SOX2-Upregulated microRNA-30e Promotes Cell Invasion in Esophageal Cancer via USP4/SMAD4/CK2 Axis

Abstract

Background: Both SOX2 and microRNA-30e (miR-30e) were reported to be involved in esophageal cancer (EC) progression. However, whether there is correlation between SOX2 and miR-30e in EC cells remains undiscovered. Herein, this study was intended to explore the correlation between SOX2 and miR-30e in EC and the effect of this correlation on EC cell invasion. Methods: Following collection of EC tissues and cells, miR-30e, SOX2, miR-30e, USP4, SMAD4, and CK2 expression was determined by RT-qPCR, western blot analysis and immunohistochemistry. Gain- and loss-of-function approaches were conducted in screened cell lines, followed by determination of cell proliferation, migration, invasion, and expression of epithelial-mesenchymal transition (EMT)-related genes. The relationship among miR-30e, USP4, SMAD4 and CK2 was assessed by dual-luciferase reporter assay, in vitro protein purification, immunoprecipitation, and ubiquitination experiments, and ChIP assay. The role of SOX2 and CK2 in EC in vivo was evaluated by tumor xenograft in nude mice. Results: EC tissues and cells contained high expression of SOX2, miR-30e, and CK2 as well as low expression of USP4 and SMAD4. miR-30e was upregulated by SOX2, and specifically targeted USP4. Additionally, USP4 induced the deubiquitination of SMAD4 to upregulate SMAD4. SMAD4 was enriched in CK2 promoter region and inhibited CK2 expression. Silencing of SMAD4 or USP4 or upregulation of SOX2, miR-30e or CK2 triggered enhancement in EC cell proliferation, migration, invasion, and EMT in vitro, and promotion of tumor growth and lung metastasis in vivo. Conclusion: Taken together, our work reveals a novel SOX2-mediated regulatory axis that plays critical roles in the invasiveness of EC cells. Funding Statement: This work was supported by Youth Foundation of the National Natural Science Foundation of China (No. 81702971), Key Scientific Research Projects of Colleges and Universities of Henan Province (No. 18A320054), Science and Technology Research Projects of Henan Province (No. 182102310116) and Youth Talent Lifting Projects of Henan Province (No. 2020HYTP050) Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The ethics committee of the First Affiliated Hospital of Zhengzhou University provided Ethical Approval for the experiments involved human beings in this study which were guided by the intentions of the Declaration of Helsinki. Ethical agreements were obtained from the donors or their relatives by written informed consent. The experiments involved animals were implemented in accordance with the principles embodied in the National Institutes of Health Guide for the Care and Use of Laboratory. Efforts were made to avoid all unnecessary distress to the animals.