| Glioma is the most common primary malignant tumor of the central nervous system in the human being.It has the characteristics of high recurrence rate and high mortality.Among them,Glioblastoma Multiforme(GBM) is the most malignant tumor,incidence rate accounts for about 50% of all glioma patients.Because of its highly aggressive growth,its clinical prognosis is very poor.Over the years,although significant progress has been made in surgery,chemotherapy and radiotherapy,the improvement of clinical prognosis is still very limited,and the median survival time has not significantly improved.It is still a serious public health problem.In recent years,some new breakthroughs in molecular targeted therapy of other tumors have also provided new ideas for the treatment of glioma.Therefore,it is necessary to study its molecular mechanism in order to provide theoretical basis for finding suitable therapeutic targets.The occurrence and development of glioma is related to abnormal gene expression,and the expression level of genes is regulated by various mechanisms,including transcription level and post-transcription level.Gene methylation can interfere with gene transcription levels.At the same time,the final expression level of the gene will be adjusted after the transcription level,and the expression level of the protein in the cell will be regulated by regulating the stability and degradation level of messenger RNA(m RNA).6-Methyladenosine(N6-methyladenosine,m~6A) is an important factor for modifying m RNA in eukaryotic cells,which can regulate the expression level of genes at the post-transcriptional level,while methyltransferase-like 3(methyltransferase-like 3,Mettl3) is an important factor that regulates m6 A.Studies have shown that Mettl3 has anti-cancer or pro-cancer effects,but its expression characteristics and mechanism in glioma is not yet completely clear.n addition,the expression level of Mettl3 protein is also targeted by micro RNA(micro RNA,miRNA).Therefore,this paper mainly analyzes the expression characteristics of mettl3 in glioma cells,and the influence of mettl3 on the biological behavior of glioma cells and its upstream and downstream regulatory mechanism from two aspects of in vitro cell experiment and animal in vivo experiment.Part ? Expression of mettl3 in glioma cells and its effect on cell biological behaviorObjective: To analyze the expression of mettl3 in human glioma cells and its effect on the biological behavior of glioma cells were analyzed,and to analyze the effect of silent or overexpression of Mettl3 on the biological behavior of glioma cells.Methods:Human glioma cell lines U87,LN229 and normal human glioma cell line HA1800 were detected the expression of mettl3 m RNA and protein by q PCR and Western blot,respectively.By plasmid transfection method,U87 and LN229 cells were used to construct Mettl3 silenced or overexpressed cell models,respectively,and then silenced Mettl3 or overexpressed Mettl3 cells were detected by CCK-8 method,clone formation experiment,cell scratch experiment,and Transwell experiment.The effects of viability,proliferation,migration and invasion ability,and the effect of silencing Mettl3 or overexpressing Mettl3 on apoptosis was detected by flow cytometry.Results: The levels of Mettl3 m RNA and protein in glioma tissues were significantly reduced.And as the stage of glioma disease increases,the level of Mettl3 gradually decreases.Silencing Mettl3 can significantly improve the viability,proliferation,migration and invasion ability of U87 and LN229 cells,and inhibit apoptosis(P<0.05).Overexpression of Mettl3 can inhibit cell proliferation,migration and invasion ability,and promote apoptosis(P<0.05).Conclusion: The m RNA and protein levels of mettl3 in glioma cells were significantly lower than those in normal human glioma cell line ha1800.Silencing Mettl3 will promote the proliferation,migration,invasion,and apoptosis of glioma cells,and overexpression of Mettl3 has a tumor suppressive effect,suggesting that Mettl3 is in glioma.The tumor may play an anti-tumor effect.Part ? Mettl3 exerts tumor suppressive effects in glioma cells through