Nuclear factor (erythroid-derived 2)-like 2 counter-regulates thymosin beta-4 expression and primary cilium formation for HeLa cervical cancer cell survival

Abstract

Abstract We investigated the function of thymosin beta-4 (TB4) expression and primary cilium (PC) formation via the underlying Nrf2-dependent mechanism for cervical cancer cell (CC) survival under conditions of serum deprivation (SD). TB4 silencing was achieved using RNA interference. The percentage of PC formation was analyzed by immunofluorescence staining. Nrf2 expression was modified by the preparation of stable Nrf2-knockdown cells with shNrf2 and the overexpression of Nrf2 with pcDNA-Nrf2 plasmids. Gene expression was measured using reverse-transcription PCR, Gaussia luciferase assay, and western blotting. Cell viability was assessed using the MTT assay or CellTiter Glo assay. Reactive oxygen species (ROS) were detected with flow cytometry. CCs incubated in SD without fetal bovine serum remained viable, and SD increased PC formation and TB4 transcription. CC viability was further decreased by treatment with ciliobrevin A to inhibit PC formation or TB4-siRNA. SD increased ROS, including H2O2. N-acetylcysteine inhibited ROS production following H2O2 treatment or SD, which also decreased PC formation and TB4 transcription. Meanwhile, H2O2 increased PC formation, which was attenuated in response to TB4 siRNA. Treatment with H2O2 increased Nrf2 expression, antioxidant responsive element (ARE) activity, and PC formation, which were inhibited by the Nrf2 inhibitor clobestasol propionate. Nrf2 knockdown via expression of Tet-On shNrf2 enhanced ROS production, leading to increased PC formation and decreased TB4 expression; these effects were counteracted by Nrf2 overexpression. Our data demonstrate that Nrf2 counter-regulates TB4 expression and PC formation for CC survival under conditions of SD, suggesting cervical CC survival could be upregulated by PC formation via Nrf2 activation and TB4 expression.