Introduction
There are two major subtypes of esophageal cancer, esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). More than 90% of all esophageal cancers in China are ESCC [
1]. Because of the lack of clear symptoms and sensitive screening methods at an early stage, most ESCC patients are at an advanced stage of diagnosis. Moreover, the 5-year overall survival (OS) rate of ESCC is only 15 – 25% due to recurrence and metastasis [
2]. Currently, the critical role of molecular mechanisms in cancer has received consistent attention, with molecular markers found progressively more involved in cancer diagnosis, risk stratification, and treatment selection. Although several typical molecular markers of ESCC have been discovered, there are many unknown molecules worth exploration and investigation.
Recently, the dysregulation of lipid metabolism during the development of ESCC has attracted increasing attention. Key enzymes and certain lipid components in lipid metabolism are abnormally expressed and dysregulated in ESCC, which can influence tumor progression, including disorders of glycerophospholipid metabolism [
3‐
6]. PLC is a membrane-associated signaling protease involved in constructing biological membranes, storing energy, signaling, and other essential life activities. PLC is activated by a variety of extracellular ligands to hydrolyze phosphatidylinositol 4,5-diphosphate to generate the second messenger inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG), leading causes phosphorylation of target proteins by increasing intracellular Ca
2+ levels and activating protein kinase C (PKC) to achieve the desired biological effect [
7]. PLCD3 is a critical metabolic enzyme in the 13 mammalian PLC subtypes, whose function in tumors is not frequently mentioned. It has been reported to be an oncogene in nasopharyngeal and thyroid cancer [
8,
9] and a prognostic marker for early-stage pancreatic ductal carcinoma [
10]. However, its role in ESCC has not yet been found. Therefore, the study of PLCD3 affecting the biological behavior of ESCC cells can provide a theoretical basis for treatment.
In this study, PLCD3 was significantly upregulated in ESCC tissues and cells. Further studies focused on its biological function in ESCC cells and explored the underlying mechanism of PLCD3 in ESCC carcinogenesis. The results suggest that PLCD3 promotes ESCC proliferation, migration and invasion via the PI3K/ AKT / P21 signaling pathway.
Materials and methods
Esophageal cancer specimens and cell lines
An initial collection of tumor and adjacent non-tumor tissues was performed on.
40 patients undergoing ESCC radical resection at the Department of Thoracic Surgery, Northern Jiangsu People’s Hospital (Yangzhou, China) between March 2020 and March 2021. Samples from 22 patients were selected for paraffin tissue embedding, which were diagnosed with ESCC by the pathology department. The clinicopathological characteristics of all patients are presented in Table
1. Informed consent has been obtained from all individuals included in this study. This study was approved by the Institutional Review Board at Northern Jiangsu People’s Hospital. HEEpiC, ECA109, KYSE150, KYSE410, and TE1 cell lines were obtained from the China Cell Resource Center (Shanghai, China). The cells were cultured in RPMI 1640 (Solarbio) supplemented with 10% fetal bovine serum (Procell). The cells were incubated in a humidified incubator (Thermo Scientific, China) with 5% CO2 at 37 °C.
Table 1
PLCD3 Relationship between expression and ESCC characteristics of clinical parameters
All | 40 | 33 (82.5) | 7 (27.5) | | |
Age(year) | | | | 0.153 | 0.55 |
≤55 | 21 | 17 (51.5) | 4(57.1) | |
>55 | 19 | 16 (48.5) | 3(42.9) | |
Gender | | | | 0.175 | 0.579 |
Male | 29 | 25 (75.8) | 4(57.1) | | |
Female | 11 | 8 (24.2) | 3(42.9) | | |
Tumor central location | | | | 0.955 | 0.62 |
Distal | 6 | 4(12.1) | 2 (28.6) | | |
Mid | 24 | 20 (60.6) | 4 (57.1) | | |
Proximal | 10 | 9 (27.3) | 1 (14.3) | | |
Histological type | | | | 3.238 | 0.198 |
Ulcerative type | 15 | 13(39.4) | 2(28.6) | | |
Medullary type | 13 | 10(30.3) | 3(42.9) | | |
Other types | 12 | 10(30.3) | 2(28.6) | | |
Pathologic stage | | | | 0.929 | 0.03* |
Stage I + II | 22 | 20(60.6) | 2(28.6) | | |
Stage III + IV | 18 | 13(39.4) | 5(71.4) | | |
Lymphatic metastasis | | | | 8.505 | 0.011* |
No | 27 | 21(63.6) | 2(28.6) | | |
Yes | 13 | 12(36.4) | 5(71.4) | | |
Immunohistochemical staining and scoring
Sliced 4 μm thick paraffin-embedded tissue was dewaxed and hydrated, then boiled in PH6.0 sodium citrate antigen repair solution for 20 min for antigen repair. Peroxidase activity was blocked during incubation in endogenous peroxise-blocking solution for 15 min. Sections were incubated with anti-PLCD3 (abmart, code:PK83857S, 1: 50 dilution) overnight. After being placed at room temperature and combined with the secondary antibody, IgG antibody (Servicebio, product number: G1215-200T), DAB was used as a chromogen, and hematoxylin was stained with the cell nucleus.
