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 obesity; however, the intricate underlying mechanisms remain elusive⁴. Given the limited understanding of TC's molecular mechanisms, increasing attention has been paid to non-coding RNAs, especially lncRNAs, for their regulatory roles in cancer.

Emerging evidence establishes lncRNAs as pivotal regulators of multifaceted biological processes, particularly in tumor development, through mechanisms such as alternative splicing, transcriptional regulation, and microRNA (miRNA) sponging^5,6,7. Plasmacytoma variant translocation 1 (PVT1) is located downstream of chromosome 8q24.21 and the oncogene C-myc⁸. Among these, lncPVT1, a lncRNA located downstream of the C-myc oncogene at chromosome 8q24.21, has garnered significant attention for its oncogenic roles in several malignancies, including liver cancer⁹and renal cell carcinoma¹⁰. Nevertheless, the functional implications of lncPVT1 in TC remain largely unexplored.

Cancer cells undergo metabolic reprogramming, a key feature of tumorigenesis, where they exhibit heightened glycolytic activity coupled with diminished oxidative phosphorylation, namely the Warburg effect¹¹. Hypoxia, a common microenvironmental feature in solid tumors, profoundly influences tumor behavior, promoting glycolysis and aggressiveness, and conferring a poor prognosis¹². Hypoxia-inducible factor 1 (HIF-1), a key factor regulating the cell's reaction to low oxygen levels, orchestrates a plethora of adaptive changes that support tumor survival and progression¹³. Previous studies have demonstrated that lncPVT1 modulates glycolysis in multiple malignancies, including breast cancer¹⁴, pancreatic ductal adenocarcinoma¹⁵, and osteosarcoma¹⁶. In addition, lncPVT1 has been shown to enhance cell proliferation by stabilizing HIF-1α in nasopharyngeal carcinoma¹⁷. However, the correlation between lncPVT1 expression and clinical parameters has not been reported in cancer. In addition, the specific function and mechanisms or unique clinical implications of lncPVT1 remain unclear in TC. Therefore, the goal of this study was to explore how lncPVT1 influences glucose metabolism in TC cell lines TPC-1 and K1 exposed to normoxia and hypoxia. We examined the impact of lncPVT1 overexpression on glucose consumption, lactate production, and cell proliferation. We further deciphered lncPVT1-governed HIF-1α stabilization mechanisms and their resultant effects on glycolysis-related transcriptional programming. This comprehensive investigation offers novel perspectives on how lncPVT1 contributes to tumorigenesis, particularly in the context of metabolic reprogramming and hypoxic adaptation.

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 corresponding negative control (NC) were generated in TPC-1 and K1 cells using a lentiviral expression system, following the manufacturer's instructions.

Evaluation of glucose levels

To assess glucose consumption, thyroid carcinoma cell lines were cultured under normoxic conditions (21% O₂) and hypoxic conditions (1% O₂) for 24 h. Glucose concentrations in the culture medium were measured using a glucose assay kit according to the manufacturer's protocol. Briefly, 2 µL of conditioned medium was mixed with freshly prepared glucose working reagent and incubated at 37 °C for 10 min. The absorbance was measured at 530 nm using a microplate reader to quantify glucose levels.

Evaluation of lactate levels

Thyroid carcinoma cell lines TPC-1 and K1 (2 x 10⁴cells/well) were cultured under normoxic conditions (21% O₂, 5% CO₂, 37 °C) and hypoxic conditions (1% O₂, 5% CO₂, 37 °C) for 24 h. After incubation, culture plates were centrifuged at 300 x g for 5 min at room temperature to pellet cells and debris. Subsequently, 2 µL of the clarified supernatant was collected from each well and mixed with 200 µL of lactate assay reagent in a 96-well plate. The reaction mixture was incubated at 37 °C for 10 min, after which a stop solution was added to terminate the reaction. Absorbance was measured at 530 nm using a microplate reader. Lactate concentrations were determined by comparing the absorbance values to a standard curve generated from known lactate standards (typically ranging from 0 to 20 nmol). All measurements were performed in triplicate.

