Endocrine Abstracts (2008) 16 P12

RNA interference inhibits VEGF and EG-VEGF expression of H295R human adrenocortical carcinoma cell line

Gloria Appolloni, Giorgio Arnaldi, Michele Di Stefano, Monica Spegni, Marina Cardinaletti, Laura Trementino & Marco Boscaro


Division of Endocrinology, Department of Internal Medicine, Ancona, Italy.


Background: The development of a vascular supply is a critical factor in the growth and metastatic spread of malignant tumors. The recognition of the vascular endothelial growth factor (VEGF) pathway as a key regulator of angiogenesis has led to a considerable efforts to exploit its potential for therapy in oncology. Endocrine gland-derived (EG-VEGF) has been recently identified as an endothelial cell mitogen, predominantly expressed in steroidogenic tissues; it represents a novel type of cell-specific effector that acts in a tissue-specific manner to promote angiogenesis. Both VEGF and EG-VEGF are over-expressed in adrenal carcinoma.

Objective: To inhibit the expression of VEGF and EG-VEGF in human adrenal carcinoma cell line H295R by RNA interference approach.

Methods: Designed oligonucleotides that contain the siRNA-expressing sequence targeting VEGF and EG-VEGF were annealed and inserted into the eukaryotic expression vectors pSuper and pSuper.neo (Oligoengine). After sequencing confirmation of the positive clones, the vectors were transfected into H295R cells and the levels of suppression of VEGF and EG-VEGF were measured by semi-quantitative RT-PCR and Real Time RT-PCR. Appropriate positive (GAPDH RNAi) and negative (scramble RNAi) controls were considered.

Results: In the H295R cells transfected with pSuper.neo-RNAi the amount of VEGF and EG-VEGF mRNAs was decreased by 55% and 40%, with respect to the cells transfected with the empty vector. Lower inhibition levels were found using the pSuper vector.

Conclusion: RNA interference inhibits the expression of VEGF and EG-VEGF in H295R providing a starting point for the biological therapy of adrenal cancer. As H295R cells express EG-VEGF receptors, we hypothesize that particularly EG-VEGF inhibition will result in decreased cellular proliferation. The effects of RNAi targeting VEGF and EG-VEGF on cells proliferation and apoptosis will be studied.

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