The evaluation of the positive rate of PLCD3 in tissues, The details are as follows: staining intensity score 0 (negative), 1 (weak), 2 (moderate), 3 (strong), and positive cell ratio score 0 (0–5%), 1 (6–25%), 2 (26–50%), 3 (51–71%), 4 (over 75%), The score reference plot is shown in Supplementary Materials (Supplementary material: Figure
S1), with the final score assigned as the product of staining intensity and positive rate cell scores.
RNA extraction and quantitative real-time PCR (qRT-PCR) assay
The TRIzol reagent (Vazyme) was used to extract RNA from tissues and cells. The cDNA was synthesized using the Hifair
® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (Yeasen Biotechnology, Shanghai, China). Hieff
®qPCR SYBR Green Master Mix (High Rox Plus) (Yeasen Biotechnology, Shanghai, China) was used to perform the quantitative real time PCR in the StepOne Plus Real-Time PCR System (Applied Biosystems). The relative expression levels of PLCD3 mRNA were normalized to GAPDH as endogenous control respectively by using the 2
−ΔΔCt method. The primer sequences are presented in Table
2.
Table 2
Primers Sequencing for qPCR.
PLCD3-Forward | CCACAACACCTATCTGACTGAC |
PLCD3-Reverse | CTGGGCAAAGGCCCTAACAT |
GAPDH-Forward | TCATTTCCTGGTATGACAACGA |
GAPDH-Reverse | GTCTTACTCCTTGGAGGCC |
PLCD3-siNC-sense | UUCUCCGAACGUGUCACGUTT |
PLCD3-siNC-antisense | ACGUGACACGUUCGGAGAATT |
PLCD3-siRNA1-sense | GCAGCUCAUUCAGACCUAUTT |
PLCD3-siRNA1antisense | AUAGGUCUGAAUGAGCUGCTT |
PLCD3-siRNA2-sense | GCCCACUACUUCAUCUCUUTT |
PLCD3-siRNA2-antisense | AAGAGAUGAAGUAGUGGGCTT |
PLCD3-siRNA3-sense | GCCACGCUCUUCAUCCAAATT |
PLCD3-siRNA3-antisense | UUUGGAUGAAGAGCGUGGCTT |
Western blot (WB) assay
The whole cell or tissue mixture was isolated using RIPA lysate (Solarbio, Cat: R0020), PMSF, protease inhibitor, and protein phosphatase inhibitor mixture, and equal amounts of protein were separated on 10%SDS-PAGE and transferred to a PVDF membrane (Immobilon-P, cat:IPVH00010).They were blocked using 5% skim milk and incubated overnight with primary antibody PLCD3 (Abmart, code:PK83857S), anti-GAPDH (Proteintech, cat:10494-1-AP), MMP2 (Cell Sinaling, cat:40,994), MMP9 (Cell Sinaling, cat:15,749), p-PI3K (Cell Sinaling, cat:17,366), t-PI3K (Cell Sinaling, cat:9655), p-AKT (Cell Sinaling, cat:13,038 S), t-AKT (Cell Sinaling, cat:4685 S), P21 (Cell Sinaling, cat:2947), Bax (Cell Sinaling, cat:5023T), Caspase3 (Cell Sinaling, cat:9664T) and Bcl2 (Cell Sinaling, cat:4223T) binding 4 °C. Then they were incubated in secondary antibody IgG (ABclonal, lot:9,300,014,001) for 2 h at room temperature. Protein blots were cut prior to hybridisation with antibodies during blotting. Protein bands were visualized using Super ECL Detection Reagent(Yeasen Biotechnology, Shanghai). Image J software performed the protein band gray-scale analysis.
