| BackgroundNon-small cell lung cancer(NSCLC)comprises 80%-85% of lung cancer cases,and is a leading cause of cancer deaths worldwide.Over the last 10 years,in spite of considerable progress made in the research on the molecular mechanisms in lung cancer,and the adoption of combined therapy,including surgical resection,chemotherapy,radiation therapy,molecular targeted therapy,and immunotherapy,its dismal 5-year survival rate has not improved substantially.Only 15.9% of lung cancer patients have survived 5 years or more,while most patients died of tumor recurrence and metastasis.At present,the cancer stem cell hypothesis is widely accepted: In malignant tumor,there exist cell subsets with stemness properties,which are called cancer stem-like cells(CSLCs)or tumor initiating cells(TICs)that are capable of self-renewal and multipotent differentiation,and are closely related to the initiation,metastasis,relapse and chemo-radio resistance of tumor.The existence of CSLCs has been confirmed in hematopoietic malignancies and many kinds of solid tumors,such as lung cancer,breast cancer,prostate cancer and glioma.CSLCs can be isolated and identified by floating sphere formation in serum-free medium or/and fluorescence-activated cell sorting(FACS)based on surface marker.The surface markers that can be used to identify lung CSLCs are CD133+,CD326+,CD44+,CD166+,ALDH1A1+,Sca1+/CD34+(in mice),etc.All these surface markers may express themselves alone or co-express themselves to show the CSLCs properties.The treatment directed at CSLCs is highly critical for the radical treatment of tumor.Epithelial-to-mesenchymal transition(EMT)is the process by which epithelial cells lose the epithelial sheet and acquire a mesenchymal phenotype.EMT is not only involved in embryonic development and organ fibrosis,but also plays an important role in tumor invasion and metastasis.In this process,the expression of E-cadherin on the surface is down-regulated,while the expression of vimentin and N-cadherin is up-regulated,and the cell-cell junctions are disassembled and actin cytoskeleton is reorganized,thus increasing cell motility and promoting the invasion and metastasis of tumor.EMT is not only regulated by the signal pathways(Wnt,TGF-?,Notch,etc),but also microRNAs,the small non-coding single-strain RNA molecules that are 19-22 nucleotides long and control the target protein expression of target DNA post-transcription by inducing mRNA degradation or inhibiting its translation.It is presently reported that,in addition to its role of oncogene and anti-oncogene,microRNAs participate in the process of EMT.For example,the miR-200 family plays an essential role in EMT suppression mainly through targeting ZEB expression in lung and breast cancers,and mi R-214 induces EMT in ovarian cancer cell by PI3K/AKT pathway.Increasing evidence suggests that microRNAs can serve as a novel therapeutic target as well as a diagnostic and prognostic marker in cancer.Because of the important role of microRNAs in EMT of both CSLCs and cancer cells,their regulation network is a research hotspot at present.Many questions remain to be answered in this field: Whether the EMT of CSLCs can be induced? Are they the root cause of tumor invasion and metastasis? Can they be regulated by micro RNAs? How do they work? And whether can the underlying mechanisms be used to guide the clinical treatment? This research aims to answer the above these questions.Objectives1.To induce EMT in CSLCs of lung adenocarcinoma.2.To select the differentially expressed microRNAs(miR-181b-5p)in CSLCs after EMT.3.To confirm that miR-181b-5p regulates the process of EMT in CSLCs in vitro and in vivo,and elucidate the underlying mechanism.Methods1.Inducing EMT in lung CSLCs(1)Suspension culture of A549 cells in serum-free medium was applied to generate floating cell spheres.(2)The CD133+/CD326+ cells were enriched by FACS,and the “stemness” was tested by xenograft assay,real-time PCR that was used to detect the expression of CD133,CD326,OCT-4 and Nanog,and confocal laser scanning microscope(CLSM).(3)EMT was induced by TGF-?1 in CD133+/CD326+ cells,and was verified by Transwell invasion assay and real-time PCR at the morphological and gene levels.2.Detecting the differentially expressed microRNAs after the induced EMT using miRNAs array,and selecting miRNAs of interest(1)The microRNA expression profiles between A549,A549+TGF-?1,CD133+/CD326+ cells and CD133+/CD326+ cells,and +TGF-?1 were acquired by real-time PCR miRNA array.