The Enhancement Effects Of VEGF-C On Radiation Resistance In Nasopharyngeal Carcinoma Cell CNE-2 And Its Mechanisms

Vascular endothelial growth factors(VEGFs),including VEGF-A,VEGF-B,VEGF-C,VEGF-D,VEGF-E and placental growth factor(PLGF),are some of the key factors responsible for tumor angiogenesis.The biological effect of VEGF is specifically mediated by the vascular endothelial growth factor receptor(VEGFR).In addition to promoting the vascular endothelial cell formation,VEGF can also stimulate tumor proliferation and migration by binding to receptors expressed on the tumor tissues.VEGF-C is one of the subtypes of VEGF.VEGF-C is a lymphangiotropic growth factor.Many malignant cells express VEGF-C and its receptor VEGFR-3.It has also been confirmed that the autocrine signal axis comprising VEGF-C and VEGFR-3 can cause proliferation,invasion,and metastasis of many tumor cells through a series of biochemical reactions.Nasopharyngeal carcinoma(NPC)is extremely prone to lymph node metastasis.Nowadays,radiotherapy is the main treatment for NPC.For the first time,here,we investigated the effects of VEGF-C on the biological behavior and radiosensitivity of NPC CNE-2cells in the current study.The underlying molecular mechanisms were also elucidated in order to provide experimental basis and theoretical support for the use of VEGF-C as a target of radiosensitization in NPC as well as new therapeutic targets for personalized treatment of NPC.ObjectiveTo elucidate the effects and underlying mechanisms of the effect of radiation resistance in NPC CNE-2 cells by VEGF-C as well as to provide theoretical support for the intervention of VEGF-C in enhancing radiosensitivity of NPC CNE-2 cells.Methods:(1)The irradiation conditions used in this study were as follows:Synergy VMAT(Elekta,Sweden),a single X-rays radiation dose of 8 Gy,dose rate:300 cGy/min.(2)CCK-8 assay was used to detect the proliferation activity of NPC cell lines.(3)The vector was constructed by genetic engineering to establish the cell model.The siRNA-VEGF-C recombinant plasmid was constructed and transfected into the human NPC CNE-2 cells.Empty vector cells as well as siVEGF-C cells were also constructed.(4)Real-time quantitative PCR assay was used to detect the mRNA expression.(5)Colony formation experiment was used to detect the radiosensitivity of CNE-2 cells.(6)Western blot assay was used to detect the protein expression of VEGF-C,cyclinD1,GADD45β,p65,p-p65,IκB,p-IκB,Bax,Bcl-2,caspase3,C-caspase3,Bim and caspase-9.(7)The level of apoptosis in CNE-2 cells was measured by flow cytometry.(8)Comet assay was used to detect the DNA fragment distribution as well as to observe DNA damage in CNE-2 cells after X-ray irradiation.(9)The co-localization of p-p65 and VEGF-C was analyzed using immunofluorescence staining.(10)Luciferase reporter assay was used to detect the binding of p65 to ATM promoter.(11)Animal model building and grouping:Establishment of a subcutaneous tumor-bearing animal model of immunodeficient nude mice.In the animal model of tumor,the tumor-bearing mice were divided into the following six groups:normal group,negative control group,inhibition group,irradiation group,negative control+irradiation group,and irradiation+inhibition group.The groups were closely observed for the tumor growth changes,and related protein expression levels.(12)Statistical analysis:Student’s t-test was used for data analysis.The difference was considered statistically significance when p<0.05.Image J software was used to perform grayscale analysis for protein electrophoresis bands.Results:1.VEGF expression affects prognosis in patients with nasopharyngeal carcinomaOur meta-analysis shows that the low-expression total survival of VEGF in patients with nasopharyngeal carcinoma is older than patients with high VEGF expression(HR 2.07,95%CI:1.32-3.25,I~2=79.1%),significant correlation between the two.The low-expression disease-free survival of VEGF in patients with nasopharyngeal carcinoma is older than patients with high VEGF expression(HR5.99,95%CI:2.66-13.48,I~2=50.2%),significant correlation between the two.The low survival rate of VEGF in patients with nasopharyngeal carcinoma was significantly correlated with the short-term overall survival of patients with high VEGF expression in serum(HR 2.47,95%CI 1.16-5.28,I~2=45.2%).Therefore,VEGF expression is associated with prognosis in patients with nasopharyngeal carcinoma.2.VEGF-C interference up-regulates the radiosensitivity of CNE-2We compared the expression of VEGF-C in different head and neck cell lines and found that it was highly expressed in CNE-2 cell line.We detected 2、4、6、8、16Gy different doses of X-rays and found that VEGF-C expression was significantly increased 24 hours after 8Gy X-ray irradiation(P<0.05).VEGF-C expression was detected at 0、6、12、24、48 hour different times after 8Gy X-ray irradiation,and it was found that VEGF-C increased significantly after 24 hours(P<0.05).Therefore,we