RNA oligo and plasmid transfection
The siRNA and BamHI RcoRI pcDNA3.1 was purchased from GenePharma (Shanghai, China). The siRNA sequences are presented in Table
1. The cells were incubated in 6-well plates, and transfection started when cell density reached 50%. GP-transfect-Mate (GenePharma) was used to perform the transfection. The transfection efficiency was detected by qRT-PCR and WB methods.
CCK-8 assay
The proliferation assay were performed on a panel of 96 wells with 1 × 103 cells per well. After 24, 48, 72, and 96 h, 10µL of CCK-8 solution (Yeasen) was added to every well and then incubated for 1 h 30 min. The absorbance (OD) of each well at 450 nm was detected by an enzyme labeling instrument(Skanlt RE 7.0).
Cells after siRNA or BamHI RcoRI pcDNA3.1 transfection were seeded at a density of 1 × 103 cells per well in 6 or 12-well plates, during which fresh medium containing 10%FBS was replaced on time. Two weeks later, the cells were fixed with 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet solution for 12 min, air-dried and photographed, and colony counts were performed with Image J software.
Wound healing assay
Wound-healing assays were performed to assess the migration ability of the cells. Transfected cells (15 × 104/well) were seeded in six-well plates and incubated continuously until the monolayer of cells were evenly distributed at the bottom of the plate. A micropipette gun head uniformly and slowly forms a scratch wound on the surface of the cell. The cells were washed with PBS several times to remove all floating cells. Then residual cells were cultured with RPMI 1640 without FBS. Wounds were then taken at 0, 24, 48 h under an inverted microscope (OLYMPUS-CKX53, China). Images were analyzed using the Image J software.
Transwell assay
Transwell assay was performed to assess the migration and invasion ability of the cells. Cells were transfected in 6-well plates and incubated after 48 h before starting the experiments. Cells were washed twice with PBS, cells were digested with 0.25% trypsin cell digestive solution (Beyotime) and centrifuged, and cells were resuspended in serum-free 1640 and counted. Then 200ul of cell suspension was added to the upper layer covered with matrix (BD Biocoat) or no matrix free chamber (Corning), while 500ul of 1640 containing 10%FBS was added to the lower chamber. The transwell chamber was grown in a cell culture incubator for 48 h. The cells were then fixed with 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet solution for 7 min, then washed twice with PBS, and the cells on the upper surface of the bottom chamber were gently wiped away with a cotton swab. Cells on the lower surface of the bottom of the chamber were left dry, and images were taken with an inverted microscope (OLYMPUS-CKX53). Images were analyzed using the Image J software.
Flow cytometry analysis
Cell cycle and apoptosis were measured using flow cytometry. For the cell cycle, the cells were washed twice with PBS and digested with trypsin, 70% ethanol for 4 h at 24°C, it was subsequently incubated in 500 µl of configured propidium iodide staining solution (PI) (Beyotime) at 37°C for 30 min. With regard to cell apoptosis, cells were washed twice with PBS and digested with trypsin, add the Annexin-FITC detection reagent (Beyotime) in turn.Then the cells were detected using a FACS flow cytometer (BD Biosciences, CA, USA), and analyzed the results using the Flow Jo software.
Functional enrichment analysis
To further investigate the function between PLCD3 and the upregulated genes caused by it, KEGG (
www.kegg.jp/kegg/kegg1.html) and GO enrichment analysis was performed by clusterProfiler (version 3.14.3) R software [
11‐
13]. Among them, GO enrichment includes biological processes (BP), molecular functions (MF) and cellular components (CC). The minimum gene set was set to 5, the maximum gene set to 5000, P value of < 0.05, and an FDR of < 0.25 were considered statistically significant. PLCD3 was divided into two groups of samples with high and low expression, and the correlation between PLCD3 expression and PI3K/AKT pathway genes was analyzed using GSEA software.
Xenograft model
The animal experiments were performed with the approval of the Experimental Animal Ethics Committee of Yangzhou University. A mouse xenograft model was established to explore the functional role of PLCD3 in vivo. The ECA109 cells were spread in six-well plates and then transfected with siNC, siRNA2 and siRNA3. 15 Balb / c nude mice (Nanjing JiBiological Co., LTD) were randomly divided into 3 groups to inject the above treated cells (6 × 105) in the axilla of nude mice. Tumor volume was monitored every 5 days, the formula was: V = (Length x Width2) x 0.5, nude mice were killed 5 weeks later, and tumor weights were recorded. Anestheia using isoflurane and subsequently nude mice were killed by cervical dislocation. Animal experiments were performed in accordance with the animal care guidelines and were approved by the ethics committee.
