| Part one The application of magnetic resonance spectroscopy in the invasion of human glioma.Objective:Glioma is the most common primary tumor in the brain,and most gliomas are invasive and malignant.The imaging evaluation of glioma invasion has been the focus of attention.Magnetic resonance imaging(MRI)is a non-invasive technique,which can obtain the information of physiological and pathological metabolites in vivo brain tissue.The changes of these metabolites can be used for qualitative diagnosis of glioma and to determine the therapeutic effect.Because of the breadth and maturity of the application of1H-MRS in magnetic resonance spectroscopy,it has become an important method for the evaluation of glioma.1H-MRS combined with traditional MRI technology can improve the accuracy of preoperative diagnosis and pathological grading of glioma,and reflect the metabolic characteristics of biochemical substances of glioma.The main purpose of this study was to analyze the pathological features of 1H-MRS in glioma and to evaluate the effect of 1H-MRS on the invasiveness of glioma.Method : There were 54 cases of glioma patients who were treated surgically and pathologically confirmed in our hospital,including male and female,with a total of 22 cases,with a total of 32 cases.The age range was20-77 years old,with an average age of 46 years,and a total of 30 volunteers were selected as the control group,with an average age of 18-64 years..Two experienced pathological doctors with double blind method to diagnose the pathological sections of patients,were divided into groups according to the WHO 2016 standard of glioma grading,the low level group(including WHO I,II)and high grade group(including WHO III,IV),which is low the level ofgroup 24 cases,30 cases of high-grade grouphe.Two experienced radiologists with double blind method of routine brain MR patients and enhanced MR image analysis,we grouped according to the following criteria: non edema group(conventional MR scan and enhanced MR scan lesions had no obvious change),this group included 18 cases of edema group(enhancement MR lesions range is significantly less than the conventional MR examination),a total of 36 cases of this group.The patients in each group and the volunteers were treated with conventional MRI、enhanced MRI scan and 1H-MRS scan after 48 hours.1H-MRS scanning of glioma patients using T2 WI image region of interest(ROI)selection,the maximum level of ROI in the lesions displayed in axial T2 WI images,should try to include gliomas,the tumor with edema,no edema around the tumor,and should try to avoid the air,bone and adipose tissue.The volunteer ROI area was selected at the center of the oval area above the lateral ventricle,including the white matter of the frontal lobe and parietal lobe.ROI imaging was performed using PRESS,and the image was processed into the SIEMENS syngo MR image workstation after scanning.The peak area of each metabolite of NAA,Cho,Cr,and the Cho/Cr,NAA/Cr,and Cho/NAA ratios of the peaks of the ROI spectra were calculated.The ratio of 1H-MRS metabolites in each group was analyzed and compared by statistical analysis software to evaluate the invasion of glioma by 1H-MRS.Result:Group of normal brain tissue metabolite ratio of high and low grade group tumor metabolite ratio and control has the difference,the difference was statistically significant,specific differences in Cho/Cr,Cho/NAA increased,NAA/Cr decreased;the high level group compared to the group of low grade tumor metabolites the ratio of high grade group Cho/Cr,Cho/NAA value is higher than the low level group,NAA/Cr value is lower than the low grade group,the difference was statistically significant.Group of normal brain tissue metabolite ratio of high and low grade group tumor metabolite ratio and control has the difference,the difference was statistically significant,specific differences in Cho/Cr,Cho/NAA increased,NAA/Cr decreased;the high level group compared to the group of low grade tumormetabolites the ratio of high grade group Cho/Cr,Cho/NAA value is higher than the low level group,NAA/Cr value is lower than the low grade group,the difference was statistically significant.High level group of peritumoral tissue metabolite ratio compared with the control group of normal brain tissue metabolite ratio,the difference was statistically significant,specific differences in Cho/Cr,Cho/NAA increased,NAA/Cr decreased;the high level group compared to the low level group of peritumoral tissue metabolites the ratio of high grade group Cho/Cr,Cho/NAA value is higher than the low level group,the difference was statistically significant,NAA/Cr value is lower than the low level group,the difference was not statistically.The tumor edema group and control group of normal brain tissue metabolite ratio compared with the difference,the difference was statistically significant,Cho/Cr,for the specific differences,Cho/NAA increased significantly,NAA/Cr decreased;non tumor edema group