| Background and ObjectiveGlioma that is the most common intracranial tumor caused a great threat for people's life and health,for its morbidity,mortality and recurrence rate are high.At present,the clinical treatment of glioma is mainly based on surgery which generally needs to use anesthetic drugs.Recent studies have shown that the anesthetics may affect the metastasis and recurrence of glioma.Studies have shown that sevoflurane and pentobarbital reduce the migration of glioma U87 cells by down-regulating the expression of(matrix metalloproteinase-2)MMP-2.miR-34a-5p is involved in the proliferation,apoptosis,migration and invasion of colorectal cancer,gastric cancer,endometrial cancer and other tumor cells.Bioinformatics software analysis showed that there are binding sites between the 3'UTR of MMP-2 and the nucleotide sequence of miR-34a-5p,suggesting that MMP-2 may be the target gene of miR-34a-5p.It may affect the migration and invasion of tumor cells by regulating the expression of MMP-2.Therefore,this study investigated whether sevoflurane inhibits the expression of MMP-2 by up-regulating miR-34a-5p,thereby inhibiting the migration and invasion of glioma cells;whether sevoflurane affects the sensitivity of glioma to cisplatin.MethodsTaking glioma LN229 and U251 cells as the research object,different concentrations of sevoflurane were used to treat LN229 and U251 cells for 6 h.Transwell was used to test the effect of sevoflurane on the migration and invasion of LN229 and U251 cells.After LN229 and U251 cells were treated with sevoflurane and cisplatin,flow cytometry was used to detect the apoptosis rate of each group to evaluate whether sevoflurane affects the sensitivity of glioma LN229 and U251 cells to cisplatin.We selected tumor tissue of patients with glioma and normal brain tissues,and detected the expression level of miR-34a-5p in glioma tissues and normal brain tissues using real-time fluorescent quantitative polymerase chain reaction(qRT-PCR).According to the mean value of the expression level of miR-34a-5p in glioma tissue,glioma patients were divided into patients with low expression of miR-34a-5p and patients with high expression.Chi-square test was used to analyzed the correlation of the expression of miR-34a-5p and age,gender,tumor diameter,tumor grade,quality of life(KPS)and tumor location in patients with glioma.Kaplan-Meier survival curve was used to analyze the relationship between the expression level of miR-34a-5p and the prognosis of glioma patients.Cox proportional regression risk model analysis of factors affecting the prognosis of patients with glioma.Compared with normal human astrocyte NHA,the expression levels of miR-34a-5p,MMP-2 mRNA and MMP-2 protein in glioma cells LN229 and U251 with different concentrations of sevoflurane or without sevoflurane were detected by qRT-PCR and Western Blot.And qRT-PCR was used to detect MMP-2mRNA levels in glioma tissue and normal brain tissue.MiR-34a-5p mimics and inhibitors were transfected into LN229 and U251 cells respectively.Transwell was used to detect the migration and invasion ability of LN229 and U251 cells with sevoflurane by up-regulating or down-regulating of miR-34a-5p.Flow cytometry was used to detect the apoptosis rate of cells in each group.We evaluated whether sevoflurane affected the sensitivity of LN229 and U251 cells to cisplatin and the migration and invasion ability of LN229 and U251 cells by regulating miR-34a-5p.The bioinformatics software predicts that the 3' UTR of MMP-2 has a nucleotide sequence that binds to miR-34a-5p.The dual luciferase reporter gene experiment verified the relationship between miR-34a-5p and MMP-2.Western blot detects the expression of MMP-2 protein in LN229 and U251 cells by up-regulating or down-regulating of miR-34a-5p.Transwell was used to detect each group cell migration and invasion ability to evaluate whether down-regulation of MMP-2 can reverse the effect of down-regulating of miR-34a-5p on cell migration and invasion.Flow cytometry was used to detect the apoptosis rate of each group to evaluate whether miR-34a-5p could affect the sensitivity of LN229 and U251 cells to cisplatin by regulating MMP-2.Statistical analysis SPSS20.0 tests the data for normal distribution and homogeneity of variance.Measurement data conforming to the normal distribution use mean ± standard deviation.The comparison between the two groups uses independent sample t test,and the comparison between multiple groups uses one-way analysis of variance.ResultsAfter sevoflurane acted on LN229 and U251 cells,their migration and invasion abilities were significantly reduced.Sevoflurane increased the rate of apoptosis induced by cisplatin.The expression of miR-34a-5p in glioma tissue was significantly lower than that in normal brain tissue;the expression of miR-34a-5p was closely related to the tumor grade and KPS score of glioma patients,and had nothing to do with the patient's age,gender,tumor diameter and tumor location.Kaplan-Meier survival curve analysis showed that the 3-year overall survival rate and recurrence-free survival rate of patients with low miR-34a-5p expression were significantly lower than those with high expression.Cox proportional regression risk model analysis results show that low expression of miR-34a-5p is an independent risk factor affecting the prognosis of patients with glioma.Compared with normal astrocytes,the expression of miR-34a-5p in LN229 and U251 cells was significantly reduced,and the expression of MMP-2 mRNA and protein were significantly increased.The expression of miR-34a-5p in LN229 and U251 cells treated with sevoflurane was significantly increased,and the expression of MMP-2 mRNA and protein were significantly reduced.Compared with normal brain tissue,the expression of MMP-2 mRNA in glioma tissue was significantly increased.The expression of miR-34a-5p in LN229 and U251 cells were significantly changed,and the miR-34a-5p mimics and miR-34a-5p inhibitors were successfully transfected.Compared with NHA cells,the expression of miR-34a-5p in LN229 and U251 cells were significantly reduced,and the expression of MMP-2 mRNA and protein were significantly increased.Compared with normal brain tissue,the expression of MMP-2 mRNA in glioma tissue was significantly increased.Compared with the LN229 and U251 cell groups,the expression of miR-34a-5p in the LN229 and U251 cells of the sevoflurane group were significantly increased,and the expression of MMP-2 mRNA and protein were significantly reduced in a concentration-dependent manner.The expression of miR-34a-5p in LN229 and U251 cells were significantly changed,and the miR-34a-5p mimics and miR-34a-5p inhibitors were successfully transfected.The migration and invasion ability of LN229 and U251 cells decreased after up-regulated miR-34a-5p,while the migration and invasion ability of cells was enhanced by down-regulating miR-34a-5p.The apoptosis rate of LN229 and U251 cells which were treated with sevoflurane and cisplatin were significantly increased.The apoptosis rate of cells were increased by up-regulation miR-34a-5p.However the apoptosis rate of cells were decreased by down-regulation miR-34a-5p.The results of the dual luciferase gene report experiment showed that miR-34a5p has a binding site with 3'UTR-WT of MMP-2.MMP-2 protein expression in LN229 and U251 cells were significantly reduced after up-regulating miR-34a-5p.MMP-2 protein expression in LN229 and U251 cells were significantly increased by down-regulating miR-34a-5p.After down-regulating of miR-34a-5p and si-MMP-2 in LN229 and U251 cells,the migration and invasion of cells were significantly reduced,and the apoptosis rate of LN229 and U251 cells were increased.ConclusionSevoflurane inhibits the invasion and migration of glioma cells and enhances its sensitivity to cisplatin chemotherapy by up-regulating miR-34a-5p to inhibit the expression MMP-2. |
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