ITGA5 Can Promote Lymphangiogenesis By Enhancing Its Own VEGF-C Expression And Secretion In Laryngeal Carcinoma Cells

Objective:To investigate the role of integrin α5(ITGA5)in promoting lymphangiogenesis in laryngeal carcinoma cells and its feedback effected on the biological activity of tumor cells.And to provide a certain experimental basis for the application of anti lymphangiogenesis in the treatment of laryngeal carcinoma.Methods:1.The expression of ITGA5 in head and neck squamous cell carcinoma(HNSC)was analyzed by UALCAN,and the correlation between expression of ITGA5 and prognosis of HNSC was analyzed by KMplot and GEPIA.2.The paraffin blocks and clinicopathological data of 80 patients with newly diagnosed laryngeal carcinoma were collected.The expression of ITGA5 and vascular endothelial growth factor-C(VEGF-C)in laryngeal carcinoma was detected by immunohistochemistry(IHC).LEC was labeled by lymphatic endothelial cell(LECs)specific marker D2-40,and the lymphatic vessel density(LVD)was counted.The correlation between ITGA5 expression and clinicopathological features,as well as the correlation with VEGF-C and LVD were analyzed.3.Hep-2 and TU212 laryngeal carcinoma cell lines were used to construct si-ITGA5.The secretion of VEGF-C was detected by ELISA,and the expression of VEGF-C was detected by RT-q PCR and Western bolt.4.A transwell(0.4μm)co-culture system of laryngeal carcinoma cell lines and human lymphatic endothelial cells(HLECs)was established to analyze the role of ITGA5 in promoting lymphangiogenesis in laryngeal carcinoma cells,and the Mono group(single HLECs culture group),Con group(wild type Hep-2/TU212 + HLECs),NC group(si-NC Hep-2/TU212 + HLECs)and si-ITGA5 group(si-ITGA5 Hep-2/TU212 + HLECs)were established.In order to further analyze whether ITGA5 can affect the tube forming rate of HLECs by promoting its own secretion of VEGF-C in laryngeal carcinoma cells,exogenous VEGF-C(5ng/m L)was added for rescue experiment.Therefore,siITGA5+VEGF-C group(si-ITGA5 Hep-2/TU212+HLECs + VEGF-C)was set at the same time.The tube forming rate of HLECs in each group was detected.5.In order to verify that ITGA5 can promote the invasion and migration of laryngeal carcinoma cells by inducing lymphangiogenesis,Transwell(8μm)co-culture system of laryngeal carcinoma cell lines and HLECs was established,and the Mono group(single Hep-2/TU212 culture group),Con group(wild type Hep-2/TU212 + HLECs),NC group(si-NC Hep-2/TU212 + HLECs)and si-ITGA5 group(si-ITGA5 Hep-2/TU212 + HLECs)were set up.The microenvironment of lymphangiogenesis was simulated by adding exogenous VEGF-C into the co-culture system HLECs,and the si-ITGA5+VEGF-C group(si-ITGA5 Hep-2/TU212+HLECs+VEGF-C)was set up.The invasion and migration ability of laryngeal carcinoma cells in each group were detected by Transwell invasion and migration assay.6.Statistical analysis method.Chi-square test or Fisher’s exact test,Mann-Whitney test and Spearman rank correlation test were used to analyze the clinicopathological features and immunohistochemical results.Cell experiments in vitro were carried out for three times independently,experiments data were expressed as means ± standard deviation.One-way analysis of variance(ANOVA)was used for the comparison between different experimental groups,and P<0.05 indicated a statistically significant difference.Results:1.Bioinformatics analysis of ITGA5 expression level in HNSC tissues(44 cases of adjacent tissue and 520 cases of cancer tissues)from TCGA database was conducted through UALCAN.The results showed that the expression level of ITGA5 in HNSC tissues was significantly higher than that in adjacent tissues(P < 0.001);The clinical data of HNSC patients were analyzed by using KMplot and GEPIA website.The results showed that the overall survival time of patients with high expression of ITGA5 in HNSC tissues is shorter than that of patients with low expression(P < 0.01).2.IHC results showed that the positive expression of ITGA5 was located in the cell membrane and /or cytoplasm in 80 cases of laryngeal carcinoma,the high expression rate was 65.0%(52 / 80),and the low expression rate was 35.0%(28 / 80);The high expression of ITGA5 was significantly correlated with lymph node metastasis and T stage(P < 0.05).The positive expression of VEGF-C was mainly located in the cytoplasm,the high expression rate was 62.5%(50 / 80),and the low expression rate was 37.5%(30 / 80);The high expression of VEGF-C was significantly correlated with lymph node metastasis and tumor size(P < 0.01),but not with other clinicopathological parameters.The median LVD of 80 laryngeal carcinoma tissues was 3.0(2.0,6.0),LVD < 3 was divided into low density group,LVD ≥ 3 was divided into high density group,high density rate was 58.8%(47 / 80),low density rate was 41.2%(33 / 80);LVD was significantly correlated with lymph node metastasis and tumor location(P < 0.05),but not with other clinical pathological parameters.The statistical results also showed that the LVD of ITGA5 high expression group was significantly higher than that of ITGA5 low expression group(P<0.05);The expression level of ITGA5 was positively correlated with the expression level of VEGF-C(r = 0.677,P < 0.001).3.The results of ELISA showed that the secretion of VEGF-C in laryngeal carcinoma cell lines with ITGA5 knockdown was significantly decreased;RT-q PCR and Western bolt showed that the expression of VEGF-C in laryngeal carcinoma cell lines was significantly decreased by down regulating the expression of ITGA5.4.The tube forming ability of HLECs was tested by tube forming experiment.It was found that the tube forming rate of HLECs co-cultured with laryngeal carcinoma cell line was significantly higher than that of HLECs alone.The tube forming rate of HLECs cocultured with si-ITGA5 transfected laryngeal carcinoma cells was significantly lower than that of HLECs co-cultured with si-NC transfected laryngeal carcinoma cells.The effect of ITGA5 knockdown on the tube forming rate of HLECs could be partially reversed by exogenous VEGF-C.5.Transwell experiment detected the invasion and migration ability of laryngeal carcinoma cells co-cultured with HLECs,and the results showed that the invasion and migration ability of laryngeal carcinoma cells was significantly enhanced after cocultured with HLECs;The invasion and migration ability of laryngeal carcinoma cells was significantly reduced by down regulating ITGA5 expression;The invasion and migration ability of laryngeal carcinoma cells co-cultured with HLECs was significantly enhanced by adding exogenous VEGF-C to simulate the microenvironment of lymphangiogenesis.Conclusion:ITGA5 in laryngeal carcinoma cells can enhance its lymphangiogenesis effect by promoting its own VEGF-C secretion,and feedback enhance the migration and invasion ability of laryngeal carcinoma cells.