CN116794325B - 敲低或抑制slc35f6的试剂在制备激活ampk的药物中的应用 - Google Patents
敲低或抑制slc35f6的试剂在制备激活ampk的药物中的应用 Download PDFInfo
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Abstract
本发明公开了敲低或抑制SLC35F6的试剂在制备激活AMPK的药物中的应用，涉及生物技术领域。本申请发明人研究发现，SLC35F6能够作为AMPK活性调控的新靶点，并通过进一步研究发现，SLC35F6‑ALPP复合体抑制AMPK的活性。本发明发现，敲低SLC35F6或ALPP能够激活AMPK，以SLC35F6或ALPP为目标，选取合适的siRNA，高效敲低SLC35F6或ALPP，为AMPK的激活提供新的技术方案。本发明首次明确了SLC35F6对AMPK活性调控作用，为与AMPK活性相关的疾病的诊断和治疗提供新的防治药物研发途径和药物作用靶点，具有十分重要的药用价值。
Description
技术领域
本发明涉及生物技术领域，具体涉及敲低或抑制SLC35F6的试剂在制备激活AMPK的药物中的应用。
背景技术
AMPK(Adenosine 5‘-monophosphate(AMP)-activated protein kinase)即AMP依赖的蛋白激酶，是生物能量代谢调节的关键分子，广泛存在在动物和植物细胞中的蛋白质分子，是研究糖尿病及其他代谢相关疾病的核心。它表达于各种代谢相关的器官中，能被机体各种刺激激活，包括细胞压力、运动和很多激素及能影响细胞代谢的物质。AMPK作为一种营养和能量传感器，它可以感知细胞内ATP的水平高低。当线粒体被抑制，低能量供应条件下，AMPK磷酸化特异性的酶和位点，增加ATP生成，降低ATP消耗，以此来恢复能量平衡。
AMPK是代谢调节中最重要的激酶，当细胞内能量缺乏时，AMPK被上游激酶磷酸化激活，进而磷酸化许多代谢中的关键酶以及控制蛋白，促进分解代谢，抑制合成代谢，增加ATP的积累，为细胞提供能量。传统观点认为，AMPK的激活是由于细胞内AMP的浓度上升，与AMPK的γ亚基结合，改变AMPK异源三聚体的构象，使其α亚基的T172位点能够被上游激酶LKB1磷酸化，进而激活AMPK全酶。近年来的研究发现，AMPK的激活在不同的亚细胞区域并不一致，AMPK能够在溶酶体上激活，其溶酶体定位依赖于AMPKβ亚基的第二位甘氨酸的豆蔻酰化修饰。另外，除了响应AMP，溶酶体上的AMPK还能响应糖代谢次级产物FBP(果糖-1,6-二磷酸)的浓度变化。溶酶体上调控AMPK活性的蛋白，以及这些蛋白是否能够作为潜在的药物靶点，影响AMPK的活性，现在还不得而知。
现有的AMPK激活剂基于的调控机制主要集中在增加AMPKα的磷酸化，所涉及的调控位点已经饱和。目前还没有针对AMPK去磷酸化的机制，去探索和发现AMPK抑制剂和激活剂。
发明内容
本发明的目的在于克服现有技术的不足，提供敲低或抑制SLC35F6的试剂在制备激活AMPK的药物中的应用。
为实现上述目的，本发明采取的技术方案为：SLC35F6在制备AMPK活性抑制剂中的应用。本申请的目的在于探索AMPK的溶酶体激活机制，进而发现新的AMPK活性调控蛋白。研究显示，溶酶体上的AMPK在能够感受糖代谢次级产物的变化，不同于整个细胞中的AMPK活性调控。所以，溶酶体上应该存在新的AMPK调节蛋白，能够更加直接的调控AMPK活性。本发明利用BiFC和临近标记技术，发现了SLC35F6-ALPP复合体在溶酶体上抑制AMPK的活性，而SLC35F6的配体UDP-GlcNAc能够解除SLC35F6-ALPP复合体对AMPK的抑制，进而激活AMPK，发挥疗效。
本发明还提供敲低或抑制SLC35F6的试剂在制备激活AMPK的药物中的应用。
作为本发明所述应用的优选实施方式，所述试剂包括敲低SLC35F6基因表达的siRNA。
作为本发明所述应用的优选实施方式，所述siRNA的核苷酸序列如SEQ ID NO.1～SEQ ID NO.3任一项所示。
本发明还提供敲低或抑制ALPP的试剂在制备激活AMPK的药物中的应用。
作为本发明所述应用的优选实施方式，所述试剂包括敲低ALPP基因表达的siRNA。
作为本发明所述应用的优选实施方式，所述siRNA的核苷酸序列如SEQ ID NO.4～SEQ ID NO.6任一项所示。
