CN113817797A - 一种研究乳酸影响肺癌细胞转移侵袭能力的方法 - Google Patents
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Abstract
本发明公开了一种研究乳酸影响肺癌细胞转移侵袭能力的方法，包括如下步骤：S1、验证乳酸诱导EMT表型更加明显；S2、验证Snail表达诱导EMT表型更加明显；S3、观察肺癌细胞H1299和A549经乳酸处理的EMT表型；S4、LKB1野生型的H1299的EMT表型明显、LKB1缺失型的A549的EMT表型不明显；S5、验证LKB1诱导了Snail表达；S6、推测过量乳酸影响了细胞内能量代谢的动态平衡；S7、推测LKB1可能通过调控下游激酶AMPK或者Src诱导了Snail表达；S8、验证AMPK磷酸化是因LKB1活性改变导致；S9、同时利用不同信号传导途径抑制剂，观察哪种信号通路抑制剂可影响乳酸对AMPK的磷酸化；S10、利用不同信号转导通路抑制剂筛选出介导LKB1调控Src激酶的信号通路。本方法能够阐明肿瘤转移的分子机制。
Description
技术领域
本发明涉及细胞治疗技术领域，具体涉及一种研究乳酸影响肺癌细胞转移侵袭能力的方法。
背景技术
肺癌目前相对的5年生存率仅为18％，造成肺癌病人远期生存率较低的主要原因是肿瘤的侵袭和转移，研究表明，约80-90％的肺癌死亡病例是由肿瘤转移引起的，而转移的分子机制尚不清楚。现有研究中发现肿瘤Warburg效应中其终产物乳酸通过诱导转录因子Snail来促进肺癌细胞EMT的发生，从而诱发侵袭转移，同时，发现能量代谢关键调控基因LKB1的相关通路介导了Warburg效应促进肺癌细胞侵袭转移的过程。
在现有技术中，缺乏一种对LKB1相关通路在介导Warburg效应促进肺癌细胞侵袭转移过程中的作用及机制的研究方法，从而无法筛选在此过程中的关键调控蛋白并明确其功能，为进一步阐明肿瘤转移的分子机制以及为肿瘤治疗提供新的分子靶点和思路。
发明内容
本发明的目的在于提供一种研究乳酸影响肺癌细胞转移侵袭能力的方法，以解决现有技术中因缺乏对LKB1相关通路介导Warburg效应的机制的研究，导致无法筛选LKB1相关通路参与调控的蛋白的技术问题。
为解决上述技术问题，本发明具体提供下述技术方案，
一种研究乳酸影响肺癌细胞转移侵袭能力的方法，包括如下步骤：
S1、通过将肺癌细胞的观察EMT表型是否明显作为判断肺癌细胞的迁移能力和侵袭能力的指标，并将肺癌细胞置于乳酸环境中，以验证乳酸诱导EMT表型更加明显；
S2、推测乳酸能够促进肺癌细胞中Snail表达，而Snail表达诱导EMT表型更加明显，然后通过在肺癌细胞株中转染Snail的siRNA观察EMT表型是否逆转，以验证Snail表达诱导EMT表型更加明显；
S3、取两种体外肺癌细胞H1299和A549，分别将H1299和A549置于相同浓度的乳酸中，并通过抑制体外肺癌细胞H1299和A549自身的乳酸，然后，观察肺癌细胞H1299和A549的EMT表型；
S4、根据步骤S3观察结果，LKB1野生型的H1299的EMT表型明显、LKB1缺失型的A549的EMT表型不明显；
S5、推测乳酸通过控制LKB1诱导了Snail表达，并通过向A549内转入LKB1的cDNA，向H1299中敲除LKB1，再重复步骤S3，以验证LKB1诱导了Snail表达；
S6、推测过量乳酸影响了细胞内能量代谢的动态平衡，以提供LKB1的活性，或者导致LKB1胚系突变；
S7、推测LKB1可能通过调控下游激酶AMPK或者Src诱导了Snail表达；
S8、在H1299细胞中，敲除LKB1，在A549细胞中转入LKB1的cDNA，然后，通过对照试验，验证AMPK磷酸化是因LKB1活性改变导致；
S9、同时利用不同信号传导途径抑制剂，观察哪种信号通路抑制剂可影响乳酸对AMPK的磷酸化；
S10、将在LKB1缺失的A549细胞中，转入LKB1的cDNA，以及在LKB1野生型的H1299细胞中转入LKB1siRNA，检测LKB1与Src激酶间的关系，利用不同信号转导通路抑制剂筛选出介导LKB1调控Src激酶的信号通路。
作为本发明的一种优选方案，在步骤S8中，所述对照试验包括如下步骤，
S801、乳酸或过氧化氢在体外肺癌细胞中，对LKB1磷酸化和AMPK磷酸化的影响；
S802、向A549内转入LKB1的cDNA，按步骤S801处理A549；
