| Part1 Expression and the Role of Nedd4-1 in Glioma CellsObjectiveThe expression of NEDD4-1 in human glioma cell line U251 was detected.The lentiviral vector targeting NEDD4-1 gene was constructed and the viral vector was packaged.The expression of NEDD4-1 in human glioma cell line U251 was interfered.Further study NEDD4-1 on glioma cell migration,invasion,proliferation and apoptosis,lay the foundation for the next experiment.MethodsIn this study,the expression level of NEDD4-1 in human glioma cell line(U251)was studied at the mRNA level,which was mainly verified by reverse-transcription PCR(RT-PCR).Furthermore,U251 cells were transfected with NEDD4-1 lentiviral expression vector and lentivirus interfering vector in vitro to investigate the effect of downregulation of NEDD4-1 gene expression on the biological characteristics of glioma cell lines.To detect the expression of NEDD4-1 mRNA and protein in transfected cells by RT-PCR and Western blot.Transwell,MTT and flow cytometry were used to further investigate the migration,proliferation,apoptosis and invasion of NEDD4-1 in glioma cells.Results1.The results of this part of the study show that NEDD4-1 mRNA is expressed in U251 lines.2.The lentiviral expression vector containing the full length of NEDD4-1 was successfully constructed,and the vector and the NEDD4-1 lentiviral vector were respectively virus-packaged and infected with human glioma cell line U251.3.The growth curve of MTT showed that the proliferation of U251-KD cells was slower than U251-NC cells(P<0.05).4.Cell cycle analysis showed that the number of cells in U251-KD group were significantly increased at GO-G1 phase(P<0.05),the number of cells in S phase were increased(P<0.05),There was no statistically significant difference in cell stage G2/M(P>0.05),and G1 arrest occurred.Compared with U251-NC cells,the apoptosis rate of U251-KD cells increased(P<0.05).5.The effect of NEDD4-1 gene on the migration and invasion of glioma cells:The invasion and migration of U251-KD cells were significantly lower than that of U251-NC group(P<0.01).ConclusionNEDD4-1 is expressed in human glioma cell lines,and NEDD4-1 gene may play an important role in regulating the malignant biological characteristics of human glioma cells in vitro such as migration,invasion,proliferation,cell cycle and apoptosis.Part 2 Expression of NEDD4-1 in Human Glioma Tissue and its Correlation with Clinicopathological FeaturesObjectiveTo study the protein and mRNA expression of E3 ubiquitin ligase NEDD4-1 in glioma tissue and its correlation with age,sex,maximal tumor diameter,pathological grade of glioma,Ki-67,p53 respectively.To investigate the relationship between MRS quantitative parameters and NEDD4-1 in different pathological grades of glioma.MethodsFifty-two patients with pathologically confirmed gliomas underwent routine MRI plain scan,enhanced sequence and MRS-related examinations under SIMENS Prisima 3.0 T magnetic resonance scanner with a 64-channel combined head and neck coil.According to the sequence of E3 ubiquitin ligase NEDD4-1,the corresponding primers were designed and the expression of NEDD4-1 mRNA was detected by RT-PCR.The expression of NEDD4-1 protein in 52 cases of glioma was detected by immunohistochemistry and western blot.The correlation between NEDD4-1 protein and the age of patient,gender,tumor diameter,pathological grade of glioma and proliferation antigen Ki-67 and p53 were analyzed.The relationships between NEDD4-1 protein and MRS signal parameters of different grades of glioma were analyzed.Results1.Magnetic resonance spectroscopy analysis of human brain gliomas revealed significant difference in Cho/Cr and Cho/NAA between tumor parenchyma and paratumor tissue.2.Human glioma tissue NEDD4-1 protein positive cells located in the cytoplasm and/or membrane staining.In 52 human glioblastoma tissue samples,the expression of NEDD4-1 was detected in 40 cases of tumor tissues,the positive rate was 76.9%.The positive rate of NEDD4-1 in different grades of gliomas was significantly different in human glioma