M. J. Metzner1, J. Mazar2, A. Rosado1, T. J. Westmoreland1 1Nemour’s Children’s Hospital; University Of Central Florida,Pediatric General Surgery,Orlando, FL, USA 2Sanford Burnham Presbys Medical Discovery Institute,Orlando, FL, USA
Introduction:
Neuroblastoma is a childhood predominant cancer, and its prognosis is greatly affected by MYCN overexpression. MYCN amplification is directly related to poor tumor prognosis and has been previously shown to attenuate RGS5, a regulator of G-protein signaling involved in tumor growth, metastasis, and angiogenesis. It has been previously shown that a decrease in RGS5 expression has been associated with increased tumor aggressiveness. Next Generation sequencing has enabled many advancements in cancer research and has enabled researchers to look at potential cancer markers to help further stratify how cell types react to treatments. We hypothesize that MYCN amplification correlates with RGS5 expression.
Methods:
To test this hypothesis we performed Next-Generation Sequencing on IMR-32 (MYCN amplified) cell line as well as MYCN knockdown in the same cell line. Next, total cellular RNA was isolated from the human neuroblastoma pre-treatment cell lines (SK-N-Be(1), SMS-KAN, IMR32) and post-treatment cell lines (LA-N-6, SK-N-As, SK-N-FI). The pre-treatment cell lines have overexpression of MYCN. The expression of RGS5 was measured using real-time reverse transcriptase polymerase chain reaction (qPCR).
Results:
Utilizing Next Generation sequencing, we demonstrated that the expression of RGS5 in MYCN amplified cell lines showed attenuated expression when compared to post-treatment cell lines where MYCN was non-amplified. The qPCR data using both pre and post-treatment cell lines followed the same pattern and showed that RGS5 expression was decreased in the MYCN amplified pre-treatment lines.
Conclusion:
RGS5 is differentially expressed in neuroblastoma cell lines with and without MYCN amplification. There is an inverse correlation of MYCN and RGS5, which follows the theory that low levels of RGS5 and high MYCN expression lead to a worse prognosis. It has been shown that RGS5 works in many cell growth, metastatic, and neovascularization pathways. This work further correlates the connection between expression of MYCN and RGS5. Because RGS5 expression levels are increased in post-treatment cell lines, RGS5 could serve in the future as a possible marker to further stratify and characterize the aggressiveness of recurrent neuroblastoma.