The Research Of MAP4K4 Regulated MAPK And NF-?B Signaling Pathways And Is Involved In The Regulation Of Proliferation,Migration And Apoptosis Of Esophageal Squamous Cell Carcinoma Cells

?Objective?The relationship between MAP4K4 expression in esophageal squamous cell carcinoma and clinicopathological characteristics and prognosis by statistical analysis.Further in vitro experiments were used to reveal the effect and molecular mechanism of MAP4K4 expression changes on the proliferation,apoptosis,migration and invasion ability of esophageal squamous carcinoma cells.To explore the potential value of MAP4K4 protein as a potential site for targeted therapy and prognosis judgment in patients with esophageal squamous cell carcinoma.?Methods?(1)Based on bioinformatics,the MAP4K4 expression in esophageal squamous cell carcinoma was statistically analyzed through GEO database,and MAP4K4 data was extracted and statistically analyzed after standardization.55 pairs of normal tissues adjacent to cancer and esophageal squamous cell carcinoma were collected, and the expression of MAP4K4 in tissues was detected by immunohistochemistry. The relationship between the expression of MAP4K4 and the clinicopathological characteristics and prognosis of patients was analyzed.Western Blotting method was used to detect the difference in MAP4K4 expression between normal esophageal epithelial cell line Het-1a and esophageal squamous cell carcinoma cell lines TE-1 and ECA-109.(2)After transfecting HEK-293 T cells with plasmid and packaging and concentrating lentivirus by PEG precipitation method,the esophageal squamous cell carcinoma ECA-109 cell line with high expression of MAP4K4 was selected for lentivirus transfection knockdown.Fluorescence microscope to observe the positive cell rate. Cells positive for GFP expression were sorted by flow cytometry.Western Blotting was used to detect and evaluate the change of MAP4K4 expression in cells before and after knockdown treatment.(3)Cell counting method and clone formation experiment were used to detect the effect of MAP4K4 knockdown on the proliferation of ECA-109 cells.The scratch experiment and Transwell experiment verified the effect of knocking down MAP4K4 expression on cell migration and invasion ability.Western Blotting revealed that the expression of MMP-2 and MMP-9 in esophageal squamous cell carcinoma cells after knocking down MAP4K4.Combined with ionizing radiation treatment,flow cytometry and Western Blotting were used to detect the apoptosis of ECA-109 cells after knocking down MAP4K4.(4)Through the Western Blotting method,the key role of knocking down the MAP4K4 protein in the activation of MAPK and NF-?B signaling pathways and their regulation of proliferation,invasion and apoptosis was revealed.?Results?(1)In the data sets GSE45670 and GSE77861,the expression of MAP4K4 mRNA in esophageal squamous cell carcinoma tissues was higher than that in normal esophageal tissues(P < 0.01).Among 55 esophageal squamous cell carcinoma tissues,37(67.3%)patients had high expression of MAP4K4 in cancer tissues,18 (32.7%)patients had low expression in cancer tissues,and 32(58.2 %)MAP4K4 was negatively expressed,and 23 cases(41.8%)showed low expression of MAP4K4.further analysis found that the abnormally high expression of MAP4K4 was positively correlated with the size of esophageal squamous cell carcinoma, lymph node metastasis,and poor prognosis,and had no significant correlation with other clinicopathological characteristics of patients.The expression of MAP4K4 protein in esophageal squamous carcinoma cell lines TE-1 and ECA-109 cells was significantly stronger than that of normal esophageal epithelial cells Het-1a.(2)After transfection and sorting of lentivirus,fluorescence microscope observation showed that the positive rate of transfection was higher than 95%.Through Western Blotting detection,the shRNA sequence with significant effect(P < 0.05)was selected as the subsequent processing condition.(3)Cell counting experiments and clone formation experiments showed that after knocking down MAP4K4,the proliferation ability of esophageal squamous carcinoma cells weakened.(P < 0.05).The scratch experiment and Transwell experiment showed that after knocking down MAP4K4,the cell migration and invasion ability weakened(P < 0.05).Meanwhile,Western Blotting results showed that the knockdown of MAP4K4 also resulted in a decrease in the expression of invasion-related proteins MMP-2 and MMP-9.In the absence of radiotherapy radiation,knocking down MAP4K4 had no significant effect on apoptosis,but after combining with 10 Gy ionizing radiation for 24 h,the apoptosis level of ECA-109 cells knocking down MAP4K4 was significantly increased compared with the control group(P < 0.05).(4)After knocking down MAP4K4,phosphorylation of Erk1/2,JNK and c-Jun in the MAPK signaling pathway was inhibited,while p38 was not significantly affected. Knockdown of MAP4K4 in the NF-?B signaling pathway leads to the inhibition of TAK1,IKK?/?,I?B?,and NF-?B(p65)phosphorylation.?Conclusions?The expression of MAP4K4 in esophageal squamous cell carcinoma was significantly higher than that in normal tissues adjacent to the cancer,and the high expression of MAP4K4 was positively correlated with larger tumor size,easy lymph node metastasis and poor prognosis of patients.MAP4K4 may affect the proliferation,migration and apoptosis of esophageal squamous cell carcinoma cells by participating in MAPK and NF-?B signaling pathways.