| Glioma is one of the most common primary malignancies in the adult central nervous system,accounting for approximately 70%of adult malignant primary brain tumors.In the clinical diagnosis and treatment process,except for a very small number of low-grade gliomas,such as pilocytic astrocytoma,which are cured by microsurgery under the microscope,the survival of most patients with malignant glioma is still relatively short,and statistics indicates that the median survival time is about 14 months.In recent years,with the development of medical technology,comprehensive treatments for patients with malignant glioma undergoing surgery,chemotherapy,radiotherapy,etc.have made great progress,but it is difficult to avoid recurrence.Only about 5%of patients have a survival period of more than 5 years,and the overall prognosis is still very poor.Exploring effective methods to inhibit the growth and invasion of glioma cells,prolonging the survival time of patients and improving the survival of patients is an urgent problem to be solved in neuroscience today.Therefore,it is urgent for us to conduct in-depth research on the biological behavior of glioma from the genetic and molecular level,in order to expect more effective treatment methods and ways for malignant tumors.The tripartite motif(TRIM)protein family is a class of proteins with RBBC structure;more than 70 TRIM family members have been identified currently.Its members are found in almost all multicellular eukaryotes.Previous studies have found that they play an important role in intracellular signaling,cell cycle,apoptosis,differentiation,development,metabolism,and viral immune responses,autophagy,and cancer.Its protein structure is characterized by the presence of a relatively conservative N-terminal region,containing one RING finger domain,one or two B-box domains,and a coiled-coil domain.However,up to now,eight TRIM proteins in human are RING-less TRIM proteins.The difference in members of the TRIM protein family lies in the composition of the C-terminus,which is the basis of human TRIM family classification,and on the basis of these domain organization TRIM proteins are classified in subfamily ?to XI(C-I?C-XI).The C-terminal domains include:COS domain,fibronectin type ? repeat(FNIII),PRY domain,SPRY domain,acid-rich region(ACID),filamin-type IG domain(FIL),NHL domain,PHD domain,bromodomain(BROMO),Meprin and TRAF-homology domain(MATH),ADP-ribosylation factor family domain(ARF),and transmembrane region(TM).The common ARF domain is formed by the fusion of SPRY and PRY domains to form B30.2 domain,and studies have shown that this domain may be involved in protein interactions.The tripartite motif(TRIM)family proteins contain the RING-finer domains,so most of them have E3 ubiquitin ligase activity,which can be used as an E3 ubiquitin ligase to ubiquitinate other proteins,thus the labeled proteins are degraded by the Ubiquitin-Proteasome Pathway.In the process of cell morphology maintenance,signal transduction,cycle regulation,DNA repair,transcriptional regulation,and protein quality control,ubiquitination-mediated protein degradation pathways play an important role in maintaining cell function stability.In theprocess of tumorigenesis and development,it has been found that its ubiquitination modification can regulate many oncoproteins and tumor suppressor proteins.For example,several TRIM proteins,TRIM15,TRIM25,TRIM29,TRIM44 and TRIM59,have been shown to be involved in tumor progression as oncogenes or tumor suppressors.TRIM37(Tripartite motif-containing protein 37)is a member of the TRIM protein family,whose genes are located on chromosome 17q22-23 and also has E3 ubiquitin ligase activity.TRIM37 expression is abnormal in many tumors or diseases.So far,it has been found that TRIM37 expression is significantly unregulated in breast cancer,liver cancer,pancreatic cancer,rectal cancer,lung cancer and other tumor tissues,which promotes the occurrence and development of tumors by silencing tumor suppressor factors and related genes and regulating signal pathways.According to literature reports,it was confirmed that the expression of TRIM37 is closely related to the stage or grade of tumors.In most tumors,TRIM37 expression increased with the increase of tumor malignancy,and its overexpression effectively induced the migration and metastasis of tumor cells.Shin SW et al.reported that TRIM37 was identified as an asthma-related gene in pernpheral blood mononuclear gene expression profiles,but found that TRIM37 was significantly down-regulated in peripheral blood mononuclear cells in asthmatic patients.In addition,Kallhjarvi J et al.reported that the mutation of TRIM37 gene led to the occurrence of Mulibrey nanism syndrome,a rare autosomal recessive genetic disease characterized by growth and development disorder,pericardial stenosis,and multiple organ dysplasia including muscle,liver,brain and eye.Human TRIM37 protein has been confirmed to have a powerful ability to inhibit the replication of HIV1 virus