MicroRNAs Screening In Lung Cancer Cell Line And The Metastasis Mechanisms Promoted By MiR-544a

The incidence and motality of lung cancer is very high in malignant tumors.Without typical early symptoms, screening sensitive mrkers and effective earlydiagnosis, most patients are in middle or advanced stage and miss effective treatmentwhen seeing a doctor. Poor prognosis of lung cancer is often due to recurrence andmetastasis. Even after complete resection, many patients die of recurrence andmetastasis. Further study of the molecular mechanisms of lung cancer metastasis cancontribute to prevent recurrence and metastasis of lung cancer. miRNAs are newmolecular tumor biomarkers. Oncogenic miRNAs induce cancer cell proliferation bydown-regulating expression of tumor suppressor genes, whereas tumor suppressormiRNAs inhibit cancer progression by targeting oncogenes post-transcriptionally.Further study of miRNAs in tumor can bring new diagnostic plans and therapeutictargets.1. miRNAs screening95C and95D are lung cancer cell lines with different metastatic ability. After totalRNA extraction, miRNA array is used to screen different miRNAs in95C and95D.Results show that miRNA expression is abnormal. miR-544a, miR-320, miR-22,miR-103, miR-565and miR-23a are up-regulated in95D, while miR-135b,miR-455-5p, miR-196a, miR-448, miR-888, miR-193a-5p, miR-28-5p andmiR-139-5p are down-regulated in95C.The sensitivity of miRNA array is very high and there are false positive results. Inorder to reduce false positive results, QPCR is used to verify the results of miRNAarray. QPCR show that the relative level of miR-544a in95D is higher and is almost3fold than that in95C. QPCR results are consistent with those of miRNA array.2. miR-544a promotes the invasion of lung cancer cells by down-regulating CDH1and up-regulating Vimentin Targetscan shows that miR-544a can combine with CDH1, GSK-3β tightlyindicating that CDH1, GSK-3β may be the target genes of miR-544a. Luciferase assayis carried out to verify Targetscan result, showing that miR-544a mimic can combinewith CDH1or GSK-3β resulting in the decrease of RLUC/FLUC ration. These results pointout that CDH1, GSK-3β are the target genes of miR-544a.miR-544a mimic is transfected to95C. Transwell, scratch assay and Western blotare carried out in transfected cells to detect invasion ability and CDH1, Vimentinexpression. Results show that invasion ability of transfected cells increases, CDH1isdown-regulated and Vimentin is up-regulated.3. Lung cancer cells obtaining features of cancer stem cells by miR-544a targetingGSK-3βmiR-544a sequence is cloned to vector. After miR-544a-vector is transfected with95C and95D, QPCR, Western blot and sphere suspension culture are carried out.QPCR show that miR-544a in tranfected cells is up-regulated.Western blot results indicate that GSK-3β is down-regulated and β-catenin, CD133are up-regulated in transfected cells. As one target gene of miR-544a, GSK-3β isdown-regulated by miR-544a and the degradation of β-catenin is inhibited resulting inhigher β-catenin level in cytoplasm. At last Wnt/β-catenin pathway is activated andcells invasion, metastasis and proliferation ability is promoted. CD133, one marker ofcancer stem cell, is also up-regulated. Sphere suspension culture shows that sphere isformed. These results show that transfected cells obtain the features of cancer stemcells.Conclusions are drawn below:1) miR-544a is up-regulated in95D.2) CDH1and GSK-3β are target genes of miR-544a.3) EMT is induced and invasion ablity is prompted by miR-544a targeting CDH1.4) Wnt/β-catenin passway is activated and CD133is up-regulated by miR-544atargeting GSK-3β. And lung cancer cells obtain the features of cancer stem cell.