Protection of Islet Beta Cells from Iron Overload-Induced Injury by Long Non-Coding RNA Maternally Expressed Gene 3

Summary

Iron overload reduced MEG3 expression in pancreatic tissue. MEG3 may play a protective role against iron-induced islet β-cell apoptosis, potentially involving modulation of the NF-ĸB pathway. This mechanism may contribute to islet dysfunction in patients with β-TM with iron overload.

Abstract

Patients with beta-thalassemia major (β-TM) commonly present with abnormal glucose metabolism, and factors such as iron overload, chronic anaemia, hormonal imbalances, liver dysfunction and inflammation-associated insulin resistance are considered important contributors to pancreatic islet dysfunction. Long non-coding ribonucleic acids (lncRNAs) are increasingly recognised for their regulatory roles in cell apoptosis and glucose homeostasis. This study explores the function of the lncRNA, maternally expressed gene 3 (MEG3), in iron-induced islet β-cell injury. An iron-overload mouse model was induced via intraperitoneal injection of iron dextran. Iron deposition and tissue damage in the pancreas were evaluated using Prussian blue and hematoxylin and eosin staining, and MEG3 expression was quantified via reverse transcription polymerase chain reaction (RT-PCR). In vitro, MIN6 cells were transfected with MEG3 small interfering RNA to examine apoptosis via flow cytometry, and NF-kappa (ĸ)B signaling pathway activity was assessed by Western blotting. Histological staining confirmed significant iron deposition and structural damage in the pancreatic tissue of iron-overloaded mice. The RT-PCR analysis revealed a marked reduction of MEG3 expression in the pancreas (p<0.05). In vitro, MEG3 knockdown significantly increased apoptosis of MIN6 cells compared with controls. Western blot analysis showed an elevated level of NF-ĸB, indicating activation of the NF-ĸB signaling pathway in MEG3-silenced cells. These findings suggest that MEG3 downregulation may enhance islet β-cell apoptosis via NF-ĸB signaling under iron overload conditions. The MEG3 gene may play a protective role against iron-induced islet β-cell apoptosis, potentially involving modulation of the NF-ĸB pathway. This mechanism may contribute to islet dysfunction in patients with β-TM with iron overload.

Introduction

Beta (β)-thalassemias are a group of inherited hematological disorders characterized by reduced or absent β-globin chain production, resulting in chronic anemia and ineffective erythropoiesis¹. In patients with transfusion-dependent β-thalassemia major (β-TM), repeated blood transfusions and hemolysis frequently lead to systemic iron overload, which can damage multiple organs and contribute to a range of endocrine complications, including abnormal glucose metabolism². Additionally, chronic anemia increases the burden on the pancreas, hormonal imbalances affecting glucose regulation, liver dysfunction impairing glucose metabolism, and insulin resistance induced by inflammatory processes may all contribute to hyperglycemia in patients with β-TM. Therefore, glucose metabolic disturbances in β-TM are multifactorial in nature^2,3. A recent meta-analysis reported that the prevalence of impaired fasting glucose, impaired glucose tolerance (IGT), and diabetes mellitus (DM) in patients with β-TM was 17.21%, 12.46%, and 6.54%, respectively, indicating an increasing burden of glucose metabolic disorders in this population³.

Despite its growing clinical relevance, the underlying mechanisms of abnormal glucose metabolism in β-TM remain poorly understood. Previous studies have demonstrated that chronic iron overload, primarily from repeated transfusions and hemolysis, can lead to excessive iron deposition in the pancreas, particularly within islet β cells. This accumulation promotes the generation of reactive oxygen species and exacerbates oxidative stress, triggering mitochondrial dysfunction, impairing insulin synthesis and secretion, and ultimately inducing β-cell apoptosis and insulin deficiency^4,6. Additionally, iron overload may contribute to peripheral insulin resistance through oxidative stress-mediated inflammation and interference with insulin signaling pathways. However, the molecular pathways linking iron overload to β-cell injury and systemic glucose dysregulation are complex and are yet to be fully elucidated. Long non-coding ribonucleic acids (lncRNAs) – a class of transcripts longer than 200 nucleotides that do not encode proteins – have emerged as important regulators in diverse biological processes, including glucose homeostasis, insulin secretion, and oxidative stress responses^7,8,9. Pancreatic islet β cells are known to express over 1,000 lncRNAs, suggesting their potential regulatory role in maintaining endocrine function¹⁰.