chose 8Gy X-ray irradiation for 24 hours as a follow-up experiment.To interfering the VEGF-C gene,negative control group and siRNA-VEGF-C group were formed.After48 hours of transfection,the expression of VEGF-C in siVEGF-C cells decreased significantly(P<0.01)compared to that in the negative control and normal cells.Successfully interfered with the expression of VEGF-C gene in nasopharyngeal carcinoma CNE-2 cells.The proliferation rate of siVEGF-C cells decreased significantly at 48 hours after transfection than that in the negative control group and the normal group(P<0.05).The cell proliferation rate of siVEGF-C+irradiation group was significantly lower than that of negative control+irradiation group and normal irradiation group(P<0.01),interfering with VEGF-C expression inhibits cell proliferation.The radii of cell clones in the siVEGF-C group were significantly lower than those in the negative control group and the normal group(P<0.05).The number and radii of cell clones in the siVEGF-C+irradiation group were significantly lower than those in the negative control+irradiation group and the normal+irradiation group(P<0.01),explain that interference with VEGF-C increases cell radiosensitivity.The apoptotic rate observed for siVEGF-C+irradiation group was significantly higher than that of negative control+irradiation and normal+irradiation groups(P<0.01),explain that interference with VEGF-C expression increases radiation-induced apoptosis.In comparison with the negative control and the normal cells,the DNA damage observed for siVEGF-C cells was more severe as revealed by the apparent comet tail phenomenon;this difference was statistically significant(P<0.01).There was no significant difference between the negative control cells and the normal cells.The degree of DNA damage in siVEGF-C+irradiation group was significantly higher than that in negative control+irradiation group and normal+irradiation group(P<0.01),explain that interfering with VEGF-C increases DNA damage.The above experiments demonstrate that interference with the VEGF-C gene in nasopharyngeal carcinoma cell CNE-2 can reduce radiation resistance by inhibiting cell proliferation,increasing apoptosis and DNA damage.3.Roles of NF-κB in radiation resistance mediated by VEGF-CThese experiments demonstrated that interfering VEGF-C could increase the radiosensitivity of CNE-2 cells as well as revealed the underlying signaling pathways.We screened differentially expressed genes by high-throughput sequencing in four libraries:siVEGF-C,negative control group,irradiation group,and siVEGF-C+irradiation group.Through GO and KEGG analyses,it was found that VEGF-C could affect the radiation sensitivity through NF-κB signaling pathways.We selected high expression of differentially genes.Four groups of specimens with high expression of differentially genes in each group were detected by PCR array.It was found that VEGF-C can affect the apoptosis by impacting caspase-3 by IκB of NF-κB.IκB of NF-κB affects cyclinD1,thus,affecting the cell proliferation.We chose the NF-κB signaling pathway as the downstream signaling pathway to study the effect of VEGF-C on radiation sensitivity.Compared with the negative control cells and normal cells,the expression levels of p-p65 in siVEGF-C cells decreased significantly(P<0.05);however,no significant difference between normal cells and negative control cells was observed.Under 8 Gy X-ray irradiation,the expression of p-p65(S536)and p-IκB in the group with siVEGF-C+irradiated cells was significantly lower than that in the irradiation group.Using cellular immunofluorescence staining,it was found that VEGF-C and p-p65 were co-expressed in the nucleus,and the expression of p-p65 in nasopharyngeal carcinoma cell CNE-2 was increased in the nucleus.These results suggest that interfering with VEGF-C can increase the expression of p-p65 protein activated by X-ray.Through luciferase reporter assay,we detected that p65 could bind to ATM promoter,thereby affecting ATM transcription.BAY11-7082 is an inhibitor of NF-κB.Compared with the irradiation group,the expression of C-caspase 3,caspase 9,Bax and Bim in siVEGF-C+irradiation group was significantly higher than that in the irradiation group(P<0.05)and decreased in Bcl-2 group(P<0.05).The level of change of the above genes was similar in the BAY11-7082+irradiated group+siVEGF-C cells compared with the irradiated group.Among normal group,irradiation group,siVEGF-C+irradiation group,and siVEGF-C+irradiation group+BAY11-7082 group,the apoptotic rate of siVEGF-C+irradiation group was the highest(P<0.01),explain that interference with VEGF-C increases radiation sensitivity.The apoptotic rate decreased when BAY11-7082 was added(P<0.01).It is confirmed that the radiation sensitization of siVEGF-C cells is achieved by up-regulating NF-κB.The above experiments showed that compared with the irradiation group,in irradiation+siVEGF-C group,ATM transcription was affected by