The Mechanism Of CAMK2N1 Inhibiting The Biological Activity Of Glioma Cells Through AR Signaling Pathway

Objective To explore the expression level of calcium/calmodulin dependent protein kinase II inhibitor(CAMK2N1)in glioma cells,identify its correlation with clinicopathologic features in glioma patients,investigate the regulatory role of CAMK2N1 in proliferation and apoptosis of glioma cells,explore the relationship between CAMK2N1 and androgen receptor(AR)signaling pathway.Therefore we expect to find new and effective molecular markers for early diagnosis and prognosis of glioma.Methods(1)The protein and mRNA levels of CAMK2N1 were investigated by qRT-PCR,immunohistochemical(IHC)staining using tissue microarray and western blotting in 8 cases of low grade gliomas,8 cases of high grade gliomas and 8 cases of normal noncancerous tissues.(2)The relationship between CAMK2N1 expression level and overall survival in glioma was detected by Kaplan-Meier survival analysis.(3)A stable and highly expressed CAMK2N1 U87 cell(U87-CAMK2N1)and U87 expressing the empty vector(U87-vector)were established by lentivirus transfection.MTT and flow cytometry were used to detect the proliferation and apoptosis of glioma cells when CAMK2N1 was highly expressed.(4)U87 cells were treated with 10 nm R1881(AR signaling activator)for 0-48 hours.The expression levels of CAMK2N1 and AR,proliferation and apoptosis of glioma cells were detected.The relationship between CAMK2N1 and AR signal pathway in glioma was investigated.Results(1)qRT-PCR showed that mRNA levels of CAMK2N1 were significantlyreduced as tumor malignancy degree increased.The protein expression of CAMK2N1 was significantly decreased in tumor tissues with high grade compared to those with low grade.CAMK2N1 protein expression in human glioma specimens was analyzed by IHC staining using tissue microarray.CAMK2N1 showed higher IHC scores in low grade than in high grade(P<0.05),consistent with mRNA expression in gliomas.(2)Kaplan-Meier survival analysis showed that patients with glioma expressing high expression of CAMK2N1 had good overall survival(P=0.003).Univariate analysis showed that age(P < 0.01),WHO grade(P < 0.01)and CAMK2N1 expression(P < 0.01)were significant predictors of OS.Multivariate analysis showed that WHO grade(HR=3.364,95%CI=2.146-4.844,P<0.01)and CAMK2N1 expression(HR=1.175,95%CI=0.371-2.597,P<0.01)were independent risk factors for OS.(3)CAMK2N1overexpressed lentivirus plasmid GV 166(Ubi-MCS-3FLAG-IRES-Purinomycin)was transfected into U87 cells,and the culture was continued for 24 hours.After observation under fluorescence microscope,more than 70% of the cells showed green fluorescence.CAMK2N1 stably overexpressing U87(U-87-CAMK2N1)and U87 expressing the empty vector(U87-vector)by lentiviral transfection was established.Western blot showed that the expression of CAMK2N1 was up-regulated in U87-CAMK2N1.MTT assay showed that CAMK2N1 inhibited proliferation in U87-CAMK2N1,compared to U87-vector.In addition,it was found that CAMK2N1 could up-regulate the proportion of apoptosis of glioma cells.Compared with the Mock cells,the proportion of apoptotic cells increased significantly after upregulation of CAMK2N1 expression(P<0.05).Lentiviralmediated transfection of CAMK2N1 overexpression induced visual increases of protein expression of BAX(P<0.01),Bcl-2(P<0.001),Cleaved-Caspase3(P<0.01),and Cleaved-PARP(P<0.05),representing cell apoptosis in the U87 cell line.(4)After treating U87 cells with R1881(10nm/L)for 0-48 h,the qRT-PCR assay showed that androgen significantly reduced the expression of CAMK2N1 mRNA(P<0.01)in a time-dependent manner.The expression of AR mRNA in U87-CAMK2N1 was significantly lower than that in U87-CAMK2N1(P<0.01).Compared withU87-CAMK2N1 untreated by R1881,U87-CAMK2N1 treated by R1881 can change the inhibitory effect of CAMK2N1 on glioma cells,and promote significantly increased cell growth rate(P<0.05).Flow cytometry after treatment of R1881 for 48 h showed that R1881 could down-regulated the apoptosis of glioma cells U87-CAMK2N1 compared with the untreated U87-CAMK2N1(P<0.01).Treatment with R1881 resulted in decreased expression of Bax(P<0.05),Bcl-2(P<0.01),Caspase 3(P<0.05)and PARP(P< 0.01),which induced apoptosis of U87 cells.Conclusion In human glioma cancer tissue,endogenous CAMK2N1 expression was positively correlated with prognosis of glioma.Overexpression of CAMK2N1 could inhibit the growth of gliomas.Patients with high CAMK2N1 levels had longer overall survival.CAMK2N1 may be a potential tumor suppressor gene.CAMK2N1 and AR signaling formed an auto-regulatory negative feedback loop.There was a possible functional link between CAMK2N1 and AR signaling in glioma.CAMK2N1-AR signaling pathway may be a new target of glioma therapy,and CAMK2N1 and AR may be a new prognostic indicator of glioma.