| Introduction:Human glioma(Glioma)is a primary central nervous system malignant tumor originating from neuroepithelial ectoderm in histological classification.It accounts for more than 50% of primary intracerebral tumors.It is the type of intracranial tumor with the highest incidence worldwide and cannot be completely cured,and it is also the most difficult intracranial tumor to overcome.In my country's five-year incidence statistics of systemic tumors,the incidence of glioma is second only to lung cancer and pancreatic cancer.In general,patients with glioma have a high recurrence rate,with a five-year survival rate of less than 5%,and the prognosis is extremely poor.Glioma does not have a true capsule,and it often manifests as a diffuse infiltrating growth that burrows into the normal brain tissue like a tree root.It is often difficult to distinguish the boundary between the tumor tissue and the surrounding normal brain tissue during clinical surgery.Therefore,the current clinical treatment for gliomas is based on the premise of preserving the basic neurological function and the treatment of the patient's life with the help of precise positioning equipment such as neuronavigation to perform surgical resection of the tumor to the greatest degree of safety,and postoperative auxiliary standard radiotherapy and simultaneous temozolomide chemotherapy and implement immunotherapy according to specific conditions.However,due to the strong heterogeneity of glioma,the different sensitivity of postoperative radiotherapy and chemotherapy,and the role of the blood-brain barrier on the natural barrier of drugs,the treatment effect is not satisfactory,and the overall prognosis of the patient is not significantly improved.Studies in recent decades have successively confirmed that tumor genomics has a significant impact on the prognosis of glioma.Changes in specific gene pathways may play an important role in the invasion and spread of glioma in the brain,but its specific biology function and mechanism of regulating downstream signaling pathways are still unclear.In-depth research on the occurrence and molecular mechanism of glioma will have important theoretical significance and clinical practical value for targeted individualized treatment and patient prognosis assessment.Scientific studies have confirmed that the abnormal activation or inactivation of key skeletal regulatory proteins in cells has largely led to the tumor transformation of these normal biological behaviors in the body's cells.Recent studies have confirmed that the family of molecules that interact with Cas L(MICALs)is involved in the regulation of cytoskeletal dynamics and related biological processes.This family is highly conserved in evolution and represents a series of multi-domain proteins,which can be divided into two sub-subsidiaries families: MICAL(MICAL-1,MICAL-2,MICAL-3)and MICALL(MICAL-Like,MICAL-L1,MICAL-L2).MICAL-L2(microtubule-associated monooxygenase-like protein 2),also known as Rab13 binding protein(JRAB),is one of the important members of this family.Studies have found that MICAL-L2 can participate in the control of cytoskeletal rearrangement and mediates endocytic recycling of proteins and formation of tight junctions.Most of the current understanding of the function of MICAL-L2 comes from cell biology research,while its role in human diseases is poorly understood.Previous studies have found that MICAL-L2 is highly expressed in a variety of tumors,including ovarian cancer,gastric cancer,non-small cell lung cancer and breast cancer,and can regulate the proliferation,migration and invasion of tumor cells.Epidermal growth factor receptor(EGFR)is a member of the Erb B family and has been proven to play an important role in the occurrence and development of a variety of tumors.In gastric cancer,MICAL-L2 inhibits lysosome-mediated EGFR degradation and enhances the migration ability of gastric cancer cells.And MICAL-L2 has the function of preventing the degradation of its downstream proteins,and may play a carcinogenic effect in gastric cancer.Based on the long-term focus of the research group on the dynamics of cytoskeleton regulation and microtubule dynamics in gliomas,we discovered MICALs,a family that has not been studied in gliomas.Through the integrated analysis of glioma data from multiple data platforms,the only gene with universal clinical prognostic value,namely MICAL-L2,was selected from the family,and verified with clinical surgical specimens.Using the comprehensive analysis results of the multi-data platform as the guide and entry point,the pathological activities of MICAL-L2 in gliomas are explained through cell function,and the specific mechanism of action and signal regulation to provide new solutions for poor prognosis.Objective:1.Assess and analyze the clinical value of MICAL-L2 in human glioma;2.To study the correlation between the expression level of MICAL-L2 in tumor tissues of patients with clinical glioma and the pathological grade;3.Explore the influence of MICAL-L2 in different types of glioma cell lines on its biological behavior;4.Further explore and analyze the relevant molecular mechanism of MICAL-L2 affecting the malignant biological phenotype of glioma.Materials and Methods:1.Bioinformatics methods Obtaining the required glioma-related data from two public data platforms,the Global Cancer Genome Atlas Database(TCGA)and the Chinese Glioma Genome Atlas Database(CGGA),and analyze the clinical value of members of the MICALs family through Kaplan-Meier survival curve and log-rank test to evaluate the prognostic difference between the high and low MICAL-L2 expression groups,and carry out the classification of differential expression analysis