Validation of gene expression (SPP1 and ITGB1) induced by knockdown of Phosphoinositide 3-Kinase delta gene (PIK3CD) in U373MG human glioblastoma cells

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Validation of gene expression (SPP1 and ITGB1) induced by knockdown of Phosphoinositide 3-Kinase delta gene (PIK3CD) in U373MG human glioblastoma cells

 

Author: Ng, Yuen Wah Joanna
Title: Validation of gene expression (SPP1 and ITGB1) induced by knockdown of Phosphoinositide 3-Kinase delta gene (PIK3CD) in U373MG human glioblastoma cells
Degree: M.Sc.
Year: 2014
Subject: Hong Kong Polytechnic University -- Dissertations
Glioblastoma multiforme -- Treatment
Phosphoinositides
Department: Dept. of Health Technology and Informatics
Pages: xii, 94 leaves : illustrations ; 30 cm
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2759019
URI: http://theses.lib.polyu.edu.hk/handle/200/7598
Abstract: Introduction: Glioblastoma Multiforme (GBM) is the most prevalent and aggressive form of brain tumor. Currently, the molecular pathways have been targeted for developing novel therapeutic approaches for GBM. One of the involved pathways is the Phosphoinositide 3-kinases (PI3Ks) signaling pathway. Previous researches indicated the knockdown of PI3K delta catalytic subunit could inhibit GBM cell proliferation and migration. Former cDNA array plate result indicated there are two genes up-regulated and seven genes down-regulated after the knockdown of PI3K delta gene (PIK3CD) in human glioma cell line U-373MG. Aims: This project focused on the up-regulated genes: ITGB1 and SPP1. ITGB1 encodes the beta 1 integrin while the SPP1 encodes the secreted phosphoprotein 1. The expression of both proteins are increased in GBM and responsible for tumor growth and invasion. The aim of this project is to validation the gene expression of ITGB1 and SPP1 after PIK3CD knockdown. Method: U373-MG glioma cells were cultured and the PIK3CD gene was knocked down by siRNA. The mRNA levels were examined by real-time PCR and the changes of mRNA between sample and control were determined by delta-delta CT method. The protein expression was further investigated by Western Blotting and the Image Lab software was used to calculate the relative protein quantities. Results: The PIK3CD gene was knocked down successfully. Both mRNA levels of ITGB1 and SPP1 were found to be increased. However, only the expression of beta 1 integrin protein was increased whereas that of secreted phosphoprotein 1 remained unchanged. Conclusion: The results of this project seemed contradictory to previous findings. Possible reasons included that the ligation status and α partner might adjust the activity of β1 integrin, and therefore inhibit the tumor metastasis. Besides, reports supported that mRNA and protein level may not be correlated in some genes, and could perhaps explain the unexpected results on SPP-1. Since the pathways contributed to GBM pathogenesis were complicated, more research is required to elucidate the interactions among these proteins.

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