CN112220925A - Mst1作为药物靶点在制备治疗结直肠癌药物中的应用 - Google Patents
Mst1作为药物靶点在制备治疗结直肠癌药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种MST1作为药物靶点在制备治疗结直肠癌药物中的应用。MST1作为药物靶点在制备治疗结直肠癌药物中的应用，该药物为通过促进MST1的表达来抑制结直肠癌迁移、侵袭和增殖的药物。本发明通过提升MST1的表达来抑制CRC细胞的增殖、侵袭和迁移，而且，MST1可以通过Hippo信号通路调节EMT，抑制CRC细胞的增殖、侵袭和迁移，从而发挥抑癌基因的作用。
Description
技术领域
本发明属于生物医学技术领域，具体涉及一种MST1作为药物靶点在制备治疗结直肠癌药物中的应用。
背景技术
结直肠癌(colorectal cancer，CRC)是胃肠道常见的恶性肿瘤，其早期症状不明显。随着病情的发展，出现排便习惯的改变，便血、腹泻、腹泻便秘交替、局部腹痛等症状。结直肠癌是中国仅次于肺癌和胃癌的第三高发病率的癌症。然而，CRC从癌前病变(腺瘤)到恶性病变的过程是一个漫长的过程，是为数不多的能够早期发现和早期治疗的恶性肿瘤之一。
巨噬细胞刺激1(macrophage stimulating 1，MST1)作为Hippo信号通路的核心激酶，通过与下游转录辅因子YAP和TAZ相互作用调节细胞增殖和凋亡。近期研究发现，MST通过加速caspase-3的激活，使细胞对死亡受体介导的凋亡敏感。此外，MST1和MST2在caspase激活的细胞凋亡中起着上游和下游的作用。在凋亡过程中，MST1被caspase切割并激活，导致细胞凋亡的形态发生改变，如染色质凝聚。此外，MST1的激活可抑制YAP的表达，通过控制JNK-MIEF1- 线粒体途径促进甲状腺癌细胞的死亡。肿瘤细胞中FGFR4的特异性敲除会导致MST1活化并诱导HER2+乳腺癌细胞凋亡。然而，CRC中MST1的表达及其临床意义的研究很少。
上皮-间充质转化(epithelial mesenchymal transition，EMT)是上皮细胞通过特定的程序转化为间充质表型细胞的生物学过程。它在胚胎发育、慢性炎症、组织重建、癌症转移和多种纤维化疾病中起到重要作用。其主要特征是细胞粘附分子(cell adhesionmolecules，E-cadherin)表达减少，细胞角蛋白细胞骨架向波形蛋白细胞骨架转化以及间充质细胞的形态特征。通过EMT，上皮细胞失去细胞极性，失去与基底膜等上皮表型的联系，获得较高的间充质表型，如迁移侵袭、抗凋亡和降解细胞外基质。
本研究检测了CRC患者中MST1的表达，并分析了MST1与CRC患者预后不良的关系。同时，研究了MST1是否通过Hippo信号通路影响CRC的EMT 和恶性生物学行为路径。结果表明，MST1可通过Hippo信号通路调节CRC的 EMT，进而影响CRC的恶性生物学行为。
发明内容
针对现有技术中的上述不足，本发明提供一种MST1作为药物靶点在制备治疗结直肠癌药物中的应用，MST1通过Hippo信号通路调节EMT，抑制CRC 细胞的增殖、侵袭和迁移，从而发挥抑癌基因的作用，表明MST1可能是一种很有前景的CRC治疗靶点。
为实现上述目的，本发明解决其技术问题所采用的技术方案是：
MST1作为药物靶点在制备治疗结直肠癌药物中的应用。
进一步地，药物为通过促进MST1的表达来抑制结直肠癌迁移、侵袭和增殖的药物。
进一步地，药物为促进MST1表达的药物。
一种用于检测个体MST1表达量的试剂在制备诊断试剂或试剂盒中的用途。
本发明提出的MST1可以作为胆管癌早期诊断标志物、药物治疗有效性判断标志物或患者预后标志物。
本发明的有益效果为：
MST1在CRC患者中低表达，而MST1低表达的患者OS和RFS较低，并且与不良预后密切相关。更为重要的是，MST1通过Hippo信号通路调节EMT，抑制CRC细胞的增殖、侵袭和迁移，从而发挥抑癌基因的作用，表明MST1可能是一种很有前景的CRC治疗靶点。
附图说明
图1为CRC和癌旁组织中MST1的表达；其中，A为TCGA数据库(大肠癌和邻近非癌组织)中MST1的mRNA表达；B为MST1在CRC及癌旁组织中的mRNA表达；C为MST1在癌旁组织及癌旁组织中的蛋白表达；D为MST1 在细胞中的表达；
