The Role Of TRPM8 In The Proliferation And Invasion Of Glioblastoma Cells And The Targeted Regulation Of MiR-433 On TRPM8

Glioblastoma is considered to be the most malignant brain tumor.Glioblastoma has a high degree of specificity and invasive growth characteristics,which makes the tumor progress fast and highly malignant.Current treatment methods are faced with the dilemma of poor treatment effect,poor clinical prognosis and easy recurrence.Therefore,the diagnosis and treatment of glioblastoma need to develop new effective ways.The in-depth development of bioinformatics technology such as big data omics research provides a platform and possibility for screening and identifying key new targets that play an important role in the pathological process of glioblastoma invasion and malignant proliferation.In this study,through the screening of multi-omics chip data combined with the clinical cases of our center,we found that the transient receptor potential ion channel TRPM8 is significantly overexpressed in glioblastoma,and is a direct related factor for the poor prognosis of patients.Using a series of cell biology methods,we deeply studied the function and mechanism of TRPM8 in cell proliferation,invasion and apoptosis of glioblastoma.Using weighted gene co-expression network analysis,GEO2 R and other bioinformatics techniques,we also found that miR-433 is a miRNA significantly reduced in glioblastoma compared with normal brain tissue.Furthermore,through molecular biology experiments,it was found that the selected target miR-433 and TRPM8 have a good targeting relationship and that high expression of miR-433 can inhibit the invasion ability of glioblastoma cells by targeting TRPM8.In summary,this study discovered the role of TRPM8,miRNA-433 and other genes in cell proliferation,invasion and apoptosis of glioma cells,and initially revealed the molecular mechanism of their role,which is a drug and Gene therapy provides potential molecular targets.PART ?.Bioinformatics screening of prognostic related g TRPM8 in patients with glioblastomaObjective: New molecules with prognostic potential in glioblastoma were screened out from the GEO database,and the expression of target gene TRPM8 in glioblastoma and its prognostic value in glioblastoma patients were verified through clinical cases in our center.Methods: Microarray data sets that meet the conditions were screened from the GEO database,and the online GEO2 R analysis tool was used to screen out differentially expressed genes between glioblastoma tumor tissues and normal brain tissue samples.Subsequently,GO function and KEGG pathway enrichment analysis were performed on these differential genes.Cytoscape software was used to construct protein mutual assistance network and screen pivotal genes.The gene expression levels of these hub genes were verified using GEPIA database and Oncomine database based on TCGA database,and the prognostic survival analysis of these hub genes was performed through CGGA database.In addition,71 glioblastoma cases and 11 normal brain tissues of our center were collected.The tissue chip immunohistochemistry and RT-PCR were used to verify whether the gene of interest was highly expressed in tumor tissues.Follow-up,Cox multivariate analysis and Kaplan-Meier survival analysis.Results: The data of the three chips?GSE50161,GSE4290,GSE68848?met the screening conditions,and there were 716 differential genes that they co-expressed,of which 188 genes were significantly up-regulated genes and 528 genes were significantly down-regulated genes.From these differential genes,ten of the most relevant pivot genes were selected.TCGA and Oncomine database analysis verified that the expression of these 10 hub genes in glioblastoma was significantly higher than that in normal tissues.By using the CGGA database to analyze the prognosis and survival of the above 10 hub genes,6 genes?TOP2A,CDK1,CDC20,BIRC5,MELK,and TRPM8?were found to be closely related to the clinical prognosis of patients with glioblastoma.Further verification of the expression level of TRPM8 through the case samples of our center also confirmed that TRPM8 expression in glioblastoma was significantly higher than that in normal brain tissue?P < 0.0001?.At the same time,the results of Cox multivariate analysis suggest that TRPM8 is an independent risk factor for glioblastoma?P = 0.012?,and the prognostic survival analysis results show that the overall survival of patients with glioblastoma highly expressing TRPM8 is shorter.Conclusion: TOP2A,CDK1,CDC20,BIRC5,MELK and TRPM8 can be used as valuable biomarkers for judging patients with glioblastoma.TRPM8 is highly expressed in glioblastoma and is an independent risk