However, little is known about the lncRNA expression profiles and their functional roles in the context of iron overload-induced islet β-cell damage, especially in β-TM. Recent advances revealed that LncRNA MEG3 (MEG3) promotes insulin secretion and inhibits pancreatic β-cells apoptosis¹¹. However, its effect on pancreatic β-cells in iron-overload conditions remains elusive. In other pathological contexts, lncRNAs have been shown to interact with key signaling pathways such as NF-kappa (ĸ)B, JNK, and Nrf2, which are also involved in oxidative stress and iron overload-related cell injury^12,13,14. Specifically, NF-ĸB is a central transcription factor activated under oxidative stress and has been implicated in β-cell apoptosis in iron-overload conditions^15,16,17. Therefore, this study investigates that the MEG3 downregulation may enhance islet β-cell apoptosis via NF-ĸB signaling under iron overload conditions. This work may provide new insights into the molecular mechanisms underlying abnormal glucose metabolism in β-TM.

Protocol

The experimental protocols employed in the present study were approved by the Ethics Committee of Hainan General Hospital (protocol no. 2021-239). The US National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Declaration of Helsinki were strictly followed in all experimental procedures. The reagents and the equipment used in this study are listed in the Table of Materials.

1. Animal Model and Experimental Design

Thirty-six male C57BL/6J mice (4 weeks old, approx. 20 g) were used in this study. All mice were housed under standard laboratory conditions (20–25 °C, 50%–65% relative humidity, 12-h light/dark cycle) with ad libitum access to food and water. Following a 7-day acclimatization period, mice were randomly divided into two groups (n=18 per group): the iron-overload group (Model group), which received intraperitoneal injections of iron dextran (0.2 g/kg) twice per week for 4 weeks; and the Control group, receiving equivalent volumes of normal saline via intraperitoneal injection. After 4 weeks, the mice were euthanized, and pancreatic tissues were harvested for subsequent analysis.

2. Serum Iron Measurement

Blood samples were collected from the retro-orbital sinus under 1% pentobarbital anesthesia during diestrus. After being left to stand at room temperature for 1 h, the blood was centrifuged at 4 °C, 3,000 rpm for 15 min. Serum was separated and stored at -80 °C. Serum iron concentrations were determined using an iron colorimetric assay kit following the manufacturer’s protocol.

3. Histological Analysis

Pancreatic tissue specimens were immersed in 4% paraformaldehyde for fixation, followed by paraffin embedding prior to section preparation. Serial tissue sections with a thickness ranging from 4–5 µm were obtained and processed through standard deparaffinization and rehydration procedures. Histomorphological examination was subsequently performed using hematoxylin and eosin staining, while iron deposition within pancreatic tissues was evaluated by Prussian blue staining according to conventional histological staining protocols.

4. Cell Culture and Small Interfering Ribonucleic Acid Transfection

The MIN6 mouse pancreatic β-cells were cultured in high-glucose Dulbecco’s modified Eagle medium supplemented with 15% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, 10 mM HEPES, 50 mM sodium pyruvate, and 50 µmol/L β-mercaptoethanol. All experiments were performed at 80% cell confluence. Each time during cell culture, 6–8 ml of culture medium is taken. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO₂. For functional studies, MIN6 cells in the logarithmic growth phase were transfected with lncRNA-specific small interfering (si)RNA (targeting selected lncRNA based on microarray results; Inhibitor group) or negative control siRNA (Inhibitor NC group) using Lipofectamine 2000, according to the manufacturer’s instructions. After culturing for 24 h, cells were analyzed by quantitative polymerase chain reaction (qRT-PCR) or western blot to determine knockdown efficiency. Target sequences of this siRNA were as follows siMEG3: GCGUCUUCCUGUGCCAUUUTTAAAUGGCACAGGAAGACGCTT; siNC: UUCUCCGAACGUGUCACGUTTACGUGACACGUUCGGAGAATT. Each experiment was conducted in triplicate and repeated three times.

