MicroRNA-200c increases radiosensitivity of human cancer cells with activated EGFR-associated signaling

Abstract

MicroRNA-200c (miR-200c) recently was found to have tumor-suppressive properties by inhibiting the epithelial-mesenchymal transition (EMT) in several cancers. miR-200c also interacts with various cellular signaling molecules and regulates many important signaling pathways. In this study, we investigated the radiosensitizing effect of miR-200c and its mechanism in a panel of human cancer cell lines. Malignant glioma (U251, T98G), breast cancer (MDA-MB-468), and lung carcinoma (A549) cells were transfected with control pre-microRNA, pre-miR-200c, or anti-miR-200c. Then, RT-PCR, clonogenic assays, immunoblotting, and immunocytochemisty were performed. To predict the potential targets of miR-200c, microRNA databases were used for bioinformatics analysis. Ectopic overexpression of miR-200c downregulated p-EGFR and p-AKT and increased the radiosensitivity of U251, T98G, A549, and MDA-MB-468 cells. In contrast, miR-200c inhibition upregulated p-EGFR and p-AKT, and decreased radiation-induced cell killing. miR-200c led to persistent γH2AX focus formation and downregulated pDNA-PKc expression. Autophagy and apoptosis were major modes of cell death. Bioinformatics analysis predicted that miR-200c may be associated with EGFR, AKT2, MAPK1, VEGFA, and HIF1AN. We also confirmed that miR-200c downregulated the expression of VEGF, HIF-1α, and MMP2 in U251 and A549 cells. In these cells, overexpressing miR-200c inhibited invasion, migration, and vascular tube formation. These phenotypic changes were associated with E-cadherin and EphA2 downregulation and N-cadherin upregulation. miR-200c showed no observable cytotoxic effect on normal human fibroblasts and astrocytes. Taken together, our data suggest that miR-200c is an attractive target for improving the efficacy of radiotherapy via a unique modulation of the complex regulatory network controlling cancer pro-survival signaling and EMT.

The relationship between miR-302b and EphA2 and their clinical significance in gastric cancer

“…Overexpressing miR-200c is reported to inhibit invasion, migration, and vascular tube formation of malignant glioma, breast cancer, and lung carcinoma cells. And these phenotypic changes are associated with EphA2 downregulation 26 , furthermore, miR-200a is demonstrated to direct repress EphA2 oncogene in triple-negative breast cancer cells 27 . MiR-26b can also inhibit cell proliferation, migration, and invasion by targeting EphA2 in hepatocellular carcinoma cells 28 .…”

“…Several studies have reported that miRNAs can regulate EphA2 in cancer cells 26 - 28 . Overexpressing miR-200c is reported to inhibit invasion, migration, and vascular tube formation of malignant glioma, breast cancer, and lung carcinoma cells.…”

The miR-200 family: multiple effects on gliomas

“… 15 Another study found that miR-200c prevented the invasion and migration of glioblastoma by activating EGFR pathways that reversed the epithelial–mesenchymal transition in glioblastoma. 36 Intriguingly, due to the homology in seed sequences, miR-200c and miR-141 share the same target, ZEB1, which is known to inhibit glioma cell growth and migration. 37 However, recent studies obtained inconsistent results.…”

“…One study demonstrated that miR-200c increases glioma cell radiosensitivity by activating EGFR-associated signaling. 36 …”

“…Importantly, in gliomas, the target genes dysregulated by miR-200 are associated with many conserved signaling pathways involved in cell processes such as cell proliferation, apoptosis, invasion and drug resistance. 20 , 24 , 36 , 37 Because miR-200 can target multiple downstream genes in glioma tissues, miR-200 might play both oncogenic and antioncogenic roles. For instance, TGF-β2 regulates many cellular processes including proliferation, differentiation, adhesion and migration.…”