ACAT2 contributes to cervical cancer tumorigenesis by regulating the expression of the downstream gene LATS1

Clinical samples

Human cervical cancer tissue microarrays were obtained from Shanghai Outdo Biotech Co., Ltd. (Catalog No.: HUteS154Su01, Lot No.: XT17-039), comprising 119 cervical cancer cases with survival data, including 35 cases with adjacent non-cancerous samples. The surgical samples were collected between January 2010 and October 2011. The Ethics Committee of Hebei North University Affiliated First Hospital approved this study (Ethics ID: K2024050), which complied with the Declaration of Helsinki.

Cells culture

Human immortalized cervical epithelial cells H8 (ATCC, United States) and cervical cancer cell lines C-33A (TCHu176), Ca Ski (TCHu137), SiHa (TCHu113), and HeLa (TCHu187) (all obtained from the Cell Bank of the Chinese Academy of Sciences) were grown in RPMI 1640 or DMEM media (10-040-CV/10-013-CV, Corning, United States) supplemented with 10 % fetal bovine serum (FBS; Wuhan Ausbian Biotechnology Co., Ltd., China). All culture media contained Penicillin-Streptomycin (100×) (Gibco; Thermo Fisher Scientific, Inc., United States). These cells were maintained in a controlled environment with 5 % CO₂ and a temperature of 37 °C. Each cell line’s authenticity was verified by its supplier, and all tested negative for mycoplasma presence.

RNA isolation and quantitative real-time PCR (qPCR)

Total RNA was isolated from the above cell lines with TRIzol reagent (15596026, Sigma-Aldrich, United States). Subsequently, cDNA was generated using the Hiscript QRT Supermix for qPCR kit (Vazyme, China). Gene expression levels of target genes and internal control GAPDH was quantified using the VII7 RT-PCR system (Thermo Fisher Scientific, Inc., United States). The specific primer utilized in this study are detailed in Table 1.

Construction and packaging of ACAT2 knockdown and overexpression plasmids

Three short hairpin RNA (shRNA) sequences targeting human ACAT2 gene loci (shACAT2-1, shACAT2-2, and shACAT2-3) were synthesized. The shRNA sequences (oligo DNA sequences synthesized by GeneScript Biotech (Shanghai) Co., Ltd., China) were cloned into vectors using T4 DNA Ligase (EL0016, Fermentas, Thermo Fisher Scientific, Lithuania). The target sequences were:

• Target sequence (38598)-KD-1: 5′-CAT​GTC​CAA​GCT​AAA​GCC​TTA-3′

• Target sequence (38599)-KD-2: 5′-GTG​CTG​CAG​CTG​TCG​TTC​TTA-3′

• Target sequence (38600)-KD-3: 5′-CAC​CTT​TAG​CAC​GGA​TAG​TTT-3′

A scrambled sequence was used as the shRNA negative control (shCtrl). For the overexpression of the target gene ACAT2, the ACAT2 gene sequence was synthesized (oligo DNA sequences synthesized by GeneScript Biotech (Shanghai) Co., Ltd., China) and cloned into vectors utilizing the ClonExpress II One Step Cloning Kit (C112, Vazyme, China). The human overexpression and knockdown sequences were cloned into fluorescent protein-based lentiviral vectors containing BamHI/NheI and AgeI/EcoRI restriction sites, respectively, to generate recombinant lentiviral vectors. The lentiviral knockdown or overexpression vectors were co-transfected with lentiviral packaging plasmids (Cat. No. #12259, Didier Trono Lab, Addgene, United States) and envelope plasmids (Didier Trono Lab, Addgene, Cat. No. #12260; United States) into cells.

Lentiviral transduction of target cells

After plating healthy cells, infection media containing the virus was added to the cells. Following 18–20 h of incubation, the fresh medium was replaced. After 72 h, infection efficiency was assessed, and fluorescence expression and cell status were visualized under a fluorescence microscope. qPCR and Western blotting were employed to screen for stable cell lines expressing the target gene. Control groups included the overexpression negative control (NC) and the overexpression group (ACAT2). After 3–5 days of lentiviral transduction, experiments on cell proliferation, migration, and apoptosis were carried out sequentially.

