Construction and Identification of Glioma Cell Lines Stably ExpressingEGFR Extracellular Domain A289V Mutation

FEI Yuchen^1,2, LIU Wenjing³, HU Xin⁴, WANG Chunlin⁴, LÜ Donglai^1,2*

(¹PLA Clinical College of Anhui Medical University, Hefei 230032, China; ²the Fifth Clinical School of Medicine of AnhuiMedical University, Hefei 230032, China; ³Center of Clinical Pathology of the First Hospital Affiliated to University of Scienceand Technology of China, Hefei 230036, China; ⁴the 901 Hospital of Joint Logistics Support Force of PLA, Hefei 230031, China)

Abstract:

The aim of this study was to construct two glioma cell lines with stable expression of EGFRECD A289V missense point mutation by lentivirus transfection technique, and to provide a cell model for the preclinical study of this mutant glioma. The recombinant plasmid was constructed using homologous recombinationenzyme linking method and the lentivirus was packaged by calcium transfer method to infect U87 and U251 gliomacells. Verification via Western blot confirmed the expression of EGFR A289V mutant protein, while Transwell invasion assay observed changes in invasion ability of both cell lines. The efficacy of TKI on CCK-8 cell viability wasinvestigated via CCK-8 cell viability assay. Results showed that constructed plasmid was identified successfully bysequencing, and the recombinant plasmid could successfully express EGFR A289V mutant protein after infectingthe target cell line. Transwell invasion assay and CCK-8 cell viability assay confirmed that the invasion ability of modified U87 and U251 cell lines was increased and could be effectively inhibited by Osimertinib. Overall, gliomacell lines expressing EGFR A289V mutation are more malignant, and Osimertinib can effectively inhibit the proliferation of glioma cells with such mutation in vitro. This experiment provides a useful cell model for the pharmacological study of this mutation.

CSTR: 32200.14.cjcb.2023.11.0009