CN117144008B - 三阴性乳腺癌生物标志物及其应用 - Google Patents
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Abstract
本发明涉及生物医药技术领域，提出了三阴性乳腺癌生物标志物及其应用，公开了生物标志物在制备诊断或预测三阴性乳腺癌的产品中的应用，所述生物标志物为MBNL3和KLHL31。本发明的MBNL3和KLHL31能够显著促进三阴性乳腺癌细胞的增殖，并且两者联合处理后发挥的促进增殖的功能更加显著，为三阴性乳腺癌的诊断、预测和治疗提供了新的靶点。
Description
技术领域
本发明涉及生物医药技术领域，尤其涉及三阴性乳腺癌生物标志物及其应用。
背景技术
乳腺癌是威胁全世界女性生命健康安全的重要因素。而三阴性乳腺癌(TNBC)是所有乳腺癌亚型治疗中最棘手的一种，死亡率最高且最容易复发。TNBC因其特殊的分子遗传背景(雌激素受体ER、孕激素受体PR及人表皮生长因子受体2HER2均为阴性)对激素和靶向治疗都不敏感，临床上治疗的方法几乎只有化疗。尽管三阴性乳腺癌对化疗具有一定的敏感性，但患者经过常规化疗治疗后预后仍然较差。研究统计发现，III-IV期三阴性乳腺癌患者五年生存率仅为13％。并且还有一部分患者在化疗治疗过程中因出现多药耐药的情况导致治疗失败。因此，深入探究三阴性乳腺癌的发病机制，挖掘新的诊断标志物和新的药物靶点对临床上预防和治疗TNBC至关重要。
盲肌样蛋白(MBNL)是一类RNA结合蛋白，能够调控RNA成熟和表达过程的多个步骤，包括靶基因pre-mRNA的剪接、降解、RNA输出、稳定性维持、修饰和翻译等，在转录后层面广泛参与调控多种基因的表达和功能。盲肌样蛋白家族包含MBNL1、MBNL2、MBNL3三个成员，其中MBNL1是目前研究最多的家族成员。MBNL1已被证实在多种肿瘤组织中异常表达，有望成为新的肿瘤诊断标志物并且靶向MBNL1能够达到肿瘤治疗的作用。而作为与MBNL1具有相同结合位点的同家族成员MBNL3虽然在肿瘤中的相关研究不多，尤其是MBNL3在三阴性乳腺癌中的功能尚未见相关报道。
KLHL31是Kelch-like(KLHL)蛋白家族中的一员。迄今为止，已被鉴定出的KLHL家族成员有42个，该家族成员均含有一个BTB/POZ结构域、一个BACK结构域以及五至六个重复的Kelch基序，而且它们在进化上高度保守。研究发现，具有BTB结构域的蛋白在细胞中可以通过与Cullin3蛋白相互作用形成E3泛素连接酶，催化特定底物蛋白质发生泛素化修饰，参与泛素-蛋白酶水解途径(UPP)，选择性降解细胞内多种具有生物活性的蛋白质，在翻译后基因表达调控中发挥着至关重要的作用。近年来，随着高通量测序技术的发展，KLHL蛋白家族成员越来越多的功能被发现，且被证实在肿瘤的发生发展中具有重要的生物学功能，但是KLHL31在乳腺癌特别是三阴性乳腺癌中的功能尚未见相关报道。
发明内容
有鉴于此，本发明提出了MBNL3和KLHL31三阴性乳腺癌生物标志物及其应用。
本发明的技术方案是这样实现的：第一，本发明提供了生物标志物在制备诊断或预测三阴性乳腺癌的产品中的应用，所述生物标志物为MBNL3和KLHL31。
在以上技术方案的基础上，优选的，所述诊断或预测三阴性乳腺癌的产品通过使用免疫法、northern杂交方法、印迹杂交、qRTPCR、基因芯片、原位杂交阵列杂交、核酶保护分析技术、二代测序方法或单分子测序方法来检测所述MBNL3和KLHL31生物标志物的水平以诊断或预测乳腺癌。
在以上技术方案的基础上，优选的，检测样本选自血液或组织。
在以上技术方案的基础上，优选的，所述MBNL3和KLHL31在三阴性乳腺癌患者血清中表达水平同时上调。