Cell proliferation assay

Thyroid carcinoma cells were seeded into 96-well plates at a density of 3 x 10³ cells per well in 100 µL of culture medium. Cells were then incubated under normoxic (21% O₂, 5% CO₂, 37 °C) or hypoxic (1% O₂, 5% CO₂, 37 °C) conditions for 0 h, 24 h, or 48 h. At each time point, 10 µL of cell proliferation reagent was added to each well of a 96-well plate. The plate was then incubated at 37 °C for 40 min. After incubation, the optical density at 450 nm (OD450) was measured using a microplate reader to quantify cell proliferation. All measurements were performed in at least three technical replicates.

qRT-PCR analysis

Total RNA was extracted from 1 x 10⁶ thyroid carcinoma cells under normoxic conditions (21% O₂, 5% CO₂, 37 °C) and hypoxic conditions (1% O₂, 5% CO₂, 37 °C) using a guanidinium thiocyanate-phenol-chloroform extraction reagent according to the manufacturer's protocol. RNA purity and concentration were assessed spectrophotometrically by measuring absorbance ratios at 260/280 nm. For complementary DNA (cDNA) synthesis, 500 ng of total RNA was reverse-transcribed in a 20 µL reaction volume containing reverse transcriptase, random hexamer primers, dNTPs, RNase inhibitor, and reaction buffer. The reverse transcription was performed at 25 °C for 10 min, followed by 42 °C for 50 min, and terminated by heating at 70 °C for 15 min.

Quantitative PCR was performed using a SYBR Green-based detection system in a total reaction volume of 20 µL containing cDNA template (1:5 to 1:20 dilution of the original cDNA synthesis), SYBR Green PCR master mix, forward and reverse primers (each at 0.2 µM), and nuclease-free water. PCR amplification was conducted on a thermocycler with the following cycling conditions: initial denaturation at 95 °C for 3 min; 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s; followed by a melting curve analysis to verify the specificity of amplification.

Primer sequences for GLUT1, HK1, HK2, PGK1, and GAPDH (Table 1) were designed based on published sequences¹⁸ and synthesized commercially. Expression levels of target genes were normalized to GAPDH and calculated using the 2^-ΔΔCt method¹⁹.

Western blot

After treatment, thyroid carcinoma cells were washed 2x with cold phosphate-buffered saline (PBS) and lysed using radioimmunoprecipitation assay (RIPA) buffer supplemented with a protease inhibitor cocktail. Cell lysates were incubated on ice for 30 min with intermittent mixing and then centrifuged at 12,000 x g for 15 min at 4 °C to remove insoluble debris. The protein concentration of the supernatant was determined using a bicinchoninic acid (BCA) assay according to the manufacturer's instructions. Equal amounts of protein (30 µg per sample) were mixed with 5x loading buffer, boiled at 95 °C for 5 min, and loaded onto 10% SDS-polyacrylamide gels for electrophoretic separation. Proteins were separated by electrophoresis at 120 V for approximately 90 min and then transferred onto polyvinylidene fluoride (PVDF) membranes using a wet transfer system at 100 V for 90 min at 4 °C. Membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature to prevent nonspecific binding. Subsequently, membranes were incubated overnight at 4 °C with primary antibodies diluted in blocking buffer: anti-HIF-1α antibody (1:3,000) and anti-beta-actin antibody (1:8,000). After washing 3x with TBST (each for 10 min), membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies diluted 1:5,000 in blocking buffer for 1 h at room temperature. Following three additional TBST washes, protein bands were visualized using an enhanced chemiluminescence (ECL) detection system and imaged with a gel documentation system.

Dual luciferase assay

To investigate the transcriptional regulation of glycolysis-related genes by lncPVT1, TPC-1 cells were seeded in 24-well plates at a density of 1 x 10⁵ cells per well and cultured in DMEM containing 4.5 g/L glucose, supplemented with 10% fetal bovine serum and 100 µg/mL penicillin-streptomycin overnight. Cells were co-transfected with 500 ng of a hypoxia response element (HRE)-driven firefly luciferase reporter plasmid and 50 ng of Renilla luciferase control plasmid using a lipid-based transfection reagent at a final concentration of 2 µL per well, according to the manufacturer's instructions. Transfection efficiency was confirmed by including a parallel transfection with a GFP-expressing plasmid and assessing GFP expression under a fluorescence microscope 24 h post-transfection. Following 24 h incubation under normoxic (21% O₂) or hypoxic (1% O₂) conditions, luciferase activities were measured using a dual-luciferase reporter assay system. Firefly luciferase activity was normalized to Renilla luciferase activity, and the ratio was used for comparative analysis across samples.

Data analysis

All data were presented as mean ± SD. Statistical evaluations were executed in statistical analysis software. Comparisons between groups were conducted using independent t-tests, and statistical significance was determined based on a p-value threshold of less than 0.05.

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