The Effect And Mechanism Of Nfe2l1 Deficiency In Vascular Endothelial Cells On Tumor Angiogenesis In Subcutaneous Tumor-bearing Mice

Objective:The complex pathological mechanism of cancer has become a serious global public health problem.The occurrence and development of cancer are mainly affected by angiogenesis.Nuclear factor erythroid-2-related factor 1(Nfe2l1)is a core transcription factor that regulates a variety of stress response and plays an important role in regulating a variety of biological functions such as redox reactions and proteasome homeostasis.Tumors need to form blood vessels to get the oxygen and nutrients they need.The proliferation and migration of endothelium promote angiogenesis.However,little is known about the role of Nfe2l1 in vascular endothelial cells.Therefore,in the present study,we established an in vivo tumor growth model of subcutaneous tumor-bearing lung cancer cells in Nfe2l1 knockout mice and an in vitro co-culture model of Nfe2l1silenced mouse vascular endothelial cell line(SVEC4-10)and tumor cells(3LL).To study the possible mechanism of Nfe2l1 regulating vascular endothelial cells affecting tumor growth,and to provide reference for clinical tumor treatment.Methods:1.Construction of Nfe2l1 gene knockout mouse model of vascular endothelial cells:Cdh5-Cre,Nfe2l1-Flox and Nfe2l1(E)-KO transgenic mice were established from C57BL/6 genetic background,and genotype identification was conducted by PCR amplification.2.A mouse tumor growth model was established:Cdh5-Cre,Nfe2l1-Flox and Nfe2l1(E)-KO mice aged 6-8 weeks were injected subcutaneously with mouse lung cancer cells(3LL),and each mouse was injected with 8×10~5cells,the weight and tumor size of mice were measured every other day,and tumor tissues were collected two weeks later.3.Evaluation and characterization of mouse tumor growth model:After collecting tumor tissues from mice,we took gross photographs and measured their weights;observed tumor vascular density by immunofluorescence staining of platelet endothelial cell adhesion molecule-1(PECAM-1/CD31)in frozen sections.The expression levels of Vascular endothelial growth factor receptor 2(VEGFR2)and p-VEGFR2 were measured to evaluate the activation of angiogenesis-related signaling pathways.4.Detection of vascular permeability of mouse subcutaneous tumor:Evans blue(30mg/kg)was injected into the blood circulation of tumor-bearing mice for half an hour through the tail vein,and mice in the same litter were collected at the same time to measure the weight of tumor tissue,extract and detect the content of Evans blue in the tissue and evaluate the vascular permeability.5.Establishment and detection of Nfe2l1 silenced vascular endothelial cells:Vascular endothelial cells were transfected with lentivirus carrying sh RNA targeting Nfe2l1(Nfe2l1-KD)and its negative control Scramble(Scr).The stable transfected cells were screened with 1μg/ml puromycin,and the silencing effect of Nfe2l1 was identified by extracting m RNA and protein.6.Cell co-culture:Scramble and Nfe2l1-KD groups were seeded in 6-well plates at a cell number of 7.5×10~5 cells/well,and 3LL cells were seeded in 0.4μm pore diameter co-culture inserts at a cell number of 1×10~6 cells/well.The cells in the co-culture system were cultured in DMEM medium containing 10%fetal bovine serum.Scramble and Nfe2l1-KD were collected for experiments after two days of co-culture.7.Cell migration assay:Scramble and Nfe2l1-KD co-cultured vascular endothelial cells were seeded into Transwell chambers(8μm microwells).The upper chamber was added with DMEM,and the lower layer was added with DMEM containing 10%FBS as a factor to induce cell migration.The number of cells penetrating the lower layer of the chamber was observed under an inverted microscope.8.Scratch healing experiment:Scramble and Nfe2l1-KD of co-cultured vascular endothelial cells were re-inoculated into the 6-well plate,and 3LL cells were inoculated into the co-culture plug-in to treat the above two kinds of cells,and the percentage of scratch healing of vascular endothelial cells at 6,12,18,24 and 30 h was observed.9.SVEC4-10 cell line treated with 3LL cell conditioned medium:vascular endothelial cell Scramble and Nfe2l1-KD were treated with 3LL cell conditioned medium at the same time,and the