by interfering with viral DNA synthesis.However,the expression pattern,biological role and mechanism of TRIM37 in the development and progression of human glioma are still not well understood.So far,there have been few reports on relevant research at home and abroad.Therefore,the purpose of this study is to explore the expression and role of TRIM37 in human glioma,and to try to understand its molecular biological mechanism.This paper includes three parts:The first part is to study the expression of TRIM37 in human glioma tissue.Subsequently,U87 glioma cells were transfected with siRNA in vitro to knock down the expression of TRIM37 protein and detect the influence on the biological behavior of glioma cells.In the second part,we construct lentiviral vector,screen stable transfected cells,and study the effect of TRIM37 on glioma in vivo level through the experiment of glioma inoculation in nude mice.In the third part,combining the previous two parts of the study,we further explore the molecular mechanism of TRIM37 on the biological behavior of glioma,in order to provide theoretical basis and practical basis for gene-targeted therapy of glioma.Part I:Expression of TRIM37 in malignant glioma and in vitro study on proliferation,migration and invasion of glioma cells Objective:1.To detect the protein expression of TRIM37 gene in normal brain tissue and malignant glioma tissue,and further explore the correlation between TRIM37 and the proliferation,migration and invasiveness of malignant glioma.2.Whether the malignant biological characteristics of glioma cells can be inhibited by knocking down the expression of TRIM37 gene in U87 glioma cells using siRNA interference technology.Methods:1.Tissue samplesClinical tissue samples of 124 patients with primary glioma who underwent surgery between June 2014 and September 2017 were collected from Department of Neurosurgery,Qilu Hospital of Shandong University.All glioma patients included in this study cohort were treated for the first time,were not treated with radiotherapy or chemotherapy before surgery.According to the classification of central nervous system tumors by the World Health Organization(WHO)in 2007,32 cases of low-grade gliomas(grade II gliomas)and 92 cases of high-grade gliomas(38 grade III gliomas and 54 grade IV gliomas)were included.The histological types and grades of gliomas in all patients were assessed and evaluated by two experienced pathology specialists.20 samples of normal brain tissue were collected from patients undergoing decompression surgery after severe traumatic brain injury and examined to ensure the absence of brain tumors.The patient or family members agrees and signs an informed consent statement for the collection and application of the tissue samples,agreeing to the collection and application of this study.This study was agreed and approved by the Ethics committee of Shandong University Qilu Hospital.2.Cell cultureThe human glioma cell line U87 was obtained from the Chinese Academy of Sciences and cultured in Dulbecco's modified Eagle's medium(DMEM)supplemented with 10%fetal bovine serum,100 U/ml penicillin and 100?g/ml streptomycin in a humidified chamber containing 5%C02 at 370C.U87 glioma cells were randomly divided into three groups:control(non-transfected U87 glioma cells),siRNA-control(transfected with scrambled negative control)and siRNA-TRIM37(transfected with siRNA targeting TRIM3 7).3.Western blot analysisWestern blot was used to detect the expression level of TRIM37 protein in glioma tissues and normal brain tissues.The expression levels of TRIM37 protein in three groups of U87 glioma cells:blank control group,siRNA control group and siRNA-TRIM37 group were analyzed by Western blot to evaluate the transfection efficiency.The protein expression of Bax,Bcl-2,Cleaved-caspase3,MMP2 and MMP9 in three groups of U87 cells was detected,using ?-actin as the internal control.4.ImmunohistochemistryThe expression levels of TRIM37 protein in glioma tissues of different WHO grades and normal brain tissues were detected by immunohistochemistry to analyze the relationship between TRIM37 protein expression and clinicopathological characteristics(age,sex,WHO grade)of glioma patients.5.qRT-PCR analysisThe expression levels of TRIM37 mRNA in human glioma tissues and normal brain tissues were detected by real-time quantitative polymerase chain reaction(RT-qPCR)and ?