图2为免疫组化法检测CRC组织中MST1的表达；其中，A为CRC及癌旁组织MST1表达的免疫组化分析；B为CRC及癌旁组织MST1表达的统计分析；
图3为MST1与CRC预后不良的关系；其中，A为Kaplan-Meier法分析 CRC患者MST1高表达与低表达；B为CRC MST1高表达与低表达RFS的 Kaplan-Meier分析；
图4为MST1对CRC细胞增殖的影响；其中，A为MST1在HCT116和SW480 细胞中的蛋白表达；B为进行CCK-8分析，以检查MST1过度表达对HCT116 和SW480细胞增殖能力的影响；C为用EdU检测MST1高表达后HCT116和 SW480细胞的增殖情况；
图5为MST1对CRC细胞迁移和侵袭的影响；其中，A为迁移实验检测 MST1过表达对HCT116细胞迁移的影响；B为迁移实验检测MST1过表达对 SW480细胞迁移的影响；C为侵袭实验检测MST1过表达对HCT116细胞侵袭的影响；D为侵袭实验检测MST1过表达对SW480细胞侵袭的影响；
图6为MST1对EMT和Hippo信号通路的影响；A为p-LATS1、p-YAP、 YAP、E-cadherin、N-cadherin和vimentin在HCT116细胞中的表达；B为p-LATS1、p-YAP、YAP、E-cadherin、N-cadherin和vimentin在SW480细胞中的表达。
具体实施方式
下面对本发明的具体实施方式进行描述，以便于本技术领域的技术人员理解本发明，但应该清楚，本发明不限于具体实施方式的范围，对本技术领域的普通技术人员来讲，只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内，这些变化是显而易见的，一切利用本发明构思的发明创造均在保护之列。
实施例1细胞培养和转染
1 材料和方法
1.1 一般资料
收集南充中心医院普外科2009年3月至2014年4月收治的81例大肠癌患者(肿瘤组织和配对正常邻近组织)。该研究已经医学伦理委员会审核通过，符合世界医学协会赫尔辛基宣言的相关要求。所有患者对研究内容均知晓并签署知情同意书。
1.2细胞培养和转染
从中国科学院(上海)细胞库中获得HCT116、SW480、LOVO和人结肠上皮细胞HCoEpiC，在RPMI-1640培养基(Gibco，美国)中用10％胎牛血清培养HCT116、SW480、LOVO细胞。按照制造商的说明(基因制药，中国上海) 转染LV-MST1和LV-NC慢病毒载体。
1.3定量实时PCR(quantitative real-time PCR，qRT-PCR)
用TRIzol试剂(TaKaRa，日本)从CRCTOMOR组织、配对正常邻近组织和细胞系中提取总RNA，用PrimeScript RT试剂(TaKaRa，日本)将提取的RNA 反转录为cDNA，用Prime
RT ReagentKit(TaKaRa，日本)进行qRT-PCR，使用光循环系统(罗氏)检测。
引物qRT-PCR设计如下：
MST1：正向ACAAA-tctctcccaca-TTCCG；
MST1：反向cactctgcaaa-TGGGTGCTG；
GAPDH：前进TGATCACAGCGCGACACCA；
GAPDH：反向CCCTGTTGCTGTAGCCAA。
GAPDH作为内部控制。相对用2-ΔΔCT法计算RNA表达水平。
1.4Western blot
用蛋白提取缓冲液(RIPA裂解缓冲液)从CRC组织、正常邻近组织和细胞系中分离总蛋白，用10％SDS-PAGE分离总蛋白凝胶。薄膜室温下1h，加入 5％的脱脂粉牛奶。膜在4℃下用抗MST1探针过夜(1:3000，Abcam,美国)， anti-p-LA TS1(1:5000，Abcam，美国)，anti-Y-AP(1:5000，Abcam，美国)， anti-p-Y-AP(1:5000，美国Abcam)，anti-E-cadherin(1:5000，Abcam，美国)， anti-E-cadherin(1:5000，美国Abcam)，anti-N-cadherin(1:5000，Abcam，美国)，抗β-肌动蛋白(1:5000，Abcam，美国)，然后，用适当的二级抗体。最后，用化学发光试剂(HyperfilmECL，美国)分析蛋白表达。