and control group of normal brain tissue metabolites than value,Cho/NAA increased,NAA/Cr decreased,Cho/Cr the ratio increased,the difference was statistically significant,edema group and non edema group tumor metabolite ratio,Cho/NAA,NAA/Cr,Cho/Cr,showed no statistically significant difference.Peritumoral edema group had difference with the control group of normal brain tissue metabolite ratio,the difference was statistically significant,specific differences in Cho/Cr,Cho/NAA increased,NAA/Cr decreased;non peritumoral edema group area compared with the control group of normal brain tissue metabolite ratio of Cho/,NAA increased,NAA/Cr decreased,the difference was statistically significant and there was no significant difference of Cho/Cr.Edema group and non edema group group peritumoral tissue metabolites the ratio of Cho/NAA increased,the difference was statistically significant,NAA/Cr and ChO/Cr difference was not statistically significantConclusion: 1H-MRS can metabolic characteristics of glioma were analyzed to invasive tumor tissue evaluation.1H-MRS judgment invasive glioma edema around on conventional brain MRI and can make a supplementary proof of no peritumoral edema of gliomas on the invasivenessof glioma at different levels of the surrounding tissue to provide the new assessment method.Part two The expression of β-catenin and Fra-1 in human glioma and its relationship with 1H-MRS spectrum.Objective:At the molecular level,the invasiveness of glioma is regulated by a variety of cytokines,including Wnt/β-catenin signaling pathway and proto oncogene Fra-1 play a key role in the development of glioma.However,the β-catenin and Fra-1 beta Wnt/ important factor in the β-catenin signaling pathway in glioma expression and mechanism study for less relationship study on 1H-MRS spectrum of invasive means and evaluation of gliomas.The purpose of this study was determined by the expression of human gliomaβ-catenin and Fra-1 related protein two associated with invasive factor,determine whether there are differences in the expression level of β-catenin and Fra-1 related protein in gliomas of different grades,so as to determine its effect on the invasion of glioma.To study the correlation between the immunohistochemical results of β-catenin and Fra-1 related proteins and the levels of 1H-MRS metabolites in glioma,in order to find the theoretical basis of 1H-MRS to evaluate the invasiveness of glioma at the molecular level.Method:a total of 87 patients with glioma confirmed by surgery and pathology were selected from 2014-1015 Hospital of Hebei Medical University in.There were a total of 51 males and a female of with an average age of 46 years(range,20-77 years).In addition,15 patients with traumatic brain injury should be removed from the brain parenchyma as the control group of immunohistochemistry.Two experienced pathological doctors with double blind method to diagnose the pathological sections of patients,were divided into groups according to the WHO2016 classification standard of glioma,the low level group(including WHO I,II)and high grade group(including WHO III,IV),the low level group in 43 cases,44 cases of high-grade group.The conventional MR and brain 1H-MRS scan were performed on glioma patients,and the images were processed into SIEMENS syngo MR image workstation,ROI was selected to calculate the ratio ofCho/Cr,NAA/Cr,and Cho/NAA in the tumor area(as far as possible to select the parenchyma of the tumor,to avoid the bad liquefaction necrosis area).The patients with gliomas and normal brain with trauma decompression were performed immunohistochemical analysis with preoperative conventional MRI and 1H-MRS images and clinical surgeons discussed sampling position.Immunohistochemistry was used to analyze the expression ofβ-catenin and Fra-1 in glioma and normal brain tissues.The results of immunohistochemistry were used to calculate the proliferation index ofβ-catenin and Fra-1 in two cases of glioma by double blind method,and the results were compared with the Fromowitz standard : The results of immunohistochemical staining = staining intensity score positive cell percentage score,the score in the 0-6 definition of negative expression,7-12 is defined as positive expression.Using statistical software for analysis of the expression of β-catenin and Fra-1 in the normal group,group of low grade and high grade group of glioma,analyze the correlation between the β-catenin and Fra-1 proliferation index PI in glioma and 1H-MRS of each metabolite ratio.Result:In the normal brain tissues,the expression of β-cateninwas only expressed in the cell membrane,which was characterized by brown staining,which was mainly expressed in cytoplasm,and was expressed in the nucleus of the high-grade glioma.Low grade glioma in 43 cases,negative β-catenin expression were 30 cases,13 cases of positive expression,the positive expression rate of 30.2%.in 44 cases of high grade gliomas,negativeβ-catenin expression were 19 cases,25 