Protein extraction and Western blot analysis

Proteins were isolated from H8, as well as cervical cancer cell lines C-33A, SiHa, and HeLa. The cells were collected, washed with PBS, and lysed using a Western and IP cell lysis buffer supplemented with PMSF (100:1 ratio). The lysates were incubated on ice for 10–15 min and then centrifugated at 12,000 rpm for 10 min at 4 °C. The supernatants were collected, combined with loading buffer, and boiled at 100 °C for 20 min. Samples were then centrifuged at 12,000 rpm for 1 min at 4 °C and preserved at −20 °C until use. The concentration of extracted proteins was quantified using a bicinchoninic acid (BCA) assay (Pierce; Thermo Fisher Scientific, United States).

A total of 20 μg of protein was loaded per lane and separated using SDS-PAGE gel containing 10 % SDS. After electrophoresis, proteins were transferred to PVDF membranes at 300 mA for 90 min at 4 °C. Membranes were blocked with 5 % non-fat milk in TBST for 1 h at room temperature, followed by incubation with primary antibodies for 2 h at room temperature or overnight at 4 °C. The primary antibodies used included anti-ACAT2 (14755-1-AP; Proteintech, China) anti-LATS1 (3477S; CST, United States), anti-SMC2 (5329S; CST, United States), anti-HAUS1 (ab243808; Abcam, United Kingdom), anti-SPC25 (26474-1-AP; Proteintech, China), and anti-RPA1 (12448-1-AP; Proteintech, China) at 1:2,000, anti-GAPDH (60004-1-lg; Proteintech, China) at 1:30,000. After washing with TBST, membranes were treated with secondary antibodies (Goat Anti-Rabbit or Goat Anti-Mouse at 1:3,000) for 1 h at 23 °C. Protein bands were detected using the Millipore immobilon Western Chemiluminescent HRP Substrate and captured with a chemiluminescence imaging system (RPN2232; Millipore, United States).

Cell counting kit-8 (CCK-8) assay for cell viability

Cell viability was evaluated using a CCK-8 assay kit (96992, Sigma-Aldrich, USA). Cells in the logarithmic growth phase were collected, counted, and the seeding density was optimized based on their growth rate over a 5-day period. A cell suspension containing 2,000 cells per 100 μL was plated in each well of a 96-well plate. Following cell attachment, 10 μL of CCK-8 solution was added to each well 2–4 h prior to the end of the incubation period. The plates were then incubated for 4 h, gently agitated, and the absorbance was recorded at 450 nm using a microplate reader (Synergy H1, BioTek, USA).

Colony formation assay

For the colony formation assay, cells in the logarithmic growth phase were seeded at 400–1,000 cells/well into 6-well plates, with three replicates per group, and maintained in culture for 14 days or until the majority of single clones contained >50 cells. The medium was replaced every three days. Cell clones were fixed with 4 % paraformaldehyde for 30–60 min and then stained with 500 μL of Giemsa solution per well (AR-0752, Shanghai Dingguo Biotech Co., Ltd., China) for 10–20 min, washed with ddH₂O, and air-dried. Images of the cell clones were then photographed under a fluorescence microscope (IX71, Olympus Company, Japan), and the number of colonies (defined as clones containing more than 50 cells) was counted.

Wound-healing assay

Cells in the logarithmic growth phase were harvested, resuspended in a complete medium, and counted. A total of 50,000 cells per well were plated in 96-well plates (100 μL/well) to achieve >90 % confluency by the next day for wound-healing assays. After 24 h, the medium was replaced with a low-serum medium, and a scratch was made using a wounding tool. Cells were gently washed with serum-free medium, and a low-serum medium (0.5 % FBS) was added. Images of the wound area were captured every 12 h under a microscope (CKX31, Olympus Company, Japan). Migration was monitored, and images were acquired at appropriate intervals using a Cellomics system (ArrayScan VT1, Thermo Fisher Scientific, Inc., United States). The migration area was analyzed using Cellomics^® vHCS Scan Software v3.0.

