C33A and Hela, types of the human cervical cancer cell lines,were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). RPMI-1640 medium which contained 10% fetal bovine serum and 0.1% penicillin-streptomycin was used to culture the CC cells.Cells were maintained in a humidified incubator at 37°C with 5% CO₂ subsequently.

The open reading frame of human VTN was integrated into the eukaryotic expression vector GFP-pCDH (System Biosciences, Inc.). The resulting recombinant plasmid, pCDH-VTN, was co-transfected with lentivirus plasmids psPAX2 and pMD2.G (Genechem Inc, Shanghai, China) into HEK293T cells using Lipofectamine 3.0 (Invitrogen) at a concentration of 60%, according to the manufacturer’s instructions. The cells were cultured for 48 hours after transfection. Subsequently, Hela and C33A cells, following lentivirus transfection, underwent selection with puromycin at a concentration of 1 μg/mL1 μg/mL (Invitrogen) for a period of two weeks.

Total RNA was extracted from cells using Trizol reagent (Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions. Then, cDNA was synthesized and qRT-PCR was performed using a cDNA Reverse Transcription kit and Step One Real-Time PCR system (Applied Biosystems, CA, USA) in an ABI Prism 7900HT instrument (Applied Biosystems) according to the supplier’s recommendations. The primers used were as follows: VTN: F: 5′-GGGTCTACTTCTTCAAGGGGAA−3′, R: 5′-AATGAACTGGGGCTGTCTGG−3′; GAPDH: F: 5′-GGAGCGAGATCCCTCCAAAA T−3′, R: 5′-GGCTGTTGTCATACTTCTCATGG-3′. All values were normalized to the level of GAPDH. The relative mRNA expression of VTN was calculated using the 2^-ΔΔCt comparative method. All experiments were repeated three times.

The cells were disrupted by RIPA buffer fortified with a protease inhibitor concoction and protein measured with a BCA protein assay kit (Beyotime). After that, the cell lysates were fractionated using SDS-PAGE, followed by the transferation of equivalent protein quantities onto polyvinylidene difluoride (PVDF) membranes (Millipore). Following blockage, the PVDF membranes were subjected to overnight incubation at 4°C in the presence of primary antibodies. Detection of the primary antibodies was achieved using secondary goat anti-rabbit or goat anti-mouse HRP-IgG (Proteintech, Rosemont, IL, USA). Subsequently, immunoreactive complexes were visualized via a chemiluminescent substrate and documented on film.

Cells were centrifugated,with a concentration of 10⁴ cells/mL, and then were seeded in a 96-well plate with 100 µL of culture medium per well. Each group was established with four replicate wells. The plate was then placed in a 37°C incubator to facilitate continued cultivation. After seeding, the cells were collected, and cell quantification was undertaken using the CCK-8 on days one, two, three, and four. CCK-8 was added to each well and subsequently layed in a 37°C incubator for further two hours. The absorbance was counted with a microplate reader. The experiment was iterated three times.

Cells (100 µL) were added to a 6-well plate with a concentration adjusting to 8×10³ cells/mL, subsequently cultured for seven days, and the culture medium were refreshed every two days. Crystal violet was used to stain the colonies. For each condition, the number of colonies within a given area was counted. All experiment was repeated three times.

Cells were seeded at a density of 1×10^{^6} cells per well in a 6-well plate and cultured at 37°C with 5% CO₂ until reaching 90% confluence, forming a uniform monolayer. A sterile 200 μL pipette tip was used to create a straight scratch across the cell layer, taking care to apply even pressure to ensure a consistent width without damaging the plate surface. The cells were then rinsed twice with phosphate-buffered saline (PBS) to remove any detached cells, followed by the addition of serum-free medium. The plates were incubated at 37°C with 5% CO₂, and the wound width was observed under a microscope at 0 and 24 hours.

Cells (50 μL) were initially prepared by suspending in serum-free RPMI-1640 medium and added to Transwell chamber inserts to ensure proper density and even distribution. For each experimental group, 200 μL of cell suspension containing 1×10^{^6} cells was carefully transferred into the upper chamber of a Transwell insert, which is designed to allow cell migration through a porous membrane. RPMI-1640 medium containing 20% fetal bovine serum (FBS) was added to the lower chamber, creating a chemoattractant gradient to encourage cell invasion. The Transwell plates were incubated at 37°C with 5% CO₂ for 24 hours to allow cells to migrate toward the serum in the lower chamber. After incubation, the non-invading cells on the upper surface of the membrane were gently removed using a cotton swab. The chambers were then rinsed twice with phosphate-buffered saline (PBS) to remove any residual medium. Invading cells attached to the lower surface of the membrane were fixed with 4% paraformaldehyde for 20 minutes, then stained with 0.1% crystal violet solution for 15 minutes at room temperature. The membrane was thoroughly rinsed to remove excess stain, and the stained cells on the lower surface were visualized and counted under a microscope. The number of invaded cells was recorded for each group, and the experiment was repeated three times for statistical validity.

Statistical analyses were performed using SPSS 21.0 (SPSS, Chicago, Illinois, USA), and the presented values were expressed as mean ± standard deviation (SD). Two-sided t-tests were used for comparisons between two groups, and one-way analysis of variance (ANOVA) was used for comparisons among multiple groups.

In order to explore the roles of VTN in cervical cancer cells, our group transfected pCDH-VTN to enhance VTN expression and transfected VTN shRNA to inhibit VTN levels in Hela and C33A. Negative controls were established by transfecting cells with scrambled pCDH or shRNA. We quantified mRNA expression using qPCR ( Figures 1A, B ) and protein level using Western blotting ( Figures 1C, D ). The over-expression and down-expression VTN models were successfully built.

pCDH-VTN transfection in HeLa and C33A promoted cell proliferation ( Figures 2A, C , P<0.001), higher pCDH-VTN transfection colony formation rate was found in both HeLa and C33A cells (Figures 2B, D), compared to the control group. Then, pCDH-VTN transfected HeLa and C33A cells resulted in faster wound closure than the control group ( Figures 3A, B , P<0.001). Furthermore, the rate of distance reduction between wound edges was increased in the VTN overexpressing group compared to the negative control group (NC group). Up-regulated VTN significantly enhanced the invasion of HeLa and C33A by the Transwell invasion assay,compared with the control group ( Figures 3C, D , control, P<0.001).

To investigate the effect of VTN on the epithelial-mesenchymal transition (EMT) of cervical cancer cells, we assessed the expression of typical EMT hallmark proteins, such as ZO-1, E-cadherin, β-catenin, and N-cadherin. Overexpression of VTN in cervical cancer cells led to a decrease in the protein expression levels of ZO-1 and E-cadherin and an increase in the expression levels of β-catenin and N-cadherin ( Figure 4A ). Conversely, the knockdown of VTN expression resulted in increased protein expression levels of E-cadherin and ZO-1, while decreased expression of N-cadherin and β-catenin ( Figure 4B , P<0.001). These results indicated that VTN played a significant role in regulating the EMT of cervical cancer cells ( Figure 5 ).