miRNAs在叶酸缺乏诱导自噬中可能的调控机制研究
摘要
叶酸是水溶性维生素B9,作为一碳单位的载体,主要参与DNA合成和DNA甲基化这两个重要的生物化学过程。叶酸缺乏在发育异常、衰老、肿瘤等各种疾病中发挥着重要作用,可诱导细胞死亡,其中细胞自噬是细胞死亡的一种类型。自噬是细胞在饥饿条件下维持存活的基本分解代谢机制。本团队前期研究发现叶酸缺乏可诱导小鼠胚胎干细胞发生凋亡且miRNAs是胚胎发育过程中增殖分化和凋亡的关键调控因素,另有研究发现自噬在脊椎动物发育中发挥关键作用。但是叶酸缺乏、自噬和miRNAs三者间的关系尚未明确。因此我们选取小鼠胚胎干细胞为研究模型,探索叶酸缺乏是否可诱发自噬,miRNAs是否参与叶酸缺乏诱导的自噬。进而研究在叶酸缺乏和正常叶酸条件下自噬的关键蛋白的表达状况,筛选、验证参与该过程的miRNAs和mRNAs。以期多角度研究叶酸缺乏,miRNA调控,自噬发生间的关联性。
目的
以小鼠胚胎干细胞为模型,研究叶酸缺乏、自噬和miRNAs三者间的关联性及可能的调控机理。
方法
1.分别在低剂量叶酸水平(叶酸缺乏组)和正常叶酸(正常对照组)条件下,培养小鼠胚胎干细胞36h,通过CCK-8实验评估叶酸缺乏对细胞增殖的影响；免疫印记杂交法和荧光定量PCR法分别在蛋白水平和mRNA水平,检测自噬关键蛋白LC3B和Beclinl的表达量变化。
2.采用全基因组表达谱芯片和miRNA表达谱芯片检测两组细胞的表达谱。
3.通过生物信息学方法,应用Targetscan软件对两组细胞的芯片结果进行靶基因预测,之后进行G0分析、Pathway分析、构建基因间调控网络以及miRNA与靶基因间的负调控网络,最终筛选出与自噬相关的miRNA和mRNA。
4.经茎环引物反转录所得产物采用TaqMan Small RNA Assays验证7个与自噬相关的差异miRNA,用SYB Green法验证4个与自噬相关的差异靶基因。
结果
1.与正常对照组相比,小鼠胚胎干细胞在叶酸缺乏组培养36h,细胞增殖减慢(P<0.05);LC3B和Beclinl在蛋白水平的表达量均上调(P<0.05),但是在mRNA水平仅LC3B表达量上调(P<0.05)。
2.与正常对照组相比,叶酸缺乏组的全基因组表达谱芯片共筛选出差异表达的基因共4607个,其中上调的基因共2599个,下调的基因共2008个。miRNA表达谱芯片共筛选出差异表达的miRNAs共188个,其中上调的miRNAs共70个,下调的miRNAs共118个。
3.通过生物信息学方法进行的G0分析提示叶酸缺乏对DNA转录、多细胞生物发育特别是神经系统发育的影响最显著；Pathway分析显示叶酸缺乏主要对多类肿瘤的信号转导通路产生影响。最终筛选结果：自噬PI3K/Akt信号通路的Pik3rl被3个miRNAs(mmu-miR-466d-3p,mmu-miR-669f-3p, mmu-miR-669m-3p)靶向下调,Pik3r3被4个miRNAs(mmu-miR-409-3p, mmu-miR-380-5p, mmu-miR-291a-3p, mmu-miR-345-3p)靶向下调,Pik3r5被1个miRNA(mmu-miR-466K)靶向下调,Akt1被3个miRNAs(mmu-miR-409-3p,mmu-miR-369-3p, mmu-miR-291a-3p)靶向下调,Foxo3同时被mmu-miR-574-5p靶向下调和mmu-miR-212-3p靶向上调。
4.荧光定量PCR验证的结果：相对于正常对照组,叶酸缺乏组中除了mmu-miR-345-3p表达下调,其他6个miRNAs(mmu-miR-212-3P、mmu-miR-291a-3p、 mmu-miR-380-5p、mmu-miR-409-3p、mmu-miR-466d-3p、mmu-miR-669f-3p)均上调；其中与芯片结果相反的是mmu-miR-212-3P、mmu-miR-345-3p。4个基因中仅Foxo3表达上调1.3倍,差异有统计学意义(P<0.01),而Pik3r1表达下调1.1倍,Pik3r3和Aktl的表达分别上调1.04倍和1.03倍均未发现具有统计学意义(P>0.05)。说明Foxo3可能参与叶酸缺乏诱导的自噬过程主要途径。
结论
叶酸缺乏条件下培养的小鼠胚胎干细胞可发生自噬,自噬的发生可能通过mmu-miR-212-3P靶向作用于PI3K/Akt信号通路中促进自噬的转录因子Foxo3,使其表达量升高,影响了DNA的转录,自噬相关基因表达上调,从而促进了自噬的发生。在生物体内则可能进一步影响胚胎发育特别是神经系统的发育。本研究首次提出了叶酸缺乏可诱导自噬的发生；首次探索了叶酸缺乏、miRNA与自噬三者间的关系,这也可能为深入研究细胞生长发育调控的机理,特别是神经管畸形的发病机制研究提供了新的思路和可靠的数据。
