Investigating the Role of lncRNA GAS5 in Aortic Valve Calcification

Yuan Zhou (Bonn)1

1Universitätsklinikum Bonn Medizinische Klinik und Poliklinik II Bonn, Deutschland


Background: The complex interplay of genetic, molecular, and environmental factors in calcification aortic valve disease (CAVD) is a major challenge in cardiovascular medicine. Long non-coding RNAs (lncRNAs) have been recognized as key regulatory molecules in cellular processes, with lncRNA GAS5 attracting attention for its potential role in cardiovascular pathologies. Prior studies have associated GAS5 with the regulation of cellular apoptosis, proliferation, and differentiation; functions that are critical in the pathogenesis of CAVD.


Objective: This study investigates the specific role of lncRNA GAS5 in CAVD, aiming to elucidate its regulatory mechanisms and to assess its potential as a biomarker or therapeutic target in calcific heart diseases.


Methods: We established calcification models using Human Coronary Artery Smooth Muscle Cells (HCASMCs), Valvular Interstitial Cells (VICs), and Human Coronary Artery Endothelial Cells (HCAECs). Calcification was induced using osteogenic medium (OM) and pro-calcific medium (PCM) for seven days. We measured GAS5 expression levels post-induction and investigated the effects of GAS5 and hnRNPU knockdown on the expression of calcification and EndMT markers, cell necrosis, and proliferation using quantitative PCR, Western blot, MTT viability assays, and bromodeoxyuridine (BrdU) incorporation experiments. Additionally, a protein array for cell necrosis factors was performed to discern the effects of GAS5 modulation on apoptosis-related proteins.


Results: Post-calcification induction, GAS5 was significantly upregulated in all cell models. Targeted knockdown of GAS5 led to alterations in calcification markers, including decreased expression of BMP2, VEGFA, and RUNX2. EndMT markers NOS3 and SM22 also displayed differential expression upon GAS5 suppression. Notably, MTT assays indicated reduced necrosis in VICs following GAS5 knockdown. The protein array analysis revealed that the suppression of GAS5 notably decreased TRAIL R1/DR4 and Pro-Caspase-3 expression, linking GAS5 to apoptosis regulation in CAVD. Furthermore, hnRNPU knockdown in HCAECs resulted in a marked downregulation of GAS5, suggesting a regulatory association. BrdU assays confirmed a significant reduction in proliferation rates in VICs with GAS5 knockdown.

Conclusion: Our comprehensive analysis positions GAS5 as a pivotal regulatory molecule in CAVD, influencing critical cellular pathways such as apoptosis, proliferation, and osteogenic differentiation. The interaction between GAS5 and hnRNPU may represent a novel post-transcriptional regulatory mechanism in CAVD pathophysiology. These insights not only enhance our understanding of the molecular mechanisms underlying CAVD but also highlight GAS5 as a promising target for therapeutic intervention.

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