-actin was used as the internal control.6.Cell TransfectionU87 cells were cultured in DMEM supplemented with 10%FBS in a humidified atmosphere of 5%C02 at 37?.When cells reached 80%confluence,they were transfected with siRNA targeting TRIM37 and a scrambled negative control was set up.Transfection of siRNAs was performed using Lipofectamine 2000 according to the manufacturer's protocol.Transfection efficiency was verified by Western blot analysis.7.Cell proliferation assayU87 glioma cells were seeded at a density of 3000 cells/well in 96-well culture plates according to the experimental design group(blank control,negative control,transfection group).The CCK-8 kit was used to detect the proliferation of glioma cells after transfection of siRNA targeting TRIM37 at 24,48,and 72 hours.8.Cell cycle and apoptosis analysisU87 glioma cells were cultured according to the experimental design(blank control,negative control,transfection group).Annexin V-FITC/PI cell apoptosis test Kit and flow cytometer were used to analyze the apoptosis and cell cycle changes of glioma cells after transfection of siRNA targeting TRIM37.9.Cell migration and invasion assayU87 glioma cells were cultured according to the experimental design(blank control,negative control,transfection group).The migration and invasion ability of glioma cells after transfection with siRNA targeting TRIM37 were detected by Transwell chamber and Matrigel chamber.10.Statistical analysisThe experimental data were statistically processed by SPSS18.0 software.Data were analyzed by two-tailed Student's t-test and one-way analysis of variance,followed by Dunnett's test for multiple comparisons of the means.?2 test was used to analyze categorical data.P<0.05 was considered to indicate a statistically significant difference.Data are expressed as the meanąSD.Result:1.The expression of TRIM37 was up-regulated in human glioma tissues:RT-PCR detection showed that TRIM37 mRNA expression was significantly up-regulated in glioma tissues compared with human normal brain tissue.Western Blot analysis showed that TRIM37 protein in glioma tissues was increased.Immunohistochemical analysis showed that the expression of TRIM37 in glioma tissues was closely related to the malignant grades of brain glioma,and the higher the degree of glial malignancy,the more abundant TRIM37 expression.2.The expression of TRIM37 was not significantly correlated with age and gender in patients with glioma,and there was no statistical difference(p>0.05).TRIM37 expression was associated with the WHO grades of gliomas,and was significantly expressed in grade? and ? gliomas,which was statistically significant(p<0.05).3.The proliferation ability of U87 glioma cells transfected with TRIM37 siRNA was significantly decreased.After 72 hours of transfection,the proliferation rate of glioma cells was 55.3%(p<0.05)lower than that of the control group,indicating that knockdown of TRIM37 could inhibit the proliferation of glioma cells.4.Knockdown of TRIM37 promotes apoptosis of U87 glioma cells.The apoptosis rate of tumor cells in the blank control group and the negative control group was 1.7%and 1.9%respectively,while in the siRNA-TRIM37 group was as high as 32.7%(P<0.01).Western Blot analysis showed that the ratio of Bax/Bcl-2 and Cleaved-caspase3 in siRNA-TRIM37 group was significantly increased,and the proportion of glioma cells arrested in S phase increased(P<0.05).5.TRIM37 knockdown inhibits glioma cell migration and invasion.Knockdown of TRIM37 suppresses the migration and invasion of glioma cells.After 48 hours of glioma cell transfection,Transwell assay showed that the migration and invasion of tumor cells in siRNA-TRIM37 group were significantly inhibited(P<0.01).Meanwhile,Western Blot analysis showed that the expression of MMP2 and MMP9 in U87 glioma cells knocked down TRIM37 gene was significantly decreased(P<0.01).Conclusion:1.The expression of TRIM37 in glioma tissues and U87 glioma cells was significantly up-regulated,which was correlated with WHO grade.2.Knockdown of TRIM37 can suppress the proliferation,migration and invasiveness of glioma cells,suggesting that TRIM37 may be a promising therapeutic target for malignant glioma.Part ?: Construction of a valid sbRNA lentiviral vector targeting TRIM37 cene and the study on Inhitian of Glioma growth h aude mice by shRNA lentiviral vector targeting TRIM37 geneobiective1. To design and construct a shRNA lentiviral vector that specifically targets amTR7gefficiency to provide a reliable experimental basis for further in vitro experiments.2. In vitro, to verify whether the shRNA lentiviral vector targeting the TRIM37gene can effectively inhibit the growth of glioma.Method:1. Design and synthesize shRNA lentiviral vector and package it.Using the public network design software, we designed the gene sequence targeting human TRIM37 and divided into 1#. 2#, 3#, a total of 3 groups, and a negative control group (NC group). After the results of sequencing comparison were correct, the lentivirus was packaged using the tool cell line(HEK293T), and the lentivirus particles were extracted successfully, and the titer of the virus wus determined.2. Glioma cell lines (U87) were transfected with shRNA lentivirus and determined the efficieney of silencing the target geneU87 glioma cells cultured in a good growth state were divided into a blank control group(oon group), a negative control group(NC group). 