to screen out the molecules with the most prognostic value and research significance.The gene enrichment analysis(GSEA)and Glio Vis glioma data platform are used to predict the biological behavior involved in differential expression of MICAL-L2,and the molecular signaling pathways that may be regulated by MICAL-L2 are analyzed through KEGG.2.Immunohistochemical methods Using immunohistochemical methods to visually express different pathological grades of glioma cells,such as low-grade grade II glioma cells and high-grade grade III or IV glioblastoma,as well as the lesion tissues are removed due to traumatic brain injury or epilepsy.The expression of MICAL-L2 in non-tumor brain tissues further shows that it`s closely related to the occurrence and development of glioma cells and the degree of malignancy.3.Cell culture Human glioma cell lines U87,U373,U251,LN229,T98 G and human astrocyte cell line NHA were cultured in a complete medium containing high glucose DMEM + 10%FBS + 1% penicillin/streptomycin double antibodies.The patient-derived primary glioma cell line PGC21,PGC2410% extracted by our laboratory,was cultured with 10%fetal bovine serum + 1% penicillin/streptomycin RPMI1640 medium.All cell lines were cultured in an incubator with a relative humidity of 90%,5% CO2,and a constant temperature of 37°C.4.Real-time QPCR The TRIzol method and Takara rapid RNA extraction kit were used to extract total RNA from different glioma tissues and cells to performRNA quantification and reverse transcription to obtain the corresponding c DNA,and then to perform a three-step PCR reaction with SYBR fluorescent labeling.5.Western bot Extracting the total protein of different glioma tissues and cells with RIPA protein lysate on ice,determine the protein concentration by BCA method,and then perform SDS-PAGE gel electrophoresis,transfer membrane,immunohybridization reaction,ECL luminescence fixation detection.6.Editing of gene expression Taking human glioma cell line LN229 and human-derived primary glioma cell line PGC21 as the research objects,RNA interference technology was used to construct a cell model of MICAL-L2 knockdown;Taking human glioma cell line U87 and human-derived primary glioma cell line PGC24 as the research object,and the lentivirus carrying the MICAL-L2 plasmid was used to construct the overexpression group cell model;puromycin was used to screen the stable knockdown and overexpression MICAL-L2 cell lines.7.Functional experiments of glioma cells in vitro Detecting the effect of MICAL-L2 on the proliferation and cell viability of different glioma models through MTS cell proliferation experiments,EDU imaging detection,and plate cloning experiments;Detecting glioma cells at different levels of MICAL-L2 expression through the apoptosis Tunel test Transwell experiment and Matrigel model were used to test the effect of MICAL-L2 on the migration and invasion ability of glioma cells in vitro under different treatment conditions.8.Statistical analysisIn this study,Graph Pad Prism 8.0 and SPSS 22.0 were used for statistical analysis,one-way analysis of variance(ANOVA),and two-tailed t test were applied to the corresponding conditions.The results of statistical analysis of all data are expressed as meanąstandard deviation SD.Unless otherwise specified,the two-tailed p<0.05 is considered as a statistically significant difference,that is,statistically significant.Where* P <0.05;** P <0.01;*** P <0.005,**** P <0.001,ns: P?0.05.Results:1.Comprehensive data analysis of multiple glioma databases shows that MICAL-L2 has extremely stable prognostic value.(1)The survival and prognosis data of glioma extracted from the TCGA data platform was used to analyze the difference of five molecules in the MICALs family of cytoskeleton regulatory proteins.The forest diagram drawn indicates that in this family,only MICAL-L2 has stable expression differences and prognostic significance,which is found in all grades of glioma and GBM.(2)All-grade survival curve and sub-grade survival curve of glioma indicate that the expression of MICAL-L2 significantly affects the survival time of patients with glioma.Regardless of whether methylation occurs in GBM,patients with high expression of MICAL-L2 and low expression have poor prognosis.Patients with low expression of MICAL-L2 and not accompanied by 1p/19 q co-deletion had a poor prognosis in the higher expression group.(3)Regardless of whether it is CGGA or TCGA database,the expression level of MICAL-L2 is positively correlated with the number of pathological grades of gliomas,and in the mesenchymal subtypes represented by GBM,the expression level of MICAL-L2 is significantly higher than other subtypes of glioma.In addition,the expression of MICAL-L2 in IDH1 wild-type gliomas in both LGGs and HGGs was significantly higher than that in mutants.2.There are differences in the expression of MICAL-L2 in glioma tissues of different clinicopathological grades.We took various grades of glioma tissues and non-tumor brain tissues for immunohistochemistry experiments,and the mRNA and protein levels of them were tested also.According to the experimental results,we found that the expression of MICAL-L2 showed an increasing trend as the malignant degree of gliomas increased.3.Predictive analysis of biological effects.GO analysis suggests that MICAL-L2 is closely related to the regulation of the cytoskeletal structure of glioma extracellular matrix formation;GSEA analysis further suggests that MICAL-L2 is mainly related to cell adhesion,cell apoptosis and necrosis.4.Interfering with the expression of MICAL-L2 can affect the proliferation ability of glioma cells from different sources.MTS cell proliferation experiments and EDU intracellular