Lymphoid-specific helicase inhibits cervical cancer cells ferroptosis by promoting Nrf2 expression

Patients sample

A total of 15 pairs of cervical cancer tumor tissues and paired normal tissues were obtained by surgical resection. Patients with other cancers and received preoperative chemo/radiotherapy were excluded. This study was approved by the Research Ethic Committee of the Ningbo Medical Centre Lihuili Hospital (DYLL2018079, 2018). All patients signed informed consent prior to participating in the study.

Immunohistochemistry (IHC)

Immunohistochemistry staining for HELLS was carried out. A total of 4-µm sections of cancer tissues were rehydrated and incubated with anti-HELLS (1:100, ab3851, Abcam, Cambridge, UK) overnight. The next day, tissue sections were washed with PBS, incubated with goat-anti mouse HRP-conjugated (1:1000, ab6721, Abcam, Cambridge, UK) for 1 h at 37 °C, and visualized by diaminobenzidine (DAB). The sections were counterstained byhematoxylin (Solarbio, Beijing, China).

Cell culture andtreatment

ICE cells and cervical cancer cell lines C4-1, HeLa, Caski, and SiHa were purchased from Chinese Academy of Science (Shanghai, China). All cells were grown in RPMI-1640 medium (Gibco, Waltham, MA, USA) supplemented with 10%FBS. All cell lines were cultured in an incubator with 5% CO₂ at 37 °C.

To overexpress HELLS, The HELLS lentiviral plasmid was purchased from Genechem (Shanghai, China), and was transfected into C4-1 cells using Lipofectamine™ 3000. The cells were selected by 3 µg/mL puromycin for one month after 48 hours’ transfection.

To silence HELLS or Nrf2, si-HELLS (siRNA ID: 145350) and si-Nrf2 (siRNA ID: 107969) were purchased from Thermo Fisher (Waltham, MA, USA), and SiHa or C4-1cells were transfected by Lipofectamine^® RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA, USA), in accordance with the manufacturer guidelines.

To inhibit apoptosis or ferroptosis, SiHa cell with si-HELLS were stimulated with Z-VAD-FMK (10 µM) or ferrostatin-1 (0.5 µM). To induce ferroptosis in cervical cancer cells, HELLS-OEC4-1cells were treated with erastin (20 µM) for 24 h.

Quantitative real-time PCR

Total RNA was extracted from cervical cancer tissues and cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The SMART PCR cDNA Synthesis Kits were used for reverse transcriptional PCR (Clontech, Mountain View, CA, USA). Quantitative real-time PCR (qRT-PCR) was carried out with SYBR Green incorporation (Thermo Fisher, Waltham, MA, USA). The 2^−ΔΔCt method was utilized to determine the fold changes of RNA transcripts, and the actin gene was employed as a reference gene.

Cell counting kit-8

The cell viability of cervical cancer cells was analyzed by using the Cell-counting kit-8 (CCK8) (Thermo Fisher, Waltham, MA, USA). Cervical cancer cells (2  × 10⁴ cells/ml) were seeded on 96-well plate (100 µl per well), and cells were cultivated for five different times under diverse treatments. The CCK-8 test was carried out at each interval of time in accordance with the manufacturer’s instructions.

5-ethynyl-2′-deoxyuridine (Edu)assay

EdU assay was performed to measure the DNA synthesis of cervical cancer cells. Cells were seeded at 2  × 10⁴ cells/ml in 96-well plates (100 µl/well) and fixed with 4% paraformaldehyde for 10 min. Then, cells were permeabilized with 0.5% Triton-X-100 in PBS for 20 min and blocked with 1% BSA in PBS for 30 min at room temperature. Next, cells were treated with 10 µM EdU for 1 h at 37 °C with 5% CO₂. After that, the Click-iT^® reaction cocktail (Thermo Fisher, Waltham, MA, USA) was added and cells were incubated for 30 min at room temperature. Finally, cells were stained with Hoechest for 5 min and imaged using a Multiskan FC microplate reader (Thermo Fisher, Waltham, MA, USA).

