Analysis of RNA Interference Targeted Against Human Antigen R (HuR) to Reduce Vascular Endothelial Growth Factor (VEGF) Protein Expression in Human Retinal Pigment Epithelial Cells

VEGF-A or vascular endothelial growth factor-A is an important factor in enabling neovascularization and angiogenesis. VEGF-A is regulated transcriptionally as well as post transcriptionally. Human antigen R (HuR) belonging to the embryonic lethal abnormal vision (ELAV) family is a key regulator pro...

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Vydáno v:Molecular biotechnology Ročník 66; číslo 10; s. 2972 - 2984
Hlavní autoři: Supe, Shibani, Dighe, Vikas, Upadhya, Archana, Singh, Kavita
Médium: Journal Article
Jazyk:angličtina
Vydáno: New York Springer US 01.10.2024
Springer Nature B.V
Témata:
Angiogenesis
Antigens
Biochemistry
Biological Techniques
Biotechnology
Cell Biology
Cell Line
Chemistry
Chemistry and Materials Science
Diabetes mellitus
Diabetic retinopathy
Down-Regulation
ELAV-Like Protein 1 - genetics
ELAV-Like Protein 1 - metabolism
Epithelial Cells - metabolism
Epithelium
family
Gene expression
Gene sequencing
Growth factors
Human Genetics
Humans
HuR protein
Original Paper
Pigments
Protein Science
protein synthesis
Proteins
Reagents
Real time
Retina
Retinal pigment epithelium
Retinal Pigment Epithelium - cytology
Retinal Pigment Epithelium - metabolism
Retinopathy
RNA Interference
RNA, Messenger - genetics
RNA, Messenger - metabolism
RNA, Small Interfering - genetics
RNA, Small Interfering - metabolism
RNA-mediated interference
siRNA
therapeutics
transcription (genetics)
Transfection
Vascular endothelial growth factor
vascular endothelial growth factor A
Vascular Endothelial Growth Factor A - genetics
Vascular Endothelial Growth Factor A - metabolism
Vascularization
vision
Western blotting
siRNA
RNA interference
VEGF-A
HuR/ELAV
ISSN:1073-6085, 1559-0305, 1559-0305
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Shrnutí:VEGF-A or vascular endothelial growth factor-A is an important factor in enabling neovascularization and angiogenesis. VEGF-A is regulated transcriptionally as well as post transcriptionally. Human antigen R (HuR) belonging to the embryonic lethal abnormal vision (ELAV) family is a key regulator promoting stabilization of VEGF-A mRNA. In this research we investigate, whether HuR targeted RNA interference would enable the reduction of the VEGF-A protein in human retinal pigment epithelial cells (ARPE-19) in-vitro, in normoxic conditions. Three siRNA molecules with sequences complementary to three regions of the HuR mRNA were designed. The three designed siRNA molecules were individually transfected in ARPE-19 cells using Lipofectamine™2000 reagent. Post-transfection (24 h, 48 h, 72 h), downregulation of HuR mRNA was estimated by real-time polymerase reaction, while HuR protein and VEGF-A protein levels were semi-quantitatively determined by western blotting techniques. VEGF-A protein levels were additionally quantified using ELISA techniques. All experiments were done in triplicate. The designed siRNA could successfully downregulate HuR mRNA with concomitant decreases in HuR and VEGF-A protein. The study reveals that HuR downregulation can prominently downregulate VEGF-A, making the protein a target for therapy against pathological angiogenesis conditions such as diabetic retinopathy.
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ISSN:1073-6085
1559-0305
1559-0305
DOI:10.1007/s12033-023-00913-w