tumor tissues(P<0.05).With the increase of clinicopathological grade,the positive rate of NEDD4-1 protein expression(P<0.05).However,the expression of NEDD4-1 was not related to the sex,age and tumor size(all P>0.05).There was a positive correlation between NEDD4-1 and Ki-67 and p53 in human glioma tissue.3.There was a positive correlation between NEDD4-1,Ki-67 and p53 in human glioma tissue.4.NEDD4-1 in human glioma tissues was positively correlated with Cho/Cr and Cho/NAA in MRS of all grades of glioma.Conclusions1.The results of this part of the study show that,with the pathological grade of glioma increased,the expression of NEDD4-1mRNA and protein in glioma tissues gradually increased.NEDD4-1 has effect on the invasion of glioma.2.There was a positive correlation between NEDD4-1 and Ki-67 and p53 in human glioma tissues.There was a positive correlation between Cho/Cr,Cho/NAA and MRS in all grades of gliomas.NEDD4-1 binding with 1H-MRS as an effective indicator of invasiveness of glioma.Part 3 Correlation Analysis between NEDD4-1 and DKI in Glioma TissuesObjectiveThe DKI was used as a non-invasive predictive method for the expression of NEDD4-1 in gliomas,and the correlation of NEDD4-1 expression in different grades of gliomas and the MK,FA,MD,Ka,Kr parameters of DKI were analyzed.MethodsProspectively collected in our hospital from June 2015 to December 2017,pathologically confirmed 52 cases of glioma patients.All patients underwent routine MRI and DKI related examination.The SIMENS Prisima 3.0 T magnetic resonance scanner and the 64-channel head-and-neck co-ordinate coil performed a scan of the conventional magnetic resonance and DKI sequences for about 30 minutes.DKI parameters:TR/TE=3500.0 ms/78.0 ms,b=0,500,1000,1500,2000,2500,dispersion direction=30,layer thickness 5.0 mm,number of layers 20,FOV 220 mm×220 mm,scan time 9 minutes 01 second.Image Processing:The DKI data were processed by the third-party software(DKE),the T2WI axial surface was enhanced and the solid components of the tumors were enhanced.The ROIs were manually drawn and contralateral white matter(NAWM,MK,Ka,Kr,FA,MD,etc.)/(corresponding white matter in NAWM area)to get MK,Ka,Kr,FA and MD values of all grades of gliomas.Combined with the second part,protein expression level of 52 cases of glioma was based on the E3 ubiquitin ligase NEDD4-1.Using SPSS(21.0,IBM)statistical software,one-way ANOVA and L-S-D test were used to compare the MK,Ka,Kr,FA and MD between the two groups.Draw a ROC curve,calculate the area under the curve,and predict the threshold for valid parameters.The correlations between MK,Ka,Kr,FA and MD and gender,age,NEDD4-1 expression and other variables were compared between groups using independent samples t test,Pearson linear correlation or Spearman rank correlation analysis.Results1.The difference of DKI parameter MK value,Ka value,Kr value of different levels of glioma was statistically significant(P<0.01).However,there was no significant difference in MD and FA between the two groups(P>0.05).2.There was a positive correlation between the age and the MK value.There was a positive correlation between the age and the Ka value.There was a positive correlation between age and Kr value.There was a negative correlation between age and FA value.There was no correlation between age and MD value.3.There was no significant difference in the values of MK,Ka,Kr,FA and MD between men and women in all grades of glioma patients(P>0.05).4.The expression of NEDD4-1 mRNA in glioma tissues was positively correlated with MK(r = 0.691 P<0.01),positively correlated with Ka(r = 0.604 P =0.000)and positively correlated with Kr(r = 0.630 P = 0.000),but negatively correlated with MD(r =-0.350 P = 0.011).There was no correlation with FA(r =-0.061 P>0.05).ConclusionsDKI contributes to the diagnosis of glioma grade malignancy and can be used as a predictive tool for the expression of NEDD4-1 in gliomas.DKI combined with NEDD4-1 can be used as a tool for glioma invasion and grading. |
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