Folic acid is a form of water-soluble vitamin B9, as a carrier of one carbon unit, which mainly involved in two biochemical process of DNA synthesis and DNA methylation. Folate deficiency can result in many diseases such as birth defects, aging and tumor. It can also induce cell death. Autophagy is a type of cell death which is a basic catabolic mechanism of survival for cells with fasting. The previous study of our team has confirmed that apoptosis can be induced by folate deficiency in mouse embryonic stem cells and miRNAs are the key factor of proliferation and apoptosis in embryo development. Other study have demonstrated that autophagy play a key role in development of vertebrates. However, the relationship between folic acid, miRNA and autophagy has not been characterized. In this study, mouse embryonic stem cells (mESCs) were used for research. The aim is to exploring whether folate deficiency could induce autophagy and whether miRNAs could involve in this process. Then we tested critical proteins of autophagy, screened and verified the miRNAs and mRNAs associated with autophagy so as to elaborate the relationship between folate deficiency, miRNA regulation and autophagy with multi-dimension.
Objective
To study the relationship between folate deficiency, autophagy and miRNAs, and the possible regulating mechanism in mESCs.
Methods
1. mESCs were treated with both normal folate levels (Control) and low folate levels (folate deficiency, FD medium)for36hours. Then CCK-8assay was applied to assessing the proliferation of mESCs in these two cultured systems. After cell harvesting, autophagy-related molecules such as LC3B and Beclinl were tested by Western Blotting and real-time PCR.
目的
以小鼠胚胎干细胞为模型,研究叶酸缺乏、自噬和miRNAs三者间的关联性及可能的调控机理。
方法
1.分别在低剂量叶酸水平(叶酸缺乏组)和正常叶酸(正常对照组)条件下,培养小鼠胚胎干细胞36h,通过CCK-8实验评估叶酸缺乏对细胞增殖的影响；免疫印记杂交法和荧光定量PCR法分别在蛋白水平和mRNA水平,检测自噬关键蛋白LC3B和Beclinl的表达量变化。
2.采用全基因组表达谱芯片和miRNA表达谱芯片检测两组细胞的表达谱。
3.通过生物信息学方法,应用Targetscan软件对两组细胞的芯片结果进行靶基因预测,之后进行G0分析、Pathway分析、构建基因间调控网络以及miRNA与靶基因间的负调控网络,最终筛选出与自噬相关的miRNA和mRNA。
4.经茎环引物反转录所得产物采用TaqMan Small RNA Assays验证7个与自噬相关的差异miRNA,用SYB Green法验证4个与自噬相关的差异靶基因。
结果
1.与正常对照组相比,小鼠胚胎干细胞在叶酸缺乏组培养36h,细胞增殖减慢(P<0.05);LC3B和Beclinl在蛋白水平的表达量均上调(P<0.05),但是在mRNA水平仅LC3B表达量上调(P<0.05)。
2.与正常对照组相比,叶酸缺乏组的全基因组表达谱芯片共筛选出差异表达的基因共4607个,其中上调的基因共2599个,下调的基因共2008个。miRNA表达谱芯片共筛选出差异表达的miRNAs共188个,其中上调的miRNAs共70个,下调的miRNAs共118个。
3.通过生物信息学方法进行的G0分析提示叶酸缺乏对DNA转录、多细胞生物发育特别是神经系统发育的影响最显著；Pathway分析显示叶酸缺乏主要对多类肿瘤的信号转导通路产生影响。最终筛选结果：自噬PI3K/Akt信号通路的Pik3rl被3个miRNAs(mmu-miR-466d-3p,mmu-miR-669f-3p, mmu-miR-669m-3p)靶向下调,Pik3r3被4个miRNAs(mmu-miR-409-3p, mmu-miR-380-5p, mmu-miR-291a-3p, mmu-miR-345-3p)靶向下调,Pik3r5被1个miRNA(mmu-miR-466K)靶向下调,Akt1被3个miRNAs(mmu-miR-409-3p,mmu-miR-369-3p, mmu-miR-291a-3p)靶向下调,Foxo3同时被mmu-miR-574-5p靶向下调和mmu-miR-212-3p靶向上调。
