MicroRNA-520a-3p induces apoptosis in lung cancer stem cells via modulation of MAP3K2

doi:10.3969/j.issn.2095-4344.0504

Abstract

Abstract:

BACKGROUND: Studies have confirmed that microRNA (miR)-520a-3p has a regulatory role in lung cancer stem cells, but the specific function and mechanism of action are still unknown.
OBJECTIVE: To investigate the influence of miR-520a-3p on the apoptosis of lung cancer stem cells, and to explore the underlying mechanism.
METHODS: The magnetic activated cell sorting method was utilized to separate the CD133⁺ lung cancer stem cells from the lung cancer A549 cell line. The expression of miR-520a-3p in the lung cancer stem cells was determined by the real-time PCR assay. Liposome transfection assay was used to up-regulate the miR-520a-3p expression level in the lung cancer stem cells, and flow cytometry assay was applied to detect the influence of miR-520a-3p expression on the apoptosis of the lung cancer stem cells. Moreover, the modulation of MAP3K2 gene by the miR-520a-3p was analyzed by the dual-luciferase reporter gene assay, and the influence of miR-520a-3p expression on the protein expression of MAP3K2, Bcl-2 and Caspase-3 was analyzed by western blot assay.
RESULTS AND CONCLUSION: The real-time PCR showed that the expression level of miR-520a-3p in CD133⁺ lung cancer stem cells was significantly lower than that in the CD133^- lung cancer cells (P < 0.05). Overexpression of miR-520a-3p significantly increased the apoptotic rate of CD133⁺ lung cancer stem cells (P < 0.05). The dual-luciferase reporter gene assay results suggested that inhibition of miRNA-520a-3p could increase the luciferase activity of the reporter plasmids containing the 3’-untranslated region (3’-UTR) of MAP3K2 gene (P < 0.05), and overexpression of miR-520a-3p could decrease the luciferase activity of the reporter plasmids containing the 3’-UTR of MAP3K2 gene (P < 0.05). Moreover, miR-520a-3p overexpression also decreased the protein levels of MAP3K2 and Bcl-2 in the CD133⁺ lung cancer stem cells, P < 0.05), and increased the Caspase-3 protein level (P < 0.05). To conclude, in the lung cancer stem cells, miR-520a-3p was in a low-expressed status. miR-520a-3p could inhibit the expression of MAP3K2 gene, thereby inducing the cell apoptosis.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Lung Neoplasms, Neoplastic Stem Cells, MAP Kinase Kinase Kinase 2, Apoptosis, Tissue Engineering

CLC Number:

R394.2

Yan Jun, Zhong Zhi-hong, Shi Hua-qiu. MicroRNA-520a-3p induces apoptosis in lung cancer stem cells via modulation of MAP3K2[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(13): 2051-2056.

Figures/Tables 1

2.1 免疫磁珠细胞分选肺癌干细胞采用免疫磁珠法在肺腺癌A549细胞系中分离CD133+肺癌干细胞，流式细胞仪检测分选前后CD133+细胞比例。分选后CD133+细胞比例显著高于分选前，差异有显著性意义(t=-45.706，P=0.000)，见图1。 2.2 miRNA-520a-3p在CD133+肺癌干细胞中的表达采用real-time PCR技术检测CD133+细胞中miRNA-520a-3p表达，以CD133-细胞为对照。结果显示，CD133+细胞中miRNA-520a-3p相对表达量显著低于CD133-细胞，差异有显著性意义(t=-9.636，P=0.000)，见图2。 2.3 miRNA-520a-3p促进CD133+肺癌干细胞凋亡转染miRNA-520a-3p mimics至CD133+细胞中48 h后，流式细胞术检测结果显示，实验组细胞的凋亡比例(52.32±4.83)%显著高于空白对照组(18.32±2.84)%和阴性对照组(19.12± 2.99)%，差异均有显著性意义(t=10.510，P=0.000；t=10.123，P=0.000)，而空白对照组和阴性对照组之间差异无显著性意义(t=-0.336，P=0.377)，见图3。 2.4 MiRNA-520a-3p靶向调节MAP3K2基因表达 HEK-293T细胞中，miRNA-520a-3p mimics+pmirGLO- MAP3K2-3’-UTR wt组的相对荧光强度(0.42±0.08)显著低于miRNA-520a-3p mimics+pmirGLO空载体对照组(1.04±0.13)和miRNA-520a-3p mimics+pmirGLO- MAP3K2-3’-UTR mut组(0.99±0.15)，差异均有显著性意义(t=-7.035，P=0.001；t=-5.807，P=0.002)，而miRNA-520a- 3p mimics+pmirGLO空载体对照组和miRNA-520a-3p mimics+pmirGLO-MAP3K2-3’-UTR mut组之间差异无显著性意义(t=0.436，P=0.343)，见图4。 2.5 miRNA-520a-3p对MAP3K2蛋白及凋亡相关蛋白Bcl-2和Caspase-3表达的影响 Western Blotting检测结果显示，与空白对照组和阴性对照组相比，实验组细胞中的MAP3K2蛋白水平显著降低，差异有显著性意义(t=11.209，P=0.000；t=11.529，P=0.000)，空白对照组和阴性对照组之间差异无显著性意义(t=-0.231，P=0.414)，见图5；与空白对照组和阴性对照组相比，实验组细胞中的Bcl-2蛋白水平均显著降低，差异有显著性意义(t=4.038，P=0.008；t=3.798，P=0.010)，空白对照组和阴性对照组之间差异无显著性意义(t=0.201，P=0.425)，见图5；实验组细胞中的Caspase-3蛋白水平(1.08±0.12)显著高于空白对照组(0.09±0.02)和阴性对照组(0.10±0.03)，差异有显著性意义(t=14.095，P=0.000；t=13.723，P=0.000)，空白对照组和阴性对照组之间差异无显著性意义(t=-0.480，P=0.328)，见图5。"

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