miR-302a对叶酸缺乏小鼠胚胎干细胞增殖和凋亡的影响

发布时间：2019-07-08 12:26

【摘要】：目的探讨miR-302a对叶酸缺乏小鼠胚胎干细胞(mESC)增殖和凋亡的影响。方法 mESC分为完全培养基组(对照组),无叶酸培养基组(无叶酸组),无叶酸培养基+miR-302amimic组(miR-302a组),通过RT-PCR进行检测miR-302a在完全培养基及无叶酸培养基中的表达。构建miR-302amimic,后转染到无叶酸培养基mESC中,采用MTT法检测miR-302a mimic对mESC活力的影响,Annexin V-FITC/PI流式细胞术检测miR-302amimic对mESC细胞凋亡的影响;流式细胞术检测miR-302amimic对mESC细胞周期的影响;Western blot检测及磷脂酰肌醇3羟激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路激活情况,以及下游分子细胞周期蛋白D1(CyclinD1)、p21、p27表达的影响。结果 RT-PCR证实无叶酸组中miR-302a表达量下降(P0.01)。与完全培养基比较,无叶酸培养基中,mESC细胞活力下降,细胞凋亡增加,细胞周期阻滞在G1期,Akt及mTOR磷酸化水平下降,CyclinD1表达下调,p21及p27表达上调,差异均具有统计学意义(P0.01)。与无叶酸组比较,miR-302a组中,mESC细胞活力上升,凋亡下降,G1期缩短,AKT及mTOR磷酸化水平提高,CyclinD1表达上调,p21及p27表达下调,差异均具有统计学意义(P0.01)。结论 miR-302a类似物能显著抑制缺乏叶酸mESC凋亡,能促进其增值,与PI3K/AKT/mTOR信号通路有关。
[Abstract]:Objective to investigate the effect of miR-302a on proliferation and apoptosis of embryonic stem cells (mESC) in folic acid deficient mice. Methods mESC was divided into complete medium group (control group), folic acid free medium group (folic acid free group) and folic acid free medium miR-302amimic group (miR-302a group). The expression of miR-302a in complete medium and folic acid free medium was detected by RT-PCR. MiR-302amimic, was constructed and transformed into folic acid free medium mESC. The effect of miR-302amimic on mESC activity was detected by MTT assay, the effect of miR-302amimic on apoptosis of mESC cells was detected by Annexin V-FITC/PI flow cytometry, and the effect of miR-302amimic on mESC cell cycle was detected by flow cytometry. Western blot was used to detect the activation of phosphatidylinositol 3-hydroxykinase (PI3K) / protein kinase B (Akt) / mammal rapamicin target protein (mTOR) signaling pathway, and the expression of downstream molecular cell cycle proteins D1 (CyclinD1), p21 and p27. Results RT-PCR confirmed that the expression of miR-302a in folic acid group decreased (P 0.01). Compared with the complete culture medium, the activity of mESC cells decreased, the apoptosis increased, the cell cycle arrest was in G 1 phase, the phosphorylation level of Akt and mTOR decreased, the expression of CyclinD1 was down-regulated, and the expression of p21 and p27 was up-regulated in folic acid-free medium, the difference was statistically significant (P 0.01). Compared with folic acid group, the activity of mESC cells was increased, apoptosis decreased, G 1 phase was shortened, AKT and mTOR phosphorylation level was increased, CyclinD1 expression was up-regulated, p21 and p27 expression was down-regulated in miR-302a group, the difference was statistically significant (P 0.01). Conclusion miR-302a analogues can significantly inhibit apoptosis of folic acid deficient mESC and promote its increment, which is related to PI3K/AKT/mTOR signaling pathway.
【作者单位】： 四川省医学科学院/四川省人民医院儿科;
【分类号】：R321

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