1# 2#.3#,and transfected with shRNA lentivirus, The mRNA and protein expression levels of TRIM37 gene were detected by qrt-pcr and western blot, and the interference effects against different targets were determined. The most effeetive group of lentiviral vector was selected.3. Screening the stable transfected U87 glioma ccll lines The U87 glioma cells cultured in a good growth state were diluted to 000 cellsiml, and then were screened in Dulbecco's Modilied Eagle's medi mcontaining puromycin at a concentration of oI wgm to 3 Hyml The minimun contration of puromycin (aEml) could be determined to kill all cells in 10-14 days After 24 hours of transtection, After 24 hours of lentiviral transtecmon, puromycin (lug/ml)was added for screening. The puromycin acrecoing solution was neplnoed every 3daya. After 14 day, the stable transfected cdll linr were obtained and continued tobe cultured in the medium coutaining puromycin(Olug/ml).4. umor formation in nude miceFifteen male BALB/cnw/mu nude mice were randomly divided into three groupa,5 in cach group. U87glioma cell were cultured in throe groups: blank controlgroup(con group). NC group and shRNA- TRIM37 group Under sterile conditions,the cells were made into a suspension, adjusted to a cell concentration of 5x10 cells/ml0. 1ml cell suspension was extracted with a medical sterile Imlsyringe and injected into the left groin of nude mice. After the subcutaneous tumor was observed in nude mice, the longest diameter L(mm) and the shortest diameterw L(mm) of subcutancous tumors were measured with vemier calipers every 5 days.The formula for calculating the tumor volume was: tumor volume V(mm~3)=(LxW~2)/2. After 45 days of feeding, nude mice were killed by cervical dislocation, and the tumors were weighed and recorded.Results:1.The 3 targets sclected had silence cffect on TRIM37. The qRT-PCR showed thatthe mRNA silencing eficiencies of the experimental groups 1#, 2#, and 3# were80.-2%, 88.1%, and 71.-5%, respectively. Westerm blot analysis showed that theexpressions of TRIM37 protein in experimental group 1#, 2#, 3# were significantly inhibited, and the silencing efficiencies were 82.1%,90.1%%, 70.3%, respectively.Compared with the blank control group and the negative control group, there was a significant diffcrence(P < 0.05). The sccond group with the highest silenceefficiency was selected for the next experiment.2. The volume, weight and inhibition rate of tumors were as follows: 35 days after tumorigenesis in blank control group (con group), negative control group(NC group), TRIM37-shRNA 2#group, the volumes of subcutaneous tumors in nude mice wer82712=141.8mm~3,992.58ą1256mm~3,231.3184.5mm~3, and theweights of tumors were 0.650+.12g, 0.686+0. IIg and 0. 174+0. 08g. respectively. TRIM37-shRNA2 group had statistical significance compared with blank control group and negative control group (P<0.05).Conclusion1. Three groups of shRNA lentiviral vectors specifically targeting TRIM37 gene were successfully constructed. According to the silencing efficiency of targetedgencs, the group 2 with the highest silencing efficieselected, which provided a reliable experimental basis for the preparation of stable transfection of U87 glioma coll lines and in vitro experiments of nude mice.2. Experiments confirmed that glioma cells transfected with shRNA lentivirus tArgeting TRIM37 were inhibited in viteo.Part ?: Study on the mechamism of knockdown of trim37 gene expressionto inhibit the growth of goma cellsobjective1. Based on the first part of the experiment, we further study on the mechanism ofinhibiting glioma cell growth by knocking down TRIM37 gene expression in U87 glioma cells2. Whether knockdown of TRIM37 gene expression can inhibit tumor cellmigration and invasion by blocking the EMT process in U87 glioma cellsMethod:1. Cell culture and transfection are the same as the first part of this paper2. Trim37-sima US7 glioma cells were treated with SC79 at the coneentration of 5uM for 24h.3. Westem blot analysis was the same as the first part of the experiment.4. CCK-8 was used to detect cell proliferation and Transwell was used to detect cell migration/invasion. The method was the same as the first part of the experment.Results:1, Western blot analysis showed that the phosphorylation levels of PI3K and Akt were decreased in U87 glioma cell line knocked down of TRIM37 gene, whilep-akt was significantly decreased, However, there was no significant change total P13K and Akt P13K. AKT signaling pathway activator (SC79) can partiallyreverse the inhibitory effeet of knockdown of TRIM37.2. Westerm blot was used to deteet the expression changes of related proteins In U87 glioma cells transfected with siRNA, the expression levels of Vimentin and n-cadherin were decreased, while the expression levels of c-cadherin were Increaseed.Conclusion1. Knockdown of trim37 expression inhibits proliferation and invasion of glioma cells by inhibiting the activation of the pi3k/akt signaling pathway.2. Knockdown of TRIM37 expression in U87 glioma cells also can inhibit tumor migration and invasion by blocking the epithelial-mesenchymal transformation(EMT) Process in glioma cells. |
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