Effect of CUB Domain-containing Protein 1 On Proliferation and Epithelial–Mesenchymal Transition In Nasopharyngeal Carcinoma Cells

Summary

This protocol aims to investigate the expression and function of CUB domain-containing protein 1 in nasopharyngeal carcinoma, using clinical tissue analysis and in vitro .assays to assess its role in the regulation of epithelial-mesenchymal transition via the ERK1/2 signaling pathway.

Abstract

This protocol describes a methodological approach to investigate the expression and functional role of CUB domain-containing protein 1 in nasopharyngeal carcinoma and its potential involvement in epithelial-mesenchymal transition. Clinical tissue samples from patients with nasopharyngeal carcinoma and rhinitis were collected to analyze CDCP1 expression using real-time quantitative PCR and immunohistochemistry. In vitro experiments were performed using CNE2 and HK1 nasopharyngeal carcinoma cell lines. CDCP1 overexpression and knockdown were achieved by transfection with a CDCP1 plasmid or specific siRNA. Cell proliferation was assessed by MTT assay, apoptosis was evaluated by Caspase-3 activity measurement, and the expression of EMT-related markers and phosphorylation levels of ERK1/2 were detected by western blot and quantitative PCR. To validate pathway involvement, rescue experiments were conducted using the ERK1/2-specific inhibitor U0126. This protocol provides a systematic in vitro and ex vivo. framework for elucidating the molecular mechanisms by which CDCP1 may regulate tumor progression in nasopharyngeal carcinoma.

Introduction

Nasopharyngeal carcinoma is the most common primary malignant tumor of the nasopharynx, originating from the nasopharyngeal epithelium, and is a head and neck malignancy with unique geographical distribution characteristics^1,2. Approximately 98% of nasopharyngeal carcinoma cases are associated with latent EBV infection. This disease has a high incidence in Asia but is rare in Europe, and its occurrence may be related to dietary habits and other factors^3,4,5. According to histological classification, nasopharyngeal car....

Protocol

This study was approved by the Ethics Committee of Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology [Approval No.: 2026IEC(RYJ016)]. All patients provided informed consent, and the collection and use of clinical samples complied with the relevant ethical guidelines of the Declaration of Helsinki. The main experimental materials and instruments used in this study are listed in the Table of Materials.

Collection of clinical tissue samples and detection of CDCP1 expression

Surgical tissue specimens were collected from seven patients diagnosed wi....

Results

CDCP1. upregulation in nasopharyngeal carcinoma tissues

The expression level of CDCP1 mRNA in seven nasopharyngeal carcinoma tissues and six rhinitis tissues was detected by real-time quantitative PCR. The results showed that CDCP1 mRNA expression in nasopharyngeal carcinoma tissues was significantly higher than that in rhinitis tissues (P < 0.05, Figure 1A). Furthermore, immunohistochemical staining was performed to .......

Discussion

This study systematically investigated, for the first time, the expression characteristics of CDCP1 in nasopharyngeal carcinoma tissues and its effects on tumor cell biological behavior, and preliminarily elucidated the molecular mechanism by which CDCP1 promotes epithelial-mesenchymal transition through activation of the ERK1/2 signaling pathway. The findings suggest that CDCP1 exerts a protumorigenic role in nasopharyngeal carcinoma, providing new experimental evidence for understanding the mechanisms of metastasis in .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BCA Protein Assay Kit	Thermo Fisher, USA	23225	For protein concentration determination
Caspase-3 Activity Assay Kit	Beyotime, China	C1403	Colorimetric assay for Caspase-3 activity
CDCP1 Overexpression Plasmid	Not specified	Not specified	For CDCP1 overexpression transfection
CDCP1 Primary Antibody	Abcam, USA	ab137706	For IHC and Western blot; dilution 1:200 (IHC) or 1:1000 (WB)
CDCP1 siRNA	Not specified	Not specified	For CDCP1 gene silencing
CNE2 Cell Line	China Center for Type Culture Collection (CCTCC), China	Not specified	Human nasopharyngeal carcinoma cell line
DMEM High Glucose Medium	HyClone, USA	SH30243.01	Cell culture
DMSO	Sigma, USA	D2650	For dissolving crystals in MTT assay
E-cadherin Primary Antibody	Cell Signaling Technology, USA	3195S	Western blot; dilution 1:1000
ERK1/2 Primary Antibody	Cell Signaling Technology, USA	4695S	Western blot; dilution 1:1000
GAPDH Primary Antibody	Abcam, USA	ab8245	Western blot loading control; dilution 1:5000
HK1 Cell Line	China Center for Type Culture Collection (CCTCC), China	Not specified	Human nasopharyngeal carcinoma cell line
Lipofectamine 2000	Invitrogen, USA	11668019	Transfection reagent
Microplate Reader	BioTek, USA	Not specified	Absorbance detection (MTT, Caspase-3)
MTT	Sigma, USA	M2128	Cell proliferation assay; prepared at 5 mg/mL in PBS
Opti-MEM	Gibco, USA	31985070	Reduced serum medium for transfection
p-ERK1/2 Primary Antibody	Cell Signaling Technology, USA	4370S	Western blot; dilution 1:2000
PrimeScript RT Master Mix	Takara, Japan	RR036B	Reverse transcription reagent for real-time PCR
PVDF Membrane	Millipore, USA	IPVH00010	Protein transfer; 0.45 μm pore size
QuantStudio 5 Real-Time PCR System	Applied Biosystems, USA	A34322	qPCR detection
RIPA Lysis Buffer	Beyotime, China	P0039	Protein extraction; complete RIPA lysis buffer
Secondary Antibody (HRP-conjugated)	Dako, Denmark	Not specified	For IHC and Western blot
Sodium Citrate Buffer	Not specified	Not specified	For IHC antigen retrieval, pH 6.0
TB Green Premix Ex Taq II	Takara, Japan	RR820A	qPCR reaction reagent; 200 reactions
Tissue Homogenizer	IKA T10, Germany	Not specified	For tissue RNA extraction homogenization
TRIzol Reagent	Invitrogen, USA	15596026	RNA extraction
U0126	Selleck, USA	S1102	ERK1/2-specific inhibitor; used at 10 μmol/L
Vimentin Primary Antibody	Cell Signaling Technology, USA	5741S	Western blot; dilution 1:1000