S803、将已构建好的SOD2和线粒体过氧化氢酶的cDNA分别转染至肺癌细胞中,观察过表达SOD2和线粒体中过氧化氢酶能否影响乳酸或过氧化氢所诱导的AMPK磷酸化以及Snail的表达；
S804、将在H1299细胞中,敲除LKB1；在A549细胞中转入LKB1的cDNA，之后利用步骤S803中的方法处理细胞，明确此p-AMPK确实来源于LKB1活性改变。
本发明与现有技术相比较具有如下有益效果，
本发明通过分子和细胞水平展开深入研究，揭示了LKB1相关通路在介导Warburg效应促进肺癌细胞侵袭转移过程中的作用及机制，能够筛选相应的关键调控蛋白并明确其功能，为进一步阐明肿瘤转移的分子机制以及为肿瘤治疗提供新的分子靶点和思路。
附图说明
为了更清楚地说明本发明的实施方式或现有技术中的技术方案，下面将对实施方式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示例性的，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，还可以根据提供的附图引伸获得其它的实施附图。
图1为本发明实施方式中的技术路线图；
图2为本发明实施方式中步骤S1的结果图；
图3为本发明实施方式中步骤S2的结果图；
图4为本发明实施方式中步骤S3中H1299的结果图；
图5为本发明实施方式中步骤S3中A549的结果图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所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做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。
如图1所示，本发明提供了一种研究乳酸影响肺癌细胞转移侵袭能力的方法，包括如下步骤：
S1、通过将肺癌细胞的观察EMT表型是否明显作为判断肺癌细胞的迁移能力和侵袭能力的指标，并将肺癌细胞置于乳酸环境中，以验证乳酸诱导EMT表型更加明显；
S2、推测乳酸能够促进肺癌细胞中Snail表达，而Snail表达诱导EMT表型更加明显，然后通过在肺癌细胞株中转染Snail的siRNA观察EMT表型是否逆转，以验证Snail表达诱导EMT表型更加明显；
S3、取两种体外肺癌细胞H1299和A549，分别将H1299和A549置于相同浓度的乳酸中，并通过抑制体外肺癌细胞H1299和A549自身的乳酸，然后，观察肺癌细胞H1299和A549的EMT表型；
S4、根据步骤S3观察结果，LKB1野生型的H1299的EMT表型明显、LKB1缺失型的A549的EMT表型不明显；
S5、推测乳酸通过控制LKB1诱导了Snail表达，并通过向A549内转入LKB1的cDNA，向H1299中敲除LKB1，再重复步骤S3，以验证LKB1诱导了Snail表达；
S6、推测过量乳酸影响了细胞内能量代谢的动态平衡，以提供LKB1的活性，或者导致LKB1胚系突变；
S7、推测LKB1可能通过调控下游激酶AMPK或者Src诱导了Snail表达；
S8、在H1299细胞中，敲除LKB1，在A549细胞中转入LKB1的cDNA，然后，通过对照试验，验证AMPK磷酸化是因LKB1活性改变导致；
S9、同时利用不同信号传导途径抑制剂，观察哪种信号通路抑制剂可影响乳酸对AMPK的磷酸化；
S10、将在LKB1缺失的A549细胞中，转入LKB1的cDNA，以及在LKB1野生型的H1299细胞中转入LKB1siRNA，检测LKB1与Src激酶间的关系，利用不同信号转导通路抑制剂筛选出介导LKB1调控Src激酶的信号通路。
用外源乳酸(不同浓度)处理肺癌细胞株，细胞的迁移速率增快，侵袭能力增强，证实乳酸对肺癌细胞株的侵袭转移能力有着明显的影响。我们同时发现，在高糖培养条件下诱导的Snail表达及EMT表型同样均可被乳酸脱氢酶的两种siRNA可逆转。由此推断，有氧糖酵解中代谢产物乳酸在促进肺癌细胞产生EMT过程中起到关键作用，且很有可能通过诱导转录因子Snail来发挥作用。随后在肺癌细胞株中转染SnailsiRNA，发现乳酸对肺癌细胞EMT表型的诱导可被部分逆转，证实了乳酸是通过诱导Snail来促进肺癌细胞EMT的发生。
用不同浓度的乳酸分别处理A549，H1299细胞株，发现在LKB1野生型的H1299细胞系中，乳酸促进了Snail的表达以及EMT表型，但在LKB1缺失型的A549中，乳酸诱导的EMT表型并不明显。细胞外乳酸可通过MCT1转运蛋白进入细胞，我们利用MCT1抑制剂CHC以及MCT1的siRNA发现，两者均可部分逆转乳酸诱导肺癌细胞中Snail的表达及EMT的表型。由此可见，重新进入细胞内的乳酸促进了肺癌细胞EMT的产生。