4.荧光定量PCR验证的结果：相对于正常对照组,叶酸缺乏组中除了mmu-miR-345-3p表达下调,其他6个miRNAs(mmu-miR-212-3P、mmu-miR-291a-3p、 mmu-miR-380-5p、mmu-miR-409-3p、mmu-miR-466d-3p、mmu-miR-669f-3p)均上调；其中与芯片结果相反的是mmu-miR-212-3P、mmu-miR-345-3p。4个基因中仅Foxo3表达上调1.3倍,差异有统计学意义(P<0.01),而Pik3r1表达下调1.1倍,Pik3r3和Aktl的表达分别上调1.04倍和1.03倍均未发现具有统计学意义(P>0.05)。说明Foxo3可能参与叶酸缺乏诱导的自噬过程主要途径。
结论
叶酸缺乏条件下培养的小鼠胚胎干细胞可发生自噬,自噬的发生可能通过mmu-miR-212-3P靶向作用于PI3K/Akt信号通路中促进自噬的转录因子Foxo3,使其表达量升高,影响了DNA的转录,自噬相关基因表达上调,从而促进了自噬的发生。在生物体内则可能进一步影响胚胎发育特别是神经系统的发育。本研究首次提出了叶酸缺乏可诱导自噬的发生；首次探索了叶酸缺乏、miRNA与自噬三者间的关系,这也可能为深入研究细胞生长发育调控的机理,特别是神经管畸形的发病机制研究提供了新的思路和可靠的数据。
Folic acid is a form of water-soluble vitamin B9, as a carrier of one carbon unit, which mainly involved in two biochemical process of DNA synthesis and DNA methylation. Folate deficiency can result in many diseases such as birth defects, aging and tumor. It can also induce cell death. Autophagy is a type of cell death which is a basic catabolic mechanism of survival for cells with fasting. The previous study of our team has confirmed that apoptosis can be induced by folate deficiency in mouse embryonic stem cells and miRNAs are the key factor of proliferation and apoptosis in embryo development. Other study have demonstrated that autophagy play a key role in development of vertebrates. However, the relationship between folic acid, miRNA and autophagy has not been characterized. In this study, mouse embryonic stem cells (mESCs) were used for research. The aim is to exploring whether folate deficiency could induce autophagy and whether miRNAs could involve in this process. Then we tested critical proteins of autophagy, screened and verified the miRNAs and mRNAs associated with autophagy so as to elaborate the relationship between folate deficiency, miRNA regulation and autophagy with multi-dimension.
Objective
To study the relationship between folate deficiency, autophagy and miRNAs, and the possible regulating mechanism in mESCs.
Methods
1. mESCs were treated with both normal folate levels (Control) and low folate levels (folate deficiency, FD medium)for36hours. Then CCK-8assay was applied to assessing the proliferation of mESCs in these two cultured systems. After cell harvesting, autophagy-related molecules such as LC3B and Beclinl were tested by Western Blotting and real-time PCR.
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