5. Reverse Transcription Polymerase Chain Reaction Assay

Total RNA was extracted from pancreatic tissue samples and MIN6 cells using a commercially available phenol-based RNA isolation reagent according to the manufacturer’s instructions. Complementary DNA was synthesized using a reverse transcription kit in a total reaction volume of 20 µL. Following reverse transcription, the cDNA concentration of most samples was approximately 1 µg/µL based on sample quantification analysis. Subsequently, 1 µL of cDNA template was used for quantitative real-time PCR amplification. qRT-PCR was carried out using a SYBR Green–based PCR detection kit under the following cycling conditions: 95 °C for 30 s, 90 °C for 15 s, 60 °C for 34 s, and 68 °C for 30 s. Relative gene expression levels were determined using the comparative threshold cycle method (2^-ΔCT method.) GAPDH was used as the internal reference gene for normalization of real-time PCR data. All experimental procedures were performed according to the respective manufacturers’ protocols, and each experiment was independently repeated at least three times. The primer sequences used in this study are listed in Supplementary Table 1 and Table 2.

6. Cell Counting Kit-8 Assay

Cell proliferation capacity was determined using a cell counting kit-8 assay in accordance with the manufacturer’s guidelines. Briefly, MIN6 cells were plated in 96-well culture plates at a density of 5 x 10³ cells per well following transfection with si-MEG3 or si-NC and maintained for 24 h. At the designated time points, 10 µL of cell viability reagent was introduced into each well, and the plates were incubated at 37 °C for 2 h. Absorbance values were subsequently measured at 450 nm using a microplate reader. Cell viability was calculated as a percentage relative to the control group.

7. Apoptosis Detection by Flow Cytometry

Apoptotic changes in MIN6 cells were examined using an Annexin V-FITC/PI apoptosis detection kit according to the manufacturer’s instructions. Following 48 h of siRNA transfection, cells were harvested, washed twice with cold phosphate-buffered saline, and resuspended in binding buffer. Thereafter, cells were incubated with 5 µL Annexin V-FITC and 5 µL propidium iodide for 15 min at room temperature under dark conditions. Immediately after staining, samples were analyzed by flow cytometry. Early and late apoptotic cell populations were quantified using flow cytometric analysis software. The proportion of apoptotic cells was subsequently calculated for intergroup comparisons. Each experiment was independently conducted three times.

8. Western Blot Analysis

Western blot experiments were performed according to previously reported protocols¹⁸. Cells were first digested with trypsin, collected, and lysed on ice using radioimmunoprecipitation assay lysis buffer. Protein concentrations were quantified, and equivalent amounts of protein were subjected to separation on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels before transfer onto polyvinylidene fluoride membranes. Following membrane blocking, samples were incubated with primary antibodies against NF-κB, (1:1000), Bcl-2 (1:1000), Bax (1:1000), caspase-3 (1:1000), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:2000) at the indicated dilution ratios, followed by incubation with horseradish peroxidase-linked secondary antibodies. Protein signals were detected using a chemiluminescence system and quantified with ImageJ software. GAPDH was used as the internal loading control, and protein expression levels were expressed as fold changes relative to the control group.

9. Statistical analysis

Data were expressed as mean ± standard deviation (SD). Comparisons between groups were conducted using the Mann–Whitney U test¹⁹ using statistical analysis. A p-value <0.05 was considered statistically significant.

Results

Evaluation of the iron-overload mouse model and measurement of maternally expressed gene 3 expression in pancreatic tissues

To investigate the role of MEG3 in vivo, we established an iron-overload model in mice via intraperitoneal injection of iron dextran. Compared with the Control group, serum iron levels in the Model group were significantly increased (p<0.05) (Figure 1A). Histological examination revealed notable iron deposition in pancreat...

