Cell migration signaling molecules in phosphoinositide 3-kinase p110 delta positive glioblastoma cells : a pilot study

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Cell migration signaling molecules in phosphoinositide 3-kinase p110 delta positive glioblastoma cells : a pilot study


Author: Ng, Kwok Wa
Title: Cell migration signaling molecules in phosphoinositide 3-kinase p110 delta positive glioblastoma cells : a pilot study
Degree: M.Sc.
Year: 2015
Subject: Glioblastoma multiforme.
Hong Kong Polytechnic University -- Dissertations
Department: Dept. of Health Technology and Informatics
Pages: xii, 97 pages : illustrations
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2816502
URI: http://theses.lib.polyu.edu.hk/handle/200/8042
Abstract: Glioblastoma multiforme (GBM) is an aggressive and malignant primary brain tumor. It is the most common primary and lethal brain tumor in adults. Although multimodal therapy has applied to patients, the survival is still unsatisfactory with poor prognosis. Study of the molecular pathway of GBM is needed for discovering innovative treatment to improve the prognosis of GBM. Recent study reported knockdown of PI3K p110δ reduced glioma cell migration as well as focal adhesion kinase (FAK) and cell division cycle (Cdc42) expression in vitro. However, the micro-relationship of PI3K p110δ, FAK and Cdc42 is unknown. This study aims to investigate the knowledge gap using formalin-fixed paraffin-embedded (FFPE) tissue and laser capture microdissection (LCM). In the pilot study using breast cancer sections, immunohistochemistry (IHC) staining showed positive PI3K p110δ expressing cells. However, very little protein was extracted from the laser capture dissected cells and no band was detected in Western blot. A series of investigation were performed using cultured glioma cells. The Western blot detection limit was found to require at least 30 μg of proteins and more than 24,000 intact cells. Protein and RNA extraction efficiencies from FFPE sections were also studied. The efficiencies varied between liver and tonsil FFPE tissues, which may be due to different storage condition. RNA purity from FFPE tissue was satisfactory. However, no amplification was obtained in real-time PCR even large amounts of FFPE tissue were used. It may be due to RNA fragmentation from fixation process and storage conditions. The micro-relationship of PI3K p110δ, FAK and Cdc42 could not be demonstrated in the pilot study. In LCM, the amount of FFPE sample is usually small which may not be sufficient for Western blot detection. The unstable nature of RNA is also a barrier to use FFPE for real-time PCR. Although real-time PCR and Western blot are not suitable for limited amount of FFPE tissue, there are alternative methods such as utilizing frozen tissue, multiplex IHC and nanostring technology to study the relationship of PI3K p110δ, FAK and Cdc42.

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