P08.53 CRISPR/Cas9 knockout of PI3K delta confirms its role in glioma cell migration and invasion

• Published in: Neuro-oncology (Charlottesville, Va.) Va.), 2017-05, Vol.19 (suppl_3), p.iii65-iii65
• Main Authors: Shao, W., Ji, T., To, S. T.
• Format: Article
• Language: English
• Subjects: POSTER PRESENTATIONS
• ISSN: 1522-8517; 1523-5866
• DOI: 10.1093/neuonc/nox036.242

Introduction: The Class1A phosphatidylinositol-3-kinase (PI3K) catalytic isoforms p110α, p110β and p110δ, have been implicated to play vital roles in tumorigenesis. In the incurable malignant brain tumor, glioblastoma multiforme (GBM), their pathogenic roles have also been focused on. Although these three isoforms have overlapping roles in cellular processes, they also exhibit distinct functions in the malignant process of cancer cells. In 2012, our group found that p110δ is the only isoform involved in the migration and invasion process of GBM cells. Two years later, a report came out with results opposite to ours - p110α and p110β but not p110δ are involved in GBM cell migration and invasion. Though both studies used siRNA to knockdown these isoforms, there might be confounding factors such as off-target effects of the siRNA chosen. Materials and Methods: To clarify this discrepancy, we employed a cleaner gene silencing technology, CRISPR/Cas9, to completely knockout p110δ gene in the U87 GBM cell line. Modified cell clones were screened by Western blotting followed by DNA sequencing. The proliferation of modified cells was evaluated by the CCK-8 assay. Cell migration was assessed by the wound-healing assay. The invasiveness of modified cells was determined using the Boyden chamber method. Results: After screening for more than 80 clones, we have successfully isolated 3 clones with different gene modifications: (i) deletions: 2 bp and 12 bp, and (ii) insertion: 1 bp in exon 3 of the PIK3CD gene. The proliferation assay result showed that all of the three modified cell clones grew slower than the normal U87 parental cells. The wound healing migration assay and the Boyden chamber invasion assay showed that the migration and invasion capacity of modified cells is significantly weaker than the U87 parental cells. Conclusions: These data confirm our previous siRNA results on p110δ knockdown. Thus, the use of gene knockout technology on other PI3K isoforms may help to clarify some of the contradicting observations reported in glioblastoma multiforme. Dr. Shao is supported by the Hong Kong Scholars Program.