| ObjectivesDistant metastasis of tumor cells is the leading cause of cancer death,accounting for over 90% of cancer deaths.Early metastasis is common in lung adenocarcinoma(LAC),but the molecular and biological mechanisms that enable LAC cells to leave primary tumors and establish new tumors in a new organ remain undefined,it is still the focus of our research.Therefore,better understanding of the molecular biological changes that leading to LAC metastasis has important clinical significance for the diagnosis,targeted treatment and prognosis of LAC.The cell surface antigen CD109 is a TGF-β co-receptor.Recent studies have shown that CD109 plays an important role in the development of tumors.It is reported that in some metastatic LAC,the expression of CD109 was significantly up-regulated,and the signaling pathway mediated by Janus kinase and the transcription factor STAT3 may be the key target molecule for CD109 to drive LAC metastasis.However,it has not been studied whether this regulatory mechanism is cell-specific and whether CD109 can interfere with the metastasis process of LAC through other regulatory mechanisms.Therefore,in order to further explore the mechanism of CD109 regulating LAC,this study proposed to simulate the overexpression of CD109 in human LAC tumor cells by A549 cells,to further reveal the transcriptional regulation mechanism of CD109 on LAC.This study provides a new clue for the diagnosis,treatment and prognosis of LAC.Methods1.The expression of CD109 in metastatic and non-metastatic lung adenocarcinoma tumor tissues from tissue microarrays(TMAs)was detected by immunohistochemical staining.2.Construction of CD109-adenovirus vector.3.Culture human LAC cells,A549,transfected A549 cells with adenovirus expression vector,and examined the expression of CD109 in A549 cells by quantitative Real-time Polymerase Chain Reaction and Western blot.4.The expression of p-STAT3 and STAT3 in A549 cells was detected by Western blot,and the phosphorylation ratio of cells was calculated to detect whether the overexpression of CD109 in A549 cells had an effect on the phosphorylation level of STAT3.5.After overexpression of CD109,A549 cells were tested for cell proliferation and cell viability using MTT,CCK-8,and trypan blue staining assays.And the effect of CD109 on the migration ability of A549 cells was detected by cell Wound healing assay and Transwell migration assay(Boyden chamber migration assay).6.After overexpression of CD109,A549 cells were pretreated with STAT3 inhibitor(Stattic)for 1 hour,detect the changes in cell proliferation,viability and migration of A549 cells.7.The conditioned medium(Con-CM and CD109-CM)derived from A549 cells transfected with CD109 was collected,and the expression of CD109 in the CM was detected by Western blot.Flag was used as a positive control,Comassie blue staining of the gel was used as loading control.8.Human umbilical vein endothelial cells(HUVECs)were co-cultured with the CM of A549 cells to establish a co-culture system,simulating the microenvironment of human LAC and detect its effect on endothelial cells(ECs).9.In the established co-culture system,detect the changes in cell proliferation,viability and migration of HUVECs,to determine the effect of CD109 secreted by A549 cells on founction of HUVECs.10.In the established co-culture system,the cell proteins were collected.The protein levels of p-STAT3,STAT3 and the components of TGF-β signaling pathway(p-Smad2/3,Smad2/3,TGF-β R1)was detected,the phosphorylation ratio was calculated.This experiments was aimed to verify whether CD109 secreted by A549 cells in the co-culture system can promote the migration of HUVECs by negatively regulating the expression of TGF-β-Smad2/3 signaling pathway,so as to promote the metastasis of LAC tumors.11.TGF-β1(100 p M)was added to CD109-CM to activate the TGF-β signaling pathway.Compared with Con-CM and CD109-CM alone,cell Wound healing assay and Transwell migration assay were used to detect the cell migration,and compare the migration changes of HUVECs in the three treatments.HUVECs were pretreated with TGF-β inhibitor SB431542(10 μM)for 1 hour to inhibit TGF-β signaling pathway.A549 CM was replaced and compared with Con-CM and CD109-CM alone,cell Wound healing assay and Transwell migration assay were used to detect the cell migration,and compare the migration changes of HUVECs in the