Expression Of TIM-4 In Nasopharyngeal Carcinoma And Its Effect On Cell Proliferation And Migration

Objective: Research on the expression of T cell immunoglobulin domain and mucin domain protein(TIM-4)in nasopharyngeal carcinoma(NPC)and its effect on cell proliferation and migration.Methods:1.Immunohistochemical analysis of the expression of TIM-4 in nasopharyngeal carcinoma tissues.At the same time,its correlation with clinicopathological parameters was analyzed.2.Fluorescence quantitative PCR and Western Blot were used to analyze the expression of TIM-4 at m RNA and protein levels in 5-8F,6-10 B and HK-1 nasopharyngeal carcinoma cell lines;3.The 5-8F nasopharyngeal carcinoma cell model with overexpression of TIM-4 was established;4.Monoclonal assay,CCK-8 assay,scratch assay and Transwell assay were used to study the effect of TIM-4 on proliferation and migration of nasopharyngeal carcinoma cells.Flow cytometry was used to detect the effect of overexpression of TIM-4 on apoptosis and cell cycle.Results:1.Immunohistochemical results showed that TIM-4 was expressed in the cytoplasm and cell membrane of nasopharyngeal carcinoma cells,and the positive rate of TIM-4 in nasopharyngeal carcinoma(91.9%,79/86)was significantly higher than that in nasopharyngeal mucosa of chronic rhinitis(19.4%,6/31),with statistical significance(P = 0.000).TIM-4 expression and clinicopathological parameters analysis showed that TIM-4 expression was positively correlated with clinical stage,depth of tumor invasion,and lymph node metastasis,with statistically significant differences(P =0.005,P = 0.001,P =0.035,respectively),but had no correlation with age and gender.2.The results of q RT-PCR and Western blotting showed that TIM-4 was expressed in 5-8F,6-10 B and HK-1 cell lines of nasopharyngeal carcinoma,and the expression of 5-8F was relatively low.3.The overexpression of TIM-4 in 5-8F NPC cells and the stable transfection of 5-8F-NC group and 5-8F-TIM4 group of negative control were successfully constructed.Fluorescence microscope count and flow cytometry analysis showed that the transfection efficiency of 5-8F-NC and 5-8F-TIM4 stable transfection lines were all over 90%.The q RT-PCR results showed that the expression level of TIM-4 in 5-8F-TIM4 cell line was significantly higher than that in 5-8F-NC(P < 0.001).Western blotting showed that the expression level of TIM-4 in 5-8F-TIM4 cell line was significantly higher than that of 5-8F-NC.These results indicated that the two stably transfected cells could be used for subsequent experiments.4.CCK-8 test showed that there was no significant difference in absorbance between 5-8F-NC group and 5-8F-TIM4 group 24 h after inoculation,but the absorbance of 5-8F-TIM4 group was significantly higher than that of 5-8FNC group 48 h and after inoculation(P < 0.05);The plate clone formation assay showed that the plate clone formation ability of 5-8F-TIM4 group was significantly higher than that of 5-8F-NC group(P < 0.05).5.The results of scratch healing experiment showed that the healing rate of 5-8F-TIM4 group at 24 h and 48 h after scratch was significantly higher than that of 5-8F-NC group(P=0.041<0.05,P=0.033<0.05,respectively);Transwell migration assay results showed that the number of 5-8F-TIM4 cells crossing the basement membrane was significantly increased compared with the 5-8FNC group(P < 0.001).6.Annexin V-PI double staining flow cytometry results showed that the apoptosis rate of cells transfected with 5-8F-TIM4 group was decreased compared with 5-8F-NC group,the difference was statistically significant(P< 0.05).The results of PI single staining flow cytometry showed that compared with the 5-8F-NC group,the proportion of 2N in the 5-8F-TIM4 transfected group was significantly decreased(P=0.001<0.01),the proportion of S phase was significantly increased(P=0.000<0.001),and the proportion of4 N was not significantly different(P > 0.05).Conclusions:1.The abnormally elevated expression of TIM-4 in NPC tissues is correlated with TNM stage,presence or absence of lymph node metastasis,and the depth of tumor invasion,suggesting that TIM-4 is related to the development and disease stage of NPC.It may be a potential tumor marker for NPC.2.Overexpression of TIM-4 can promote the proliferation and migration of NPC cells,and enhance the malignant biological behavior of NPC tumors.