| Background and objectiveLung cancer is the leading cause of cancer deaths,accounting for 18%of cancer deaths worldwide.It has become the most malignant tumor that threatens human health and life.The reason for this is that,besides the complex biological characteristics of lung cancer and the high degree of malignancy,it is more important that most of the lung cancer patients have had local or extensive metastasis at the time of diagnosis.Compared with other types of lung cancer,the composition of lung adenoearcinoma in lung cancer is increasing year by year,and it is more prone to metastasis.In patients with advanced lung adenocarcinoma with metastases,the 5-year survival rate after treatment is less than 10%.Lymphatic metastasis is one of the main causes of poor prognosis and death in lung cancer.Therefore,effective prevention and treatment of lymphatic metastasis of lung adenoearcinoma is one of the difficult problems to be solved in clinical practice.In our previous study,we choosed multiple lung adenocarcinoma cell lines in the process of studying the relationship between TGF-?1 and lung adenocarcinoma with lymph node metastasis.After treatment with TGF-?1,some cell lines were transformed into epithelial cells,while another part of the cells still retain the morphology of epithelial cells.It is suspected that two types of lung adenocarcinoma cells that respond differently to TGF-?1 may be two types of lung adenocarcinomas that are biologically different from EGFR mutant lung adenocarcinoma and EGFR wild-type lung adenocarcinoma.If the conjecture is true,the treatment and prognosis may be different.Therefore,we divided the lung adenocarcinoma cells into TGF-?1 sensitive group and insensitive group according to the morphological changes after TGF-?1 treatment.The biological behaviors of EMT markers,stem cell markers such as OCT4,SOX2,Nanog and invasion and proliferation ability were analyzed.If valuable,further study on the expression of VEGF-C and VEGFR3 and the mechanism by how TGF-?1 affects the expression of the above molecules.Further exploration of TGF-?1 to classify lung adenocarcinoma into sensitive and non-sensitive types provides new research ideas for the prevention and treatment of patients with lymphatic metastasis from lung adenocarcinoma.MethodsA number of different lung adenocarcinoma cell lines(NCI-H1993,A549,NCI-H358,NCI-H1975,NCI-H1650,HCC827)were selected and observed for morphological changes after 2.5 weeks treatment with 2.5 ng/ml TGF-?1.According to the morphological changes of EMT after treatment,the above cell lines were divided into TGF-?1 sensitive group and insensitive group.Cell invasion assay and colony formation assay were used to analyze whether the proliferation and invasion ability of the two cell lines were different;PCR and western blot were used to detect the expression of EMT markers like vimentin and cadherin,stem cell markers like SOX2,OCT4,Nanog and VEGFR3 in two cell lines;then detecting the activation of ERK signaling pathway in VEGF-C in two groups;TGFpRI kinase inhibitor and siRNA that targeted to inhibit the expression of TGF-?1 were used to verify whether this pathway is involved in the up-regulation of VEGF-C expression in the two cell lines.Result1.TGF?1 induces EMT in certain lung adenocarcinoma cell lines.A549 andNCI-H1993 cell images were taken before and after incubation with 2.5 ng/mlhuman recombinant TGF?1 for two weeks.They transited from epithelial tomesenchymal like cells.Morphology changes of NCI-H1650 and HCC827 cells were not noted after TGFpl treatment.Real-time PCR revealed that 2.5 ng/ml TGF?1 treatment for two weeks significantly reduced E-cadherin mRNA expression in A549 and NCI-H1993 cells(compared with NCI-H1975,**P<0.01,***P<0.001)but not in NCI-H358,NCI-H1975,NCI-H1650 and HCC827 cells.At the same time,Vimentin mRNA expression was significantly enhanced in A549,NCI-H358 and NCI-H1993 cells but not in NCI-H1975,NCI-H1650 and HCC827 cells.Western blotting confirmed that TGFpl increased Vimentin and reduced E-cadherin protein expression in TGF?1 sensitive NSCLC line.2.TGF?1 reduces cell viability and increases cell invasion and colony formation ability in TGFpl sensitive lung adenocarcinomacell lines.CellTiter-Glo luminescent assays showed that 2.5 ng/ml TGF?1 treatment for two weeks significantly reduced A549,NCI-H358 and NCI-H1993 cell viability but had no effects on NCI-H1975,NCI-H1650 and HCC827 cell growth(compared to control without treatment,**P<0.01,***P<0.001).Cell invasion assays revealed that 2.5 ng/ml TGF?1 treatment for two weeks significantly increased the number of A549,NCI-H358 and NCI-H1993 cells which migrated through the chambers.NCI-H1975,NCI-H1650 and HCC827 cells did not exhibit the similar properties.2.5 ng/ml TGFpl treatment for two weeks significantly enhanced anchorage-dependent colony formation ability of A549,NCI-H358 and NCI-H1993 cells but not for NCI-H1975,NCI-H1650 and HCC827 cells(*P<0.05,**P<0.01).3.TGF?1 increases the gene expression of lung adenocarcinoma stem-like cell markers in TGFpl sensitive lung adenocarcinoma cell lines.Real-time PCR showed that 2.5 ng/ml TGF?1 treatment for two weeks significantly increased Oct4 and Sox2 mRNA expression in A549,NCI-H358 and NCI-H1993 cells compared to untreated cells(**P<0.01,***P<0.001).Oct4,Nanog and Sox2 expression remained same before and after TGFpl treatment.4.TGF? pathway is involved in the regulation of VEGF-C expression in TGF?1 sensitive lung adenocarcinoma cell lines.Real-time PCR showed that VEGFR3 mRNA expression levels were much higher in A549,NCI-H358 and NCI-H1993 cells than that in NCI-H1975,NCI-H1650 and HCC827 cells(fold expression as compared to control NCI-H1975,***P<0.001).Immunoblotting showed that human recombinant VEGF-C 10 ng/ml treatment for 30 and 60 minutes activated ERK pathway in NCI-H1993 cells but not in NCI-H1975 cells.Real-time PCR revealed that 2.5 ng/ml TGF?1 treatment significantly increased the VEGF-C mRNA expression in NCI-H1993 cells.The presence of 0.1?M LY2157299 significantly reduced TGF?1-induced VEGF-C expression.Similarly,siRNA targeting TGF?R1 significantly decreased TGF?1-induced VEGF-C expression compared to scramble control(***P<0.001).Conclusion1.TGF?1 can induce EMT in some lung adenocarcinoma cell lines,and promote the expression of stem cell characteristics and proliferation and invasion ability in such cell lines;2.Different lung adenocarcinomacells have different sensitivity to TGF?1,which may be related to cell lines derived from lung cancer patients with different stages and cell variability,thus introducing the concept of sensitivity;3.The expression of VEGF-C and VEGFR3 in TGF?1-sensitive cell lines was consistent with the expression of stem cell characteristics and cell function,suggesting that TGF?1 pathway may be involved in the regulation of VEGF-C expression in sensitive lung adenocarcinoma cell lines;4.This study explored the correlation between TGF?1 and stem-like properties,cell function and VEGF-C,which can be used as a new standard type for lung cancer and provide ideas with exploring targets for inhibiting lymphatic metastasis. |
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