cases of positive expression,the positive expression rate was 56.8%.There was significant difference in the expression of β-catenin in gliomas of high and low grade gliomas.The negative expression of Fra-1 in low grade glioma was 34 cases,the positive expression was in 9 cases,the positive rate was about 20%,The negative expression of Fra-1 in high grade gliomas was 18 cases,the positive expression was in the presence of,the positive expression rate was 59.1%,and the difference of Fra-1 expression in high and low grade gliomas was statistically significant.1H-MRS metabolites were detected by Pearsoncorrelation test with β-catenin and Fra-1 in glioma cell proliferation index(PI).1H-MRS,Cho/Cr was positively correlated with β-catenin PI,NAA/Cr was negatively correlated with β-catenin PI,and Cho/NAA was positively correlated with β-catenin PI.Similar to the above results,Cho/Cr was positively correlated with Fra-1 PI,NAA/Cr was negatively correlated with Fra-1 PI,and Cho/NAA was positively correlated with Fra-1 PI.Conclusion:In this study,immunohistochemical analysis was performed on specimens of glioma patients.The differential expression of β-catenin and Fra-1 protein in gliomas with different grades of glioma,It was confirmed that the two factors of-catenin and Fra-1 protein were involved in the development and invasion of glioma.The correlation between 1H-MRS results and the expression of β-catenin and Fra-1 was studied,The expression of Cho/NAA and Cho/Cr was positively correlated with the expression of β-catenin and Fra-1 protein in glioma,and the expression of NAA/Cr was negatively correlated with the expression.The expression of β-catenin,Fra-1 protein and1H-MRS can be used to evaluate the invasiveness of tumor cells from different angles,and they are related to each other from two aspects of cell proliferation and cell metabolism.Part three The molecular mechanism of Wnt3a/ β-catenin and Fra-1 regulation of glioma invasion.Objective:Through the above studies on the invasiveness of human glioma,it has been shown that the expression of β-catenin and Fra-1 in different grades of gliomas is different,and can be evaluate the invasiveness of glioma.Previous studies have shown that epithelial mesenchymal transition is an important process of metastasis and invasion of malignant tumors,and Fra-1 is a major factor in epithelial mesenchymal transition(EMT).However,the regulatory mechanism of Fra-1 in gliomas remains unclear.Wnt/β-catenin pathway affects cell survival,proliferation,metastasis and cell polarity 。Although the Wnt/ β-catenin signaling pathway has been a lot of studies,but due to the important influence the invasion ability of Wnt/β-catenin signaling in glioma glioma tumor,so identification of the signal transduction pathway oftarget genes contribute to a better understanding of the mechanisms of signal conduction pathway.The aim of this study was to investigate the effects of Fra1 and Wnt/β-catenin on the invasiveness of glioma by a series of molecular biological experiments,and to demonstrate the key role of Wnt/β-catenin and Fra-l as signal transduction axis in the development of glioma invasion ability,and to provide a new therapeutic target for malignant glioma.Method:1 U-251 and U-87 cells were treated with different concentrations of Wnt3 a,The expression of mRNA and protein of epithelial marker E-cadherin and ZO-1,as well as interstitial marker Vimentin and Fibronectin were detected by QT-PCR and western blotting.2 The expression of Vimentin and protein of mRNA in glioma cells were studied by immunofluorescence,QT-PCR and western blotting method in the presence or absence of exogenous Wnt3 a and the presence of silent-β-catenin.3 To study the expression of EMT related factors in the presence or absence of exogenous Wnt3 a detected by QT-PCR and western blotting.4 By exposing glioma cells to GSK-3 beta specific inhibitors(LiCl and CHIR99021),The mRNA and protein levels of Fra1 were detected by QT-PCR andwestern blotting.5 The luciferase activity of Fra1 promoter(-2500/+1)under different concentrations of Wnt3 a was verified by luciferase assay.Through the construction of Fra1 promoter with different lengths and the presence of the Tcf/Lef binding site mutation gene,the luciferase activity was detected in the presence or absence of exogenous Wnt3 a.6 The presence or absence of exogenous Wnt3 a in glioma cells was treated by adding β-catenin and IgG antibody to chromatin immunoprecipitation.The expression of Fra1 promoter in different conditions was studied by QT-PCR technique.7 The expression of EMT related proteins in the glioma cells was examined by Western blot experiments with over expression of Fra1 plasmid and Fra1-siRNA.8 Fra1-siRNA and β-catenin-siRNA were transfected into glioma cells without addition of exogenous Wnt3 a cells,then the invasion ability of glioma cells under different conditions was studied through the experiment of cell wound healing and cell invasion assay.9 