HMGA1 And HAND1 Expression And Their Significance In Laryngeal Cancer Tissues

Summary

This research discovered increased levels of HMGA1 and decreased levels of HAND1 expression in laryngeal cancer, indicating an inverse relationship and significant associations with metastasis and clinical stage, suggesting that both contribute to tumor progression.

Abstract

This study investigated the expression profiles of the HMGA1 and HAND1 genes in laryngeal squamous cell carcinoma (LSCC) as well as in adjacent normal laryngeal mucosal tissues through the application of immunohistochemistry and RT-PCR methodologies. The objective was to investigate their possible roles in the initiation, advancement, invasion, and metastasis of LSCC. Both HMGA1 and HAND1 were detected in cancerous and adjacent normal tissues. Nevertheless, HMGA1 demonstrated a significantly elevated expression in LSCC tissues, while HAND1 displayed considerably reduced expression levels. Elevated HMGA1 expression was positively associated with lymph node metastasis (100.0% vs. 50.0%) and advanced clinical stage (91.7% vs. 44.4%), but was not associated with tumor histological grade, clinical type, or patient age. In contrast, reduced HAND1 protein expression was associated with lymph node metastasis, clinical stage, and pathological grade, suggesting a role in tumor progression and metastasis. Furthermore, a linear negative correlation was noted between the expressions of HMGA1 and HAND1, suggesting a potential regulatory interaction in which HMGA1 may suppress HAND1 expression. These results imply that HMGA1 and HAND1 have complementary functions in the pathogenesis of LSCC, with HMGA1 potentially facilitating tumor development and HAND1 serving as a suppressor. The interaction between these two genes may offer new perspectives on the molecular mechanisms that drive LSCC progression and metastasis.

Introduction

Laryngeal cancer is the second most common primary malignant tumor of the head and neck, following nasopharyngeal cancer, accounting for 26%–30% of all head and neck tumors worldwide¹. Over 90% of laryngeal cancers are classified as laryngeal squamous cell carcinoma (LSCC)². The primary treatments for laryngeal cancer are surgery and radiotherapy. Local recurrence and lymph node metastasis are the leading causes of death in laryngeal cancer patients. Therefore, effective management of recurrence and metastasis remains a major clinical challenge. With the advancement of molecular biology, various genes with abnormal....

Protocol

This study was reviewed and approved by the Research Ethics Committee of Shijiazhuang People’s Hospital (Approval No. YKLS 2026 No. 041; March 27, 2026). The study was conducted in accordance with the Regulations on Ethical Review of Biomedical Research Involving Human Subjects, the Regulations on Ethical Review of Life Science and Medical Research Involving Human Subjects, the Declaration of Helsinki, and the International Ethical Guidelines for Health-Related Research Involving Humans. Human tissue samples were obtained from archived surgical specimens in compliance with institutional ethical requirements, ensuring anonymization and confidentiality of patient ....

Results

Immunohistochemical detection results

Representative hematoxylin–eosin (HE) staining images illustrating the histological morphology of adjacent normal laryngeal mucosa and LSCC with varying degrees of differentiation (well, moderate, and poor) are shown. Specifically, Figure 1A shows adjacent normal laryngeal mucosa, Figure 1B shows well-differentiated LSCC, Figure 1C shows moderately di.......

