Long Non-Coding RNA PVT1 Exacerbates Hypoxia-Induced Cardiomyocyte Injury via the miR-135a-5p/FOXO1 Axis
Myocardial infarction (MI) remains a leading cause of global morbidity and mortality, primarily due to prolonged ischemia-induced cardiomyocyte injury. Hypoxia, a critical pathological feature of MI, triggers oxidative stress, apoptosis, and mitochondrial dysfunction in cardiomyocytes. While long non-coding RNAs (lncRNAs) have emerged as key regulators of cardiovascular diseases, their roles in hypoxia-driven myocardial injury remain underexplored. This study elucidates the mechanistic role of lncRNA plasmacytoma variant translocation 1 (PVT1) in hypoxia-induced injury in rat H9c2 cardiomyocytes, identifying its interaction with microRNA-135a-5p (miR-135a-5p) and downstream regulation of forkhead box O1 (FOXO1).
Hypoxia Induces PVT1 Upregulation in H9c2 Cardiomyocytes
To model hypoxia-induced injury, H9c2 cells were exposed to a hypoxic environment (1% O₂, 5% CO₂, 94% N₂) for 3–24 hours. Hypoxia significantly reduced cell viability in a time-dependent manner, with viability dropping to 52% at 12 hours and 38% at 24 hours compared to normoxic controls (P < 0.05). Apoptosis, assessed via Annexin V/PI staining and flow cytometry, increased from 5.8% under normoxia to 29.4% after 12 hours of hypoxia (P < 0.05). Western blot analysis confirmed elevated pro-apoptotic Bax protein levels (2.8-fold increase) and reduced anti-apoptotic Bcl-2 levels (60% decrease) under hypoxia. Strikingly, PVT1 expression, quantified by qRT-PCR, rose by 3.5-fold in hypoxic cells, suggesting its potential role in hypoxia-induced injury.
PVT1 Knockdown Attenuates Hypoxia-Induced Apoptosis
To investigate PVT1’s functional role, H9c2 cells were transfected with short hairpin RNA targeting PVT1 (shPVT1), achieving a 65% reduction in PVT1 mRNA levels compared to scrambled controls (P < 0.05). PVT1 silencing restored hypoxia-suppressed cell viability to 78% (vs. 52% in shControl-treated hypoxic cells) and reduced apoptosis to 12.3% (vs. 29.4% in controls). Western blot analysis mirrored these findings: Bax levels decreased by 50%, while Bcl-2 increased 1.8-fold in shPVT1-transfected cells. These data demonstrate that PVT1 exacerbates hypoxia-induced cardiomyocyte injury.
PVT1 Acts as a Sponge for miR-135a-5p
Bioinformatics analysis (LncBase Predicted v.2) identified miR-135a-5p as a potential PVT1-interacting miRNA. Luciferase reporter assays confirmed direct binding: co-transfection of miR-135a-5p mimics with wild-type PVT1 (PVT1-WT) reduced luciferase activity by 40% (P < 0.05), while mutations in the miR-135a-5p binding site (PVT1-MT) abolished this effect. RNA immunoprecipitation (RIP) assays using an Ago2 antibody revealed 4.2-fold and 3.8-fold enrichments of PVT1 and miR-135a-5p, respectively, in Ago2-bound fractions compared to IgG controls, confirming their interaction within the RNA-induced silencing complex (RISC).
PVT1 overexpression (via pcDNA3.1/PVT1) reduced miR-135a-5p levels by 55%, whereas PVT1 knockdown increased miR-135a-5p expression by 2.1-fold (P < 0.05). Hypoxia itself suppressed miR-135a-5p expression by 60%, positioning PVT1 as a negative regulator of miR-135a-5p under hypoxic stress.
miR-135a-5p Overexpression Mitigates Hypoxic Injury
Transfection of miR-135a-5p mimics into hypoxic H9c2 cells increased miR-135a-5p levels by 3.3-fold, restoring cell viability to 85% and reducing apoptosis to 14.6% (P < 0.05 vs. NC-miRNA). Conversely, miR-135a-5p inhibition elevated hypoxia-induced apoptosis to 34.7%. Co-transfection of PVT1 overexpression vectors with miR-135a-5p mimics reversed these protective effects, reducing viability to 63% and increasing apoptosis to 25.8%, confirming PVT1’s dependence on miR-135a-5p regulation.
FOXO1 is a Direct Target of miR-135a-5p
TargetScan analysis predicted FOXO1, a transcription factor linked to apoptosis and oxidative stress, as a miR-135a-5p target. Luciferase assays validated this interaction: miR-135a-5p mimics reduced FOXO1-WT luciferase activity by 45% (P < 0.05), while FOXO1-MT (mutant binding site) remained unaffected. Hypoxia increased FOXO1 protein levels by 2.5-fold, which was attenuated by miR-135a-5p mimics (60% reduction) and exacerbated by miR-135a-5p inhibitors (1.8-fold increase). These findings establish FOXO1 as a miR-135a-5p target negatively regulated under hypoxia.
PVT1/miR-135a-5p Axis Regulates FOXO1-Mediated Apoptosis
FOXO1 knockdown (shFOXO1) in hypoxic H9c2 cells reduced apoptosis to 11.2% and restored viability to 82%, paralleling miR-135a-5p overexpression effects. Co-transfection of shFOXO1 with miR-135a-5p inhibitors reversed this protection, increasing apoptosis to 28.5% and reducing viability to 58%. PVT1 overexpression upregulated FOXO1 by 2.2-fold, while PVT1 silencing reduced FOXO1 levels by 50% (P < 0.05). Critically, PVT1’s pro-apoptotic effects were abolished by FOXO1 knockdown: in PVT1-overexpressing cells, shFOXO1 restored viability to 80% and reduced apoptosis to 13.4%, confirming FOXO1 as the downstream effector of the PVT1/miR-135a-5p axis.
Mechanistic Insights and Therapeutic Implications
This study delineates a novel pathway in hypoxic cardiomyocyte injury: hypoxia upregulates PVT1, which sequesters miR-135a-5p, relieving its repression of FOXO1. Elevated FOXO1 promotes mitochondrial apoptosis via Bax upregulation and Bcl-2 suppression. The PVT1/miR-135a-5p/FOXO1 axis thus serves as a critical regulatory node, offering potential therapeutic targets. For instance, PVT1 inhibitors or miR-135a-5p mimics could mitigate FOXO1-driven apoptosis, preserving cardiomyocyte viability post-MI.
Experimental Validation and Technical Rigor
Key experiments included:
- CCK-8 Assays: Quantified viability after hypoxia and genetic manipulations.
- Flow Cytometry: Measured apoptosis using Annexin V/PI dual staining.
- qRT-PCR: Validated PVT1, miR-135a-5p, and FOXO1 mRNA levels, normalized to GAPDH or U6.
- Western Blotting: Assessed Bax, Bcl-2, and FOXO1 protein expression.
- Dual-Luciferase Reporters: Confirmed miRNA-mRNA interactions.
- RNA Immunoprecipitation: Verified PVT1/miR-135a-5p binding within RISC.
Statistical analyses employed Student’s t-tests or ANOVA, with P < 0.05 considered significant.
Conclusion
This study establishes lncRNA PVT1 as a hypoxia-inducible driver of cardiomyocyte apoptosis via miR-135a-5p sponging and FOXO1 activation. The findings illuminate the pathophysiological interplay between non-coding RNAs and transcription factors in MI, providing a foundation for RNA-targeted therapies to ameliorate ischemic heart disease.
doi.org/10.1097/CM9.0000000000001147
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