Transwell assays for cell migration and invasion

During the logarithmic growth phase, cells were trypsinized, suspended in a low-serum culture medium, and counted. The upper chambers of a 24-well Transwell plate were pre-incubated with 100 μL of serum-deprived medium for 1–2 h. After removing the medium, medium (600 μL) supplemented with FBS (30 %) was introduced into the basolateral compartment. Matrigel (3422, Corning, United States) was added in advance to assess invasion capability. Cell suspensions, containing 100,000 to 200,000 cells in 100 μL of medium without serum, were placed into the top compartments, which were then placed into the lower chambers. Cells were incubated for 4–24 h at 37 °C with 5 % CO₂. Cells that did not migrate were removed with a cotton swab, while the migrated cells on the lower surface of the membrane were stained for 5 min, washed, and air-dried. Microscopic images (IX73 Olympus Company, Japan), and migrated cells were quantified.

Flow cytometry for cell apoptosis detection

C-33A and HeLa cells were gathered for apoptosis analysis. When cells reached 70 % confluency, apoptosis was induced. Culture supernatant and PBS-washed cells were harvested, followed by trypsinization and centrifugation at 1,500 rpm for 5 min. The cell pellet was rinsed with PBS and resuspended in 1× binding buffer. Cells (1 × 10⁵–1 × 10⁶) were stained with 5 μL annexin V-PE in the dark for 15 min, followed by centrifugation and resuspension in 1× binding buffer. Subsequently, 5 μL PI (diluted 10×) was added, and the volume was adjusted to 300 μL with 1× binding buffer. Samples were analyzed within 15 min using a flow cytometer (ABC250-22INT, Guava, Luminex Corp., USA) following the manufacturer’s guidelines (10010-09, Southern Biotechnology Associates, Inc., United States).

The TRULI treatment

To inhibit LATS1 gene expression, the LATS1 inhibitor TRULI was used for subsequent treatments. TRULI (E1061, Selleck, United States) was applied at a concentration of 30 µM for 24 h in the CCK-8 assay, colony formation assay, wound-healing assay, Transwell assays, and flow cytometry for cell apoptosis detection.

Hematoxylin and eosin (HE) staining and immunohistochemistry (IHC) staining

The tissue microarray was stained with HE (Baso Diagnostics, Inc., China). For IHC staining, paraffin was removed from tissue sections using xylene, followed by rehydration in a series of graded ethanol solutions. For antigen unmasking, heat-induced epitope retrieval was conducted in either citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) using pressurized heating. To block endogenous peroxidase activity, endogenous peroxidases were incubated with 3 % H₂O₂ treatment, and non-specific antibody interactions were blocked with 5 % normal serum. The tissue was then incubated with the primary anti-ACAT2 antibody (14755-1-AP, Wuhan Sanying, China) at a dilution of 1:100 (optimized based on preliminary experiments), followed by the application of a secondary antibody. Detection was performed with DAB chromogen, and the tissue sections were counterstained with hematoxylin. Staining results were independently assessed by two pathologists to ensure consistency and accuracy.

Bioinformatics analysis

To validate the role of ACAT2 in cervical cancer, we conducted bioinformatics analyses using expression profile data from multiple tumor-related databases such as TCGA (https://www.cancer.gov/ccg/research/genome-sequencing/tcga), GEO (https://www.ncbi.nlm.nih.gov/geo/), and Oncomine (https://www.oncomine.com/). Additionally, to further elucidate how ACAT2 contributes to the development and progression of cervical malignant tumors, we performed a comprehensive investigation of data from multiple tumor-related databases, including TCGA, GEO, and Oncomine, to identify the potential gene functions and downstream pathways of ACAT2. Gene ontology (GO) were grouped into three classification systems: biological process (BP), cellular component (CC), and molecular function (MF).

