The Regulatory Role Of MicroRNAs On Biological Behaviors Of Lung Cancer Cells

Background:Lung cancer,the leading cause of cancer deaths,has the most rapidly increasing incidence rate in the developed country as well as in China.Clinical data have showed that most lung cancer patients eventually suffered relapse and/or metastasis after complete excision of the cancer,even if they were at stage IA.Despite the great progress that has been made in the past decades,the prognosis for lung cancer patients is poor. So,it's urgent for us to further illuminate the mechanism of lung cancer development and progression,including relapse and metastasis.MicroRNAs(miRNAs) are a class of small,non-coding RNA that play important roles in various biological processes through negatively regulating targeted genes.Bioinformatic methods predicted that in approximately one-third of all mammalian genes,especially those in down-stream of various signal transduction pathway,were targeted by miRNAs.Interestingly, accumulating evidence has implicated miRNAs in human cancer, including tumor invasion and metastasis.However,the role of miRNAs in tumor development,including mediating tumor metastasis,has been only recently investigated and still remains largely elusive.Objects:1 To determine whether there was a relationship between miRNAs and lung cancer metastasis.2 To explore the mechanism of miRNAs in mediating lung cancer metastasis.3 To study whether miRNAs expression was correlated to activation of EGFR signaling pathway in lung cancer cells.4 To explore the function of EGFR-induced miRNAs in lung cancer cells.Methods:1 MicroRNA array and quantitative real time RT-PCR were used to find different expression of miRNAs between Paired high and low-metastatic human pulmonary giant cell carcinoma cell,801D and 95C,and to identify EGFR-induced miRNAs in lung cancer cell lines. 2 Functional analysisThe impact of miRNA on lung cancer cell biological behaviors was studied by Transwell Insert,wound healing experiment,tube formation assay,cell proliferation,apoptosis,and cycle analysis.3 Targets of miR-183 were bioinformatically predicted and verified by Western Blot and Luciferase reporter gene assay, respectively.4 Affymetrix U133 plus 2 GeneChip was used to detect metastasis-related genes which responded to ectopic miR-183 expression.Results:1 miRNA-183 expression in lung cancer cellsAmong 711 miRNAs screened,92 miRNAs exhibited significantly different expression level between 801D and 95C cells. MiR-183 was one of the most obviously altered miRNAs. Consistent with the array results,real time RT-PCR showed that miR-183 expression level in 801D was less than half of that in 95C.2 The impact of miR-183 on cell migration and invasionTranswell Insert results showed that additional miR-183 significantly inhibited both migratory and invasive numbers of 801D cells.We then assayed the polarized migration of cells using scratch-wound model.Similar to Transwell Insert analysis,cells transfected with miR-183 closed the scratch-wounds more slowly than cells un-treated or transfected with negative control.3 Targets of miR-183 prediction and verificationBioinformatic methods predicted that VIL2,which contained the binding site of miR-183 in its 3'UTR,could be regulated by miR-183.To verify experimentally,we first examined Ezrin protein,encoded by VIL2,expression by Western Blot.Our data showed that Ezrin expressed at a significantly higher level in 801D than in 95C.Interestingly,after transfection with miR-183,Ezrin expression in 801D cell was almost abolished. To further confirm VIL2 as a direct target of miR-183,the 3' VTR of VIL2,containing binding site of miR-183,was fused with a luciferase reporter gene,and the resultant luciferase activities were examined.As expected,over-expression of miR-183 resulted in significant decrease in the luciferase activity.4 Microarray detection and GO analysisWith the criterion of a Pvalue＜0.05 and at least 2-fold changes of expression level,we identified 424(258 repressed and 166 up-regulated) genes that were differentially expressed.Genes,whose expression level was significantly altered, were grouped by their assigned biological functions using the GO database.Interestingly,we found that among the 424 miR-183-responding genes,9.69%(25/258) of down-regulated genes and 7.23%(12/166) of up-regulated genes were functioning in cell adhesion,migration,invasion,and/or metastasis, including:CCL5,which enhanced motility,invasion,and metastasis of breast cancer cells through the chemokine receptor CCR5;CTHRC1,which promoted melanoma cells migration and were aberrant in human solid cancers;CYR61 and various adhesion,migration,invasion and angiogenesis-associated proteins.5 Identification of EGFR-induced miRNAWith the criterion of a Pvalue＜0.01,39 miRNAs expression were significantly altered after EGF stimulation,Among them, 5 miRNAs,let-7i,miR-24,miR-25,miR-29b,and miR-125a-5p, could be revised by gefitinib,a tyrosine kinase inhibitor of EGFR.Real time RT-PCR confirmed that expression of the above 4 miRNAs,except for let-7i,were remarkably influenced by EGF addition,and could be resumed by gefitinib.The most pronounced change in miRNA abundance after EGFR activation was observed for miR-125a-5p.6 Role of miR-125a-5pThe migratory and invasive cell numbers and proliferous ratio of PC9 lung cancer cell lines and abilities of tube formation of ECV304 vascular endothelium cell were significantly enhanced after ectopic transfection of antisense of miR-125a-5p.However,it didn't influence apoptosis,and cell cycle of PC9.Conclusions:1 Expression level of miR-183 was reversely correlated with the metastatic potential of different lung cancer cells.2 MiR-183 was a potential metastasis-inhibitor,by which directly down-regulated Ezrin expression.3 Expression of 4 miRNAs,miR-125a-5p,miR-24,miR-25,and miR-29b,were regulated by of EGFR signaling activation.4 MiR-125a-5p could negatively regulate the biological behaviors of lung cancer cells.