Discussion

Abnormal glucose metabolism is a common and serious complication in patients with β-TM, often manifesting as IGT or DM. In this study, we found that iron overload led to pancreatic injury and significantly decreased the expression of lncRNA MEG3 in pancreatic tissue, accompanied by increased apoptosis of islet β cells. Moreover, knockdown of MEG3 in MIN6 cells promoted β-cell apoptosis and was associated with NF-ĸB activation, indicating a potential mechanistic link between MEG3 and iron overload-indu...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1M Tris-HCL (pH=6.8)	Beijing Solarbio Science & Technology Co.,Ltd	T1020
50bp DNA Ladder	TIANGEN	MD108
50bp DNA Ladder	TIANGEN	MD108
6×DNA Loading Buffer	TIANGEN	GH101-01
6×DNA Loading Buffer	TRANS	GH101-01
-80ºC refrigerator	BIOBASE	BDF-86V348
ABI 7500 real-time PCR system	Applied Biosystems, USA
Annexin V-FITC/PI Apoptosis Kit	MULTI SCIENCES	AP101-100-kit
BCA Protein Assay Kit	Elabscience Biotechnology Co.,Ltd.	E-BC-K318-M
Biopulverizer	biospec
BSA	Beijing Solarbio Science & Technology Co.,Ltd	A8020
C57BL/6J male mice	Gempharmatech Co,Jiangsu,China
CCK-8 Cell Proliferation Assay Kit	KeyGEN BioTECH	KGA317
ChamQ Universal SYBR qPCR Master Mix	Vazyme	Q711-02
ChamQ Universal SYBR qPCR Master Mix	Vazyme	Q711-02
DMEM	KeyGEN BioTECH
Flow cytometry	ACEA Biosciences, USA	NovoCyte 2060R
Fluorescence PCR instrument	Bio-Rad Laboratories, Shanghai
Glycine	Beijing Solarbio Science & Technology Co.,Ltd	G8200
High-capacity cDNA reverse transcription kit	Applied Biosystems, Carlsbad, CA, USA
HiScript II Q RT SuperMix for qPCR (+gDNA wiper)	Vazyme	R223-01
HiScript II Q RT SuperMix for qPCR (+gDNA wiper)	Vazyme	R223-01
HRP conjugated Goat Anti-Mouse IgG (H+L)	Servicebio	GB23301	1/2000
HRP conjugated Goat Anti-Rabbit IgG (H+L)	Servicebio	GB23303	1/2000
Human LncRNA Array V5.0	Arraystar,Rockville, Maryland, USA
Lipofectamine 3000 Transfection Reagent	Invitrogen	L3000015
Microarray scanner	Agilent Technologies ,California, USA
MIN6 cell	procell	KGM12800N
Mini-Bead-Beater-16	biospec
Mouse Anti-GAPDH	TransGen Biotech	HC301	1/2000
NanoDrop spectrophotometer	NanoDrop Products, Wilmington, DE, USA	ND-2000
PAGE Pre-Solution	Beijing Solarbio Science & Technology Co.,Ltd	A1010
Primers	Generay Biotech,Anhui, China
PrimeScript RT Reagent Kit with gDNA Eraser	CWBIO,Jiangsu, China
PVDF	Millipore	IPVH00010
Rabbit Anti Bax	proteintech	50599-2-ig	1/5000
Rabbit Anti Bcl-2	abcam	ab194583	1/1000
Rabbit Anti Caspase3	abcam	ab184787	1/2000
Rabbit Anti P65	abcam	ab32536	1/8000
Rabbit Anti p-P65	Affinity	af2006	1/1000
TB Green Premix Ex Taq II	TaKaRa, Japan
TRIzol	Invitrogen life technologies, Carlsbad, CA, USA
TRIzol reagent	CWBIO,Jiangsu, China
Ultra-sensitive chemiluminescence imaging system instrument	Bio-Rad Laboratories, Shanghai	Chemi DocTM XRS+
Ultraviolet spectrophotometer	NanoPhotometer	NP80