Statistical analysis

All experiments were conducted with a minimum of three biological replicates. Quantitative data are expressed as mean values±standard deviation. All statistical analyses were conducted using SPSS version 20.0. The expression and knockdown efficiency of ACAT2 in various groups, along with CCK-8 assay, colony formation assay, flow cytometry analysis, wound healing assays, and Transwell migration assays, were assessed using T-tests or Mann-Whitney U tests. The distribution of ACAT2 expression levels in cervical cancer tissues and adjacent tissues, as well as the characteristics of patients with cervical cancer, were analyzed using chi-square tests. Kaplan–Meier curves were employed to evaluate OS and progression-free survival (PFS) among patients based on ACAT2 expression. p<0.05 was considered statistically significant.

Upregulation of ACAT2 in cervical cancer tissues and cell lines

By intersecting the differential expression genes identified from these databases, we found that expression of the ACAT2 gene is significantly upregulated in cervical cancer (p=0.000984<0.01, Figure 1A).

Figure 1:

The expression of ACAT2 in cervical cancer and normal tissue or cell lines. (A) RNA-seq counts data of squamous carcinoma of the cervix and human normal tissue specimens from the TCGA, GEO, and oncomine databases were normalized to compare expression levels. (B) Quantitative polymerase chain reaction (qPCR) analysis of ACAT2 relative expression in human cervical cancer cell lines C-33A, CASKI, SiHa, and HeLa compared to human immortalized cervical epithelial H8 cells. GAPDH was used as the internal reference gene. (C) ACAT2 protein expression levels were assessed by western blotting in human cervical epithelial cell line H8 and the human cervical cancer cell lines C-33A, SiHa, and HeLa. (D) HE staining in cervical cancer tissues (sample F9) is more intense than in adjacent noncancerous tissues (sample A6). Representative images at 200× and 400× magnifications are shown. Scale bar, 200 and 100 μm. (E) ACAT2 staining in cervical cancer tissues (sample F9) is stronger than in adjacent noncancerous tissues (sample A6). Representative IHC images at 200× and 400× magnifications are shown. Scale bar, 50 and 20 μm. (F) Corresponding statistical analyses for IHC. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

To further examine the expression of ACAT2 in cervical cancer cell lines, qPCR and Western blot analyses were performed to assess ACAT2 expression in H8 cells and C-33A, CASKI, SiHa, and HeLa cell lines. As expected, ACAT2 expression was significantly higher in C-33A, CASKI, SiHa, and HeLa cell lines compared to H8 cells (Figure 1B and C).

To further investigate ACAT2 expression in cervical cancer tissues, we compared ACAT2′ expression in 88 cervical cancer tissues as well as partially matched 25 adjacent non-cancerous tissues from a total of 119 samples due to incomplete pathological data for some specimens (Supplementary Figure S1). IHC analysis revealed a higher ACAT2 levels in tumor tissues compared to normal tissues (p<0.001, Table 2; p<0.0001, Figure 1D–F; Supplementary Figure S2).

Upregulation of ACAT2 is associated with pathological grading and survival in cervical cancer patients

To further elucidate the role of ACAT2 in cervical cancer, we used the median expression level of ACAT2 in cervical cancer tissues as a threshold to assess its correlation with clinical-pathological attributes of cervical cancer. It was found that the proportion of high ACAT2 expression was notably higher in cervical cancer compared to normal tissues, and high ACAT2 expression was markedly associated with tumor pathological grading (p=0.041, Tables 2 and 3).

