Abstract: Background and purpose: Ubiquitin-specific protease 9x (USP9x) is related to the development and radioresistance of multiple cancers. Currently, it is reported that the USP9x expression is associated with invasive depth and lymphatic metastasis of esophageal squamous cancer. However, the effect of U5P9x on the radioresistance of esophageal cancer remains unknown. This study aimed to investigate the role of USP9x in the radiosensitivity of radioresistant esophageal cancer Ec9706-R cells and its mechanism. Methods: The mRNA expression and protein level of USP9x and myeloid cell leukemia-1 (Mcl-1) were detected by real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) and Western blot in Ec9706-R cells and the parental Ec9706 cells after irradiation. Then, Ec9706-R cells were randomly divided into 3 groups: irradiation (IR) group, IR+control siRNA group (IR+si-NC, transfected with control siRNA), and IR+USP9x siRNA group (IR+si-USP9x, transfected with USP9x siRNA). All cells were exposed to designated dose of 6 MV-X irradiation. MTT analysis was used to determine the viability of cells following 6 MV-X irradiation with different doses (0, 2, 4, 6 and 8 Gy). The migration, apoptosis and expression levels of Mcl-1, proliferating cell nuclear antigen (PCNA) and excision repair cross-complementing gene 1 (ERCC1) of Ec9706-R cells under 6 Gy irradiation were analyzed using Transwell, flow cytometry, real-time PCR and Western blot, respectively. Results: The mRNA expression and protein level of USP9x and Mcl-1 were elevated in both Ec9706-R and Ec9706 cells after irradiation, and their expression levels were more obvious in Ec9706-R cells (P<0.05). Compared with the IR group, cell viability, migrated cell numbers and the expression levels of PCNA, Mcl-1 and ERCC1 were decreased, whereas cell apoptosis was increased in the IR+si-USP9x group (P<0.05). However, compared with the IR group, these indexes were not changed in the IR+si-NC group (P>0.05). Conclusion: Inhibition of USP9x enhances the radiosensitivity of esophageal cancer Ec9706-R cells, which may be dependent on the downregulation of Mcl-1.

Key words: Ubiquitin-specific protease 9x, Radioresistant esophageal cancer cells, Radiosensitivity, Myeloid cell leukemia-1