Cinobufotalin Powerfully Reversed EBV-miR-BART22-Induced Cisplatin Resistance via Stimulating MAP2K4 to Antagonize Non-Muscle Myosin Heavy Chain IIA/Glycogen Synthase 3β/β-Catenin Signaling Pathway

Background: Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-related tumor. The role of EBV-encoding miR-BART-22 is still unclear in NPC. This study aimed to identify the detailed mechanisms by which EBV-miR-BART22 functions as a tumor-promoting factor and evaluate the action of cinobufotalin in treating EBV-miR-BART22-overexpressing NPC cells. Methods: Flow cytometry was used to identify the EBV-miR-BART22- overexpressing NPC stem cells. Sphere formation assay, western blotting, qRT-PCR assay, transwell, boyden, xenograft assay and immunohistochemistry staining analysis were utilized to evaluate the effect of EBV-miR-BART22 on the cells metastasis and DDP chemoresistance. qRT-PCR assay, immunofluorescence staining, Immunofluorescence, luciferase reporter assay, CHIP, EMSA and Co-IP assay were performed to explore the detailed molecular mechanism of EBV-miR-BART22 and its target gene. Findings: We observed that EBV-miR-BART22 not only promoted tumor stemness and metastasis, but also enhanced the resistance to Cisplatin (DDP) in vitro and in vivo. Mechanistic analyses indicated that EBV-miRBART-22 directly targeted the MAP2K4 and upregulated non-muscle myosin heavy chain IIA (MYH9) expression by PI3K/AKT/c-Jun-induced transcription. Further, MYH9 interacted with glycogen synthase 3β(GSK3β) protein and induced its ubiquitin degradation by activating PI3K/AKT/cJun-induced ubiquitin transcription and the latter combined with increased TRAF6 E3 ligase, which further bound to GSK3β protein. Reductions in the GSK3β protein thus promoted β-catenin expression and nuclear translocation, which induced tumor stemness and the epithelialto-mesenchymal transition (EMT) signals. Furthermore, we observed that cinobufotalin, a new chemically synthesized compound, significantly suppressed EBV-miR-BART-22-induced DDP chemoresistance by upregulating MAP2K4 to suppress MYH9/GSK3β/β-catenin and its downstream tumor stemness and EMT signals in NPC. Finally, clinical data revealed that increased miR-BART-22 and reduced MAP2K4 expression caused the poor prognoses of NPC patients. Interpretation: Our study provides a novel mechanism that cinobufotalin reversed the DDP chemoresistance and EMT induced by EBV-miR-BART22 in NPC. Funding Statement: China (No.81572643,81772872), People's Livelihood Science and technology grant of Guangzhou Municipal Science and technology project(No. 201803010023), Guangzhou science and technology plan(No. 201804010023), The Supporting plan for Special Talents in Guangdong Province (No. 2016TQ03R466), Project of Guangdong Province (No.2016A020215233), National Science Foundation for Young Scientists of China (No.81702672). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: For the use of the clinical materials for research purpose, approval from the Ethics Committee of the Nanfang Hospital were obtained. All animal experiments were conducted in accordance with a protocol approved by the Animal Care and Use Committee of Integrated Hospital of Traditional Chinese Medicine of Southern Medical University.