Inhibitory effect of miR-34a on lung cancer stem cells via Notch1 signaling pathway

doi:10.3969/j.issn.2095-4344.2016.23.001

Abstract

Abstract:

BACKGROUND: It has been proved that miR-34a plays an inhibitory role in the growth of lung cancer stem cells, but the underlying mechanism remains unclear.
OBJECTIVE: To explore the inhibitory effect of miR-34a on lung cancer stem cells and the underlying mechanism.
METHODS: The CD133⁺ lung cancer stem cells were separated from lung cancer A549 cell lines using magnetic activated cell sorting method. And miR-34a-overexpressing CD133⁺ lung cancer stem cells were established by liposome transfection technology. Besides, the targeted relationship between miR-34a and Notch1 was analyzed by the dual-luciferase reporter. Afterwards, Notch1 silencing was performed by gene knockout, and its effect on lung cancer stem cells was determined.
RESULTS AND CONCLUSION: After sorted and detected by immunomagetic selection and flow cytometry assay respectively, a high rate of CD133⁺ lung cancer stem cell was obtained. And qRT-PCR detected that the expression level of miR-34a in CD133⁺ lung cancer stem cells was significantly lower than that in CD133^- lung cancer stem cells. Moreover, miR-34a-overexpressing CD133⁺ lung cancer stem cells were successfully constructed and miR-34a significantly inhibited proliferation and induced apoptosis of lung cancer stem cells. Dual-luciferase reporter assay indicated that Notch1 mRNA was a target of miR-34a. In addition, Notch1 silencing obviously inhibited proliferation and induced apoptosis of lung cancer stem cells. These findings suggest that miR-34a can inhibite lung cancer stem cells via the Notch1 signaling pathway.

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, MicroRNAs, Receptors, Notch1, Tissue Engineering

CLC Number:

R394.2

Han Ji-chang, Zhang Yi-jie, Li Hong-bing, Yang Cun-bao, Ma Chao-nan, Qi Guan-bin. Inhibitory effect of miR-34a on lung cancer stem cells via Notch1 signaling pathway[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(23): 3349-3356.

Figures/Tables 4

2.4 miR-34a对细胞增殖的影响为了验证miR-34a对肺癌干细胞增殖的作用，采用MTT法检测了过表达miR-34a对细胞增殖的影响。结果发现，转染了miR-34a模拟物的细胞增殖速度明显低于miRNA模拟物对照组细胞(P < 0.01)，说明miR-34a对肺癌干细胞具有明显的抑制作用(图4)。 2.5 miR-34a对细胞凋亡的影响运用流式细胞术检测了过表达miR-34a对肺癌干细胞凋亡的影响。如图5所示，miR-34a模拟物组的凋亡率明显高于miRNA模拟物对照组(P < 0.01)。 2.6 miR-34a抑制Notch1表达 miR-34a模拟物及miRNA模拟物对照转染肺癌干细胞48 h后，用qRT-PCR方法检测各组细胞中Notch1 mRNA的表达水平(图6A)。结果显示，与对照组相比，miR-34a模拟物转染的细胞中Notch1 mRNA的表达水平明显降低(P < 0.01)。又用Western blot检测Notch1蛋白水平的表达(图6B)。结果显示，与对照组相比，miR-34a模拟物转染的细胞中Notch1的表达水平明显降低(P < 0.05)。 2.7 双荧光素酶报告基因检测结果 WT-3’UTR和miR-34a模拟物共转染组的相对荧光素酶活性明显低于miRNA模拟物对照共转染组(P < 0.05)。MUT-3’UTR和miRNA模拟物对照共转染组与MUT-3’UTR和miR-34a模拟物共转染组相比，两者差异无显著性意义(P > 0.05)，见图7。结果说明miR-34a可以与Notch1 mRNA 3'UTR的特定位点结合，Notch1可能是miR-34a的靶向基因。 2.8 基因敲除Notch1对肺癌干细胞的影响为了进一步检测Notch1与肺癌干细胞的增殖、凋亡的关系，首先利用基因敲除技术将肺癌干细胞中Notch1的基因进行敲除，图8A显示，基因敲除后干细胞中Notch1的表达显著降低。MTT法检测结果显示(图8B)，基因敲除后干细胞的增殖明显受到抑制(P < 0.05)。流式细胞仪检测结果显示(图8C)，si-Notch1显著增加了肺癌干细胞的凋亡(P < 0.05)。这些结果说明，基因敲除Notch1能够抑制肺癌干细胞的增殖，诱导细胞凋亡。"

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