我们在LKB1缺失的A549细胞及NCI-H460细胞中转入LKB1cDNA以及在LKB1野生型H1299细胞中转入LKB1shRNA发现LKB1诱导Src抑制位点527磷酸化，同时在高糖条件下LKB1siRNA促进H1299细胞的迁移可被Src抑制剂PP2所逆转。结果提示LKB1可能通过调控Src活性抑制细胞迁移。
本发明通过分子和细胞水平展开深入研究，揭示了LKB1相关通路在介导Warburg效应促进肺癌细胞侵袭转移过程中的作用及机制，能够筛选相应的关键调控蛋白并明确其功能，为进一步阐明肿瘤转移的分子机制以及为肿瘤治疗提供新的分子靶点和思路。
在步骤S8中，所述对照试验包括如下步骤，
S801、乳酸或过氧化氢在体外肺癌细胞中，对LKB1磷酸化和AMPK磷酸化的影响；
S802、向A549内转入LKB1的cDNA，按步骤S801处理A549；
S803、将已构建好的SOD2和线粒体过氧化氢酶的cDNA分别转染至肺癌细胞中,观察过表达SOD2和线粒体中过氧化氢酶能否影响乳酸或过氧化氢所诱导的AMPK磷酸化以及Snail的表达；
S804、将在H1299细胞中,敲除LKB1；在A549细胞中转入LKB1的cDNA，之后利用步骤S803中的方法处理细胞，明确此p-AMPK确实来源于LKB1活性改变。
以上实施例仅为本申请的示例性实施例，不用于限制本申请，本申请的保护范围由权利要求书限定。本领域技术人员可以在本申请的实质和保护范围内，对本申请做出各种修改或等同替换，这种修改或等同替换也应视为落在本申请的保护范围内。
Claims (2)
1.一种研究乳酸影响肺癌细胞转移侵袭能力的方法，其特征在于，包括如下步骤：
S1、通过将肺癌细胞的观察EMT表型是否明显作为判断肺癌细胞的迁移能力和侵袭能力的指标，并将肺癌细胞置于乳酸环境中，以验证乳酸诱导EMT表型更加明显；
S2、推测乳酸能够促进肺癌细胞中Snail表达，而Snail表达诱导EMT表型更加明显，然后通过在肺癌细胞株中转染Snail的siRNA观察EMT表型是否逆转，以验证Snail表达诱导EMT表型更加明显；
S3、取两种体外肺癌细胞H1299和A549，分别将H1299和A549置于相同浓度的乳酸中，并通过抑制体外肺癌细胞H1299和A549自身的乳酸，然后，观察肺癌细胞H1299和A549的EMT表型；
S4、根据步骤S3观察结果，LKB1野生型的H1299的EMT表型明显、LKB1缺失型的A549的EMT表型不明显；
S5、推测乳酸通过控制LKB1诱导了Snail表达，并通过向A549内转入LKB1的cDNA，向H1299中敲除LKB1，再重复步骤S3，以验证LKB1诱导了Snail表达；
S6、推测过量乳酸影响了细胞内能量代谢的动态平衡，以提供LKB1的活性，或者导致LKB1胚系突变；
S7、推测LKB1可能通过调控下游激酶AMPK或者Src诱导了Snail表达；
S8、在H1299细胞中，敲除LKB1，在A549细胞中转入LKB1的cDNA，然后，通过对照试验，验证AMPK磷酸化是因LKB1活性改变导致；
S9、同时利用不同信号传导途径抑制剂，观察哪种信号通路抑制剂可影响乳酸对AMPK的磷酸化；
S10、将在LKB1缺失的A549细胞中，转入LKB1的cDNA，以及在LKB1野生型的H1299细胞中转入LKB1siRNA，检测LKB1与Src激酶间的关系，利用不同信号转导通路抑制剂筛选出介导LKB1调控Src激酶的信号通路。
2.根据权利要求1所述的一种研究乳酸影响肺癌细胞转移侵袭能力的方法，其特征在于，在步骤S8中，所述对照试验包括如下步骤，
S801、乳酸或过氧化氢在体外肺癌细胞中，对LKB1磷酸化和AMPK磷酸化的影响；
S802、向A549内转入LKB1的cDNA，按步骤S801处理A549；
S803、将已构建好的SOD2和线粒体过氧化氢酶的cDNA分别转染至肺癌细胞中,观察过表达SOD2和线粒体中过氧化氢酶能否影响乳酸或过氧化氢所诱导的AMPK磷酸化以及Snail的表达；
S804、将在H1299细胞中,敲除LKB1；在A549细胞中转入LKB1的cDNA，之后利用步骤S803中的方法处理细胞，明确此p-AMPK确实来源于LKB1活性改变。
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