如权利要求5所述的验证LZTR1过表达质粒逆转肝癌细胞对仑伐替尼耐药的方法,其特征在于,所述实时荧光定量PCR中使用的引物序列如SEQ ID No.3~ SEQ ID No.8所示,具体为:
LZTR1 F:5’-GCGGGGAGATGTACAAGGTT-3’;
LZTR1 R:5’-CCCGTAGTCCTCGTGCAG-3’;
KARS F:5’-AGTCATGGTCACTCTCCCCA-3’;
KARS R:5’-GCAGTCTGACACAGGGAGAC-3’;
GAPDH F:5’-CCCTCAGATGCCTGCTTC-3’;
GAPDH R:5’-CATGCCTTCCGTGTTCC-3’。
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