expression levels of VEGFR2 and p-VEGFR2 were detected after 5,10 and 20 min of treatment.Results:1.The successful construction of Nfe2l1 gene knockout mouse models of vascular endothelial cells:Cdh5-Cre,Nfe2l1-Flox and Nfe2l1(E)-KO mouse models were confirmed by genotype identification.2.Accelerated tumor growth in vascular endothelial Nfe2l1 knockout mice:3LL cells were inoculated into the back of three groups of mice,the tumor growth volume was recorded every other day,and the tumor was harvested two weeks later.Compared with the other two groups,Nfe2l1(E)-KO mice had faster tumor growth(P<0.05)and larger tumor weight(P<0.05).3.Vascular endothelial Nfe2l1 knockout mice had faster tumor cell proliferation:To assess whether Nfe2l1 loss affects tumor cell proliferation,paraffin sections of tumor tissue were subjected to immunohistochemical staining for the tumor cell proliferation marker Ki-67.The quantitative results showed that Ki-67 expression in transplanted tumors of Nfe2l1(E)-KO mice was higher than that of Nfe2l1-Flox mice(P<0.05).4.Vascular endothelial Nfe2l1 knockout mice showed increased tumor angiogenesis:Frozen sections of tumor tissues were stained with immunofluorescence for vascular endothelial cell marker CD31 to evaluate tumor angiogenesis.Compared with the Nfe2l1-Flox group,the Nfe2l1(E)-KO group had a significantly higher vascular density in tumor tissues(P<0.05).5.Increased permeability of tumor microvessels in Nfe2l1 knockout mice:To detect tumor vascular permeability,Evans blue was injected into mice through the tail vein,and then Evans blue was extracted from tumor tissue and quantized by spectrophotometry.It was found that the content of Evans blue in tumor tissue of Nfe2l1(E)-KO mice was higher than control mice(P<0.05).Thus,tumors transplanted into Nfe2l1(E)-KO mice were characterized by increased microvascular permeability.6.Elevated VEGFR2 and p-VEGFR2 expression in endothelial Nfe2l1 knockout mice:The effect of Nfe2l1 deficiency on tumor angiogenesis was evaluated by extracting tumor tissue protein and detecting the protein expression levels of tumor angiogenesis-related proteins VEGFR2 and p-VEGFR2.The protein expression levels of VEGFR2 and p-VEGFR2 in the transplanted tumor of Nfe2l1(E)-KO group were higher than those in the control group(P<0.05).7.Nfe2l1 silencing cells of vascular endothelial cells were successfully constructed:The Nfe2l1 silencing SVEC4-10 cell line was established to study the possible mechanism of Nfe2l1 involved in tumor angiogenesis.The m RNA and protein were extracted to verify the successful construction of Nfe2l1 silencing vascular endothelial cell line Scramble and Nfe2l1-KD cells.8.Nfe2l1 silencing of vascular endothelial cells promotes cell migration in a tumor cell co-culture environment:A co-culture system of tumor cells and vascular endothelial cells was established,in which 3LL cells were inoculated in the upper chamber and Scramble and Nfe2l1-KD cells were inoculated in the lower chamber,respectively,to evaluate the migration ability of vascular endothelial cells in the tumor environment.The results showed that after two days of co-culture with 3LL cells,the number of endothelial cells migrated out of the chamber in Nfe2l1-KD group was significantly higher than that in the Scramble group(P<0.05).9.Nfe2l1 silencing of vascular endothelial cells promotes cell proliferation and migration in tumor cell co-culture environment:SVECs treated with co-culture for two days were re-inoculated into 6-well plates and the scratch healing ability was observed at6,12,18,24,and 30 h.The results showed that the scratch healing ability of the co-culture Nfe2l1-KD group was stronger than that of the Scramble group.10.Nfe2l1 silencing promotes VEGFR2 phosphorylation in vascular endothelial cells in tumor cell co-culture:3LL cell conditioned medium treated Scramble and Nfe2l1-KD vascular endothelial cells for the following time.The phosphorylation level of VEGFR2was significantly higher in the Nfe2l1-KD group than in the Scramble group at 5,10,and20 min(P<0.05).Conclusions:Vascular endothelial cell-specific Nfe2l1 deletion may promote vascular endothelial cell migration to accelerate tumor angiogenesis by up-regulating VEGFR2phosphorylation,and ultimately leading to accelerated tumor growth.