Long Non-Coding RNA PVT1 Enhances Glycolysis in Thyroid Carcinoma by Stabilizing HIF-1α and Activating Glycolytic Gene Transcription

Summary

Here, we present a protocol to examine how lncPVT1 regulates HIF-1α stability and glycolytic gene expression in thyroid cancer cells, enabling the study of metabolic regulation and potential therapeutic targets.

Abstract

As the foremost endocrine system cancer, thyroid carcinoma exhibits an accelerating epidemiological trend across populations. Despite advances in treatment modalities, recurrence and metastasis remain challenges. The long non-coding RNA PVT1 (lncPVT1) has emerged as a conserved oncogenic regulator in multiple cancer types, yet its specific function in TC requires further exploration. Our research focused on examining how lncPVT1 affects glycolysis under both normoxic and hypoxic conditions using TC cell lines TPC-1 and K1. Overexpression of lncPVT1 significantly enhanced glucose consumption and lactate production under hypoxia, corroborating its ability to augment glycolysis. Further, lncPVT1 upregulated key glycolytic genes such as GLUT1, HK1, HK2, and PGK1, and facilitated TC cell proliferation. Mechanistically, lncPVT1 stabilized hypoxia-inducible factor 1α (HIF-1α) by impeding its degradation and leading to its accumulation. This accumulated HIF-1α then bound to specific regulatory sequences that control the expression of genes involved in glycolysis, ultimately activating these genes and boosting glycolytic activity. Our findings demonstrate that lncPVT1 regulates HIF-1α stability and glycolytic gene expression, advancing our understanding of TC metabolism and potentially revealing novel therapeutic targets.

Introduction

Thyroid cancer (TC), a highly prevalent endocrine malignancy, has experienced a rapid increase in incidence rates¹. Despite advancements in therapeutic strategies, including surgical procedures, radiation therapy, and thyroid hormonal suppression therapy, a substantial proportion of TC patients still face the risk of tumor recurrence and metastasis, emphasizing the need for a deeper understanding of its molecular pathogenesis^2,3. TC development is associated with several established risk factors, including ionizing radiation exposure, contact with chemical carcinogens, tobacco use, and ....

Protocol

Cell culture and transfection

Human thyroid carcinoma (TC) cell lines TPC-1 and K1 were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 4.5 g/L glucose, supplemented with 10% fetal bovine serum and 100 µg/mL penicillin-streptomycin. Cells were maintained at 37 °C in a humidified incubator with 5% CO₂. For hypoxia treatment, 1 x 10⁵ cells were transferred to a sealed hypoxic chamber equilibrated with 1% O₂, 5% CO₂, and 94% N₂ at 37 °C for 24 h, unless otherwise specified. Stable overexpression of lncPVT1 (lncPVT1 OE) and the co....

Results

Enhanced glycolysis in TC cell lines mediated by lncPVT1

We initially established lncPVT1 OE and NC in TPC-1 and K1 cells using a lentiviral expression system. Successful overexpression of lncPVT1 was confirmed by qRT-PCR (Figure 1A-B). To evaluate the impact on glycolysis, cells were cultured under normoxia and hypoxia for 24 h. Our findings indicated that hypoxia .......

Discussion

As the most common endocrine malignancy, TC poses significant challenges in terms of its recurrence and metastasis²². The mechanistic landscape of thyroid carcinogenesis remains incompletely defined, especially regarding lncRNA-mediated regulation of oncogenic metabolic reprogramming and fundamental cellular behaviors²³. In this study, we focused on the oncogenic lncPVT1 and its influence on glycolysis and HIF-1α stability in TC cells.

The W.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BCA Protein Assay Kit	meilunbio	MA0082-2	WB Protein Quantification Assay
Biosafety Cabinet	Lishen	HFsafe-1200LC	Cell Passage and Expansion
Centrifuge	Baiyang	B320A	Centrifuge Cells
Chemiluminescence Imaging System	BIO-RAD(USA)	ChemiDoc Touch	WB Development
CO2 Incubator	Thermo(USA)	311	Cell Culture
ECL Chemiluminescence Detection Kit	meilunbio	MA0186-1	WB Development
Flow Cytometer	BD	FACSCalbur	Flow Cytometry Apoptosis Assay
Fluorescence Inverted Microscope	NIKON(Japan)	Ts2-FL	Cell Observation
Fluorescence Quantitative PCR Instrument	ABI(USA)	7300	PCR Experiment
GAPDH	proteintech	60004-1-Ig	WB Internal Reference Antibody
Glucose Assay Kit	Jiancheng	A154-1-1	Biochemical Assay
HIF1A	BOSTER	A00013	WB Antibody
HRP-conjugated Affinipure Goat Anti-Mouse IgG(H+L)	proteintech	SA00001-1	Immunohistochemistry (IHC) Experiment
HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L)	proteintech	SA00001-2	Immunohistochemistry (IHC) Experiment
K1 cell line	Cell Bank of the Chinese Academy of Sciences (Shanghai, China)	NA	Human papillary thyroid carcinoma cell line
Lactic Acid Assay Kit	Jiancheng	A019-2-1	Biochemical Assay
Microplate Reader	Thermo(USA)	K3	ELISA Detection
PAGE Gel Ultra-Fast Preparation Kit (15%)	meilunbio	MA0384	WB Electrophoresis
PCR Instrument	BIO-RAD(USA)	PTC100	PCR Experiment
Pre-stained Rainbow Protein Marker	meilunbio	MA0342	WB Electrophoresis
Protein Vertical Electrophoresis System	BIO-RAD(USA)	POWER PAC 200	WB Electrophoresis
SDS-PAGE Protein Loading Buffer (5X)	Beyotime	P0015L	WB Electrophoresis
Sodium Dodecyl Sulfate (SDS)	sigma	151-21-3	WB Electrophoresis
TPC-1 cell line	Cell Bank of the Chinese Academy of Sciences (Shanghai, China)	NA	Human thyroid carcinoma cell line