Investigation of subcellular proteomic changes that associate with SH-SY5Y neuroblastoma cell differentiation

• Main Authors: Wen-Ling Hsu, 許雯菱
• Other Authors: Yeou-Guang Tsay
• Format: Others
• Language: en_US
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/2p9d75

碩士 === 國立陽明大學 === 生化暨分子生物研究所 === 107 === Neuroblastoma is an aggressive embryonal malignancy arising from neural crest tissues of sympathetic nervous system. Until now, several gene mutations have correlations with congenital neuroblastoma, but the molecular changes underlying this tumor are mostly unknown. Although neuroblastoma is severely malignant in general, surprisingly, there is a considerably proportion of neuroblastoma that can self-differentiate into benign tumors or even vanish completely. For example, stage 4S is designated to a group of neuroblastoma tumors with spontaneous regression, and the long-term outcome has been excellent. Due to poor prediction of the prognosis, markers predicting self-differentiation can help clinicians to avoid unnecessarily aggressive treatment. Given that proteomics only found minimal changes in ATRA-induced differentiation, we would like to examine whether protein interaction changes are better predictors of neuroblastoma differentiation. In Chapter II, we used a modified in situ fractionation procedure as a protein interactomics tool to uncover proteins with all-or-none changes in a set of subcellular fractions during differentiation. When adapting this approach, we surprisingly found that detergent-permeabilized SH-SY5Y cells stably attached to the culture plate only under acidic pH like pH 4.2. Using two-dimensional differential gel electrophoresis, we found color changes in 37, 23, 18, 33 and 4 spots for the Triton X-100, DNase I, RNase A, high-salt and cell-architecture fractions, respectively. There were four proteins showing >10-fold increase in the Triton X-100 fraction from ATRA-treated cells, while other fractions still had only modest or minimal changes. These include GRP78, ERP29, NCS1 and CRABP2 proteins. Intriguingly, all four proteins are from ER-Golgi system, suggestive of an unnoticed role of this system in regulation of cell differentiation. Some cytoskeletal proteins with high expression were identified in certain subcellular fractions, implicating their participation in morphological changes like neurite formation. Ingenuity pathway analyses of proteins with significant changes imply that carbohydrate metabolism is involved in cell differentiation. We will further characterize whether these all-or-none protein interactions can be seen in vivo and how they can be used in choosing the strategy for neuroblastoma treatment.