proliferation-related product imaging experiments were used to detect the proliferation ability of glioma cell line LN229 and human glioma primary cell line PGC21 in vitro before and after interference with MICAL-L2 expression.The experimental results reveal that the cell proliferation activity of the MICAL-L2 knockdown interference group is significantly weaker than that of the control group.Colony formation experiments showed that after MICAL-L2 gene interference editing,compared with the control group,the tumor cell clone colony formation in the interference treatment group was significantly reduced,which intuitively provides evidence that high expression of MICAL-L2 can promote cell proliferation.5.Inhibition of MICAL-L2 expression can promote the apoptosis of glioma cells from different sources.The results of the Tunel apoptosis experiment suggest that the knockdown treatment of MICAL-L2 expression makes tumor cells increase undergoing apoptotic phenomenon indicates that MICAL-L2 can inhibit the apoptosis of glioma cells.6.Inhibition of MICAL-L2 expression can reduce the migration and penetration and invasion ability of glioma cells from different sources in vitro.The Transwell chamber model and Matrigel chamber model were used to simulate the living environment of tumor cells in vitro.The experimental results suggest that the expression of MICAL-L2 in glioma cells is up-regulated and can significantly promote its migration and penetration and invasion ability in vitro.7.MICAL-L2 can regulate the expression levels of key proteins involved in the proliferation,apoptosis,invasion and migration of different types of glioma cells.The relevant proteins involved in the life activities of cell proliferation,apoptosis,invasion and migration in the organism can be detected by Western blot experiment.We found that the expression of PCNA,Bcl-2,Cyclin D1,FAK,ICAM1,MMP2 and MMP9 are significantly reduced after the MICAL-L2 interference knockdown.However,the expression of Bax and Cleaved-caspase-3 was significantly higher than that of the negative control group.8.MICAL-L2 can induce epithelial-mesenchymal epithelial transition(EMT)in glioma cells.In order to reveal the relationship between MICAL-L2 and EMT in gliomas,we used WB to detect the expression level of the key proteins E-cadherin,N-cadherin and Vimentin in the EMT process.The results showed that MICAL-L2 is closely related to the decrease of epithelial characteristics of glioma cells and the increase of mesenchymal traits.9.MICAL-L2 can activate the PI3K/AKT signaling pathway in glioma cells.KEGG signaling pathway analysis suggests that differentially expressed MICAL-L2 is closely related to the activation of PI3K/AKT signaling pathway.Using Western blot to analysis PI3K/AKT signaling pathway related proteins,the results suggest that the expression levels of phosphorylated PI3 K and AKT,and phosphorylated m TOR and GSK3? downstream of PI3K/AKT signaling pathway were significantly reduced in the MICAL-L2 knockdown glioma cell model compared to the control group.10.There is a positive correlation between the expression of MICAL-L2 and EGFR protein levels.The mRNA and protein levels of EGFR were detected by q PCR experiment and Western blot experiment.The results showed that with the decrease of MICAL-L2 in glioma cells,the protein expression level of EGFR also decreased,but the abundance of its mRNA did not change greatly.11.EGFR inhibitors can inhibit the activation of PI3K/AKT signaling pathway caused by overexpression of MICAL-L2.After treating the U87 and PGC24 cell lines with EGFR inhibitor in overexpressing MICAL-L2 group and the corresponding control group,the expression of PI3K/AKT signaling pathway related proteins was detected.The data suggest that MICAL-L2 can participate in the activation of PI3K/AKT signaling pathway through the mediation of EGFR.12.EGFR inhibitors can restore the changes in the malignant biological behavior of glioma cells in vitro caused by the overexpression of MICAL-L2,and prevent glioma cells from undergoing EMT transformation.Using the MTS method,EDU imaging and clone formation experiments to detect the proliferation ability of the overexpressing MICAL-L2 cell line,the Tunel method for apoptosis detection,and the chamber model to observe the cell migration and invasion ability in vitro after EGFR inhibitors process these two overexpression cell models.Experimental data indicate that MICAL-L2 can indeed transmit relevant regulatory signals through EGFR to participate in the regulation of the malignant biological effects of glioma cells and affect their mesenchymal transformation in vitro.Conclusion:In this study,we used biological big data analysis to screen out MICAL-L2 genes with stable clinical prognostic value and research value from MICALs,a family of cytoskeleton regulatory proteins.And it is confirmed in clinical specimens that the significant up-regulation of MICAL-L2 is related to the pathological grade and histological subtype of glioma.Abnormal activation of MICAL-L2 in glioma can affect the proliferation,apoptosis,migration,and invasion activities of tumor cells,and participate in the cell's epithelial-mesenchymal transition process.In addition,our findings reveal the relationship between the cytoskeletal protein MICAL-L2 and the endogenous epidermal growth factor receptor EGFR and PI3K/AKT signaling pathway,which is confirmed that MICAL-L2 in glioma cells can maintain the activity of PI3K/AKT signaling pathway through EGFR regulate the proliferation,apoptosis,migration and invasion behavior of glioma cells,and affect the progress of malignant transformation of glioma cells. |
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