MicroRNA-520a-3p induces apoptosis in lung cancer stem cells via modulation of MAP3K2 / 中国组织工程研究

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.