Study On The Expression And Regulation Mechanism Of Vwf In Neovascularization Of Lung Adenocarcinoma

Objectives:Lung adenocarcinoma is the leading cause of cancer-related death worldwide and poses great threat to the health and life of the population.However,there is still a lack of biomarkers for early diagnosis and prognosis of lung adenocarcinoma.It was reported that the number of new vessels is significantly correlated with the development of lung adenocarcinoma.Aberrant expression of genes expressed preferentially in the lung tumor vasculature may yield clues for prognosis and treatment.Von Willebrand factor(vWF)is a large multifunctional glycoprotein with a well-known function in hemostasis.However,vWF has been reported to exert an anti-tumor effect,independent of its role in hemostasis.However,in lung adenocarcinoma,the expression pattern and regulation mechanism of vWF are not clear.Some studies have shown that the transcription factor GATA regulates the basal expression of vWF in vascular endothelial cells.Whether or not GATA regulates the expression of vWF in the microvessels of lung adenocarcinoma has not been studied.In order to further explore the expression pattern and regulation mechanism of vWF in lung adenocarcinoma,we detected the expression of vWF in different lung adenocarcinoma tissues.In addition,a co-culture system of A549-conditioned medium and endothelial cells was established to further reveal the transcriptional regulation mechanism of transcription factor GATA3 on vWF.This study provides a new clue for the diagnosis,treatment and prognosis of lung adenocarcinoma.Methods1.The expression of vWF in the blood vessels of lung adenocarcinoma was detected by immunohistochemical staining on tissue microarrays(TMAs).2.Samples of fresh lung adenocarcinoma tissues were collected.And the expression of vWF in the blood vessels of lung adenocarcinoma was detected by qRT-PCR and immunofluorescence staining.3.Establish a anmial model of lung adenocarcinoma by injecting with Lewis lung carcinoma cells into C57BL/6 mice.The expression of vWF in the blood vessels of lung adenocarcinoma was detected by qRT-PCR and immunofluorescence staining.4.The conditioned medium derived from A549 cells was used to establish a co-culture system in HUVECs to simulate the effects of lung adenocarcinoma microenvironment on blood vessels.QRT-PCR,WB,immunofluorescence and ELISA were used to detect the effect of the microenvironment of lung adenocarcinoma on the expression of vWF in HUVECs.5.In the co-culture system,qRT-PCR,WB and immunofluorescence were used to detect if ERG is the transcription factor that upregulates the the expression of vWF.6.In the co-culture system,qRT-PCR and WB were used to detect the expression of GATA3 in the microenvironment of lung adenocarcinoma.7.The expression of GATA3 was interfered using si-RNA in the co-culture system.And the interference efficiency was detected by qRT-PCR and WB.The effect of the lung adenocarcinoma microenvironment on the expression of vWF after GATA3 silenced was also detected.8.ChIP assay was used to detect whether GATA3 binds to the binding site of vWF promoter and whether A549 conditioned medium enhanced the interaction between GATA3 and vWF promoter in co-culture system.Results1.Immunohistochemical staining of TMAs suggested that vessels in both lung adenocarcinoma and adjacent tissues were positive for vWF,whereas microvessels in all normal lung tissues examined were negative for vWF.However,the staining of vWF in lung adenocarcinoma vessels was even more pronounced than that in adjacent tissues,suggesting a gradient enhanced expression pattern from normal lung tissues to lung adenocarcinoma.2.QRT-PCR was used to exam the mRNA level of vWF in fresh samples.Significantly elevated mRNA levels for vWF were found in lung adenocarcinoma tissues compared to paired normal tissues from each patient.Similarly,immunofluorescent staining of vWF in fresh-frozen sections was more intense in the lung adenocarcinoma vasculature than that of adjacent tissue vasculature.3.VWF expression in a mouse lung adenocarcinoma model was examined.QRT-PCR analysis showed a significant increase of vWF mRNA expression in LLCs,compared to that in the normal mouse lungs.Immunofluorescence analysis demonstrated stronger vWF immunofluorescence staining in LLC vessels compared to normal mouse lungs.4.Collected A549-derived conditioned medium(A549-CM)was treated on HUVECs to establish a co-calture system.The mRNA level of vWF in A549-CM treated HUVECs was significantly increased.vWF protein level was higher in HUVECs treated with A549-CM than in HUVECs treated with control media.ELISA showed that A549-CM significantly promoted the secretion of vWF from HUVECs.Taken together,vWF expression was promoted in HUVECs by A549-conditioned medium.5.In the co-culture system,both the mRNA levels of ERG,evidenced by qRT-PCR assay,and the protein levels of ERG,as measured by Western blot and immunofluorescence,were not altered by A549-CM.This suggests that ERG was not responsible for elevated vWF in HUVECs in response to A549-CM.6.In the co-culture system,both qRT-PCR and WB showed a rise in the expression of GATA3.Si-RNA interference was used to confirm whether GATA3 was the factor that upregulated the transcription of vWF.In the GATA3 silenced HUVECs,no increase in vWF expression was observed in response to A549-CM as expected.These findings suggested that GATA3 is the transcriptional regulator that mediating the elevation of endothelial vWF after treatment with the A549-CM.7.ChIP assay showed that GATA3 bound to the +220 GATA motif of the vWF promoter in HUVECs.And A549-CM further enhanced the amount of GATA3 binding at the +220 GATA-binding motif.Therefore,GATA3 is induced by A549-CM and is the factor responsible for transcription enhancement of vWF expression in lung adenocarcinoma.Conclusion1.VWF is highly expressed in lung adenocarcinoma blood vessels,which is not dependent on the vessel type,but instead may be associated with the lung adenocarcinoma microenvironment.2.In the co-culture system,vWF expression was promoted in HUVECs by A549-conditioned medium,suggesting enhanced vWF expression is regulated by the lung adenocarcinoma secretome.3.The transcription factor GATA3,but not ERG,mediated the epression of vWF.4.GATA3 binds directly to the +220 GATA binding motif on the human vWF promoter and A549 conditioned media significantly increases the binding of GATA3.Taken together,we demonstrate that vWF expression in endothelial cells of lung adenocarcinoma is elevated by the cancer cell-derived secretome through enhanced GATA3-mediated transcription.