The Mechanism Of CD109 Released By Lung Adenocarcinoma Cells On Lymphatic Endothelial Cells

Background:Lung adenocarcinoma(LUAD),which belongs to the non-small cell lung cancer(NSCLC),accounting for about 40%of the lung cancer.The incidence of adenocarcinoma has exceeded that of squamous cell carcinoma in many countries.The metastasis of tumor cell is the leading cause of mortality in lung adenocarcinoma patients.Therefore,identifying the underlying molecular mechanisms of metastasis will improve diagnoses and treatments for lung cancer.The first stage of cancer cell metastasis usually starts at lymphatic vessels.Tumor cells release various cytokines to induce lymphangiogenesis.It will cause endothelial cells sprouting from the existing lymphatics and generating new lymphatic vessels,which facilitate tumor cells escape from the primary tumor and disseminate through the new lymphatic vessels.Despite the rapid development of tumor biology,the molecular mechanisms of lymphatic invasion and metastasis are largely unknown.Previous studies demonstrated that CD 109 was up-regulated in lung adenocarcinoma,and the over-expression of CD 109 was positively correlated with tumor progression,distant metastasis and a poor prognosis of lung adenocarcinoma.However,the molecular mechanism by which CD 109 influences lymphangiogenesis in cancer and promotes tumor cell metastasis remains to be elucidated.In the present study,we identified that the released form of CD 109 promotes the migration of lymphatic endothelial cells by constructing a CD109-overexpressed cell model.Mechanically,we found that Jak/Stat3 mediated signaling pathway was a molecular target of CD 109 to induce the migration of HDLECs.Meanwhile,Hes family plays as the only known Notch effector in mammals.The released form of CD 109 may affect the normal function of endothelial cells by activating Notch signal to regulate the expression of Hesl.Although radiotherapy and chemotherapy show responses during the early treatment of lung adenocarcinoma,molecular changes are the major problem of drug resistance and distant metastasis.Targeting the signaling pathway of lymphangiogenesis provide an effective therapeutic strategy to limit the spread of lung adenocarcinoma metastasis.Part Ⅰ:Regulating CD 109 expression and the effects of CD109 on proliferation and migration of HDLEC cellsObjective:Regulating the expression of CD109 in lung adenocarcinoma,the secretion of CD109 in culture medium was detected and CD 109 acted on lymphatic endothelial cells.We explored the effects of CD 109 on the functional changes of HDLECs,including the proliferation and migration.Methods:1.Construction of CD109-adenovirous expression vectorCD 109 overexpressing adenovirus vector was made by Jikai Company.2.The expression and release of CD109 in lung adenocarcinoma cells2.1 Regulating the expression of CD109 and detecting the expression of CD109 in lung adenocarcinoma cellsHuman lung adenocarcinoma cells-A549 were infected with the packaged adenovirus above and they were divided into negative control group and experimental group.Cells were collected and we examined the CD 109 expression with Western Blot.We extracted the RNA from transfected A549 cells,and designed the primers for CD 109 and GAPDH respectively.The expression of CD 109 mRNA was detected by Real Time PCR.2.2 Detecting the release of CD109 after adenovirus transfection of A549CD 109 could be released into surrounding culture medium from cell surface.We collected the conditional supernatant culture medium after A549 transfected with adenovirus and divided them into Con-CM and CD109-CM.Then we use Western Blot to detect the secretion of CD 109 in conditional medium,use flag as positive control and use Coomassie blue staining as the loading control.3.Effects of CD109 on the functional changes of human dermal lymphatic endothelial cells(HDLECs)3.1 Establishing the co-culture system and detecting the effects of CD109 on the proliferation of HDLECsThe conditioned culture medium of A549 was treated on human dermal lymphatic endothelial cells,simulating the microenvironment of HDLECs in lung adenocarcinoma and establishing the co-culture system.We examined the proliferation activity of HDLEs with MTT and CCK-8 assays.3.2 Detecting the effects of released form of CD109 on the migration ability of HDLECsWe collected the conditioned culture medium and established the co-culture system with HDLEC cells.And we use cell Scratch assay and Transwell migration assay to detect the effect of soluble CD 109 in culture medium on the migration ability of HDLECs.Results:1.CD109 overexpression adenovirus were successfully established.2.Compared with the negative control group,the expression of CD 109 protein and mRNA were significantly increased in the experimental group after A549 transfected with adenocarcinoma.It illustrated that CD 109 overexpression adenocarcinoma was successfully transfected into A549 cells and expressed.3.The results of MTT and CCK8 assay showed that CD109 secreted by A549 cells had no effect on the proliferation of HDLECs.The results of cell Scratch assay and Transwell migration assay demonstrated that the migration area and number of HDLECs in CD109-CM group was dramatically increased in contrast with negative control group.The released form of CD 109 could promote the migration of endothelial cells but had no effect on their proliferation ability.Conclusions:The lung adenocarcinoma cell line A549 cells could release CD109 from the cell surface,act on endothelial cells,promote the migration of HDLECs,affect lymphangiogenesis,and further promote the distant metastasis of tumor through lymphatic vessels.Part Ⅱ:The molecular mechanism of CD109 action on lymphatic endothelial cellsObjectives:To further study the specific molecular mechanism of CD 109 acting on HDLEC cells to regulate their migration ability,and to explore the effect of CD109 on endothelial cell functionMethods:1.Effects of CD 109 on Jak/Stat3 signaling pathway in human dermal endothelial cells1.1 Effects of CD 109 on the expression of JAK and STAT3 in HDLECsCD109-CM supernatant of the conditional culture medium above were treated on lymphatic endothelial cells respectively.We collected proteins in HDLECs and examined the expression of P-Jak2/Jak2 and P-Stat3/Stat3 with Western Blot.The corresponding phosphorylation ratio was calculated.We analyzed the effects of CD 109 secreted by lung adenocarcinoma on the expression of Jak and Stat3 in endothelial cells.1.2 After adding the Stat3 inhibitor,the effects of CD109 on HDLEC migration ability and Stat3 expression were detectedTo further verify CD 109 positive regulation of Stat3 signaling pathway to promote HDLECs migration,we use Stattic with final concentration of 20 μmol for 1 hour.2.Regulating the expression of CD109 and detecting the effects of CD109 on Notch-Hesl signaling pathway in lung adenocacinoma and HDLECs2.1 CD109 regulates Notch-Hesl signal in lung adenocarcinomaWe collected the A549 cells transfected with adenovirus and detected the effects of CD 109 on the expression of Hesl at protein and mRNA levels with Western Blot and RT-PCR assays.2.2 Effects of CD109 on the expression of Hesl in endothelial cells.We collected HDLEC cells treated with conditioned culture medium for 24 hours and examined the effects of CD 109 on Notch-Hesl signaling pathway in endothelial cells with Western Blot and qPCR assays.Results:1.The results show that the phosphorylation level of Jak2 and Stat3 in CD109-CM group were significantly increased when compared with the control group.The increase in CD109-induced endothelial cell migration was eliminated and the expression of P-Stat3 was also reduced after the addition of Stattic.It demonstrated that CD 109 positively regulated the Stat3 signaling pathway to promote endothelial cell migration.2.Compared with the control group,the expression of Hesl in A549 and HDLECs was significantly increased in the CD 109-overexpressing adenovirus transfected group.The results indicated that CD 109 could activate Notch-Hesl signaling pathway.Conclusions:CD 109 which released from cell surface had bioactivity and promoted endothelial cell migration by inducing Jak/Stat3 signaling pathway.Meanwhile,the released form of CD 109 could active Notch signaling pathway.