RNA Transfection with Hypoxia Inducible Factor-1alpha

ABSTRACT

Introduction: Ischemic necrosis of dermal flaps is a devastating complication of reconstructive surgery. The increasing prevalence of diabetes, obesity, and an aging population adds to this concern. Hypoxia-inducible factor-1alpha (HIF-1alpha), a master regulator of the adaptive response to hypoxia, controls the expression of angiogenic growth factors. The development of biologically active, gene-specific mRNAs, especially in COVID-19 vaccines, has shown the ability for intracellular protein expression. We sought to express HIF-1alpha through mRNA transfection and determined its biological activity by measuring the upregulation of selected downstream targets. Method(s) 5'-methyl-capped poly-A tailed mRNA was generated using T7 RNA polymerase and verified by gel electrophoresis. Predominant and variant HIF-1alpha mRNA were transfected into primary human dermal fibroblasts via Lipofectamine in triplicate, and RNA levels were assessed using RT-qPCR. All gene expression levels were normalized to beta-actin expression levels Results: At one day after transfection, the levels of HIF-1alpha transcript were significantly higher in the cells transfected with predominant (p = 0.0104) and variant (p = 0.0007) HIF-1alpha transcripts relative to the control. Additionally, the expression of HIF-1alpha transcription product genes VEGF (p = 0.0274) and ANG-1 (p = 0.05) were significantly higher in the cells transfected with the HIF-1alpha transcripts than the control. Conclusion(s) Our approach led to the successful transfection of HIF-1alpha mRNA into human fibroblasts, resulting in upregulation of HIF-1alpha downstream angiogenic targets. Thus, the use of biologically active HIF-1alpha mRNA transfection offers a promising approach to inhibit ischemic necrosis.