第二，本发明提供了检测MBNL3和KLHL31表达水平的试剂在制备诊断或预测三阴性乳腺癌的产品中的应用。
在以上技术方案的基础上，优选的，所述产品为制剂、芯片或试剂盒。
第三，本发明提供了MBNL3和KLHL31的抑制剂在制备治疗三阴性乳腺癌的药物中的应用。
在以上技术方案的基础上，优选的，所述抑制剂包括抑制MBNL3和KLHL31基因的功能、下调MBNL3和KLHL31基因mRNA水平、减少MBNL3和KLHL31基因mRNA有效作用时间的物质。
第四，本发明提供了一种治疗三阴性乳腺癌的药物，所述药物包括MBNL3和KLHL31的抑制剂。
在以上技术方案的基础上，优选的，所述抑制剂是针对MBNL3和KLHL31的siRNA
本发明的三阴性乳腺癌生物标志物及其应用相对于现有技术具有以下有益效果：
(1)本发明的MBNL3和KLHL31能够显著促进三阴性乳腺癌细胞的增殖，并且两者联合处理后发挥的促进增殖的功能更加显著，本发明为三阴性乳腺癌的诊断、预测和治疗提供了新的靶点。
(2)本发明揭示了MBNL3和KLHL31调控三阴性乳腺癌细胞增殖的分子作用机制，MBNL3通过与抑癌基因PTEN mRNA的3`-UTR结合降低其mRNA的稳定性，在转录后层面上抑制PTEN的表达。而KLHL31则是通过与PTEN蛋白直接结合，介导PTEN蛋白的泛素化降解，在翻译后层面上抑制PETN的表达。MBNL3和KLHL31分别通过转录后层面和翻译后层面调控PTEN的表达，发挥促进三阴性乳腺癌细胞增殖的作用。由此可见，抑制MBNL3和KLHL31的组合药物或基因疗法可用于对三阴性乳腺癌的诊断和治疗。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为MBNL3和KLHL31对三阴性乳腺癌MDA-MB-231细胞活力的影响结果图；
图2为MBNL3和KLHL31过表达抑制PTEN的mRNA水平示意图；
图3为MBNL3和KLHL31过表达抑制PTEN的蛋白水平示意图；
图4为MBNL3和KLHL31通过抑制PTEN的表达促进TNBC细胞增殖的示意图。
具体实施方式
下面将结合本发明实施方式，对本发明实施方式中的技术方案进行清楚、完整地描述，显然，所描述的实施方式仅仅是本发明一部分实施方式，而不是全部的实施方式。基于本发明中的实施方式，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施方式，都属于本发明保护的范围。
本发明提供一种能够用于三阴性乳腺癌诊断、预测和治疗的新靶点：MBNL3和KLHL31，通过使用免疫法、northern杂交方法、印迹杂交、qRTPCR、基因芯片、原位杂交阵列杂交、核酶保护分析技术、二代测序方法或单分子测序方法来检测MBNL3和KLHL31生物标志物的水平来诊断或预测乳腺癌，检测结果为：MBNL3和KLHL31在三阴性乳腺癌患者血清中表达水平同时上调，抑制MBNL3和KLHL31的组合药物或基因疗法可用于对三阴性乳腺癌的诊断和治疗。具体检测方法见实施例1-3。
实施例1MBNL3和KLHL31促进TNBC细胞的增殖
