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 Center Lihuili Hospital (DYLL2018079). All patients signed informed consent prior to participating in the study.
Immunohistochemistry (Ihc)
Immunohistochemistry staining for HELLS carried out. 4-µm sections of cancer tissues were rehydrated and incubated with anti-HELLS (1:100, ab3851, Abcam) overnight. The next day, tissues sections were washed by PBS, and incubated with goat-anti mouse HRP-conjugated (1:1000, ab6721, Abcam) for 1 h at 37°C, and visualized by DAB. The sections were counterstained by haematoxylin (Solarbio).
Cell culture and treatment
Human epidermal cells (HaCaT) 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, USA) supplemented with 10% FBS. All cell lines were cultured in an incubator with 5% CO2 at 37℃.
To overexpress HELLS, The MALAT1 lentiviral plasmid was purchased from Genechem (Shanghai, China), and were transfected into C4-1 cells using Lipofectamine™ 3000. The cells were selected by 3µg/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 ThermoFisher (Shanghai, China), and SiHa or C4-1cells were transfected by Lipofectamine® RNAiMAX Transfection Reagent (Invitrogen, USA), in accordance with the manufacturer guidelines.
To inhibit apoptosis or ferroptosis, SiHa cell with si-HELLS were stuimulated with Z-VAD-FMK (10 µM) or ferrostatin-1 (0.5 µM). To induce ferroptosis of cervical cancer cell, HELLS-OE C4-1cells were treated with erastin (20 µM) for 24 h.
Quantitative Real-time Pcr (Qrt-pcr)
Total RNA was extracted from cervical cancer tissues and cells using Trizol reagent (Invitrogen). The SMART PCR cDNA Synthesis Kits were used for reverse transcriptional PCR (Clontech). qRT-PCR was carried out with SYBR Green incorporation (ThermoFisher). The 2−ΔΔCt method was utilized to determine the fold changes of RNA transcripts, and the actin gene was employed as a reference gene.
Enzyme Linked Immunosorbent Assay (Elisa)
The ROS and iron level in C4-1cells HELLS-OE C4-1cells were treated with erastin (20 µM) analyzed by ROS Elisa kit (A098751-96T, affandi, shanghai, china) or iron assay kit (MAK025, Sigma) as the manufacturer’s instructions.
Cell Counting Kit-8 (Cck8)
The cell viability of cervical cancer cells was analyzed by using the CCK8 (Thermo fisher). Cervical cancer cells (2×104 cells/ml) were seeded on 96-well plate (100µl 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.
Western-blotting Analysis
Western-blotting was performed as our previous study [14], and the antibodies used were as follows: anti-HELLS (1:1000, ab3851, Abcam), anti-Nrf2 (1:1000, ab137550, Abcam), and anti-actin (1:5000, ab8224, Abcam) overnight at 4°C.
Colony Formation Assay
Colony formation was performed as our previous study[14], Briefly, cervical cancer cells were seeded in 10 cm-plates at a concentration of 1000 cells/well and cultured normally for 14 days in RPMI 1640 with 10% FBS. The colonies were fixed and stained with 0.4% crystal violet (Sigma) and methanol before being counted.
Statistical analysis
In the current study, statistical analyses were carried out by SPSS 22.0 software (SPSS, USA). The mean ± standard deviation is used to represent all the data, the differences between two groups were analyzed by Student’s t-test, and one-way ANOVA was used to investigate differences more than two groups. P values < 0.05 were considered significant.