the PI3K/AKT/m TOR pathwayObjective: To explore the mechanism by which Mettl3 regulates the progression of glioma through PI3K/AKT/m TOR pathway through in vitro and in vivo experiments.Methods: U87 and LN229 cells were used to construct Mettl3 silenced or overexpressed cell models by plasmid transfection,respectively,and the phosphorylation level of PI3K/AKT/m TOR pathway protein was detected by Western blot.Then LY294002 and GSK690693 were used to inhibit PI3 K and AKT proteins,respectively,and cell viability and cell migration ability were detected in vitro.Then use overexpressed Mettl3 cells to construct a tumor-bearing nude mouse model,detect changes in tumor volume and mass,and detect the phosphorylation level of proteins in the PI3K/AKT/m TOR pathway in tumor tissues.Results: Silencing Mettl3 significantly increased the phosphorylation levels of PI3 K,AKT and m TOR protein(P<0.05).After using LY294002 and GSK690693 to inhibit PI3 K and AKT,the ability of silencing Mettl3 to promote cell viability and migration was significantly reversed(P<0.05).The results of tumor-bearing nude mice also showed that overexpression of Mettl3 inhibited the tumorigenic ability of U87 and LN229 cells,and inhibited the phosphorylation levels of PI3 K,AKT and m TOR proteins(P<0.05).Conclusion: Silencing Mettl3 can significantly increase the phosphorylation level of proteins in the PI3K/AKT/m TOR pathway.The use of LY294002 and GSK690693 will block the cancer-promoting effect of silencing Mettl3,suggesting that in glioma,the downregulation of Mettl3 may be through PI3K/AKT/ The m TOR pathway plays a role in promoting cancer.Part ? Micro RNA-135b-5p through the targeted regulation of METTL3Objective: To explore the mechanism of miR-135b-5p regulating the progression of glioma by targeting the expression of Mettl3.Methods: Firstly,the target miRNA upstream of Mettl3 was predicted by Starbase,and then the untranslated region(3'-UTR)at the 3'end of miR-135b-5p and Mettl3 messenger RNA(m RNA)was verified by dual luciferase report.Then the expression levels of miR-135b-5p in human glial cell line HEB and human glioma cell lines U87 and LN229 cells were analyzed.By co-transfection,U87 cells were constructed mimic-NC,Mir-135b-5p mimic and Mir-135b-5p mimic+Mettl3 groups,and LN229 cells were constructed inhibitor groups,Mir-135b-5p inhibitor and Mir-135b-5p inhibitor+sh-Mettl3 groups,respectively.The expression levels of Mettl3 m RNA and protein in each group were detected by q PCR and Western blot,and cell proliferation,clone formation and invasion ability were also detected.Results: The predicted results showed that mir-135b-5p could bind to the 3 '-UTR region of mettl3 m RNA.The double luciferase report results confirmed the targeting relationship between mir-135b-5p and mettl3 m RNA.The expression levels of miR-135b-5p in human glial cell line HEB and human glioma cell lines LN229 and U87 cells were detected,and the results showed that the levels of miR-135b-5p in LN229 and U87 cells Significantly higher than HEB cells(P<0.05),and the m RNA and protein levels of mettl3 in mir-135b-5p mimic group were significantly decreased(P < 0.05),and overexpression of mettl3 could reverse the inhibitory effect of mir-135b-5p on mettl3.In mir-135b-5p mimic group,cell proliferation,clone formation,migration and invasion were significantly increased,while the apoptosis rate was significantly decreased(P < 0.05),but overexpression of mettl3 could reverse the effect of mir-135-5p.The m RNA and protein levels of mettl3 in mir-135b-5p inhibitor group were significantly higher than those in mir-135b-5p inhibitor group(P < 0.05),and Silencing mettl3 could reverse the promotion of mettl3 by down regulating mir-135b-5p.In mir-135b-5p inhibitor group,cell proliferation,clone formation,migration and invasion were significantly decreased,while the apoptosis rate was significantly increased(P < 0.05),but silencing mettl3 could reverse the inhibitory effect of mir-135-5p on the cells.Conclusion: miR-135b-5p can promote glioma cells through targeted regulation of Mettl3. |
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