The findings of this study support the potential utility of miR-378, miR-20a, and miR-520a-3p as promising prognostic biomarkers of cervical cancer. However, further large-cohort validation studies in various populations are needed to verify the prognostic accuracy and clinical significance of these miRNAs. Functional studies are required to explore the molecular mechanisms through which these miRNAs regulate cervical cancer pathogenesis and elucidate their potential as treatment targets.

Women who underwent primary tumor resection at the Hongqi Hospital Affiliated with Mudanjiang Medical University were recruited for this study from January 2019 to January 2021. Preoperative serum specimens and postoperative tumor tissues were obtained from 127 patients with cervical cancer, 62 with a good prognosis, and 65 patients with a poor prognosis. The inclusion criteria were: (1) cervical cancer as the first primary tumor; (2) cervical cancer diagnosed by preoperative biopsy or postoperative histopathology; (3) complete clinicopathological, demographic, and follow-up information. The exclusion criteria were: (1) underwent clinical therapy before collecting the serum samples; (2) had confirmed distant metastasis before surgery; (3) had other malignant tumors; (4) incomplete clinical data or interrupted follow-up. This study followed the ethical standards stipulated in the Declaration of Helsinki. The Ethics Committee of the Chinese Academy of Sciences and the Hongqi Hospital Affiliated with Mudanjiang Medical University approved this study (No. 2024028), and the patients signed informed consent before sample collection.

The included patients were followed up regularly for three years at 3-month intervals from the first day after surgery. Follow-up was terminated at the time of disease progression, recurrence, metastasis, or cervical cancer-related death. The follow-up information was collected through a medical history review and telephone interview until January 31, 2024. The following information was collected: (1) basic information, such as age, tumor diameter, TNM stage, differentiation grade, depth of invasion, metastasis, pathological type, and myometrial invasion; (2) follow-up information, including disease progression, time of recurrence or metastasis, location of metastasis, time of occurrence of a second or additional primary malignant tumor, time and cause of death, and last follow-up time. Patients with a stable postoperative condition, no recurrence, no metastasis, or no death were defined as having a good prognosis. Patients who experience disease progression, recurrence, metastasis, or cervical cancer-related death were defined as having a poor prognosis.

We used the optimal cut-off values derived from the receiver operating characteristic (ROC) curves to divide the patients into high (≥ 0.132) and low (< 0.132) miR-378, high (≥ 0.075) and low (< 0.075) miR-20a, and high (≥ 0.810) and low (< 0.810) miR-520a-3p groups.

The human cervical epithelial cell line (CRL2614) and cervical carcinoma cell lines (SiHa, C33a, HeLa, CasKi, MS751, ME180, and AV3) were obtained from the Chinese Academy of Sciences Cell Bank of Type Culture Collection. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) and 100 IU/mL penicillin-streptomycin (Thermo Fisher Scientific, Inc.) at 37 °C in a humidified atmosphere with 5% CO. All experiments were conducted with early-passage cells (passages 3-10).

Tissues and serum samples were transferred to sterile tubes, and total RNA was extracted immediately using TRIzol reagent (Sigma Aldrich) following the manufacturer's instructions. RNA yield and purity were measured by NanoDrop One (Thermo Fisher Scientific), using samples with an A260/A280 absorption ratio greater than 1.8. The isolated RNA was reverse-transcribed using the Primescript Reverse Transcription kit and miScript SYBR Green PCR kit (Qiagen, Valencia, CA, USA). Using U6 as an internal reference gene, the levels of miR-378, miR-20a, miR-95-3p, miR-330-3p, miR-520a-3p, miR-155-5p, miR-651-3p, and miR-331-3p in the tissue and serum samples were measured. The following reaction procedure was used: 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min, and 72 °C for 1 min. The relative amounts of the miRNAs were calculated using the 2 method. The primer sequences for the miRNAs and U6 are listed in Table 6.

The lentivirus gene transfer vectors carrying the miR-378 (Gene ID: 100314258), miR-20a (Gene ID: 777935), and miR-520a-3p precursor (Gene ID: 574467) and encoding green fluorescence protein (GFP) were constructed by GenePharma Corporation (Shanghai, China). Synthetic miRNAs mimics, mimic controls, inhibitors, and inhibitor controls for miR-378, miR-20a, and miR-520a-3p were purchased from GenePharma. The vector transfections were conducted using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. Cells were seeded in six-well plates and grown to 70 ~ 80% confluency. The cells were transfected and then cultured in a complete medium for 4 h. The final miRNA mimic concentration was 50 nM.

The cell viability was assessed by the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Inc.). Briefly, after undergoing the relevant treatment for 48 h, cells (5 × 10 cells/mL) were seeded into 96-well plates and incubated for another 48 h. Subsequently, 10 µL CCK-8 solution was added to each well, and the plates were incubated for 4 h at 37 °C. The absorbance at 450 nm was determined using a BioTek Synergy H1 microplate reader (BioTek Instrument Inc.).

The invasion of cells was assessed using Transwell chambers (6-well plates, 8-µm pore size; Corning Incorporated). The chambers were pre-coated by placing 100 µL Matrigel (Beijing Solarbio Science & Technology Co., Ltd.) into them. After undergoing the relevant treatment for 48 h, starved cells (5 × 10 cells/mL) were added into the upper chamber, while 500 µL of DMEM supplemented with 20% FBS was added into the lower chamber. After 36 h, the invasive cells were fixed with 70% methanol for 30 min at ambient temperature and stained with 0.1% crystal violet. The number of invading cells was counted under a light microscope (magnification, ×200; Olympus Corporation).

The migratory ability of C33a cells was evaluated using the wound-healing assays. After undergoing the relevant treatment for 48 h, the C33a cells were grown to 80%-90% confluency in 6-well plates. The cell monolayer was scratched in a straight line (time point 0). The scratch width was photographed under a light microscope (magnification, ×200; Olympus Corporation) at time 0 and after 48 h.

The C33a cell apoptosis was measured using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection Kit (Vazyme Biotech Co., Ltd.). After undergoing the relevant treatment for 48 h, cells (5 × 10 cells/mL) were seeded into 96-well plates and incubated for another 48 h. C33a cells (5 × 10 cells/mL) were harvested and incubated with 5 µL of Annexin V-FITC and 5 µL of PI for 20 min in the dark at ambient temperature. The rate of apoptotic cells was calculated using a FacsCalibur flow cytometer (BD Biosciences), and the data were analyzed using CellQuest, Version 3.3 (BD Biosciences) software.

Statistical analysis was performed using IBM SPSS Statistics for Windows, Version 20.0 (IBM Corp., Armonk, NY, USA). Quantitative results are expressed as means ± standard deviations and were compared by unpaired Student's t-test (two groups) or one-way analysis of variance (three or more groups). ROC curve analysis and the area under the ROC curve (AUC) were used to assess the prognostic predictive value of miR-378, miR-20a, and miR-520a-3p for patients with cervical cancer. The optimal cut-off values were determined from the ROC curves using Youden's index. The Kaplan-Meier curves were used to analyze the patient's progression free survival (PFS). Univariate and multivariate Cox proportional hazards regression were used to identify independent risk factors associated with cervical cancer prognosis. Pearson's correlation analysis was used to assess the correlation between miR-378, miR-20a, and miR-520a-3p and squamous cell carcinoma antigen (SCC-Ag) and carcinoembryonic antigen (CEA). A two-sided P-value < 0.05 was considered statistically significant.