选择生长状态良好，处于对数生长期的三阴性乳腺癌患者细胞MDA-MB-231以1×104个/孔的密度接种于96孔板中(每组设置6个复孔)，另设实验对照组(含培养液和细胞)、空白对照组(只加正常培养液，不加细胞)，细胞铺板后置于37℃、5％CO2培养箱中培养24h，24h后进行转染，按照表1所示的分组分别取质粒加入10μL无血清培养基中混匀作为A液；取0.3μLPEI转染试剂于10μL无血清培养基中混匀作为B液；混匀后的A液和B液室温放置5min，随后将B液加入A液中涡旋震荡10s，混匀后的转染复合物室温孵育25min。在孵育的过程中，弃去待转染细胞的上清并加入100μL无血清的DMEM高糖培养基，待转染复合物孵育完毕后螺旋式转圈加入待转染的细胞中，放置于细胞培养箱中培养6h之后将无血清培养基更换为完全培养基，培养72h后加入CCK-8试剂(10μL/孔)，37℃孵育2h后用酶标仪在450nm波长处测得吸光值，并按照细胞相对活力(％)＝(实验组吸光度-空白组吸光度)/(对照组吸光度-空白组吸光度)×100％公式计算细胞相对活力值并进行统计分析。
表1转染质粒的量
图1结果显示：MBNL3和KLHL31对MDA-MB-231细胞的生长具有显著促进作用，并且相比于MBNL3和KLHL31单独处理组，两者联合处理组的促进效果更加显著。
实施例2MBNL3和KLHL31抑制PTEN的表达
一、MBNL3和KLHL31过表达抑制PTEN的mRNA水平
选择生长状态良好，处于对数生长期的三阴性乳腺癌患者细胞MDA-MB-231以1×106个/孔的密度接种于6孔板中，细胞铺板后置于37℃、5％CO2培养箱中培养24h，24h后进行转染，按照表2所示的分组分别取质粒加入100μL无血清培养基中混匀作为A液；取6μLPEI转染试剂于100μL无血清培养基中混匀作为B液；混匀后的A液和B液室温放置5min，随后将B液加入A液中涡旋震荡10s，混匀后的转染复合物室温孵育25min。在孵育的过程中，弃去待转染细胞的上清并加入2mL无血清的DMEM高糖培养基，待转染复合物孵育完毕后螺旋式转圈加入待转染的细胞中，放置于细胞培养箱中培养6h之后将无血清培养基更换为完全培养基，培养72h后弃原培养基，弃原培养基，每孔加入500μL 1×PBS溶液洗1次，弃PBS，每孔加入1mLTrizol试剂，置于4℃冰箱的摇床中摇20min。20min后反复吹打孔板底部，并将Trizol移至无核酸酶的1.5mL EP管中，加入1/5Trizol体积的氯仿即200μL氯仿，上下轻轻颠倒混匀，冰上放置10min。随后12000g 4℃离心7min。离心结束后，取水相上清加入预冷的等体积异丙醇，上下颠倒混匀，置于-20℃静置1h。1h后12000g 4℃离心10min，EP管管底会出现白色沉淀。弃上清，加入1mL 75％的乙醇上下颠倒清洗白色沉淀，12000g 4℃离心10min。弃尽上清，EP管敞口室温放置5min挥发残留的乙醇，随后加入适量DEPC水溶解RNA沉淀。
用微量定量仪测定RNA含量并电泳鉴定RNA结构完整后，用逆转录试剂盒(南京诺唯赞生物科技股份有限公司)合成cDNA。以合成的cDNA为模板，依据ChamQ SYBR qPCRMasterMix试剂盒说明书进行实时荧光定量PCR检测(GAPDH为内参基因)。Realtime PCR引物序列如下：PTEN，F：5′-AGACCATA ACCCACCACAGC-3′，R：5′-ACCAGTTCGTCCCTTTCCAG-3′；β-actin，F：5`-TCA AGAAGGTGGTGAAGCAG-3`，R：5`-AGGTGGAGGAGTGGGTGTCG-3`。
表2转染质粒的量
图2结果显示：与对照组相比，MBNL31的过表达能显著抑制PTEN的mRNA水平，但是PTEN的mRNA水平不受KLHL31表达的变化。
二、MBNL3和KLHL31过表达抑制PTEN的蛋白水平
