Differential mRNA Expression and Detection of Alternatively Spliced Forms of VEGFA in different Neuroblastoma Cell Lines




Neha Patel

Introduction

Neuroblastoma is a malignant, solid tumor that develops from embryonic neural crest cells found in several areas of the body (most commonly originating in and around adrenal glands). It usually occurs in children younger than five years and sometimes forms before a child is born. It is slightly more common in males. (5)

There are three phenotypes of neuroblastoma cell lines. They are N (neuroblastic), S (substrate adhesive) and I (intermediate). (4) I type cells are precursor cells because N type cells and S type cells are thought to be derived from I type cells. I type cells share properties of both N and S cell lines. I type cells are the most malignant cells followed by N type cells. S type cells are considered non malignant.

Vascular Endothelial Growth Factor (VEGF) is a sub family of growth factors, to be specific, Platelet Derived Growth Factors. The members of this subfamily are VEGFA, VEGFB, VEGFC, VEGFD and Placenta Growth Factor (PlGF). The most important member is VEGFA. VEGFA is a signaling protein, associated with angiogenesis and vasculogenesis. It is also referred to as Vascular Permeability Factor (VPF) for its vascular permeability enhancing activities.
VEGFB makes vascular endothelial cells pump free fatty acids from the blood stream into nearby muscles and adipose tissue. Thus VEGFB has a critical biological function in stimulating fatty acid transport through endothelial cells of the vasculature.(9) Like VEGFA, VEGFC and VEGFD stimulates cell functions through selective binding and phosphorylation of tyrosine kinase receptors followed by activation of downstream signaling molecules. However, they all bind to different receptors. VEGFC binds to flt4 receptors while VEGFD binds to flk1 and flt4 receptors. VEGFA binds to flt1 and flk1 receptors. VEGFC also plays a role in lymph angiogenesis.

VEGFA is a disulfide linked homodimer but it is also found as a heterodimer with Placenta Growth Factor (PGF), homolog of VEGFA. VEGFA gene is located on the short arm of chromosome 12. It has 8 exons and 8 transcript variants.



Results

Primers 1F/1R are on exons 3 and 4 respectively and primers 2F/2R are on exons 4 and 8 respectively which amplifies variant section of VEGFA (Fig 1). Primer set 1F/1R amplified an invariant part (between exon 4 and exon 8) of VEGFA (Fig 2). Two variants of VEGFA were detected in all four neuroblastoma cell lines with primer set 2F/2R (Fig 3). Sequencing identified the larger band as TV 4 and the smaller band as TV 6. Differential mRNA expression of VEGFA was studied in four different neuroblastoma cell lines using RT-PCR. Two TV were expressed in all neuroblastoma cell lines. No significant difference was found in expression levels of these two transcripts in N and S cell lines. However, there was higher relative expression of the TV 6 in both I type cell lines (JMN and Be2C)(Fig 3) when compared with expression level of TV 4. GAPDH was used as a loading control (Fig 4).


Discussion

Normally, angiogenesis in adult human body occurs less frequently, usually during wound healing or during menstruation in women. But abnormally, angiogenesis plays an important role in tumor development. In tumors, insufficient oxygen activates the expression of a transcription factor, Hypoxia Inducible Factor (HIF) which then stimulates the release of VEGFA. VEGFA then binds to VEGF1 and VEGF2 receptors (VEGFR or Tyrosine kinase receptors) on endothelial cells. The receptors are then dimerized and activated triggering a tyrosine kinase pathway. This pathway contributes to endothelial cell proliferation and migration, and formation of tubular structures. These new vessels provide the tumor with additional nutrients and oxygen. It also facilitates the metastasis of tumors.

The differential mRNA expression levels of VEGFA was studied in 4 different neuroblastoma cell lines using RT-PCR. Two transcript variants were expressed in all the neuroblastoma cell lines. There was no significant difference found in the expression levels of these two transcripts in N and S cell lines. However, there was relatively higher expression of TV 6 in both I type cell lines (JMN and Be2C).



Literature Cited

1. Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature. 1992 Oct 29.

2. Napoleone Ferrara and Terri Davis-Smyth The Biology of Vascular Endothelial Growth Factor Department of Cardiovascular Research, Genentech, Inc., South San Francisco, California 94080

3. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science. 1989

4. Ciccarone, V., Spengler, B. A., Meyers, M. B., Biedler, J. L., Ross, R. A. (1989). Phenotypic Diversification in Human Neuroblastoma Cells: Expression of Distinct Neural Crest Lineages. Cancer Research 49, 219-225.

5. Hagberg, C.E. et al. Vascular endothelial growth factor B controls endothelial fatty acid uptake Nature; published online March 14, 2010; doi:10.1038/nature08945.


Figures


Figure 1-Transcript Variants of VEGFA, Positions of primer sets 1F/1R and 2F/2R, Expected product lengths of each transcript variant with primer set 2F/2R


Figure 2-Primers 1F and 1R amplifies invariant section (between exon 3 and exon 4) of all transcript variants of VEGFA in all four neuroblastoma cell lines


Figure 3-Detection of two alternatively spliced variants of VEGFA with primers 2F and 2R in all four neuroblastoma cell lines. The levels of TV 4 and TV 6 are equivalent in both S and N cell lines studied. The level of TV 6 is relatively higher in both I type cell lines when compared with the levels of TV 4


Figure 4-GAPDH was used as a loading control


Abstract

Neuroblastoma is a malignant, solid tumor that develops from neural crest cells and usually occurs in children younger than 5 years. The neuroblastoma cell lines exhibit 3 phenotypes N ( Neuroblastic), S (Substrate adherent) and I (Intermediate). I type cells are the most malignant. The mRNA expression of VEGFA was studied in these cell lines. VEGFA is a signaling protein, a member of Platelet Derived Growth Factor (PDGF), associated with angiogenesis and vasculogenesis. Angiogenesis plays an important role in tumor development. Differential expression of VEGFA mRNA in a few neuroblastoma cell lines was found. Two transcript variants were expressed in all four cell lines. Transcript variant four and six were detected. No significant differences in the expression of these two transcript variants were found in the N and S cell lines. However, the levels of transcript variant six appeared to be higher in both I type cell lines studied when compared to transcript variant 4.


Full Paper

Acknowledgments

I would like to thank Dr. Berish Rubin for his guidance and giving me an opportunity to accomplish this project. I would also like to thank Alex and Xie Xie for their consistent support and patience throughout the project. Further I would like to thank Dr. Ross for generously providing neuroblastoma cell lines. Additional thanks to Dr. Sylvia Anderson, Bo Liu and Class of 2011 for their help and encouragement throughout the project.


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