Additionally, regarding the impact of ACAT2 expression on prognosis, Kaplan-Meier survival analysis indicated that patients with high ACAT2 expression exhibited significantly worse OS compared to those with low ACAT2 expression (93.99 months vs. not reached, HR=1.83 (95 % CI, 1.11–3.01), p<0.05; Figure 2A), while there was no significant difference in PFS (HR=1.37 (95 % CI, 0.85–2.22), p=0.198; Figure 2B). These findings imply that high ACAT2 expression may indicate a higher pathological grade and poorer survival outcomes in cervical cancer patients.

Figure 2:

Kaplan–Meier curves for OS and PFS among patients based on ACAT2 expression. (A) OS and (B) PFS among patients stratified by ACAT2 expression level. HR, hazard ratio; p value by log-rank analysis.

ACAT2 positively regulates proliferation in cervical cancer

To explore the biological functions of ACAT2 in cervical cancer, lentiviruses for ACAT2 knockdown were first constructed and these viruses were used to infect C-33A cells and HeLa cells. Subsequently, the effect of ACAT2 knockdown was validated using Western blot and qPCR. The findings revealed that both the gene and protein expression levels of ACAT2 were notably decreased in C-33A and HeLa cells (p<0.01; Figure 3A–D). Additionally, fluorescence imaging indicated that the infection efficiency of the viruses exceeded 80 %, and the infected cells maintained normal morphology (Figure 3E and F).

Figure 3:

ACAT2 mediates proliferation in cervical cancer cell lines. qPCR analysis showing the knockdown efficiency of ACAT2 in the C-33A cell line (A) and HeLa cell line (B). Evaluation of ACAT2 protein expression levels via Western blot (WB) in C-33A cells (C) and HeLa cells (D). Lentiviral transduction of ACAT2 knockdown in C-33A cells (E) and HeLa cells (F). Scale bar, 100 μm. ACAT2 knockdown significantly inhibited the proliferation of C-33A cells (G) and HeLa cells (H), as measured by the CCK-8 assay. Colony formation assay showed that ACAT2 knockdown reduced the colony-forming ability of C-33A cells (I) and HeLa cells (J). *p<0.05, **p<0.01, ***p<0.001.

To thoroughly assess the impact of ACAT2 on cell viability, proliferation, apoptosis, and migration, we conducted CCK-8 assays, which revealed that ACAT2 knockdown notably decreased the viability of C-33A and HeLa cells (p<0.01; Figure 3G, and H). Additionally, colony formation assays demonstrated a significant reduction in the colony-forming capacity of both cell lines following ACAT2 knockdown (p<0.01; Figure 3I and J).

To elucidate the role of ACAT2 in apoptosis, we conducted flow cytometry analysis. The results demonstrated that ACAT2 knockdown promoted apoptosis in C-33A and HeLa cells (p<0.05; Figure 4A and B).

Figure 4:

ACAT2 mediates proliferation in cervical cancer cell lines. Flow cytometry analysis of apoptosis levels in ACAT2-knockdown C-33A cells (A) and HeLa cells (B). Wound healing assays showed attenuated migration abilities in ACAT2-knockdown C-33A cells (C) and HeLa cells (D). Scale bar, 100 μm. Transwell migration assays indicated reduced migratory capacity in ACAT2-knockdown C-33A cells (E) and HeLa cells (F). Scale bar, 50 μm *p<0.05, **p<0.01, ***p<0.001.

To investigate the impact of ACAT2 knockdown on cell migration in C-33A and HeLa cells, wound healing and Transwell assays were performed. The wound healing assays indicated that ACAT2 knockdown significantly inhibited the migration of C-33A and HeLa cells (p<0.01; Figure 4C and D). Furthermore, Transwell assays confirmed that ACAT2 knockdown reduced the migratory capability of C-33A and HeLa cells (p<0.01; Figure 4E and F).