1.5免疫组织化学
采用免疫组织化学方法检测大肠癌石蜡包埋切片中MST1的表达。用二甲苯使样品脱水。抗原回收在10mmol/L柠檬酸钠溶液(PH 6.0)中，100℃，16min，并将样品冷却30min。用3％过氧化氢抑制内源性过氧化物酶活性，用胎牛血清封闭30min。用抗MST1兔多克隆抗体(1:200，Abcam，美国)在4℃下培养过夜。然后，用生物素化二级抗体在37℃孵育30最小值样品用DAB(3，3-二氨基联苯胺)和迈耶苏木精染色。
MST1染色分类如下：0～3:0为阴性；1为弱；2为中等；3为强，阳性细胞的百分比范围为0～4:0为阴性或<5％；1为6％～25％；2为26％～50％；3为 51％～75％；4为76％～100％。使用阳性细胞的百分比和强度来确定最终的得分。 <4级为MST1低表达，而≥4级为MST1高表达。
1.6细胞计数试剂盒-8(cell-counting Kit-8,CCK8)法检测
采用CCK8法检测MST1对细胞增殖的影响。转染细胞后接种于96孔板。用CCK8检测试剂对96孔板接种转染细胞。吸收性在450nm处记录每个孔的细胞增殖情况。
1.7 5-乙炔基-2′-脱氧尿苷(EdU)检测
转染细胞后接种于96孔板中。4％聚甲醛固定，0.5％tritonx-100溶液固定细胞核。在96孔板中分别加入EdU(50μM)、1×ApolloR反应鸡尾酒(100μL)和 1×Hoechst 33342(100μL)。用各组细胞平均数分析增殖情况。
1.8细胞侵袭和迁移实验
将细胞转染并接种于上腔。使用Transwell chambers(Corning Costar,Cambridge,MA,美国)进行细胞迁移分析。然后，将500μL含10％FBS的高糖 DMEM添加到匹配的下室。对于侵入试验，插入物预先涂有基质凝胶(1 mg/mL)。培养48h后，细胞移到培养箱的下侧，用甲醇固定，用0.1％结晶紫染色。
1.9统计学处理
采用SPSS 23.0软件(SPSS Inc.,Chicago,IL,USA)和GraphPadPrism version6.0(CA,USA)进行数据分析。用Fisher检验分析MST1与临床病理因素的相关性。采用单因素方差分析，随后采用Newman-Keuls检验，评价各组间差异，并进行重复测量方差分析。Kaplan-Meierand对数秩检验用于评估OS和RFS。用Cox回归分析计算MST1的临床诊断意义。
实施例2MST1在CRC组织中的表达
MST1在CRC组织中呈低表达，首先对275例CRC组织和349例正常组织进行分析(http://gepia.cancer-pku.cn/index.html)评价CRC中MST1的水平，其结果见图1。
图1中A为TCGA数据库(大肠癌和邻近非癌组织)中MST1的mRNA表达；B为MST1在CRC及癌旁组织中的mRNA表达；C为MST1在癌旁组织及癌旁组织中的蛋白表达；D为MST1在细胞中的表达；*P<0.05，**P<0.01。
根据图1检测结果可知，与正常组织相比，MST1在CRC组织中的表达较低(图1A)，采用qRT-PCR和Western bolt法检测CRC组织中MST1的表达细胞。MST1在CRC组织中的mRNA水平明显低于配对正常邻近组织(图1B)。 Western bolt结果也证实了MST1在CRC组织中的表达低于配对正常邻近组织 (图1C)。此外，与正常肝细胞系相比，CRC细胞系中MST1的表达也较低，恶性程度较高的细胞中MST1的表达也较低(图1D)。
实施例3免疫组化检测MST1与CRC临床病理特征相关性
免疫组化结果如图2所示，图2中，A为CRC及癌旁组织MST1表达的免疫组化分析；B为CRC及癌旁组织MST1表达的统计分析。
根据图2检测结果可知，MST1在CRC中的低表达率为61.1％(51/81)(图 2A和B)。此外，MST1的阳性表达主要集中在细胞质中。通过分析MST1与 CRC患者临床病理特征的关系，我们发现低MST1表达与肿瘤大小(P＝0.011) 和分化(P＜0.001)呈正相关。
实施例4MST1在CRC中的表达和预后
