Human EC cell lines (Ishikawa, AN3CA, HEC-1-A, and HEC-1-B) were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China). CTTN-RNAi, HitransG A, and short-hairpin RNAi (shRNAi) were purchased from Genechem Biotech Co. Ltd (Shanghai, China). TRIzol reagent (3101-100) was bought from Pufei Biotechnology Co. Ltd (Shanghai, China). M-MLV Reverse Transcriptase (M 1705) was bought from Promega (Madison, USA). Mouse monoclonal anti-Rac1 antibody (ab33186), rabbit monoclonal anti-Cortactin antibody (ab81208) and primary antibody-like anti-F-actin (ab130935) were obtained from Abcam (Cambridge, MA, USA) and primary antibody anti-Rac1 (bs-4186R) was purchased from Biosynthesis Biotechnology Co. Ltd (Beijing, China). Secondary antibody goat anti-mouse (A-21424) and McCoy’s 5A medium (16600-082) were purchased from ThermoFisher Scientific (Massachusetts, USA), and secondary antibody HRP-goat anti-rabbit (AS1107) was purchased from ASPEN Biotechnology Co. Ltd (Wuhan, China). Transwell Permeable supports(353097), Corning BioCoat cell culture inserts (354480), Dulbecco’s Modified Eagle Medium (DMEM) (10-013-CVa), and Minimal Essential Medium (MEM) (10-010-CV) were purchased from Corning (NY, USA). Fetal bovine serum (FBS) (VS500T) was bought from Bovogen (Victoria, Australia). A real-time PCR instrument (LightCycler 480 II) (Roche, Basel, Switzerland) and a confocal quantitative image cytometer (CQ1) (Yokogawa, Tokyo, Japan) were used. Tris-EDTA antigen retrieval solution (PR30002) was bought from Proteintech (Rosemont, USA). Polymer Detection system for immuohistological stainning (PV-9000) and DAB kit (ZLI-901) were bought from ZSGB-Bio (Beijing, China).
Endometrial tissues were obtained from The First Affiliated Hospital of Guangxi Medical University (Nanning, China) between June 2019 and December 2020 containing 47 patients of EC (EC group) and 45 patients of normal endometrium (NE group). All of the patients didn’t receive any hormone therapy, chemotherapy or radiotherapy before surgery. All tissues were taken from hysterectomy specimens. The surgical indications of patients in NE group were leiomyoma and uterine prolapse. EC staging was assessed based on the International Federation of Gynecology and Obstetrics (FIGO) system . All patients signed informed consent, and the study was approved by the Ethics Committee of First Affiliated Hospital of Guangxi Medical University. The ethics number is 2021 (KY-E-309).
Total RNA was extracted using Trizol Reagent. A Moloney murine leukemia virus (M-MLV) kit was used for reverse transcription to obtain single-stranded complementary DNA (cDNA). PCR was then performed with the cDNA serving as the template. The primer sequences of CTTN-1 and CTTN-2 were both designed according to the gene CTTN (NM_005231). The CTTN-1 was applied to the experiment in vitro and CTTN-2 was used in clinical tissues (Table 1). For the two-step real-time PCR, PCR conditions used were 95 ℃ for 30 seconds, followed by 40 cycles of 95 ℃ for 5 seconds, and 60 ℃ for 30 seconds, and a dissociation program of 95 ℃ for 15 seconds, 60 ℃ for 30 seconds and 95 ℃ for 15 seconds. The gene expression levels were calculated using the 2−ΔΔCT method. The experiment was performed three times.
Table 1. PCR primers and sequences
Primer sequence (5’à 3’)
CTTN (cortactin), ACTB (actin beta). The ACTB gene was selected as the internal reference gene.
Immunohistochemical staining (IHC)
The specimens were fixed with 10% formalin and embedded in paraffin. The experiments were performed using Polymer Detection system for IHC according to the manufacturer’s instructions. The mouse monoclonal anti-Rac1 antibody (1:200, ab33186) and rabbit monoclonal anti-Cortactin antibody (1:200) were added to the sections. Positive staining percentage was graded as follows: 0, no positive cells, 1, 25% or fewer positive cells, 2, 26% to 50% positive cells, 3, 51% to 75% positive cells, and 4, 76% or more positive cells. Intensity of staining was graded as follows: 0, no intensity, 1, weak intensity, 2, moderate intensity, and 3, strong intensity . The total score was equal to the product of two scores. The total score less than 6 was low expression, and the total score greater than or equal to 6 was regarded as high expression.
Construction of a lentiviral system
The complete CTTN gene sequence (NM_005231) was obtained from the GeneBank database. Then, using the Invitrogen BLOCK-iT RNAi Designer (Invitrogen, Carlsbad, CA, USA), shRNA interference target sequences were identified. Using the basic local alignment search tool (BLAST) for comparison with the genome database, homologous coding sequences were ruled out to eventually determine three shRNA interference target sequences, as follows:
CTTN-KD2 (5'-gaAAGACTACTCCAGTGGTTT-3'), and
The plasmids were synthesized by Shanghai Genechem Biotech Co. Ltd (Shanghai, China).
