Inducible Knockout of 14-3-3β Attenuates Proliferation and Spheroid Formation in a Human Glioblastoma Cell Line U87MG

Kellie Gallo; Bhairavi Srinageshwar; Avery Ward; Carlos Diola; Gary Dunbar; Julien Rossignol; Jesse Bakke

doi:10.3390/brainsci13060868

Inducible Knockout of 14-3-3β Attenuates Proliferation and Spheroid Formation in a Human Glioblastoma Cell Line U87MG

Brain Sci. 2023 May 27;13(6):868. doi: 10.3390/brainsci13060868.

Authors

Kellie Gallo¹, Bhairavi Srinageshwar^{2

3}, Avery Ward³, Carlos Diola³, Gary Dunbar^{2

4}, Julien Rossignol^{1

2

3}, Jesse Bakke^{1

2}

Affiliations

¹ Biochemistry, Cellular, and Molecular Biology, College of Science and Engineering, Central Michigan University, Mount Pleasant, MI 48559, USA.
² Program of Neuroscience, Central Michigan University, Mount Pleasant, MI 48859, USA.
³ Foundational Sciences Department, College of Medicine, Central Michigan University, Mount Pleasant, MI 48859, USA.
⁴ Department of Psychology, Central Michigan University, Mount Pleasant, MI 48859, USA.

Abstract

Glioblastomas (GBs) are the most common and malignant brain tumors in adults. A protein encoded by the gene YWHAB, 14-3-3β, is commonly found to be upregulated throughout the initiation and progression of GB. The 14-3-3β has oncogenic roles in several different types of cancer cells through interactions with proteins such as Bad, FBI1, Raf-1, Cdc25b, and others. Previous RNA interference studies have shown that 14-3-3β promotes proliferation, cell cycle progression, and migration and invasion of GB cells. However, despite the many oncogenic functions of 14-3-3β, a CRISPR/Cas9 knockout model of 14-3-3β has not been investigated. This study confirmed previous findings and showed that siRNA inhibition of 14-3-3β results in reduced cellular proliferation in a human glioblastoma cell line, U87MG. We also used a YWHAB Tet-On CRISPR/Cas9 U87MG cell line that, upon doxycycline induction, leads to robust Cas9 expression and subsequent knockout of 14-3-3β. Using this model, we show that loss of 14-3-3β significantly reduces cellular proliferation and spheroid formation of U87MG cells.

Keywords: 14-3-3β; Tet-on CRISPR; YWHAB; glioblastoma; tumor spheroid.

Grants and funding

This work was supported by the Central Michigan University College of Medicine Start-up funds to J. Bakke and J. Rosssignol, Office of Research and Graduate Studies at Central Michigan University for an Graduate Research Grant to K. Gallo, and from the John G. Kulhavi Professorship and the E. Malcolm Field Endowed Chair in Neuroscience.