Effects Of Mettl9Gene On Proliferation And Apoptosis Of U251Glioma Cells

Glioma is the most common type of primary malignant brain tumor. It is.essentially a kind of polygenic disorders, with excessive expression of oncogenes and gene mutation of some tumor suppressor and which leads to the abnormal signal transduction pathways, accompanied with the tumor cells escaping the normal growth regulation and control mechanism, the abnormal cell proliferation, invasion, angiogenesis, and other malignant phenotype. Mettl9is a newly discovered gene which located in the downstream of the p53gene. Mettl9is specifically expressed during the development of the central nervous system of SD rats, mouse fetal lung and lung cancer. And the expression of Mettl9is positive correlated with cell apoptosis, and its role during embryonic development is much likely to achieve through apoptosis. Therefore, in this study, the influence of different expression levels of Mettl9gene on U251glioma cell proliferation and apoptosis were first observed, and its mechanism was further studied. Objective: 1. To observe the influence of different expression levels of Mettl9gene on the growth of U251glioma cell;2. To study the mechanism of the effect of Mettl9gene on U251glioma cell’s growth.Methods:Mettl9gene cDNA was amplified using RT-PCR method, cloned into pGEM (?)-T vector and sequenced, and then subcloned into the lentiviral vector pLenti6.3-MCS-IRES-EGFP, packaged in293T cells, and finally Mettl9lentiviral gene over-expression vector were constructed; Double-stranded DNA oligo including interference sequence which was designed according to the RNA interference principle was synthesized, and then the Mettl9RNA interference vector lentivirus was constructed by digestion and linkage. The whole experiment was divided into five groups:normal cell cultured group, lentiviral vector group (NC), lentiviral overexpression vector group (Mettl9group), interference lentiviral vector infection control group (NC-RNAi group), Mettl9lentiviral interference vector infection group (Mettl9-RNAi group). U251cells were infected with the above constructed vectors, and the green fluorescence was detected to observe the transfection efficiency after48h, and Mettl9gene expressionwas detected by qRT-PCR as well. The proliferation of U251was detected by MTT, apoptosis cell apoptosis was of apoptosis of U251was detected by flow cytometry, and the expression of apoptosis-related factors Bcl-2and Bax was detected by qRT-PCR. Results:1. Mettl9cDNA was amplified by RT-PCR and ligated into pGEM (?)-T vector, the sequence was submitted to the GenBank and obtain a record number HQ898855;2. Mettl9lentiviral gene overexpression vector and RNAi interference carrier was constructed successfully.3. The positive expression rate of green fluorescence GFP was about50%under a fluorescence microscope; The Mettl9expression level after infect with Mettl9Lentiviral overexpression plasmid was significantly higher than the control group NC; While, the Mettl9expression level in the group of Mettl9-RNAi was significantly lower than NC-RNAi group.4. MTT results showed that after infect with Mettl9Lentiviral overexpression plasmid, cells proliferation ability was significantly decreased; While, cells proliferation ability in the group of Mettl9-RNAi was significantly increased.5. Flow cytometry showed that after infect with Mettl9Lentiviral overexpression plasmid, cell apoptosis rate increased significantly; While, cell apoptosis rate in the group of Mettl9-RNAi was significantly decreased.6. qRT-PCR results showed that after infect with Mettl9Lentiviral overexpression plasmid, the mRNA expression of apoptosis-related factors Bcl-2was greatly reduced and Bax was significantly increased; While, the mRNA expression of apoptosis-related factors Bcl-2in the group of Mettl9-RNAi was greatly increased and Bax was significantly reduced.Conclusions:Mettl9gene overexpression inhibited U251cell proliferation, promoted U251cell apoptosis, which may correlate with the downregulation of Bcl-2mRNA and upregulation of Bax mRNA.