Regulation and mechanism of miR-9-5p on proliferation,invasion and migration of lung adenocarcinoma cells
Objective To observe the regulation of microRNA(miR)-9-5p on the proliferation,invasion and migra-tion of lung adenocarcinoma cells,and to explore the related mechanisms based on SPG-20 gene and Notch signaling path-way.Methods Lung adenocarcinoma cell line A549 was cultured and divided into the inhibitory group,negative control group and untransfected group,respectively.Cells in the inhibitory group and negative control group were transfected with miR-9-5p inhibitor and negative control group,respectively.Cells in the untransfected group were not transfected.Cell proliferation ability was detected by MTT assay,cell invasion ability was detected by Transwell chamber assay,cell migra-tion ability was detected by scratch healing assay,and Notch signaling pathway-related proteins(E-cadherin,Notch1,Snail,and MMP-2)were detected by Western blotting.TargetScan(http://www.targetscan.org/vert_80/)was used to predict binding sites between miR-9-5p and SPG20,which was identified by the dual luciferase report gene analysis.The mRNA and protein of miR-9-5p and SPG20 in lung adenocarcinoma tissues and cells were detected.A549 cells were divid-ed into the inhibitor group,negative control group and untransfected group,and SPG20 mRNA in each group was detected by RT-PCR.Results The cell proliferation ability was lower in the miR-9-5p inhibitor group than in the negative control group and untransfected group at 12,24,36 and 48 h(all P<0.05).The number of transmembrane cells and cell migra-tion distance in the inhibitor group were lower than those in the negative control group and transfection group(all P<0.05).The expression levels of Notch1,Snail and MMP-2 protein in the inhibitor group were lower than those in the nega-tive control group and non-transfected group,and the expression level of E-cadherin protein was higher than those in the negative control group and non-transfected group(all P<0.05).SPG20 gene had a continuous nucleotide sequence that complemented miR-9-5p.The relative luciferase activity of A549 cells co-transfected with SP2020-WT and miR-9-5p was lower than that of other cells(all P<0.05).The expression of miR-9-5p was high and the expression levels of SPG20 mRNA and protein were low in the lung adenocarcinoma tissues and cells(all P<0.05),and the expression of miR-9-5p was negatively correlated with SPG20 mRNA in lung adenocarcinoma tissues(r=-0.914,P<0.05).The expression of SPG20 mRNA was higher in the inhibitor group than in the negative control group and non-transfected group(all P<0.05).Conclusion MiR-9-5p can inhibit the proliferation,invasion and migration of lung adenocarcinoma cells,and its mechanism may be related to the negative regulation of SPG20 expression and Notch signaling pathway.
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