Objective To investigate the role of hypoxia-induced ubiquitin carboxyl-terminal hydrolase isozyme L5 (UCHL5) in the regulation of radiosensitivity of Hela cells in cervical cancer.

Methods UCHL5 levels were detected in Hela cells cultured under 1% O₂ condition. Western blot and qRT-PCR analyses verified the efficiency of lentiviral vector infection on stable UCHL5 modulation in HeLa cells, including oe-vector, oe-UCHL5-1, oe-UCHL5-2, oe-UCHL5-3, oe-UCHL5-4 and sh-vector, sh-UCHL5-1 and sh-UCHL5-2. The cells used in the experiment were divided into the following groups: oe-vector, oe-UCHL5, sh-vector and sh-UCHL5. Cell colony-formation rate and radiosensitivity were detected by colony-formation assay combined with single-dose (0, 2, 4 and 6 Gy) γ-ray irradiation after culturing for 2 weeks. The effect of radiation on cell apoptosis was determined by flow cytometry after 48 h of 8 Gy γ-ray irradiation. 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay was used to detect the cell viability of down- or up-regulated UCHL5 cells before and after 0, 2, 4, 6, 8 and 10 Gy of γ-ray irradiation. Gene expression profiling interactive analysis was used to analyze the correlated expression between UCHL5 and hypoxia inducible factor-1α (HIF-1α). The transcriptional activation effect of UCHL5 by HIF-1α was detected using dual-luciferase reporter-gene assay. Differences between groups were compared by single-sample t test, and Pearson test was used for correlation analysis.

Results UCHL5 was significantly induced by hypoxia after culturing at different time points. Hela cell lines with stably overexpressed and silenced UCHL5 were successfully constructed, with oe-UCHL5-2 and sh-UCHL5-2 having the highest regulatory efficiency. These two groups were selected for subsequent experiments. Compared with the control group that received the same dose of irradiation, significant differences existed at doses of 0, 2, 4 and 6 Gy (t=14.16, 19.22, 8.76, 6.79, all P<0.05), respectively. Knock-down of UCHL5 promoted apoptosis (t=10.29, P<0.05) and radiation-induced apoptosis (t=52.01, P<0.05). UCHL5 up-regulation promoted cell proliferation, and the proliferation rate was statistically significant on the third day (t=3.905, P<0.05). Furthermore, UCHL5 strengthened the viability (t=3.40, 4.06, 3.68, all P<0.05) of irradiated Hela cells, with significant differences at doses of 6, 8 and 10 Gy (t=3.40, 4.06, 3.68, all P<0.05), respectively. The expression levels of HIF-1α and UCHL5 in cervical cancer tissues were positively correlated (R=0.31, P<0.01). Additionally, HIF-1α was a potential transcriptional activator of UCHL5 in Hela cells, and its activity increased 2.5 times (t=30.47, P<0.05). Conclusions The induced expression of UHCL5 in cervical cancer Hela cells under hypoxia condition can reduce the radiation sensitivity of cells. The underlying mechanism may be related to the HIF-1α transcriptional activation of UCHL5 expression.