different treatments,to further verify whether CD109 secreted by A549 cells can promote the migration of HUVECs by negatively regulating the expression of TGF-β-Smad2/3 signaling pathway,so as to promote the metastasis of LAC tumors.12.Statistical analysis,Graphed data express mean ± standard error.Statistically significant differences were evaluated using paired-sample t-test.Statistically significant differences were evaluated using unpaired-sample t-test or paired-sample t-test.P < 0.05 was considered statistically significant.All the data represented are compiled from at least 3independent experiments.Results1.Immunohistochemical staining of lung adenocarcinoma TMAs suggested that the expression level of CD109 in metastatic lung adenocarcinoma was significantly higher than that in non-metastatic lung adenocarcinoma.2.The gene expression level and protein expression level of CD109 in A549 cells were significantly increased in the experimental group after transfection with adenovirus(ADV)-CD109.3.After overexpression of CD109 in A549 cells,the phosphorylation level of STAT3 was detected,and it showed no significantly difference from that of the control group,CD109 had no significant effect on the phosphorylation level of STAT3.4.After overexpression of CD109 in A549 cells,it had no effect on cell proliferation,cell viability and cell migration compared with control group,it proved that CD109 had no significant effect on cell proliferation,cell viability and cell migration of A549 cells.5.After overexpression of CD109 in A549 cells,pretreatment with STAT3 inhibitor,cell proliferation,viability and cell migration levels remained unchanged,demonstrating that CD109 cannot affect A549 cell function through STAT3 signaling pathway.6.After transfection of adenovirus in A549 cells,cell-derived CM was collected and the expression level of CD109 in the CM was detected.The expression level of CD109 in the experimental group(CD109-CM)was significantly higher than that in the control medium(Con-CM).It turns out that CD109 can be released from the cell surface into the cell supernatant,and overexpression of CD109 in the cells leads to a corresponding increase in the expression level of CD109 in CM.7.A549 cell-derived CM was collected and applied to HUVECs to establish a coculture system.MTT and CCK-8 assay showed that CD109 secreted by A549 cells had no effect on the proliferation of HUVECs.The results of trypan blue staining showed that CD109 secreted from A549 cells had no effect on viability of HUVECs.Cell wound healing and Transwell migration assay showed that the migration level of HUVECs was significantly enhanced in CD109-CM group.These results indicate that CD109 secreted by A549 cells can significantly promote HUVECs migration without affecting its proliferation level.8.In the co-culture system,by the detection,it was found that the phosphorylation level of STAT3 showed no difference in the Con-CM and CD109-CM groups.However,the expression level of TGF-β R1 in the experimental group was significantly decreased,and the phosphorylation levels of Smad2 and Smad3 in the downstream pathways were also significantly decreased in CD109-CM group.The results indicated that CD109 secreted by A549 cells can negatively regulate the expression of TGF-β signaling pathway in HUVECs.9.To confirm the role of the TGF-β signaling in CD109-induced ECs migration,Con-CM,CD109-CM and CD109-CM+TGF-β1 were co-cultured with HUVECs,TGF-β1addition eliminates CD109-induce increase of migrated area in cell wound healing assay and migrated cells in Transwell migration assay.In addition,HUVECs were pretreated with 10 μM SB431542 for 1 hour to inhibit TGF-β signaling pathway.Upon treatment of SB431542,CD109-CM still induces an increase of HUVEC migration in cell wound healing assay and Transwell migration assay,however the CD109-CM induced increase of cell migration was significantly reduced.In conclusion,A549 cell-secreted CD109 promotes HUVECs migration via inhibition of the TGF-β signaling pathway.ConclusionsCD109 in LAC cell line A549 can not promote tumor metastasis through JAK-STAT3 signaling pathway;CD109 was released into the culture medium to stimulate ECs,and promotes the migration of ECs by negatively regulating the TGF-β-smad2/3 signaling pathway,thus promoting the metastasis of LAC. |
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