NF-κB signaling pathway.ATM down-regulation increased Bax expression and decreased Bcl-2 expression.At the same time,such the downstream apoptotic genes of NF-κB as caspase 3,Bax,Bim and caspase-9,increased,while the expression of Bcl-2 decreased significantly.These changes resulted in the apoptosis and increased the radiosensitivity.It was found that the level of GADD45βincreased after interfering with vascular endothelial growth factor C(P<0.05).Compared with the irradiation group,the level of GADD45βincreased in siVEGF-C+irradiation group(P<0.05),which caused the increased DNA damage.Luciferase reporter assay confirmed that p65 could bind to ATM promoter.The results of immunohistochemical showed that compared with the irradiation group,the ATM of siVEGF-C+irradiation group decreased,which resulted in a decrease in DNA repair.Comet assay showed that the DNA damage was the most significant in siVEGF-C+irradiation group compared with that in irradiation group(P<0.05),and the DNA damage decreased when BAY11-7082 was added(P<0.01).The results showed that VEGF-C interfering increased DNA damage by increasing GADD45βand decreased DNA repair by ATM down-regulation,which resulted in increased DNA damage and increased radiosensitivity.It was found that the level of cyclin D1 decreased after interfering with vascular endothelial growth factor C(P<0.05).Compared with the irradiation group,the level of cyclin D1 decreased in siVEGF-C+irradiation group(P<0.05),which caused cell cycle arrest.The colony formation experiments showed that the number of clones and cloning radius of siVEGF-C cells+irradiation group were significantly lower than that of the irradiated group(P<0.05),indicating that the interference with VEGF-C increased the radiation sensitivity;the number of clones and the cloning radius of BAY11-7082 increased(P<0.05).The results showed that interference with VEGF-C decreased cyclin D1,leading to cell cycle arrest,ATM down-regulation also blocked cell cycle,cell cycle arrest caused cell proliferation,and increased radiosensitivity.4.In vivo animal experiments verify that VEGF-C in nasopharyngeal carcinoma CNE-2 cells enhances radiation resistance through NF-κB signaling pathwayThe nasopharyngeal carcinoma CNE-2 cells were injected into nude mice.When the tumors grew to about 0.7 cm,the mice were randomly divided into six groups,4 per group:the first group was tumor group,second group was intratumoral injection of siRNA-NC,50μl of siRNA-NC injection at a concentration of 50 nM was injected daily for 4 consecutive days.and third group was injected with siRNA-VEGF-C-1,50μl per day of 50 nM siVEGF-C-1 injection,continuous injection for 4 days;the fourth group was the irradiated group,our previous cell experiments showed that 8Gy irradiation had the highest expression of VEGF-C,tumor was irradiated with 2 Gy daily dose for 4 days.The fifth group was siRNA-NC+irradiation group where in the irradiation with 2 Gy daily dose for 4days,50μl of siRNA-NC injection at a concentration of 50 nM was injected daily for 4 consecutive days.The sixth group was siRNA-VEGF-C-1+irradiation group,the tumor was irradiated with 2 Gy every day for 4 days,50μl per day of 50 nM siVEGF-C-1 injection,continuous injection for 4 days.The tumor growth was observed every day and the tumor volume was measured once every three days with a Vernier caliper.Mice were sacrificed after 15 days of tumor growth and tumor size was compared.We found that the tumors in the interfered VEGF-C group were smaller than those in the negative control group and the normal group;the tumors in the interfering+irradiation group were smaller than those in the irradiation group and the negative control+irradiation group.The results of immunohistochemistry showed that after VEGF-C was interfered,VEGF-C and ATM decreased whereas p-65increased.This result suggests that intervention with VEGF-C increases radiosensitivity and is consistent with in vitro results.Conclusions1.VEGF-C could affect the radiosensitivity of NPC CNE-2 cells:VEGF-C interference led to inhibition of proliferation,repair of DNA damage,and anti-apoptotic ability of CNE-2 cells after X-ray irradiation,consequently increase the sensitivity of radiotherapy.2.VEGF-C interference in NPC CNE-2 cells enhanced NF-κB signaling pathway through increasing radiation,thereby reducing the downstream ATM of NF-κB,and ATM through the influence of apoptotic genes,leading to increased apoptosis in CNE-2 cells,increase the sensitivity of radiotherapy.3.VEGF-C interference in NPC CNE-2 cells could down-regulate cyclin D1 and ATM resulted in cause cell cycle arrest,cell proliferation is blocked,increased sensitivity to radiotherapy.4.VEGF-C interference in NPC CNE-2 cells increased GADD45βand ATM decreased,resulting in DNA damage aggravated,affecting radiosensitivity.5.In xenograft model,after VEGF-C interference,the growth rate of transplanted tumors decreased,and the radiation sensitivity increased.