MiR-940 Affects The Metabolism, Proliferation And Invasion Of Glioma Cells And Its Mechanism Through MTHFD2 Gene

Part ? Effect of mir-940 on proliferation,invasion and apoptosis in glioma cells in vitro and in vivoGlioma is the most common type of malignant tumor in the nervous system,the aggressiveness and recurrence of glioma are major obstacles for the treatment of this type of tumor.At present,the comprehensive treatments of glioma include surgery,postoperative chemotherapy and postoperative radiotherapy,but the consequence of this treatment is still unsatisfactory with the high rate of disability and fatality.Further understanding of the molecularmechanisms of glioma is necessary to improve the efficacy of therapy.Micro RNAs have been widely studied in many human cancers.Here,we found that miR-940 was one of the primarydownregulated miRNAs in clinical samplesand glioma cell lines through bioinformatics analysis and q RT-PCR.Upregulating miR-940 expression significantly inhibited the proliferation and invasion and promoted apoptosis of U87 and U118 cells.Then I found that miR-940 mainly extended the G1 phase of the cell cycle to reduce the proliferation of glioma cells during the study of mechanism.Finally,we verified that mir-940 inhibited the proliferation of glioma cells in vivo.Part ? Mir-940 downregulating the expression of MTHFD2 inhibits biological effect of gliomasMTHFD2,plays an important role in cell apoptosis,proliferation and invasion,is is a multifunctional metabolic enzyme.MTHFD2 was predicted to be a direct target of miR-940 in Target Scan,miRWalk and miRDB.Luciferase reporter technique was used to detect the action site of mir-940 on MTHFD2 gene.To further verify that MTHFD2 was the direct target of miR-940,we co-transfected U87 cells with MTHFD2 overexpression plasmids(pc DNA3.1-MTHFD2)and miR-940 mimics.And CCK-8 assay,q RT-PCR,Transwell assay,Western blot,colony formation assays,IHC,Flow cytometry and Flow apoptosis were performed to examine the cell biological activity of MTHFD2,in U87 contained miR-940 and MTHFD2,Furthermore,the relationship between miR-940 and MTHFD2 was verified in vivo.At last,we verified that MTHFD2 is highly expressed in clinical samples of glioma samples and glioma cell lines;miR-940 binds mainly to site 1(1611-1617)of the MTHFD2 3'-UTR;By down-regulating the expression of MTHFD2,miR-940 arrests gliomas in G1 phase,then reduce proliferation;overexpression of MTHFD2 can reduce the inhibitory effect of miR-940 in U87 cells including cells' proliferation and invasion but promote the apoptosis of glioma cells.Finally,we confirmed that mir-940 inhibited the expression of MTHFD2 in vivo.Part?mir-940 inhibits the mitochondrial metabolic pathway in glioma by down-regulating the expression of MTHFD2In the previous two parts,we have confirmed that miR-940 expression is low in glioma and miR-940 directly targets MTHFD2 to inhibit the proliferation and invasion of glioma and promote apoptosis.The stably expression of MTHFD2 in mitochondria of tumors is a key enzyme.Further study on the relationship between mir-940 down-regulating the expression of MTHFD2 and metabolism can provide theoretical basis and reference for clinical treatment of glioma with a important significance.In this part,we used Western blotting to detect the expression of metabolic enzymes in the mitochondria of glioma cells when mir-9jiau0 was overexpressed.The amount of amino acid was detected by gas chromatography-mass spectrometry during the different expression of miR-940.Finally,glycine sensitivity test,folate sensitivity test and formate rescue test were used to test the cell response and sensitivity to the failure of metabolic pathway.Through these studies we found that the expression of MTHFD2 was down-regulated by miR-940,that was closely related to metabolism,can block folate and one carbon unit metabolize and nucleotide synthesis,then resulting in reducing cell biological activity and enhancing apoptosis.And we predict that Mir-940 may be a potential tumor marker and MTHFD2 may be a potential drug target of glioma.