The Role And Mechanism Of MiR-493-5p In Promoting The Invasion And Metastasis Of Lung Cancer Cells Induced By CCL19

Objectives:The role of chemokines in the development of non-small cell lung cancer has received increasing attention.As a member of this group,CCL19 can promote lung cancer invasion and metastasis by binding to its receptor CCR7,but its specific mechanism is still unclear.In this experiment,chip hybridization was used to screen differentially expressed miRNAs after CCLl9 stimulation.From the perspective of miRNA,the molecular mechanism of CCL19 promoting invasion and metastasis of lung cancer cells was explored,which provided new biological indicators and potential targets for the diagnosis and treatment of non-small cell lung cancer.Methods:(1)Cell culture:Human lung adenocarcinoma cell line A549 was routinely cultured in F12 medium.(2)Screening of differentially expressed miRNAs:A549 cells were treated with 400 ng/ml CCL19 factor for 12 hours,cells were harvested,total cellular RNA was extracted,and Exiqon miRNA microarray hybridization was performed,and differentially expressed miRNAs were screened against A549 cells without CCL19 factor treatment,verified by q-PCR.The starBase and TargetScan databases were used to predict target genes for differential miRNAs.Selected miR-493-5p with significant differential expression for further study.(3)Construction of stable transgenic cell lines:Lentiviral expression vector was constructed by LV-hsa-mir-493-5p(42841-1)virus and GV309 vector(component sequence hU6-MCS-Ubiquitin-EGFP-IRES-puromycin),transfected into A549 cells,filtered by puromycin and got the cell line LV-has-miR-493-5p-A549 stably overexpressing miR-493-5p and the negative control cell LV-NC-A549.(4)Western blotting was used to detect the expression of E-cadherin,N-cadherin,Snail,c-Met and AKT proteins.(5)Scratch test and Transwell test were used to detect the migration and invasion ability of lung cancer cell line A549.(6)Analysis of clinical tissue samples of non-small cell lung cancer:58 cases of postoperative paraffin specimens and 6 specimens of paracancerous lung tissue were collected from lung cancer patients,serial sections were taken,total RNA was extracted,and differential miRNA expression was detected by q-PCR.(7)Statistical analysis was performed using GraphPad Prism 6 statistical software.The measurement data were expressed as mean ± Standard error.The t test was used for comparison between the two groups.The multivariate measurement data were analyzed by one-way ANOVA.The difference was statistically significant at p<0.05.Learning meaning.Results:(1)Several miRNAs were differentially expressed after CCL19 factor treated A549 cells,and there were 11 miRNAs up-regulated by 2 times or more with statistically significant difference,and there were 17 miRNAs down-regulated by 0.5-fold or less and the difference was statistically significant.(2)Chemokine CCL19 down-regulated the expression of E-cadherin protein in A549 cells(p<0.05),up-regulated the expression of N-cadherin protein(p<0.01)and Snail protein(p<0.05);overexpressed miR-493-5p can attenuate the down-regulation of E-cadherin protein expression by CCL 19 factor(p<0.01)and up-regulate the expression of N-cadherin protein(p<0.05)and Snail protein(p<0.01).(3)The chemokine CCL19 could significantly increase the migration and invasion ability of A549 cells(p<0.05).Overexpression of miR-493-5p could attenuate the promotion of CCL19 factor on the migration and invasion of A549 cells(p<0.05).(4)The chemokine CCL 19 could down-regulate the expression of c-Met and AKT protein in A549 cells(p<0.05).The overexpression of miR-493-5p could attenuate the effect of CCL19 on the expression of c-Met protein and AKT protein in A549 cells(p<0.05).(5)Compared with normal lung tissues,the expression of miR-493-5p was down-regulated in non-small cell lung cancer tissues(p<0.05);miR-493 in cancer tissues with lymph node metastasis or vascular tumor thrombus The content of-5p was significantly lower than that of non-metastatic patients(p<0.05);male and female lung cancer patients,Xuanwei lung cancer and non-Xuanwei lung cancer patients,miR-493-5p in tissues aged 60 years and older and patients under 60 years old There was no significant difference in expression(p>0.05).Conclusions:CCL19 can down-regulate the expression of miR-493-5p and abolish the inhibitory effect of miR-493-5p on c-Met,which may activate AKT signaling pathway,promote epithelial-mesenchymal transition in lung cancer cell A549,and increase the migration and invasive ability of A549 cells.