Theranostics 2019; 9(19):5497-5516. doi:10.7150/thno.33800

Research Paper

The miR-26a/AP-2α/Nanog signaling axis mediates stem cell self-renewal and temozolomide resistance in glioma

Wenhuan Huang1,2,3*, Zhe Zhong4*, Chang Luo5, Yuzhong Xiao6, Limin Li7, Xing Zhang8, Liu Yang1,2,3, Kai Xiao9, Yichong Ning1,2,3, Li Chen1,2,3, Qing Liu1,2,3, Xiang Hu1,2,3, Jian Zhang1,2,3, Xiaofeng Ding1,2,3✉, Shuanglin Xiang1,2,3✉

1. State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Science, Hunan Normal University, Changsha, 410081, China;
2. Key Laboratory of Protein Chemistry and Development Biology of State Education Ministry of China, College of Life Science, Hunan Normal University, Changsha, Hunan, 410081, China;
3. The National & Local Joint Engineering Laboratory of Animal Peptide Drug Development, College of Life Science, Hunan Normal University, Changsha, 410081, China;
4. Department of Neurosurgery, Hunan Provincial Tumor Hospital, The Affiliated Tumor Hospital of Xiangya Medical School of Central South University, Changsha, Hunan, 410013, China;
5. Aier School of Ophthalmology, Central South University; Aier Eye Institute, Changsha, Hunan, 410015, China;
6. Department of Endocrinology, Endocrinology Research Center, Xiangya Hospital of Central South University, Changsha, Hunan 410008, China;
7. College of Engineering and Design, Hunan Normal University, Changsha, Human, 410081, China;
8. Department of Biochemistry and Molecular Biology, University of New Mexico Health Sciences Center, Albuquerque, NM, 87131, USA;
9. Department of Neurosurgery, Xiangya Hospital of Central South University, Changsha, Hunan, 410008, China.
* These authors equally contributed to this work.

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Citation:
Huang W, Zhong Z, Luo C, Xiao Y, Li L, Zhang X, Yang L, Xiao K, Ning Y, Chen L, Liu Q, Hu X, Zhang J, Ding X, Xiang S. The miR-26a/AP-2α/Nanog signaling axis mediates stem cell self-renewal and temozolomide resistance in glioma. Theranostics 2019; 9(19):5497-5516. doi:10.7150/thno.33800. Available from http://www.thno.org/v09p5497.htm

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Abstract

Aberrant expression of transcription factor AP-2α has been functionally associated with various cancers, but its clinical significance and molecular mechanisms in human glioma are largely elusive.

Methods: AP-2α expression was analyzed in human glioma tissues by immunohistochemistry (IHC) and in glioma cell lines by Western blot. The effects of AP-2α on glioma cell proliferation, migration, invasion and tumor formation were evaluated by the 3-(4,5-dimethyNCthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) and transwell assays in vitro and in nude mouse models in vivo. The influence of AP-2α on glioma cell stemness was analyzed by sphere-formation, self-renewal and limiting dilution assays in vitro and in intracranial mouse models in vivo. The effects of AP-2α on temozolomide (TMZ) resistance were detected by the MTT assay, cell apoptosis, real-time PCR analysis, western blotting and mouse experiments. The correlation between AP-2α expression and the expression of miR-26a, Nanog was determined by luciferase reporter assays, electrophoretic mobility shift assay (EMSA) and expression analysis.

Results: AP-2α expression was downregulated in 58.5% of glioma tissues and in 4 glioma cell lines. AP-2α overexpression not only reduced the proliferation, migration and invasion of glioma cell lines but also suppressed the sphere-formation and self-renewal abilities of glioma stem cells in vitro. Moreover, AP-2α overexpression inhibited subcutaneous and intracranial xenograft tumor growth in vivo. Furthermore, AP-2α enhanced the sensitivity of glioma cells to TMZ. Finally, AP-2α directly bound to the regulatory region of the Nanog gene, reduced Nanog, Sox2 and CD133 expression. Meanwhile, AP-2α indirectly downregulated Nanog expression by inhibiting the interleukin 6/janus kinase 2/signal transducer and activator of transcription 3 (IL6/JAK2/STAT3) signaling pathway, consequently decreasing O6-methylguanine methyltransferase (MGMT) and programmed death-ligand 1 (PD-L1) expression. In addition, miR-26a decreased AP-2α expression by binding to the 3' untranslated region (UTR) of AP-2α and reversed the tumor suppressive role of AP-2α in glioma, which was rescued by a miR-26a inhibitor. TMZ and the miR-26a inhibitor synergistically suppressed intracranial GSC growth.

Conclusion: These results suggest that AP-2α reduces the stemness and TMZ resistance of glioma by inhibiting the Nanog/Sox2/CD133 axis and IL6/STAT3 signaling pathways. Therefore, AP-2α and miR-26a inhibition might represent a new target for developing new therapeutic strategies in TMZ resistance and recurrent glioma patients.

Keywords: AP-2α, glioma, glioblastoma stem cells (GSCs), TMZ resistance, Nanog, STAT3, miR-26a