(2)The target genes of differentially expressed microRNAs were predicted and their function was analyzed,the microRNA-Gene-Network was constructed,the microRNAs of interest were selected,and were verified by real-time PCR.3.Confirming that miR-181b-5p regulates the process of EMT in CSLCs in vitro and in vivo,and elucidating the underlying mechanism(1)MiR-181b-5p agomir and antagomir were constructed and transfected into A549 and CD133+/CD326+ cells.The expression of miR-181b-5p was tested by real-time PCR.(2)The expression of E-cadherin was tested by real-time PCR and Western blot assay.(3)The invasion ability was tested after transfection by Transwell invasion assay.The metastasis formation ability was confirmed by injecting agomir into xenograft in nude mice.(4)The expression of mi R-181b-5p in peripheral circulation was compared between normal persons and tumor sufferers.Results1.The EMT model of CSLCs of lung adenocarcinoma was established.We successfully induced a lot of lung adenocarcinoma floating cell spheres by suspension culture of A549 cells in serum-free suspension medium and selected CD133+/CD326+ cells by fluorescence-activated cell sorting.The identification experiments demonstrated that CD133+/CD326+ cells have “stemness” and can be recognized as CSLCs.Next,we induced EMT in CD133+/CD326+ cells by TGF-?1,which resulted in the formation of mesenchymal cells.Quantitative real-time PCR identified the significantly positive change in the genes associated with EMT,and Transwell invasion assay confirmed that invasion and metastasis ability of cells have been improved.Based on the above results,we can confirm that we have successfully established the EMT model of CSLCs of lung adenocarcinoma.2.MiR-181b-5p involved in the EMT of the CSLCs was selected.We acquired the differentially expressed microRNA profiles between A549,A549+TGF-?1,CD133+/CD326+cells and CD133+/CD326+cells,and +TGF-?1 by real-time PCR mi RNA array.In order to find the targeted microRNAs,we analyzed the data by Venn diagram.Through the Targetscan and miRanda database,the target microRNAs and their binding sites were predicted.Based on the KEGG database,the targeted microRNAs involved in the signal pathways were found by Pathway analysis.We constructed the gene-protein-pathway map and selected core microRNAs in this net.Using quantitative real-time PCR to verify the expression of microRNAs,we found that only the expression tendency of miR-181b-5p is consistent with the array data.In addition,the expression of mi R-181b-5p was up-regulated in the process of EMT induced by A549 and CSLCs.Therefore,we chose miR-181b-5p as the experimental object.3.MiR-181b-5p promotes the EMT of the CSLCs and the metastasis formation by down-regulating the expression of E-cadherin.We transfected agomir and antagomir of miR-181b-5p into A549 and CD133+/CD326+ cells,and detected the expression of miR-181b-5p by quantitative real-time PCR.Results showed that agomir could enhance miR-181b-5p expression,while antagomir could inhibit it.We tested the invasion ability by Transwell invasion assay,and the results indicated that after the transfection with agomir,the invasion ability was enhanced in the four kinds of cells.In contrast,after the transfection with antagomir,their invasion ability was reduced.It indicates that the expression of miR-181b-5p is positively correlated with the invasion ability of cells.In this process,by quantitative real-time PCR and Western blot,we found that only the expression of E-cadherin gene and protein was consistent with the tendency of miR-181b-5p expression,suggesting that E-cadherin gene might be a downstream target gene of the mi R-181b-5p.By injecting agomir into xenograft in nude mice,we found that agomir can increase the incidence of tumor metastasis.We compared the expression of miR-181b-5p in peripheral circulation between normal persons and tumor sufferers,and found that the expression of miR-181b-5p in peripheral blood of tumor sufferers was higher than that of normal persons.Thus,miR-181b-5p may be a potential therapeutic target and a new prognostic marker in NSCLC.ConclusionMiR-181b-5p enhances the formation of EMT of CSLCs and the invasion ability of cancer cells,and promotes the metastasis formation in xenograft model,by down-regulating the expression of E-cadherin.Clinical experiments also show that miR-181b-5p expression is correlated with tumor metastasis in lung adenocarcinoma patients.Therefore,it is valuable to further study and apply miR-181b-5p in lung cancer diagnosis and staging. |
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