Fra1-siRNA transfected glioma cells in the presence or absence of Wnt3 a,QT-PCR and Western blot experiments were performed to study the expression of EMT related factors.10 CCK assay was used to detect the presence of exogenous Wet3 a and the survival of the cells exposed to cisplatin in the presence of silence or over expression of Fra1.11 Flow cytometry was used to detect the difference of apoptosis after cisplatin treatment in glioma cells which stable transfectioned by over expression Wnt3 a and silencing of Fra-1.12Glioma cells were stably transfected with Wnt3 a overexpression and silenced Fra-1 glioma cells were injected into mice to produce glioma,and then xenograf tumor asssy was used to study the difference of the resistance of glioma to different concentrations of cisplatin.Result:1 In U-251 and U-87 cells,decreased E-cadherin and ZO-1accompanied by induction of Vimentin and Fibronectin at the mRNA level were observed in a Wnt3 a dose-dependent manner.Consistently,the protein levels of epithelial markers(E-cadherin and ZO-1))were repressed while mesenchymal markers(Vimentin and Fibronectin)increased after exogenous Wnt3 a treatment.Immunofluorescence showed that activation of Wnt/β-catenin signaling by Wnt3 a promoted the expression of Vimentin,while blocking Wnt/β-catenin pathway by silencing β-catenin suppressed the mesenchymal marker in glioma cells.As expected,knockdown of β-catenin attenuated Wnt3 a induced upregulation of Vimentin.Furthermore,several EMT-related transcription factors(EMT-TFs)were screened by QPCR and western blot after activation of Wnt/β-catenin signaling.Although all the factors were elevated to different extent after Wnt3 a treatment,both the mRNA and protein levels ofFra1 showed most significant upregulation.2 Activating Wnt/β-catenin by targeting GSK-3βincreased the mRNA and protein levels of Fra1.Consistently,luciferase reporter assayshowed that Wnt3 a exposure increased the transcriptional activity of Fra1 promoter(-2500/+1)in a dose-dependent manner,and the accumulation of Fra1 was further confirmed at the protein level..It was shown that the Fra1 promoter without the region between-1500 and-500 lost the ability to response to Wnt3 a treatment.The DNA fragment(-1500/-500)contained two putative Tcf/Lef binding sites.Mutaion in the latter site(Site3:-660/-647)obviously decreased the reporter activity induced by Wnt3a(Figure 2G).ChIP experiment further demonstrated that β-catenin/Tcf/Lef transcriptional complex directly bound to the Fra1 promoter around-700 bp upstream of the transcription start site,and the binding was strengthened after Wnt3 a stimulation.3 As expected,overexpression of Fra1 decreased the expressions of epithelial markersand increased the expressions of mesenchymal markers,while Fra1 knockdownhad the opposite effect.Knockdown of either Fra1 orβ-catenin weakened the migratory and invasive abilities of U-251 cells.In U-87 cells,knockdown of β-catenin impaired the promotive effect of Wnt3 a on the aggressive phenotypes.Moreover,the acquisition of migratory and invasive capacities in the U-251 cells.stimulated with Wnt3 a was completely attenuated by Fra1 silencing.Deprivation of Fra1 significantly disrupted the upregulation of EMT-TFs induced by Wnt3 a.4 The cisplatin resistance of glioma cells with Wnt3 a stimulation was1.67 fold higher than that of the control group,as measured by the IC50 values for cisplatin over 48 h treatment.Similarly,overexpression of Fra1 promoted cisplatin-resistance,with IC50 increased 1.44 fold as compared with control.However,knockdown of Fra1 sensitized glioma cells to cisplatin,with IC50 decreased 0.69 fold.Furthermore,the U-251 cells with stable Fra1 knockdown and Wnt3 a overexpression were constructed and examined for cisplatin sensitivity.Wnt/β-catenin signaling activation decreased cisplatin-inducedapoptosis in U-251 cells compared with control but failed to protect from apoptosis after Fra1 knockdown,which was also observed in the U-87 cells with Fra1 siRNA transfection and Wnt3 a treatment.Xenograft tumor assay showed that overexpressed Wnt3 a increased resistance to cisplatin,and knockdown of Fra1 sensitized glioma cells to cisplatin.Notably,silence of Fra1 inhibited the protectiveeffect of Wnt3 a against cisplatin.Conclusion:This study demonstrate that Wnt/β-catenin signaling can induce the EMT program of glioma cells by upregulating Fra1 expression.Further results reveal that Wnt/β-catenin signaling activates the transcription of Fra1 by directly binding.β-catenin-Tcf/Lef transcriptional complex to the WRE located at the proximal region of Fra1 promoter.Phenotype experiments and clinical analysis support the critical role of Fra1 in mediating the glioma aggressiveness induced by Wnt/β-catenin signaling.Therefore,the correlation between Wnt/β-catenin signaling and Fra1 raises the possibility of developing novel biomarkers and therapeutic targets for glioma. |
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