Page 209 - EJMO-9-2

Eurasian Journal of
Medicine and Oncology Tetramethyl thyroxine boosts bladder cancer

V-FITC kit (Sigma-Aldrich, USA) was used according antibody and horseradish peroxidase were incubated at
to the instructions. The cells were mixed with 5 μL of room temperature. After 2 h, the membrane was washed
Annexin V-FITC and 10 μL PI and incubated and stained 5 times with 1× TBST buffer for 5 min each time. Finally,
in the dark for about 15 min. After staining, the solution the protein expression was observed using the enhanced
was transferred to a flow cytometry tube for detection chemiluminescence kit (PE0010, Solarbio, China). The
within 1 h, and the effect of T4 treatment on cell apoptosis band density was quantified using Image J. The grayscale
was evaluated using FlowJo V10 software (BD Biosciences, values of the protein bands in the experimental group were
USA). Three independent biological replications of this compared with those in the PBS group, with all proteins in
experiment were performed. the PBS group normalized.
2.4. Determination of cell migration rate by cell 2.7. Xenograft BC mouse model
scratch assay Immunodeficient mice were purchased from SiPeifu
4
2 × 10 T24 and EJ-1 cells were seeded in equal amounts (Beijing) Biotechnology Co., Ltd. (China). EJ-1 cells
in a 24-well plate and cultured. After the cells adhered, a were injected subcutaneously into the right shoulder of
6
200 μL pipette tip was used to scratch the well plate covered each mouse at a dose of 1 × 10 cells per mouse. All mice
with cells, and then it was washed 2 – 3 times with PBS to were randomly divided into two groups (n = 5). The
wash away the suspended cells. The migration of T24 and experimental group was intraperitoneally injected with
EJ-1 cells was observed and recorded using microscopy 2 mg/mouse of T4 every day, and the control group was
before the addition of T4 treatment and at 24 h and 48 h intraperitoneally injected with the same amount PBS.
post-T4 treatment. Image J software was used to calculate During this period, changes in mouse tumor volume
and analyze the effect of T4 treatment on cell migration were recorded every 3 days, and tail blood was collected
rate. and stored every 7 days. After undergoing T4 treatment
for 30 days, they were immediately euthanized, and the
2.5. Quantitative real-time polymerase chain tumors were collected and photographed and weighted.
reaction (qRT-PCR) The formula for calculating tumor volume is as follows:

First, treat T24 and EJ-1 with T4 and collect the cells, (length × ) (width )2
then use The RNAprep pure cell kit (DP430, Tiangen, Tumor volume = 2
China) to extract total RNA. FastKing RT Kit (KR116-
02, Tiangen, China) was used to reverse transcribe cDNA 2.8. Enzyme-linked immunosorbent assay
and then analyze it quantitatively using Real-Time PCR
System (QuantStudio1-A40425, Thermo Fisher Scientific, Blood was taken from the mouse tail and centrifuged for
USA) and PowerUp SYBR Green Master Mix (01000439, serum separation, and the concentrations of T3, T4, and
Thermo Fisher Scientific, USA) for quantitative analysis. TSH in the mouse serum were detected using Jianglai Bio-
GAPDH was selected as the internal reference gene, and enzyme-linked immunosorbent assay kit.
expression changes were analyzed computationally using 2.9. Statistical analysis
the δδCT algorithm. Primers were purchased from Tsingke
Biotechnology Co., Ltd. (China), and details are shown in The statistical analysis for this study was conducted using
Table 1. GraphPad Prism 8. The data were presented as mean
± standard deviation of three independent biological
2.6. Western blotting replicates. The Student t-test was used to analyze statistical
differences between two groups, while one-way analysis
T24 and EJ-1 cells were treated with T4 for 48 h. The cells of variance was utilized for comparisons involving three
were collected and the total protein amount was evaluated or more groups. All statistical tests were two-sided, and a
using BCA reagent (PC0020, Solarbio, China). After protein p<0.05 was considered statistically significant.
separation by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, the samples were transferred to PVDF 3. Results
membrane (Millipore, USA), shaken well in TBST containing
5% skim milk, and after 1 h, the following monoclonal 3.1. Tetramethyl thyroxine promoted proliferation
antibodies were added: anti-human β-actin (1:5000; of T24 and EJ-1 cells
Cell Signaling Technology, USA), anti-TP53 (1:1000; T24 and EJ-1 cells were treated with varying concentrations
Cell Signaling Technology, USA), anti-VEGF (1:1000; Cell of T4 to examine its effects on BC cell proliferation. The
Signaling Technology, USA), and anti-αV (1:1000; Cell results indicated that after 24 h and 48 h of treatment, the
Signaling Technology, USA). Subsequently, the secondary proliferation rates of T24 and EJ-1 cells in the experimental

Volume 9 Issue 2 (2025) 201 doi: 10.36922/EJMO025080037