factor affecting the prognosis of glioblastoma.PART ?.The function and mechanism of TRPM8 in cell proliferation,invasion and apoptosis of glioblastomaObjective: The function and mechanism of TRPM8 in the proliferation,invasion and apoptosis of glioblastoma cells were clarified by molecular biology experiments.Methods: RT-PCR and Western blot were used to detect the m RNA and protein expression levels of gene TRPM8 in glioblastoma cell lines?U251,U87-MG,A172?and normal astrocyte HA1800.Patch clamp whole cell recording and Ca²⁺ imaging were used to detect the activity characteristics of TRPM8 channel in glioblastoma cell lines.Design and synthesize small interfering RNA?siRNA?targeting TRPM8,use Lipofectamine 2000 to transfect the plasmid into cells,and transfect the plasmid overexpressing TRPM8 in U251 cells.The proliferation,invasion,and apoptosis of glioblastoma cells were detected by CCK-8 proliferation experiment,scratch experiment,Transwell invasion experiment,and cell flow cytometry experiment.The expressions of extracellular singnal-regulated kinase?ERK?,cyclin D1?Cyclin D1?and Bcl-2 in U251 cells were detected by Western blot and cellular immunofluorescence after the expression level of TRPM8 was changed.Results: The expression of TRPM8 in glioblastoma cell lines was significantly higher than that in normal astrocyte cell lines?P < 0.05?.The results of patch clamp electrophysiology and Ca²⁺ fluorescence imaging indicated that the TRPM8 channel in glioblastoma cells was Functional and has the characteristics of outward rectification.CCK-8 test results show that TRPM8 can positively regulate the cell proliferation ability of glioblastoma.The scratch test and Transwell invasion test results showed that the cell invasion and migration ability of glioblastoma was positively correlated with the expression level of TRPM8?P < 0.001?.The results of flow cytometry found that the expression level of TRPM8 reversely regulated the apoptosis rate of U251?P < 0.01?.Western blot and cellular immunofluorescence results suggested that the expression level of TRPM8 can reduce the expression levels of ERK,Cyclin D1 and Bcl-2 in U251 cells.Conclusions: Ca²⁺-permeable non-selective cation channel TRPM8 promotes cell proliferation and invasion of glioblastoma,while TRPM8 reduces the sensitivity of glioblastoma cells to apoptosis,the mechanism is through the regulation of MAPK signaling pathway carry out.PART ?.miR-433 inhibits cell invasion of glioblastoma via direct targeting TRPM8Objective: To study the targeting relationship between miR-433 and TPM8,and to explore the effect of miR-433 on the cell invasion ability of glioblastoma.Methods: By screening out eligible miRNA expression data sets from the GEO database,the WGCNA package based on the R language is further used to construct a co-expression network model of the chip data set,to identify modules related to disease states and perform pivotal miRNA screening on the related modules;Use the differential miRNA obtained by another chip GSE13030 and take the intersection with the hub miRNA to obtain the target miRNA.The target relationship between miRNA and TRPM8 was verified by bioinformatics and luciferase gene report,and the effect of miRNA overexpression on TRPM8 protein level was analyzed by Western blot.The effect of miR-433 overexpression on the invasion ability of glioblastoma cells in vitro was examined by scratch test and Transwell invasion test.Results: In this study,the qualified chip GSE25631 was screened for miR-433 using bioinformatics.The dual luciferase reporter gene report confirmed that TRPM8 gene is a potential target gene of miR-433.Western blotting further confirmed that miR-433 can regulate the protein expression level of TRPM8.Scratch experiments showed that the migration ability of glioma cells?A172,U251,U87-MG?in miR-433 inhibit group was significantly higher than that in control NC group,while the migration ability of miR-433 mics group was significantly worse than that in NC group?P < 0.01?,there was no significant difference between the NC groups?P > 0.05?.Transwell invasion experiments showed that the invasive ability of A172,U251 and U87-MG cells in miR-433 inhibit group was significantly higher than that in NC group?P < 0.01?,but the number of invasive cells in miR-433 mics group was significantly less than that in NC group?P < 0.01?,there was no significant difference between the NC groups?P > 0.05?.Conclusions: The selected target miR-433 has a good targeting relationship with TRPM8.The high expression of miR-433 can inhibit the invasion ability of glioblastoma cells by targeting TRPM8.This mechanism provides a new strategy for gene therapy of glioblastoma.