MiR-433 Suppressed A549 Cell Proliferation And Migration Through Down-regulating PAK4

ObjectiveIn recent years,the incidence of lung cancer has become increasingly serious,and the most serious form of small cell lung cancer.It has been reported that miR-433 is related to gastric cancer and oral squamous cell carcinoma.No effects of miR-433 on human non-small cell lung cancer A549 has been found.Therefore,this study focused on the role of miR-433 in the proliferation and migration of human non-small cell lung cancer A549 cells,thus providing a scientific basis for the diagnosis of non-small cell lung cancer.MethodFirst,the expression of mi R-433 in A549,NCI-H1299,NCI-H358 and human lung fibroblast cell line HFL1 was detected by Real-time PCR.Different concentrations of miR-433 mimic were transfected into A549 cells,and the optimal concentration was selected for subsequent analysis;MTT assay was used to detect cell proliferation;Transwell chamber was used to detect cell migration;dual luciferase reporter gene method was used to detect the target relationship between miR-433 and PAK4.Western blotting was used to detect the expression of P21-activated kinase 4(PAK4),LIMK1,p-LIMK1,Cofilin and p-Cofilin.Results1.The expression of miR-433 in three kinds of NSCLC cells decreased significantly.The average expression level of mi R-433 in A549 group was(0.42 ± 0.047).The difference was statistically significant after one-way analysis of variance(F=20.36,P<0.003).2.The relative expression of miR-433 in the miR-433 mimic(80 nM)group was significantly higher than that in the other three groups(142.02±10.25).There was a statistically significant difference between the four groups after one-way ANOVA(F=26.32,P=0.004).Tip overexpression of miR-433 can significantly inhibit cell proliferation.3.In order to understand the effect of overexpression of miR-433 on the migration of A549 cells,a Transwell test was performed.Compared with the blank control group,the miR-433 mimic group showed a significant decrease in A549 cells.MiR-433 mimic significantly inhibited A549 cell migration(P=0.011).The mimic control and miR-433 mimic luciferase activity in the WT group were 1.00±0.120 and 0.42±0.024,respectively,suggesting that miR-433 mimic significantly inhibited luciferase activity(P=0.015)in order to observe the PAK4 gene and the protein expression was related to the three cells of the NSCLC cell line.The PAK4 protein was 3.25±0.21,3.35±0.39,and 3.38±0.15 in A549,NCI-H1299,and NCI-H358,respectively.It can also be seen from the bands that A549,NCI-H1299,and NCI-H358 are significantly thickened,which suggests that the PAK4 gene may be the target gene of the cells of NSCLC cells.5.Analysis of the effects of the blank control group,mimic control,and mi R-433 mimic PAK4,p-LIMK1,LIMK1,p-coflilin,coflilin,and GAPDH signaling pathways revealed that miR-433 mimic transfection significantly inhibited PAK4,pThe expression of LIMK1 and p-coflilin was significantly sparse and thin.Conclusion1.miR-433 is downregulated in non-small cell lung cancer cell lines.2.Over-expression of miR-433 can inhibit the proliferation and migration of A549 cells.3.miR-433 inhibits the LIMK1/cofilin signaling pathway and inhibits the proliferation and migration of A549 cells through targeted down-regulation of PAK4 expression.4.The further study of miR-433 mechanism provides new research directions for the rational prevention,scientific diagnosis and prognosis of non-small cell lung cancer.