Statistical analysis
All experimental data were treated with GraphPad Prism8.0 and displayed with the mean value plus or minus the standard deviation. Student’s test was used to compare differences between data from the two groups, and Dunnett’s test in Ordinary one-way ANOVA to compare differences between multiple groups. P < 0.05 were considered statistically significant.
Discussion
As was previously described, PLCD3 plays an oncogenic role in a variety of cancers, such as nasopharyngeal, thyroid, and early pancreatic ductal carcinoma [
8‐
10]. However, whether PLCD3 has the same effect on ESCC progression and how it works is unclear. The current study demonstrated that PLCD3 is significantly upregulated in ESCC tissues and cell lines. PLCD3 promotes the development of ESCC cells by regulating malignant behaviors such as proliferation, migration, and invasion, suggesting the tumor-promoting role of PLCD3 in ESCC.
In previous studies, Lizhi Lin et al. [
9] found that the increased expression of PLCD3 was associated with the metastatic stage of thyroid cancer. Nakamura et al. [
15] reported that loss of PLCD1 and PLCD3 in mouse embryos increased apoptosis. Our findings are consistent with its report that PLCD3 knockdown inhibited proliferation, migration, invasion and promoted apoptosis of ESCC cells. In vivo experiments show that PLCD3 promoted ESCC tumorigenesis.
Dysregulation of cell migration and apoptosis are two major drivers of cancer progression that are regulated by multiple oncogenic pathways [
16‐
19]. Metastasis is the major cause of cancer-related death, and the migration of and invasion of cancer cells into surrounding tissues and vasculature is an important step in cancer metastasis [
20]. Cell migration is a dynamic, complex process that requires multiple interdependent steps, such as cancer cells exhibiting cytoskeletal reorganization, reduced adhesion function, and loss of cell polarity [
21]. Migration-induced changes in classical molecular features include increases in mesenchymal markers and changes in matrix metalloproteinases, such as MMP2, and MMP9 [
22,
23]. In this study, we found that the expression level of metastasis markers was affected by the changes in PLCD3 expression. Downregulation of PLCD3 decreased the expression of MMP2 and MMP9, while upregulation of PLCD3 showed the opposite results. In addition, we found that the expression levels of Bax and Caspase3 increased while Bcl-2 decreased when PLCD3 knockdown. It is known that apoptosis is one of the key features of cancer [
24]. Apoptosis mainly includes the endogenous apoptotic pathway, exogenous apoptotic pathway, and execution pathway, where the key markers of the endogenous apoptotic pathway include Bax and Bcl-2, and Caspase3 proteins belong to the key proteins of the executive pathway [
25]. This is fully demonstrated by our study that PLCD3 downregulation caused increased apoptosis.
Furthermore, we sought to investigate the molecular mechanism of PLCD3 as an ESCC oncogene. The PI3K / AKT pathway is frequently over-activated in various tumor types [
26]. Several current studies [
27‐
31] showed that the PI3K / AKT pathway can promote ESCC cell growth and metastasis, and the phosphorylation level of both was significantly and positively correlated with the invasion and metastatic ability of cancer cells. This is in line with our finding that PLCD3 downregulation led to a significant reduction in PI3K / AKT phosphorylation, versus PLCD3 upregulation (Fig.
6a,b). P21 is a cyclin-dependent kinase inhibitor involved in cell cycle progression, conducting G1 / S phase arrest and simultaneously inhibiting the proliferation and migration of ESCC cells [
32‐
36]. Our findings suggest that downregulation of PLCD3 caused an increase in P21 expression, and the upregulation of PLCD3 caused a decrease of P21 expression. Meanwhile, PLCD3 downregulation induced a cell cycle GI / S arrest, which was consistent with the elevated P21 expression. Finally, the AKT inhibitor MK2206 significantly impaired the promoting effect of PLCD3 upregulation on cell growth, migration and invasion (Fig.
6c-e). The results suggest that PLCD3 may promote cell proliferation, migration and invasion by activating the PI3K / AKT / P21 pathway.
However, our study still has some limitations. For example, in vivo experiments are needed to further validate the biological function of PLCD3 in ESCC. Meanwhile, PLCD3 acts as a phospholipid metabolism enzyme and its related mechanism of action need to be specifically elucidated.
In conclusion, these results indicate that PLCD3 is upregulated in ESCC, high expression of PLCD3 is closely related to the malignant biological behavior of ESCC, and PLCD3 may play an oncogenic role in ESCC via the PI3K / AKT / P21 pathway. This study may provide potential markers and target genes for the treatment of ESCC.
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