Gain-of-function Mutant P53 Regulates GSK-3β To Affect Migration And Invasion Of Human Glioma Cells

Background and purposesLocal invasion is the intractable hallmark of malignant gliomas and the major cause of recurrence and worse outcomes.Therefore,it is particularly important to further explore the molecular mechanisms of aggressiveness.Mounting evidence indicates that hotspot p53 mutant proteins often possess gain-of-function（GOF）property in promoting cell mobility.In previous study,we found that the GOF of mutant p53 in the human glioma cell regulates GSK-3βthereby promoting the proliferation of glioma cells.In this study,we aimed to identify p53 mutations in glioma and the role of mutant p53 in the regulation of GSK-3βactivity in glioma,and to explore whether mutant p53/GSK-3βaffects the migration and invasion ability of glioma cells and the mechanism of action.Method1.Using Immunohistochemistry to detect p53 expression and p-GSK-3β^Tyr216（active form）in glioma tissue microarray.2.The mutant p53 gene in human glioma U251 cell line and U-118MG cell line was amplified by RT-PCR.3.The mutant p53 gene knockdown cell line was stably expressed by RNAinterference technology,and the expression of p53 and p-GSK-3β^Ser9 before andafter knockdown were detected,and the growth ability of the cells。4.Transwell assay and cell wound healing assay were used to detect cell migration andinvasion.And downstream regulatory molecule of GSK-3βwere detected by Western blotting.5.GSK-3βinhibitor 1-Azakenpaullone（1-AKP）was used alone to treat U251 andU-118MG cell lines to verify the ability of the cells to grow,and the ability ofmigration and invasion after the activity of GSK-3βwas decreased.And the GSK-3βdownstream related potential mechanisms were detected by Western blotting.Results1.Glioma tissue microarray showed that the expression of p53 and GSK-3βwere positively correlated with glioma grade.2.By means of direct sequencing of PCR products of U251 and U-118MG cells andalignment analysis using IARC gene database,the mutation sites of p53 were R273H（U251）and R213Q（U-118MG）.3.Two cells were treated by lentiviral interference vector sh-p53,and it was found thatthe expression of GSK-3βwas subdued with the decrease of p53,and reduce of cellmigration and invasion ability were also detected after treated by sh-p53,and themechanism may lie in the reduction of EMT-like changes through MMP-2 and Snail levels.4.1-AKP inhibited the activity of GSK-3β,which verified the reduced proliferationability of the two cell lines,as well as the reduced cell migration and invasion ability. This process also involved the mechanism mentioned earlier.Conclusion1.Both p53 R273H and R213Q hot spot mutations in human glioma cells could regulate the activity of GSK-3β.2.Mutant p53 enhanced the ability of glioma migration and invasion by upregulation of GSK-3βactivity.3.The mechanism by which mutant p53/GSK-3βsignaling pathway regulates invasion and migration may be related to EMT-like changes.Innovation1.Gain-of-function p53 R273H and R213Q hot mutation sites can regulate the activity of GSK-3β.2.Elucidated the cancer-promoting effect of mutant p53/GSK-3βpathway,andenriched the connotation of gain-of-function mutant p53.