本发明还提供一种AMPK激活剂，所述激活剂包括敲低SLC35F6或ALPP中至少一种的试剂。
本发明还提供敲低SLC35F6或ALPP中至少一种的试剂在制备预防和/或治疗肿瘤、炎症或糖尿病的产品中的应用。
作为本发明所述应用的优选实施方式，所述试剂包括UDP-GlcNAc。
本发明的有益效果：本发明提供敲低或抑制SLC35F6的试剂在制备激活AMPK的药物中的应用。本申请发明人研究发现，SLC35F6能够作为AMPK活性调控的新靶点，并通过进一步研究发现，SLC35F6-ALPP复合体在溶酶体上抑制AMPK的活性。本发明发现，敲低SLC35F6或ALPP能够激活AMPK，以SLC35F6或ALPP为目标，选取合适的siRNA，高效敲低SLC35F6或ALPP，为AMPK的激活提供新的技术方案。本发明首次明确了SLC35F6对AMPK活性调控作用，为与AMPK活性相关的疾病的诊断和治疗提供新的防治药物研发途径和药物作用靶点，具有十分重要的药用价值。
附图说明
图1为实施例1敲低SLC35F6后p-AMPK，AMPK，SLC35F6，tubulin的表达结果图；
图2为实施例2敲低ALPP后p-AMPK，AMPK，p-ACC1，ACC1，ALPP，tubulin的表达结果图；
图3为实施例3细胞裂解液和免疫沉淀后beads中GST，p-AMPK，AMPK，tubulin的表达结果图；
图4为实施例4敲低SLC35F6和饥饿处理后的p-AMPK，AMPK，p-ACC1，ACC1，SLC35F6，tubulin的表达结果图；
图5为实施例4敲低SLC35F6和H2O2处理后的p-AMPK，AMPK，p-ACC1，ACC1，SLC35F6，tubulin的表达结果图；
图6为实施例4敲低SLC35F6和AICAR处理后的p-AMPK，AMPK，p-ACC1，ACC1，SLC35F6，tubulin的表达结果图。
具体实施方式
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
实施例1
本实施例通过实验验证敲低SLC35F6对AMPK活性的影响，具体实验步骤如下：
(1)将生长状态良好的HEK293T细胞加入六孔板中，于37℃，5％CO2培养；(2)将终浓度为100nM对照siRNA(由广州锐博生物提供,货号
#siN0000001-1-5)、100nM用于敲低SLC35F6的实验组siRNA分别与10ul的RNAimax试剂、200ul的optimen混匀，得混合液；所述实验组siRNA的序列分别为：
实验组一(#1)：5′-GCAUGGUGUUGGACAGCUU-3′；
实验组二(#2)：5′-GCAUUGCCUUCUUCAACUU-3′；
实验组三(#3)：5′-GAGGAGAAGUUCGUCUACA-3′。
(3)将混合液室温静置20min后，加入六孔板的HEK293T细胞中，6小时后更换新鲜培养基；48h后，收集并裂解细胞，1000gx10min离心，收集上清，并提取蛋白。
(4)免疫印迹检测：将步骤(3)得到的蛋白进行聚丙烯酰胺凝胶电泳，电泳参数为：恒压80V，20min加上120v，45min，然后恒流0.2A，1h转膜，5％脱脂牛奶封闭1h；用一抗p-AMPK，AMPK，SLC35F6，tubulin孵育过夜，第二天再敷育对应的二抗，检测相应蛋白的表达水平。
实验结果如图1所示。p-AMPK(T172)表明AMKP的磷酸化程度，代表AMPK的活性。由图1可知看出，实验组没有表达SLC35F6，说明实验组成功敲低SLC35F6；对照组的p-AMPK的表达低于实验组，说明敲低SLC35F6后AMPK被激活。
实施例2
本实施例通过实验验证敲低ALPP对AMPK活性的影响，具体实验步骤如下：
(1)将生长状态良好的HEK293T细胞加入六孔板中，于37℃，5％CO2培养；
(2)将终浓度为100nM对照siRNA、100nM用于敲低ALPP的实验组siRNA分别与10ul的RNAimax试剂、200ul的optimen混匀，得混合液；所述实验组siRNA的序列分别为：
实验组一(#1)：5′-CUACGCAGCUCAUCUCCAA-3′；
实验组二(#2)：5′-CUGGAGACAUGAAAUACGA-3′；
实验组三(#3)：5′-CGGUCCUCCUAUACGGAAA-3′。
(3)将混合液室温静置20min后，加入六孔板的HEK293T细胞中，6小时后更换新鲜培养基；48h后，收集并裂解细胞，1000gx10min离心，收集上清，并提取蛋白。