选择生长状态良好，处于对数生长期的三阴性乳腺癌患者细胞MDA-MB-231以1×106个/孔的密度接种于6孔板中，细胞铺板后置于37℃、5％CO2培养箱中培养24h，24h后进行转染，按照表2所示的分组取质粒分别加入100μL无血清培养基中混匀作为A液；取6μLPEI转染试剂于100μL无血清培养基中混匀作为B液；混匀后的A液和B液室温放置5min，随后将B液加入A液中涡旋震荡10s，混匀后的转染复合物室温孵育25min。在孵育的过程中，弃去待转染细胞的上清并加入2mL无血清的DMEM高糖培养基，待转染复合物孵育完毕后螺旋式转圈加入待转染的细胞中，放置于细胞培养箱中培养6h之后将无血清培养基更换为完全培养基，培养72h后弃原培养基，每孔加入500μL 1×PBS溶液洗1次，弃PBS，每孔加入200μL含有蛋白酶抑制剂的IP裂解液，冰上裂解30min。随后用细胞刮子收集裂解液，12000g 4℃离心10min，取上清，并利用BCA蛋白浓度试剂盒进行蛋白定量。随后上清液加入5×上样缓冲液并置于沸水浴中处理10min变性，每个样本按照20μg/孔的蛋白量上样，通过10％的SDS-PAGE凝胶电泳分离蛋白并转印至0.45μm的PVDF膜上。随后将膜放入5％BSA(牛血清白蛋白)溶液中在37℃恒温箱中封闭1h。依据PTEN和GAPDH的抗体说明书稀释一抗，4℃孵育过夜。次日用1×TBST溶液洗膜3次(10min/次)后，室温孵育HRP标记的二抗1h，孵育结束后1×TBST溶液洗膜3次(10min/次)，随后通过ECL发光液进行化学发光成像并采集图像(以β-actin为内参蛋白)。
表3转染质粒的量
图3结果显示：与对照组相比，MBNL3和KLHL31的过表达均能显著抑制PTEN的蛋白水平。
实施例3MBNL3和KLHL31通过抑制PTEN的表达促进TNBC细胞的增殖
选择生长状态良好，处于对数生长期的三阴性乳腺癌患者细胞MDA-MB-231细胞以1×104个/孔的密度接种于96孔板中(每组设置6个复孔)，另设实验对照组(含培养液和细胞)、空白对照组(只加正常培养液，不加细胞)，细胞铺板后置于37℃、5％CO2培养箱中培养24h，24h后进行转染，按照如下图所示的分组分别取质粒加入10μL无血清培养基中混匀作为A液；取0.3μLPEI转染试剂于10μL无血清培养基中混匀作为B液；混匀后的A液和B液室温放置5min，随后将B液加入A液中涡旋震荡10s，混匀后的转染复合物室温孵育25min。在孵育的过程中，弃去待转染细胞的上清并加入100μL无血清的DMEM高糖培养基，待转染复合物孵育完毕后螺旋式转圈加入待转染的细胞中，放置于细胞培养箱中培养6h之后将无血清培养基更换为完全培养基，24h后每个孔转染0.1μg pcDNA3.1-PTEN质粒(对照组转染0.1μg的pcDNA3.1)，转染6h之后将无血清培养基更换为完全培养基，培养48h后加入CCK-8试剂(10μL/孔)，37℃孵育2h后用酶标仪在450nm波长处测得吸光值，并按照细胞相对活力(％)＝(实验组吸光度-空白组吸光度)/(对照组吸光度-空白组吸光度)×100％公式计算细胞相对活力值并进行统计分析。
表4转染质粒的量
图4结果显示：MBNL3和KLHL31对MDA-MB-231细胞的生长具有显著促进作用，并且相比于MBNL3和KLHL31单独处理组，两者联合处理组的促进效果更加显著，而利用回复实验上调PTEN的表达后能显著抑制MBNL3和KLHL31的功能，抑制TNBC细胞的增殖，说明MBNL3和KLHL31的确是通过抑制PTEN的表达促进TNBC细胞的增殖。
以上所述仅为本发明的较佳实施方式而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (2)
1.检测MBNL3和KLHL31表达水平的试剂在制备诊断三阴性乳腺癌的产品中的应用。
2.如权利要求1所述的应用，其特征在于：所述产品为试剂盒。
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