Western-blotting analysis

Western-blotting was performed as our previous study (Tie & Ge, 2021), and the antibodies used were as follows: anti-HELLS (1:1000, ab3851, Abcam, Cambridge, UK), anti-Nrf2 (1:1000, ab137550, Abcam, Cambridge, UK), and anti-actin (1:5000, ab8224, Abcam, Cambridge, UK) overnight at 4 °C.

Colony formation assay

Colony formation was performed as our previous study (Tie & Ge, 2021), Briefly, cervical cancer cells were seeded in 10 cm-plates at a concentration of 1,000 cells/well and cultured normally for 14 days in RPMI 1,640 with 10% FBS. The colonies were fixed and stained with 0.4% crystal violet (Sigma, Burlington, MA, USA) and methanol before being counted.

Statistical analysis

In the current study, statistical analyses were carried out by SPSS 22.0 software (SPSS, Chicago, IL, USA) (Duricki, Soleman & Moon, 2016). The mean ± standard deviation is used to represent all the data, the differences between two groups were analyzed by Student’s t-test, and a one-way ANOVA was used to investigate differences between more than two groups. P values < 0.05 were considered significant.

HELLS was upregulated in cervical carcinoma

To explore the genes closely related to the occurrence of cervical cancer, four cervical carcinoma high-throughput sequencing datasets (GSE63678, GSE122697, GSE63514, and GSE192897) were obtained from the Gene Expression Omnibus (GEO)database, and the common different expression genes were obtained through Venn overlap analysis. Figure 1A showed that seventeen common genes (HELLS, MELK, EZH2, CENPF, KIF2C, CCNB1, TYMS, MAPK10, CEP55, BIRC5, STAT1, CDC20, ESR1, CXCL8, CXCL9, CXCL11, and CXCL1) were obtained. Among these genes, HELLS is the one with the most significant differences. To confirm the function of HELLS, the expression of HELLS in pan-cancer was assessed by the Tumor Immune Estimation Resource (TIMER) database (http://timer.cistrome.org/) (Li et al., 2020b). Results showed that HELLS was significantly upregulated in most cancers, including cervical carcinoma (Fig. S1). Then, the HELLS expression was further assessed in the TCGA database. Figure 1B showed that HELLS was significantly upregulated in the cervical carcinoma group (N = 306) compared with the control group (N = 13). Next, HELLS expression was assessed in fifteen cervical carcinomas and paired normal tissues. In line with the results of bioinformatics analysis, we also found that HELLS was significantly up-regulated in the cervical carcinoma group compared with the normal control (Fig. 1C). Then survival analysis was performed on HELLS in Cervical squamous cell carcinoma (CESC) by GEIPA2 software, which revealed high HELLS expression was unfavorable to patient prognosis, regarding OS (overall survival), DFS (disease free survival) (Figs. 1C and 1D). Moreover, IHC staining also showed that HELLS was significantly up-regulated in the cervical carcinoma group compared with the normal control (Fig. 1E).

HELLS facilitates cervical cancer cell proliferation

To verify the function of HELLS in cervical cancer, the expression of HELLS was analyzed in four cervical cancer cell lines (Caski, SiHa, HeLa, c4-1). Figures 2A and 2B showed that HELLS was significantly increased in all cervical cancer cell lines compared with the normal ICE cells. Among these cell lines, SiHa cells showed the highest HELLS level, whereas C4-1 cells showed a relatively lower HELLS level. Therefore, to investigate the function of HELLS in cervical cancer in vitro, we overexpress HELLS (HELLS-OE) in C4-1 cells by lentivirus. Figures2C and D showed that HELLS expression was significantly increased by lentivirus. Next, the cell viability was assessed by CCK8. Figure 2E showed that C4-1 cell viability was significantly increased in the HELLS-OE group. Next, cell proliferation was assessed by the colony formation assay. Similarly, colony formation data showed that the proliferation abilities of C4-1 cells were significantly increased in the HELLS-OE group (Fig. 2F). Moreover, the cell proliferation was analyzed by EdU assay. Figure 2G showed that HELLS markedly promote C4-1 cells proliferation.