(4)免疫印迹检测：将步骤(3)得到的蛋白进行聚丙烯酰胺凝胶电泳，电泳参数为：恒压80V，20min加上120v，45min，然后恒流0.2A，1h转膜，5％脱脂牛奶封闭1h；用一抗p-AMPK，AMPK，p-ACC1，ACC1，ALPP，tubulin孵育过夜，第二天再敷育对应的二抗，检测相应蛋白的表达水平。
实验结果如图2所示。由图2可知看出，实验组没有表达ALPP，说明实验组成功敲低ALPP；对照组的p-AMPK的表达低于实验组，说明敲低ALPP后AMPK被激活。
实施例3
本实施例通过实验验证降低SLC35F6和ALPP的相互作用对AMPK活性的影响，具体实验步骤如下：
(1)将生长状态良好的HEK293T细胞加入六孔板中，于37℃，5％CO2培养；采用PEI将Flag-SLC35F6和GST-ALPP顺转入HEK293T细胞，6小时后换液；
(2)48h后，裂解细胞，在细胞裂解液中加入UDP-GlcNAc孵育1h；
(3)2h后在裂解液中加入GST beads，旋转孵育2小时；
(4)beads沉淀后，用裂解液洗涤三次；
(5)采用免疫印迹检测细胞系裂解液和免疫沉淀后beads中GST，p-AMPK，AMPK，tubulin的水平。
实验结果如图3所示。Flag和GST代表过表达的Flag-SLC35F6和GST-ALPP。5％input组里面，代表总的含量，IP组里面，代表免疫共沉淀下拉下来的成分，IP/Input的比值越高，代表相互结合越强。由图3可知，相比于Veh的对照组(仅含有1XPBS)，UDP-GlcNAc的加入使得p-AMPK增加，表明UDP-GlcNAc能够提升AMPK的活性(对比第二泳道和第三泳道)。同时，UDP-GlcNAc能够降低GST磁珠免疫沉淀GST-ALPP过后的Flag-SLC35F6，表明UDP-GlcNAc能够降低GST-ALPP和Flag-SLC35F6的相互作用。综上，UDP-GlcNAc能够通过降低SLC35F6和ALPP的相互作用，进而提升AMPK的活性。
实施例4
本实施例验证敲低SLC35F6联合常规AMPK激活剂对AMPK活性的影响，具体实验步骤如下：
(1)将生长状态良好的HEK293T细胞加入六孔板中，于37℃，5％CO2培养；
(2)将终浓度为100nM对照siRNA、100mM用于敲低SLC35F6的实验组siRNA分别与10ul的RNAimax试剂、200ul的optimen混匀，得混合液；
(3)将混合液室温静置20min后，加入六孔板的HEK293T细胞中，6小时后更换新鲜培养基；
(4)48h后，分别进行饥饿处理：使用无葡萄糖培养基代替正常培养基16h、双氧水处理：正常培养的细胞中加入浓度为1mM的H2O2,5min、AICAR处理：正常培养的细胞中加入AMPK激活剂2mM AICAR 4h；
(5)收集并裂解细胞，1000gx10min离心，收集上清，并提取蛋白；
(6)将蛋白进行免疫印迹检测：聚丙烯酰胺凝胶电泳，电泳参数：恒压80V，20min加上120v，45min，然后恒流0.2A，1h转膜，5％脱脂牛奶封闭1h；用一抗p-AMPK，AMPK，p-ACC1，ACC1，SLC35F6，tubulin孵育过夜，第二天再敷育对应的二抗，检测相应蛋白的表达水平。
结果如图4～6所示。由图4～6可知，采用常规AMPK激活剂(如葡萄糖饥饿16小时，双氧水处理5分钟，AMP类似物AICAR处理4小时)都能够提升AMPK的磷酸化(对比第一泳道和第二泳道)。敲低SLC35F6联合常规AMPK激活剂，能在本来已经激活的AMPK基础上更近一步激活(对比第二泳道和第四泳道)，由此表明敲低SLC35F6与常规AMPK激活剂联用能够更好的提升AMPK的活性。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
Claims (2)
1.敲低SLC35F6的试剂在制备激活AMPK的药物中的应用；所述试剂包括敲低SLC35F6基因表达的siRNA；所述siRNA的核苷酸序列如SEQ ID NO.1~SEQ ID NO.3任一项所示。
2.敲低ALPP的试剂在制备激活AMPK的药物中的应用；所述试剂包括敲低ALPP基因表达的siRNA；所述siRNA的核苷酸序列如SEQ ID NO.4~SEQ ID NO.6任一项所示。
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