Discussion

Laryngeal cancer, one of the most common tumors of the head and neck, has shown an increasing incidence in recent years, likely associated with environmental and lifestyle-related factors. Like other malignant tumors, its recurrence and metastasis remain the leading causes of death. With advancements in molecular biology, many factors associated with the development of laryngeal cancer have been identified, but the mechanisms of onset and metastasis are still not fully understood. HMGA1, as a transcriptional regulator, h.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Automatic Tissue Embedding Machine	Sakura Finetek, Tokyo, Japan	Tissue-Tek TEC 5	Paraffin embedding
Microtome	Leica Microsystems, Wetzlar, Germany	RM2135	Sectioning (4 μm)
Slide Dryer	Xiaogan Hongye Medical Instrument Co., Ltd., China	CS-VI	Slide drying
Heating Incubator	Shanghai Yangguang Experimental Instrument Co., Ltd., China	303-4A	Temperature-controlled incubation
Optical Microscope	Olympus Corporation, Tokyo, Japan	BX41	Histological observation
Image Capture System	QImaging, Canada	QCapture Pro 7	Microscopy imaging
Clean Bench	Beijing Xicheng Semiconductor Equipment Factory, China	SW-CJ-1FD	Sterile workspace
Refrigerated Centrifuge	Beijing Medical Instrument Repair Factory, China	LXJ-64-01	RNA separation
Tabletop Centrifuge	Shanghai Peiqing Technology Co., Ltd., China	PQ-1600A	RNA/DNA processing
Magnetic Stirrer	Jiangsu Jintan Medical Instrument Factory, China	HJ-3	Solution preparation
Mini Mixer	Shanghai Peng’s Industrial Co., Ltd., China	H-1	Sample mixing
Electronic Balance	METTLER Toledo, Switzerland	PB3002-S	Sample weighing
PCR Amplifier	Eppendorf AG, Germany	Mastercycler 5333	PCR amplification
Electrophoresis Power Supply	Beijing Liuyi Instrument Factory, China	DYY-6C	Voltage control
Electrophoresis Tank	Beijing Liuyi Instrument Factory, China	DYCZ-24DN	Gel electrophoresis
Microwave Oven	Shanghai Zhongda Medical Application Research Institute, China	WD800SL23	Agarose preparation
UV Transilluminator	Beijing Liuyi Instrument Factory, China	WD-9403B	DNA visualization
Gel Imaging System	Anlai Co., Ltd., China	AlphaImager HP	Gel documentation
Refrigerator (4 °C)	Haier Biomedical, China	BC-165D	Sample storage
Ultra-Low Temperature Freezer	Haier Biomedical, China	DW-86L388	Storage (-80 °C)
Ice Box	Shanghai Fuzhi Technology Co., Ltd., China	FZ-IB-20L	Sample transport
Cryogenic Storage Bags	Shanghai Yili Technology Co., Ltd., China	YL-ULT-50	Sample preservation
Research Pipettes	Eppendorf AG, Germany	Research Plus	Liquid handling
RNA Extraction Reagent (TRIzol)	Invitrogen (Thermo Fisher Scientific), USA	15596026	RNA extraction
Reverse Transcription Kit	Takara Bio Inc., Japan	RR037A	cDNA synthesis
PCR Master Mix	Takara Bio Inc., Japan	RR820A	PCR amplification
dNTP Mix	Takara Bio Inc., Japan	4030 (typical)	PCR component
RNase Inhibitor (RNasin)	Promega, USA	N2111	RNA protection
Oligo(dT) Primer	Takara Bio Inc., Japan	3806 (typical)	cDNA synthesis primer
Agarose	Sigma-Aldrich, USA	A9539	Gel preparation
TAE Buffer (50×)	Sigma-Aldrich, USA	TAE50X-1L (typical)	Electrophoresis buffer
DNA Marker (Ladder)	Takara Bio Inc., Japan	DL2000 (3421A)	Size reference
Ethidium bromide (EtBr)	Bio-Rad, USA	1610433	DNA visualization
PBS Buffer	Gibco (Thermo Fisher Scientific), USA	10010023	Washing buffer
DEPC-treated Water	Invitrogen, USA	AM9916	RNA protection
Chloroform	Sigma-Aldrich, USA	C2432	RNA extraction
Isopropanol	Merck, Germany	109634	RNA precipitation
Ethanol (absolute)	Merck, Germany	100983	Dehydration
Methanol	Merck, Germany	106009	Peroxidase blocking
Hydrogen Peroxide (H2O2)	Sigma-Aldrich, USA	H1009	Endogenous peroxidase blocking
Xylene	Sigma-Aldrich, USA	534056	Deparaffinization
Citrate Antigen Retrieval Buffer	Abcam, UK	ab93678	Antigen retrieval
Antibody Diluent	Dako, Denmark	S3022	Antibody dilution
HMGA1 primary antibody	Abcam, UK	ab129153	Dilution 1:150
HAND1 primary antibody	Abcam, UK	ab200040	Dilution 1:100
Secondary antibody (HRP-conjugated)	Dako, Denmark	K5007	Detection system
DAB Substrate Kit	Dako, Denmark	K3468	Chromogenic detection
Hematoxylin Stain	Sigma-Aldrich, USA	H9627	Nuclear staining
Eosin Stain	Sigma-Aldrich, USA	E4009	Cytoplasmic staining
Neutral Balsam Mounting Medium	Sinopharm Chemical Reagent Co., China	10004160	Slide mounting
HMGA1 Primers	Custom synthesized (e.g., IDT/Sangon)	N/A	Gene amplification
HAND1 Primers	Custom synthesized (e.g., IDT/Sangon)	N/A	Gene amplification
GAPDH Primers (internal control)	Custom synthesized	N/A	Housekeeping gene
APES (Aminopropyltriethoxysilane)	Sigma-Aldrich	N/A	Slide coating
RNase-free Microcentrifuge Tubes	Eppendorf	N/A	RNA handling
RNase-free Pipette Tips	Eppendorf	N/A	Liquid handling
Agarose Gel Casting Tray and Comb	Generic	N/A	Gel preparation
Primer Design Software	Integrated DNA Technologies, USA	PrimerQuest Tool	Primer design
Image Analysis Software	Media Cybernetics, USA	Image-Pro Plus 6.0	Band quantification
Statistical Analysis Software	IBM Corp., USA	SPSS Version 13.0	Statistical analysis