Study design and patient cohort
This was a prospective multicenter clinical study, including patients with a diagnosis of cervical cancer between October 2017 and May 2019. Inclusion criteria are as follows (1) patients with histologically confirmed cervical cancer, (2) patients with tumor staged IB1-IVa (FIGO 2009 definition), (3) patients who were not previously treated with any anti-cancer treatment, and (4) patients who underwent a pre-treatment contrast-enhanced computed tomography (CT) scan. Exclusion criteria were (1) patients with a history of previous chemotherapy or radiotherapy, (2) patients with a diagnosis of other cancers, or (3) patients with distant metastatic disease (para-aortic nodes involvement was not included). Institutional ethics review board approval was acquired for this study and written informed consent was waived.
All patients were treated with image-guided external beam radiotherapy (EBRT) and brachytherapy (BT), to a total dose of 85–90 Gy (EQD2, equivalent dose in 2 Gy single dose fractions). EBRT was delivered in 1.8–2.0 Gy/fraction, to a range of 45–50 Gy, using a 3D conformal technique. BT boost was volumetrically planned and delivered as weekly high-dose-rate fractions of 8 Gy EQD2 each after 15 times of EBRT. Concurrent weekly chemotherapy with cisplatin (40 mg/m2) was delivered for 4–6 weeks or carboplatin (AUC = 2) when feasible. We proposed ZNF582 methylation performance in a two-stage group sequential design as described in the following text:
The first stage: cervical exfoliated cells were collected pre-treatment (0 Gy) and periodically during treatment (after 24, 30, 36, 48, and 64 Gy). All liquid-based cytology samples were clinician-collected using the Cytoprep Brush (Hospitex Diagnostics SRL, Sesto Fiorentino, Italy) and preserved in CytoFast Solution (Hospitex Diagnostics SRL, Sesto Fiorentino, Italy). The cells were centrifuged and stored in phosphate-buffered saline (PBS) at −20°C from the day of collection.
The response to radiotherapy was evaluated by two skilled radiologists according to Response Evaluation Criteria in Solid Tumors (RECIST) Version 1.1 with the following specific standards: 1. complete remission (CR), defined as disappearance of all target foci; 2. partial response (PR), defined as a ≥30% reduction in total lesion diameter from baseline; 3. progressive disease (PD), defined as a ≥20% increase in total lesion diameter from baseline; 4. stable disease (SD), defined as an increase of <20% or a reduction of <30% in total lesion diameter from baseline. In this study, CR and PR were defined as satisfied response, SD as a modest response, and PD as non-response. ROC curve analysis was used to determine which M-index of the target gene ZNF582 would have the highest accuracy for discerning satisfied response from modest response and non-response.
The second stage, we confirmed the research result by an independent cohort at three centers (Hunan Cancer Hospital, Shandong Cancer Hospital, and Liaoning Cancer Hospital).
The study protocol was approved by the Institutional Review Board of Hunan Cancer Hospital. Informed consent was obtained from all participants in accordance with the Declaration of Helsinki.
Genomic DNA (gDNA) was extracted from the collected cells using the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany). A BioSpec-nano spectrophotometer (Shimadzu Corporation, Tokyo, Japan) was used to quantify the amount of extracted DNA.
DNA methylation tests
For DNA methylation tests, quantitative methylation specific PCR (QMSP) using TaqMan-based technologies was performed using the Lightcycler LC480 real-time PCR system (Roche Applied Science, Penzberg, Germany). Briefly, 500 ng gDNA was subjected to bisulfite conversion using the EZ DNA Methylation-Gold™ Kit (Zymo Research, CA, USA). The methylation levels of ZNF582 were then determined using the qPCR kit (Hongya Gene Technology Co., Ltd); the type II collagen gene (COL2A) was designed as the internal reference and tested with each specimen. The crossing point (Cp) value for COL2A, which is also the validity indicator of the test, should not be >35. For each sample, two Cp values were obtained: one from ZNF582 and another from COL2A. The DNA methylation level was assessed based on the methylation index (M-index) using the formula: 10,000 × 2 [(Cp of COL2A) − (Cp of ZNF582)][16, 19].