Subsequently, HELLS was knocked down in SiHa cells by siRNA interference. Figures 3A and 3B showed that HELLS expression was significantly decreased by si-HELLS. Next, cell viability and proliferation were assessed by CCK8, colony formation, and EdU assays. As expected, the knockdown of HELLS significantly reduced the cell viability (Fig. 3C) and proliferation ability (Figs. 3D and 3E) of SiHa cells.

HELLS facilitates cervical cancer cell proliferation by inhibiting ferroptosis

To verify the function of HELLS in cervical cancer cell proliferation, the cell viability of HELLS knockdown SiHa cells was treated with or without the apoptosis inhibitor ZVAD-FMK or the ferroptosis inhibitor ferrostatin-1. Figure 4A showed that the decline in SiHa cell viability induced by HELLS knockdown is reversed by these two inhibitors. Moreover, we found that ferrostatin-1 was more efficient than ZVAD-FMK in restoring SiHa cell viability, which was decreased by HELLS knockdown (Fig. 4A). To further confirm the results of Fig. 4A, we performed EdU assay to measure the DNA synthesis and cell proliferation of SiHa cells with or without HELLS knockdown and inhibitors treatment. As shown in Fig. 4B, EdU incorporation was significantly reduced in SiHa cells transfected with HELLS siRNA compared with control siRNA. However, this reduction was partially reversed by ZVAD-FMK and ferrostatin-1, indicating that both apoptosis and ferroptosis were involved in the inhibition of SiHa cell proliferation by HELLS knockdown. Our data suggest that ferroptosis may play an important role in HELLS-mediated cell proliferation. To further confirm that HELLS promotes cell proliferation by inhibiting ferroptosis, HELLS-OE C4-1 cells were treated with or without the ferroptosis inducer erastin. Figure 4C showed that C4-1 cell viability was significantly decreased by erastin, while the effect was restored by HELLS-OE. This data was also confirmed by EdU assay (Fig. 4D). Taken together, these data suggest that HELLS facilitates cervical cancer cell proliferation by inhibiting ferroptosis.

HELLS suppress cervical cancer cell ferroptosis by regulating Nrf2

Previous studies have confirmed that Nrf2, GPX4, and SLC7A11 are key regulators of ferroptosis. Therefore, we analyzed the correlation between the expression of HELLS and the expression of Nrf2, GPX4, and SLC7A11in cervical cancer using TIMER2.0 (http://timer.cistrome.org/). Figure 5A showed that HELLS expression was more significantly positively correlated with Nrf2, but not with GPX4 and SLC7A11, suggesting that HELLS may suppress cervical cancer cell ferroptosis by regulating Nrf2. To further confirm this conclusion, HELLS-OE C4-1 cells were treated with or without si-Nrf2. Figures 5B–5D showed that Nrf2 expression was significantly increased in HELLS-OE C4-1 cells and was markedly inhibited by si-Nrf2. Next, cell viability and cell proliferation were analyzed. Figure 5E showed that the promotion of cell viability by HELLS was significantly inhibited in the si-Nrf2 group. Similarly, the promotion of cell proliferation by HELLS was also significantly inhibited in the si-Nrf2 group (Figs. 5F and 5G). To further confirm the effect of Nrf2 on cell proliferation of HELLS-OE C4-1 cells, we performed EdU assay to measure the cell proliferation of HELLS-OE C4-1 cells with or without si-Nrf2 treatment. As shown in Fig. 5H, EdU incorporation was significantly increased in HELLS-OE C4-1 cells compared with control cells. However, this increase was partially reversed by si-Nrf2, indicating that Nrf2 was required for the promotion of cell proliferation by HELLS. These results suggest that HELLS enhances cervical cancer cell proliferation by regulating Nrf2 expression.