Cell transfection and grouping
HEC-1-A and HEC-1-B cells were passaged, inoculated into 6-well plates at 2 × 105 cells/well, and cultured for 2 days. Each cell line was divided into experimental groups (the KD1, KD2, and KD3 groups) and a control group (the CON group). The KD1, KD2, and KD3 groups were transduced with CTTN-KD1, CTTN-KD2, and CTTN-KD3 lentiviral vector plasmids, respectively, and the CON groups were transduced with an empty vector (lentiviral vector without siRNA), for 24 hours in 40 uL 1X HitransG A. The cells were then incubated with McCoy’s 5A medium (HEC-1-A) or MEM (HEC-1-B), both containing 10% FBS, for 72 hours. The relative mRNA expression of CTTN was assessed by Quantitative PCR to determine the transfection efficiency in EC cells, and cells were subjected to cell migration, invasion, scratch assays，immunofluorescence staining and Western blot analysis.
Transwell migration and invasion assay
For the cell migration assay, serum-starved transduced cells (HEC-1-A 2×105 cells/ well, HEC-1-B 1×105 cells/well) were suspended and added to the upper chambers of 24-well Transwell plates with 8.0-μm pores. Full-serum medium containing 10% FBS was then added into the lower chambers as a chemoattractant. After incubation at 37 °C with 5% CO2 for 24 hours, cells that had attached to the lower surface of the membrane were fixed with methanol and stained with 0.1% crystal violet. The migratory cells were then observed under a microscope (magnification ×200, XDS-100, Shanghai Caikon Optical Instrument Co. Ltd, Shanghai, China), and the number of invading cells in nine randomly selected fields was counted and averaged. The cell invasion assay was performed in the same way as the cell migration assay, except that the upper chambers of the 24-well Transwell plates were precoated with Matrigel membrane (Corning, USA).
After 72 hours of transfection, cells in each group were cultured to the logarithmic growth phase, and the cell density was adjusted to 1×106 cells/ml in a 96-well plate until the cells reached more than 90% confluence. The cells were inoculated into a marked 96-well plate. After they had adhered to the wall and were growing, the cells with good growth and uniform density were taken for a scratch assay. To perform the scratch assay, a scratch instrument was aimed at the center of the upper end of the 96-well plate and gently pushed upward to form scratches. Residual cells in the scratch area were washed away using phosphate-buffered saline (PBS). After culture, cell migration was detected using a Celigo scanning plate at 0 hours and 48 hours (for HEC-1-A cells) and at 0 hours and 24 hours (for HEC-1-B cells) after scratching. The cell areas at the detection times were analyzed, and cell mobility was calculated. The migration area was calculated as the cell area at 24 hours/48 hours minus the cell area at 0 hours. Mobility was calculated as the migration area/1-cell area at 0 hours. Each group was provided with three compound holes.
After 72 hours of transfection,, the total proteins of cells in each group were extracted, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed. After transmembrane transfer, skimmed milk powder was added to the cells for 1 hour of blocking. The first antibody (rabbit anti-human Rac1 antibody, 1:500,bs-4186R) was then added for incubation overnight, following which the secondary antibody (1:10000) was added for 1-hour incubation, with β-actin serving as the internal control. The band intensity was quantified with ImageJ software. The target protein level was expressed as the gray value of target protein band/β-actin band. The experiment was repeated three times.
After transfection, HEC-1-A and HEC-1-B cells were cultured to the logarithmic phase, and a 1×104 single-cell suspension was prepared and inoculated into a 96-well glass-bottom plate (a special laser confocal culture plate). The plate was then placed in an incubator until the cells formed a monolayer. After being rinsed twice with PBS, the cells were fixed with 4% paraformaldehyde for 10 minutes and then rinsed again with PBS for 5 minutes. Permeabilization was performed using 0.2% Triton for 5 minutes, after which the cells were rinsed twice with PBS, for 5 minutes each time. A blocking solution (PBS containing 5% BSA and 10% sheep serum) was added, and the plate was placed in a wet box at room temperature for 30 minutes. F-actin antibody (1:200) was added, and the plate was refrigerated at 4 °C overnight before being rinsed three times in PBS, for 5 minutes each time. Fluorescein isothiocyanate (FITC)-labeled goat anti-mouse fluorescent IgG secondary antibody (1:400) was added, and the plate was incubated in the dark at 37 °C for 45 minutes. The secondary antibody was discarded, and the cells were rinsed three times with PBS, for 5 minutes each time, and then once with distilled water. The cells were then incubated with DAPI (4’,6-diamidino-2-phenylindole) at room temperature for 10 to 15 minutes, before being rinsed twice with PBS, for 5 minutes each time. Finally, 100 µl PBS was added to each well for preservation and observation.
Laser confocal microscopy
A quantitative image cytometer (CQ1, Yokogawa, Japan) was used to obtain the best tomographic images of cells with excitation filter wavelengths of 405 nm, 488 nm, and 561 nm. Each of the four quadrants of the culture dish was observed, and the cell morphology was recorded.
The software SPSS 22.0 (IBM, USA) was used for statistical analysis. A t-test was used for measurement data. A one-way analysis of variance (ANOVA) method was used for counting data. Correlation analysis was performed by the Pearson or Spearman correlation analysis method. A P-value < 0.05 was considered statistically significant.