Kaplan-Meiersurvival和log-rank结果如图3所示，图3中，A为Kaplan-Meier 法分析CRC患者MST1高表达与低表达；B为CRC MST1高表达与低表达RFS 的Kaplan-Meier分析。
根据图3检测结果可知，在CRC中，低MST1表达与高MST1表达相比显著缩短(P＝0.046；图3A)，分化(P＝0.016)和MST1(P＝0.007)与CRC的 OS显著相关。此外，多变量分析显示肿瘤大小(P＝0.015)、分化程度(P＝0.012) 和MST1(P＝0.013)是CRC的OS独立预后因素。
此外，Kaplan-Meiersurvival和log-rank分析显示，在CRC中，低MST1表达与高MST1表达相比显著的缩短(P＝0.026；图3B)，进行Cox比例风险回归分析，结果显示肿瘤大小(P＝0.033)、分化程度(P＝0.011)和MST1(P＝0.009) 与CRC中的RFS显著相关。此外，多变量分析显示肿瘤大小(P＝0.037)、分化程度(P＝0.016)和MST1(P＝0.011)是CRC的RFS独立预后因素。
实施例5MST1过表达抑制CRC细胞增殖
为探讨MST1对CRC生物学功能的影响，采用LV-shRNA介导MST1的上调表达使用，之后用LV-MST1处理，其结果见图4。
图4中A为MST1在HCT116和SW480细胞中的蛋白表达；B为进行CCK-8 分析，以检查MST1过度表达对HCT116和SW480细胞增殖能力的影响；C为用EdU检测MST1高表达后HCT116和SW480细胞的增殖情况；*P<0.05。
如图4所示，HCT116和SW480细胞株中MST1的表达显著增加(图4A)。另外，在HCT116和SW480细胞系中进行了功能获得实验。CCK-8检测显示，增加MST1的表达显著抑制HCT116和SW480细胞系的细胞增殖(图4B)。如图4C所示，MST1过度表达组中含有EdU的RC细胞数量少于对照组。
实施例6MST1过表达抑制大肠癌的迁移和侵袭
接下来，检测MST1对SW480和HCT116细胞迁移和侵袭的影响，其结果见图5。图5中A为迁移实验检测MST1过表达对HCT116和SW480细胞迁移的影响；B为侵袭实验检测MST1过表达对HCT116和SW480细胞侵袭的影响； *P<0.05，**P<0.01。
如图5所示，MST1的上调抑制了SW480和HCT116细胞的迁移(图5A 和B)。此外，MST1的上调显着抑制了HCT116和SW480细胞株的细胞侵袭(图 5C和D)。
实施例7MST1的过度表达对Hippo信号通路抑制CRC细胞EMT的作用
MST1作为Hippo信号通路的关键调控因子，可以影响hippo信号通路的功能。此外，Transwell分析结果表明，MST1调节CRC细胞中的细胞迁移和侵袭，表明MST1表达与EMT之间存在联系。因此，本研究检测了Hippo信号通路的效应分子和关键EMT蛋白的表达，其结果见图6。
图6中A为p-LATS1、p-YAP、YAP、E-cadherin、N-cadherin和vimentin 在HCT116细胞中的表达；B为p-LATS1、p-YAP、YAP、E-cadherin、N-cadherin 和vimentin在SW480细胞中的表达。
如图6A和B所示，MST1的上调导致E-cadherin表达急剧下降，而p-LATS1、 p-YAP、YAP、N-cadherin和vimentin的的表达显著增加。
综上所述，MST1在CRC患者中低表达，而MST1低表达的患者OS和RFS 较低，并且与不良预后密切相关。更为重要的是，MST1通过Hippo信号通路调节EMT，抑制CRC细胞的增殖、侵袭和迁移，从而发挥抑癌基因的作用，表明 MST1可能是一种很有前景的CRC治疗靶点。
Claims (4)
1.MST1作为药物靶点在制备治疗结直肠癌药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述药物为通过促进MST1的表达来抑制结直肠癌迁移、侵袭和增殖的药物。
3.根据权利要求1所述的应用，其特征在于，所述药物为促进MST1表达的药物。
4.一种用于检测个体MST1表达量的试剂在制备诊断试剂或试剂盒中的用途。
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