Investigating The Mechanism Of MiRNA-451 Regulation On Epithelial-mesenchymal Transition Of Human Glioma Cells

Objective: The study mechanism of mi RNA-451 overexpression in the regulation of epithelial mesenchymal transition(EMT)in human LN229 and U251 glioma cell lines.And the effects of overexpression of mi RNA-451 on proliferation,invasion and migration in glioma cell lines.Methods:(1)To investigate the effect of mi RNA-451 on epithelial mesenchymal transition and pathways in human glioma cell lines.The human glioma cell lines LN229 and U251 were cultured in vitro.Glioma cells were transfected with Roche transfection reagent.The experiment was divided into control group,negative control group and mi RNA-251 mimics group.In a word,the control group without any treatment;the negative control transfection with antisense oligonucleotide 5-ACGUGACACGUUCGGAGAATT-3,5-UUCUCCGAACGUCACGUTT-3;the mi RNA-451 mimics group transfection with mi RNA-451 mimics 5-AAACCGUACCAUUACUGAGUU-3,3-CUCAGUAAUGGUAACGGUUUU-5.Real-time quantitative polymerase chain reaction(RT-PCR)was used to detect the expression level of mi RNA-451 in glioma cell lines after transfection.Western Blot was used to detect the expression of E-cadherin,N-cadherin,Vimentin,Twist,Snail.The expression of N-cadherin and Vimentin in mi RNA-451 treated group and control group were analyzed by immunofluorescence assay.In our previous study,We used the STRING computer software to construct mi RNA-451 to regulate the signaling pathway of EMT-associated proteins.Western blot was used to verify the relative protein expression of related target proteins(CAB39,AMPK,PI3 K,AKT,phosphorylated AKT)in mi RNA-451 treated group.(2)To investigate the effect of mi RNA-451 on human glioma cells.The experiment group is the same as before,quantitative polymerase chain reaction(RT-PCR)was used to detect the expression level of mi RNA-451 cells in each group of glioma cell lines;Transwell assay was used to detect the invasive ability of glioma cell lines;Cell scratches were used to detect the migration ability of glioma cell lines;The proliferation of glioma cell lines was verified by cell counting cassette(CCK-8)and monoclonal assay;Western blot was used to detect the proterin expression of glioma cell lines relates to biological behavior.Results:(1)The expression of mi RNA-451 in treated group was significantly increased when compared with the control group and the negative group.Western Blot showed that the expression of E-cadherin in mi RNA-451 treated group was significantly increased,and the protein expression of Twist,Snail,N-cadherin and Vimentin were significantly decreased,the difference was statistically significant(P <0.05).The immunofluorescence showed that the expression of N-cadherin and Vimentin in the mi RNA-451 treated group was significantly lower than that in the control group(P <0.05),and the results were compared with those in the Western Blot.The mi RNA-451 target CAB39 gene can influence the expression of EMT-related protein through AMPK and PI3K/AKT pathway in the specific signaling pathway,and then the effect on the behavior of cancer cells.Western blot showed that mi RNA-451 regulated the expression of CAB39,AMPK,PI3 K and phosphorylated Akt in EMT-related protein signaling pathway in mi RNA-451 treated group,and the results showed that the expression of mi RNA-451 was decreased(P <0.05).(2)RT-PCR experiment showed that the expression of mi RNA-451 treatment group increased significantly when compared with control group.Transwell experiments demonstrated that mi RNA-451 treatment group decreased the ability of invasive.Cell migration was performed on mi RNA-451 treated cells;CCK-8 and monoclonal assay showed that the cytokine capacity of mi RNA-451 treated group was weakened.Western blot analysis showed that the expression of Cyclin D1,MMP-2 and MMP-9 protein in mi RNA-451 treated group was significantly decreased(P <0.05).Conclusion: The results showed that mi RNA-451-targeted CAB39 could significantly inhibit the EMT in glioma cell lines through the AMPK and PI3 K / AKT signaling pathway.Overexpression of mi RNA-451 can inhibit the proliferation,invasion and migration in glioma cells.