Establishment of ZNF852 overexpression cell line
HeLa (CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030) cells were transfected with 20 µg of ZNF582 expression adenoviral vector (Vigene Biosciences, Rockville, MD, USA; NM_144690) or control adenoviral vector (Vigene Biosciences, Rockville, MD, USA; pLent-GFP-Puro-CMV), screening of transferred cells with puromycin.
Cell viability assay
Cultured cells in the logarithmic growth phase were harvested using trypsin and seeded onto 96-well plates at 5000 cells/well. The culture medium was aspirated off and 100 µL XTT solution (20-300-1000, Biological Industries) was added at the indicated times, followed by incubation for 3 h. Optical density values were measured at 450 nm as an estimate of viable cell number using a microplate reader.
Cell cycle analysis by flow cytometry
Cells were collected, washed with cold PBS, fixed in 70% ethanol, and stored at 4°C overnight. The fixed cells were washed with PBS and stained with 500 µL propidium iodide (PI; BD Biosciences, Franklin Lakes, NJ, USA) in dark conditions for 30 min. The distribution of cell cycle stages was measured by flow cytometry within 1 h. The cell debris and fixation artifacts were gated out.
Quantitative RT-PCR (Q-PCR)
Total RNA was extracted from cultured cells using Trizol (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA using oligo (dT) primers and a cDNA synthesis kit (Invitrogen, USA) according to the manufacturer’s protocol. Gene expression was assessed by qRT-PCR using SYBR Premix Dimer Eraser (Perfect Real-Time) assay kits and the following primers:
Real-time PCR was performed using the Roche LC480 PCR System and results were analyzed using the comparative Ct (threshold cycle) method.
Western blotting and immunofluorescence assays
Crude cellular proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat milk and probed with primary antibodies against ZNF582 (ABclonal, A14453;1:500) p21/Waf (Cell Signaling Technology, 9932kit, 1:1000), p27(KIP1) (Cell Signaling Technology, 9932kit, 1:1000), p21 (Cell Signaling Technology, 9932kit, 1:1000) CyclinD1 (Cell Signaling Technology, 9932kit, 1:1000), CDK2(Cell Signaling Technology, 9932kit, 1:1000),CDK4(Cell Signaling Technology, 9932kit, 1:1000), PI3K (Abcam, ab191606;1: 1000)), Akt (Cell Signaling Technology, 9272,1:1000), and α-tubulin (Abcam, ab7291,1:5000) overnight at 4°C. Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1.5 h and immunolabeling detected using a Bio-Rad imaging system. Alternatively, fixed cells were hydrated, blocked, incubated with primary antibody against ZNF582 (Biorbyt, 1:200 dilution) and Akt (Cell Signaling Technology, 1:200 dilution) at room temperature for 1 h, incubated with fluorphore-labeled secondary antibody for 10 min, and then examined under fluorescence microscopy.
Female BALB/c nude mice (RRID:IMSR_ORNL:BALB/cRl) at 6 weeks of age were obtained from SLA Laboratory Animal (Changsha, China) and housed in a specific pathogen-free facility. Individual mice were subcutaneously injected with 5 × 106 HeLa cells. Four weeks after inoculation, the mice were sacrificed and the tumors dissected out, weighed, and photographed. The tumor tissues were then fixed in 10% formalin, paraffin-embedded, sectioned at 3 µm, and stained with hematoxylin and eosin. The experimental protocol was approved by the Animal Care Committee of Hunan Tumor Hospital and the Affiliated Tumor Hospital of Xiangya Medical College (Changsha, China).
Statistical analyses were performed using SPSS(RRID:SCR_002865)18.0 for Windows (IBM, Inc., Chicago, IL, USA) and PLINK(RRID:SCR_001757)1.07. Continuous variables are presented as mean ± standard deviation. Qualitative variables are described by frequencies and percentages. The comparison between groups was conducted by t-test, ANOVA, Chi-squared test or non-parametric test (Man-Whitney Test) (selected according to the data type) and a p-value of <0.05 was considered to indicate significance.