The mechanism through which ACAT2 drives the development and progression of cervical cancer

To further elucidate how ACAT2 influences the development and progression of cervical cancer, we conducted an integrated examination from multiple tumor-related databases, including TCGA, GEO, and Oncomine, to identify the potential gene functions and downstream pathways of ACAT2. Bioinformatics analysis based on GO in three aspects, BP, CC, and MF, revealed that ACAT2 frequently participates in mitosis-related biological processes and cellular components, particularly those associated with chromosomes (Supplementary Figure S3). It is well-known that LATS1/2 serves as central regulators of cell fate, playing crucial roles in modulating a range of oncogenic and tumor-suppressive agents, participating in the canonical Hippo effectors [14]. Interestingly, the enriched GO biological process CHROMOSOME_SEGREGATION includes the LATS1 gene in this analysis. Previous investigation has indicated that the Hippo signaling pathway is regulated by ACAT2 deficiency [15]. Thus, we hypothesized that LATS1 might be an important downstream gene of ACAT2.

Therefore, we decided to use Western blotting to validate the LATS1 protein expression in ACAT2 knockdown C-33A and HeLa cells. The results revealed a marked reduction in LATS1 expression compared to control group (Figure 5A and B). This observation from protein-level experiments supports our hypothesis that ACAT2 promotes the development of cervical cancer by positively regulating LATS1 expression in mitosis-related biological processes.

Figure 5:

Inhibition of LATS1 suppresses proliferation and growth of ACAT2-overexpressing C-33A and HeLa cells. LATS1 protein expression was validated by Western blotting in ACAT2-knockdown C-33A cells (A) and HeLa cells (B). The proliferation of ACAT2-overexpressing C-33A (C) and HeLa (D) cells was significantly inhibited by TRULI treatment, as determined by the CCK-8 assay. The colony-forming ability of ACAT2-overexpressing C-33A (E) and HeLa (F) cells was reduced following TRULI treatment, as determined by colony formation assays. The protein levels of downstream genes (SMC2, HAUS1, and SPC25) identified via bioinformatics analysis were also compared with LATS1 expression. Ns, no significance; *p<0.05, **p<0.01, ***p<0.001.

To further clarify how ACAT2 influences cell viability, proliferation, apoptosis and migration in C-33A and HeLa cells through the regulation of LATS1 expression, we constructed an ACAT2-overexpressing lentivirus to infect both cell lines. Next, the cells were treated with the LATS1 inhibitor TRULI, and CCK-8 assays was conducted. TRULI significantly inhibited the proliferation of ACAT2-overexpressing C-33A and HeLa cells (p<0.001; Figure 5C and D). Similarly, the results from the colony formation assays indicated that TRULI significantly reduced the colony-forming capability of ACAT2-overexpressing C-33A and HeLa cells (Figure 5E and F). Additionally, Flow cytometry analysis showed that TRULI treatment significantly promoted apoptosis in ACAT2-overexpressing C-33A and HeLa cells (p<0.01; Figure 6A and B).

Figure 6:

Inhibition of LATS1 suppresses proliferation and growth of ACAT2-overexpressing C-33A and HeLa cells. Flow cytometry analysis demonstrated that apoptosis was promoted in ACAT2-overexpressing C-33A (A) and HeLa (B) cells treated with the LATS1 inhibitor TRULI. Transwell migration assays revealed decreased migratory capacity in ACAT2-overexpressing C-33A (C) and HeLa (D) cells treated with TRULI. Scale bar, 50 μm. Wound healing assays indicated attenuated migration ability in ACAT2-overexpressing C-33A (E) and HeLa (F) cells treated with TRULI. Scale bar, 100 μm. Ns, no significance; *p<0.05, **p<0.01, ***p<0.001.

To examine the impact of TRULI on the migration levels of ACAT2-overexpressing both cell lines, we again conducted wound healing and Transwell assays. The transwell assays confirmed that TRULI inhibited the migratory capacity of ACAT2-overexpressing both cells (p<0.001; Figure 6C and D). Furthermore, the wound healing assays demonstrated that TRULI led to a marked reduction in the migration ability of ACAT2-overexpressing both cell lines (p<0.05; Figure 6E and F).