Li Zhi-Hui, Zhang Yan-Ping, Xing Peng-Tao, Zhan Xin-Rong
Department of Hematology, Xinxiang Central Hospital, Xinxiang 453000, Henan Province, China.
Department of Hematology, Xinxiang Central Hospital, Xinxiang 453000, Henan Province, China,E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2020 Feb;28(1):104-109. doi: 10.19746/j.cnki.issn.1009-2137.2020.01.018.
To investigate the effect and mechanism of miRNA-145 on leukemic cell apoptosis.
After transfection of miRNA-145 mimic and negative control mimic in leukemia cells by Lipofectamine 2000 liposome, the MTT assay was used to detect the effect of miRNA-145 on cell proliferation. Flow cytometry was used to detect the effect of miRNA-145 on cell cycle and apoptosis. Western blotting assay was used to detect the expression levels of BCL-2, CDK6, Cyclin D1, BAX, PI3K p-PI3K, p-AKT and AKT.
The relative level of microRNA in HuT 78 cells transfected with miRNA-145 was 2.3±02, which was significantly higher than that in blank control group and miRNA-NC group (P＜0.05). MTT assay showed that the proliferation level of HuT 78 cells transfected with miRNA-145 mimic was significantly lower than that of blank control and miRNA-NC group (P＜0.05). Flow cytometry showed that the cells at G/G, S and G2/M phase of HuT 78 cells were significantly decreased after transfection with miRNA-145 mimic (P＜0.05). Annexin V/PI double staining assay showed that the apoptosis rate of HuT 78 cells was 17.6%±3.4%，which was significantly higher than that in blank control group and miRNA-NC group (P＜0.05). Western blot showed that the expression levels of BCL-2, CDK6 and Cyclin D1 in HuT 78 cells were significantly lower than those in blank control and miRNA-NC group (P＜0.05), and BAX expression in HuT 78 cells was significantly higher than that in blank control and miRNA-NC group (P＜0.05). Western blot showed that expression of PI3K, p-PI3K, AKT and p-AKT in HuT 78 cells transfected with miRNA-145 mimic were significantly lower than that in blank control and miRNA-NC group (P＜0.05).
Upregulation of miRNA-145 may inhibit the proliferation of leukemia cells and promote the apoptosis, which may be related with the inhibition of PI3K/AKT signaling pathway.
探讨微小RNA-145（miRNA-145）对白血病细胞凋亡的影响及其机制。
采用Lipofectamine 2000脂质体将miRNA-145模拟物和阴性对照模拟物转染至白血病细胞后，用MTT法检测miRNA-145对细胞增殖的影响；用流式细胞术检测miRNA-145对细胞周期和凋亡的影响；用蛋白质免疫印迹法检测BCL-2、CDK6、细胞周期蛋白D1（Cyclin D1）、BAX、磷脂酰肌醇-3激酶（PI3K）、磷酸化PI3K（p-PI3K）、磷酸化蛋白激酶B（p-AKT）和蛋白激酶B（AKT）的表达水平。
转染miRNA-145的HuT 78细胞中微小RNA的相对水平为2.3±0.2，明显高于空白对照组和miRNA阴性对照（miRNA-NC）组（P＜0.05）。MTT法检测结果显示，转染miRNA-145模拟物的HuT 78细胞增殖水平明显低于空白对照组和miRNA-NC组（P＜0.05）。流式细胞术检测结果显示，转染miRNA-145模拟物后，HuT 78细胞处于G0/G1期、S期和G2/M期的细胞明显减少（P＜0.05）。膜联蛋白V/碘化丙啶（Annexin V/PI）双染法检测结果显示，HuT 78细胞凋亡率为17.6%±3.4%，明显高于空白对照组和miRNA-NC组（P＜0.05）。蛋白质免疫印迹法检测结果显示，HuT 78细胞中BCL-2、CDK6和Cyclin D1的表达水平明显低于空白对照组和miRNA-NC组（P＜0.05），而BAX的表达明显高于空白对照组和miRNA-NC组（P＜0.05）。蛋白质免疫印迹法检测结果显示，转染miRNA-145模拟物的HuT 78细胞中PI3K、p-PI3K、AKT和p-AKT的表达明显低于空白对照组和miRNA-NC组（P＜0.05）。
上调miRNA-145可能抑制白血病细胞增殖并促进其凋亡，其机制可能与抑制PI3K/AKT信号通路有关。
