Experimental study on the effects of AURKA inhibitor on glioblastoma cell proliferation and B7-H3 protein expression
Objective To investigate the effects of aurora kinase A(AURKA)inhibitor on the proliferation of glioblastoma(GBM)cells and expression of immune checkpoint B7-H3.Methods The Chinese Glioma Genome Atlas(CGGA)dataset(325 cases)was used to analyze the correlation between expression levels of AURKA mRNA and clinical data including histopathology,World Health Organization(WHO)grade,primary/recurrent status,overall survival(OS),06-methylguanine-DNA-methyltransferase(MGMT)methylation,isocitrate dehydrogenase(IDH)mutation and chromosome 1p19q co-deletion.The expression levels of AURKA mRNA in different histopathology types,WHO grades,and primary/recurrent status gliomas were analyzed.We compared the OS of glioma patients with different expression levels of AURKA mRNA using Kaplan-Meier survival curves.The effects of AURKA mRNA expression level,IDH mutation,chromosome 1p19q co-deletion,age,radiotherapy,chemotherapy,WHO grade and primary/recurrent status on the OS of glioma patients were assessed through univariate and multivariate Cox regression analyses.Receiver operating characteristic(ROC)curve was plotted and area under the curve(AUC)was used to evaluate the performance of AURKA mRNA expression level in predicting the OS of glioma patients.Tissue samples of gliomas were collected from patients undergoing surgical treatment at the Neurosurgery Center of Beijing Tiantan Hospital,Capital Medical University,including 4 low-grade gliomas(LGG)and 4 GBM tissue samples.The expression of AURKA in the tumor tissue samples was detected by Western blot(WB).The human GBM cell line U87-MG was treated with AURKA inhibitor alisertib and U87-MG cells were divided into a control group(treated with DMSO)and an alisertib-treated group(treated with 5 μmol/L alisertib).The effects of alisertib on GBM cells proliferation and colony formation were detected by cell counting Kit-8(CCK-8)test and crystal violet staining,respectively.The effects of alisertib on the expression of AURKA,phosphorylation Thr288 of AURKA and B7-H3 were detected by WB.Flow cytometry was applied to detect the expression of membrane protein B7-H3 in GBM cells.Results In the CGGA dataset,the expression level of AURKA mRNA in GBM was significantly higher than that in astrocytoma and oligodendroglioma(both P＜0.001).The expression level of AURKA mRNA increased with WHO grades(P＜0.001).The expression level of AURKA mRNA was higher in recurrent gliomas than in primary gliomas(t=4.50,P＜0.001).Kaplan-Meier survival curve analysis showed that patients in the high AURKA mRNA expression group had shorter survival than those in the low expression group(all P＜0.05).The univariate Cox regression analysis showed that AURKA mRNA expression level,IDH mutation,chromosome 1 pl9q co-deletion,age,radiotherapy,chemotherapy,WHO grade and primary/recurrent status were all influential factors in the survival(all P＜0.05).The multivariate Cox regression analysis showed that high expression level of AURKA mRNA,recurrent status,high WHO grade,age,chemotherapy and chromosome 1p19q co-deletion were independent risk factors(all P＜0.05).ROC curve analysis showed that the AUC of AURKA mRNA expression level to predict 1-year,3-year and 5-year survival glioma patients was 0.78(95％CI:0.72-0.83),0.85(95％CI:0.80-0.89)and 0.84(95％CI:0.79-0.89)respectively.WB results based on clinical samples showed that the protein level of AURKA in GBM was significantly higher than in LGG(t=2.62,P=0.040).CCK-8 results indicated that alisertib significantly inhibited the activity of U87-MG cells after 24 h,48 h and 72 h of treatment(all P＜0.05).Similarly,colony formation experiment results showed that alisertib significantly inhibited the clone formation of U87-MG cells(t=9.30,P＜0.001).WB results showed that the phosphorylation level of AURKA in alisertib-treated group was lower than in control group(t=10.98,P＜0.001),while the expression level of B7-H3 protein was higher than in the control group(t=7.55,P=0.002).Flow cytometry showed that the alisertib-treated group had increased B7-H3 expression on the cell membrane compared to control group(t=20.04,P＜O.OO1).Conclusion AURKA inhibitor alisertib can effectively inhibit GBM cells proliferation and increase the expression of immune checkpoint B7-H3.
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