The Role And Mechanism Of MiR-3127-5p In EMT,Migration And Invasion Of Non-small-cell Lung Cancer

Background and Objective Lung cancer remains the highest incidence and mortality of malignancies worldwide,in which NSCLC accounts for approximately 80~85%.Despite that considerable progress has been made in lung cancer treatment during recent years,however,the overall prognosis of lung cancer is still unsatisfactory,5-year survival rate is less than 15%.Most patients are diagnosed at late stage and with distantmetastasis mainly contribute to the poor prognosis.EMT refers to the phenomenon that the epithelial cells transformed to mesenchymal cells,which is the key step in tumor metastasis.Wnt/b-catenin signaling pathway is highly conservative in biological evolution.Aberrant activation of this pathway is closely related to tumor occurrence,development,invasion and metastasis,therapeutic resistance of several cancers.microRNAs(miRNAs),one of hot spots in cancer research during last decades,play a very important role in the progression of cancer.In our previous study,we demonstrated that miR-3127-5p functions as tumor-suppressive miRNA and the expression level is associated with tumor recurrence and poor prognosis of NSCLC.However,the underlying mechanism of miR-3127-5p involved in tumor metastasis is not fully clear.Therefore,we aimed to investigate the function and molecular mechanism of miR-3127-5p in NSCLC metastasis and provide theoretical basis for miRNAs-based strategy for cancer treatment.Methods 1.miR-3127-5p inhibits EMT,migration and invasion in NSCLC.(1)Using qRT-PCR to determine the expression of miR-3127-5p in NSCLC tissues and cell lines,and analyze the relationship between miR-3127-5p expression and tumor metastasis;established stably transduced NSCLC cells using lenti-viral vectors;Based on the induction of EMT after TGF-b1 stimulation in stably transfected NSCLC cells,the effect of miR-3127-5p on EMT markers was assessed by RT-PCR and Western Blot;(2)Using wound-healing assay,Transwell invasion assay and cell adhesion assay to assess the abilities of cell migration,invasion and adhesion,respectively;Using BALB/c nude mice to detect the effect of miR-3127-5p during the tumor invasion in vivo;2.The mechanism of miR-3127-5p inhibits EMT,migration and invasion in NSCLC.(1)Using dual luciferase reporter assay to verify the direct binding of miR-3127-5p to target gene;Using RT-PCR and Western Blot to determine the expression of FZD4 in NSCLC cells and tissues;using immunohistochemistry to detect the expression of FZD4 in xenografted tumors;(2)Using functional complementation assays to verify the interaction between miR-3127-5p and FZD4;(3)Using TOPflash/FOPflash luciferase reporter gene assay to assess b-catenin signaling activity;Using Western Blot to detect the protein levels of b-catenin,p-b-catenin,cyclin D1 and c-myc;using immunofluorescence and confocal laser scanning microscope to detect the expression and location of b-catenin;Results 1.miR-3127-5p suppresses EMT and inhibits NSCLC migration and invasion(1)miR-3127-5p is down-regulated in NSCLC tissues compared with normal tissues(p < 0.05);miR-3127-5p is significantly decreased in metastatic NSCLC tissues compared with non-metastatic tissues(p < 0.05).RT-PCR verified the successful establishment of stably transduced A549 and H1299 cells.Cells underwent EMT translation after 5 ng/ml TGF-b1 treatment and miR-3127-5p could reverse the effect induced by TGF-b1 on EMT.(2)Ectopic expression of miR-3127-5p significantly inhibited cell migration and invasion,enhanced cell adhesion as measured(p < 0.05).H&E staining showed that miR-3127-5p overexpressed cells generally formed oval-shaped intracranial tumors and exhibited sharp edges.By contrast,tumors formed by the miR-3127-5p-inhibited cells exhibited highly invasive potential,with the blurry borders displaying a chaotic distribution.2.The mechanism of miR-3127-5p inhibits EMT,tumor migration and invasion(1)By using bioinformatics software and considering biological functions,we screened out potential miR-3127-5p’s target gene FZD4.Dual luciferase reporter gene analysis showed that both two sites in FZD4 3’ UTR are functional binding sites of miR-3127-5p.The mRNA and protein levels of FZD4 were reduced in miR-3127-5p-overexpressed cells.The expression of FZD4 was negatively associated with miR-3127-5p in clinical NSCLC tissues(R =-0.3232,p < 0.001).In addition,FZD4 was much higher in metastatic NSCLC tissues than non-metastatic tumors(p < 0.05).Immunohistochemistry showed that FZD4 expressive intensity of tumors derived from miR-3127-5p-overexpressed cells was much weaker than control group,while expressive intensity of tumors derived from miR-3127-5p-inhibited cells was much stronger than control group.(2)Ectopic expression of FZD4 reversed the effect of miR-3127-5p,while FZD4 inhibition restored the effect of miR-3127-5p.FZD4 overexpression reduced E-cadherin expression,but increased N-cadherin and Vimentin expressions,and it also resulted in increased cell migration and invasion as well as decreased cell adhesion.(3)miR-3127-5p overexpression significantly reduced the luciferase activity of TOPflash/FOPflash.In miR-3127-5p-overexpressed cells,non-active phosphorylated b-catenin was increased and downstream genes including cyclin D1 and c-myc were reduced.Immunofluorescence and confocal microscope indicated that miR-3127-5p inhibition significantly increased active b-catenin expression and induced b-catenin translocation to nucleus.Conclusions miR-3127-5p is down-regulated in metastatic NSCLC and the expression level is associated with EMT markers.miR-3127-5p exerts its role on EMT,cell migration and invasion through regulating Wnt/b-catenin signaling pathway by directly targeting FZD4 gene.Our results revealed the function and mechanism of miR-3127-5p on NSCLC progression,which can provide theoretical basis to the prevention and miRNAs-based therapy of NSCLC metastasis.