The Role And Mechanism Of GPNMB In The Progression Of Esophageal Squamous Cell Carcinoma

Objective: To explore the expression of Glycoprotein nonmetastatic melanoma protein B(GPNMB)in esophageal squamous cell carcinoma,its downstream target gene,its possible biological function and signaling pathway.To study the molecular function of GPNMB in esophageal squamous cell carcinoma.To clarify the mechanism of GPNMB in promoting malignant progression of esophageal squamous cell carcinoma by regulating PI3K/AKT signaling pathway.Methods: The first part: Isobaric tags for relative and absolute quantitation(ITRAQ)proteomics technology was used to analyze the tissue samples of 6 patients undergoing radical resection of esophagus cancer in the First Affiliated Hospital of Xinjiang Medical University.The differentially expressed proteins between esophageal squamous cell carcinoma tissue samples and paired paracancerous tissue samples were counted.Esophageal squamous cell carcinoma samples from TIMER,UALCAN,and GEO databases were used to initially verify the expression level of GPNMB in esophageal squamous cell carcinoma;bioinformatics techniques were used to analyze the biological functions and signaling pathways that GPNMB may participate in in esophageal squamous cell carcinoma.At the histological level,immunohistochemistry(IHC)staining was used to detect the expression of GPNMB,EGFR,p-PI3 K,and Ki-67 in clinical samples of ESCC,to analyze their correlation with clinicopathological features,and to evaluate their prognostic value.The second part: At the cellular level,TE-1esophageal squamous cell cell lines silenced GPNMB were constructed by si RNA,and transfection efficiency was detected by Western blot and q RT-PCR.The effect of silencing GPNMB on the biological behavior of esophageal squamous cell carcinoma cells was observed by CCK-8,Transwell and flow cytometry.At the animal level,lentivirus transfected GPNMB overexpressed stable strain was used to establish a mouse model of subcutaneous graft tumor.By monitoring the volume and weight of tumor tissue,HE staining and immunohistochemical staining were used to observe the effect of GPNMB on tumor formation ability in mice.The third part: At the cellular level,a GPNMB overexpressed KYSE-150 esophageal squamous cell cell line was constructed by plasmid transfection and treated with PI3 K inhibitor LY294002.The control group,GPNMB overexpression group and GPNMB overexpression +LY294002 group were established to compare the differences of cell proliferation,apoptosis,cycle,migration and invasion among all groups.To further verify the effect of GPNMB on the function of esophageal squamous cell carcinoma cells,and analyze whether LY294002 can reverse the effect of GPNMB on esophageal squamous cell carcinoma cells.Western blot was used to detect the key proteins of PI3K/AKT signaling pathway,and to explore the possible signaling pathways involved in the role of GPNMB in promoting cancer in esophageal squamous cell carcinoma.Results: The first part:(1)After proteomic analysis,GPNMB was selected as the main research index of this project;(2)UALCAN database showed that the expression of GPNMB was increased in esophageal squamous cell carcinoma;(3)The high expression of GPNMB in esophageal squamous cell carcinoma samples of GEO(GSE69925 data set)is related to the processes of mismatch repair(MISMATCH＿REPAIR),cell cycle(CELL＿CYCLE)and cancer pathway(PATHWAY＿IN＿CANCER);(4)Immunohistochemical results suggest that the expression of GPNMB is related to AJCC stage,lymph node metastasis,and degree of differentiation,and the prognosis of patients with high expression is worse;(5)GPNMB was positively correlated with EGFR,p-PI3 K and Ki-67 expression in esophageal squamous cell carcinoma.The second part:(1)The activity of cells in the GPNMB silencing group was significantly lower than that of the control group;(2)Compared with the control group,the proportion of G1 phase cells in the GPNMB silencing group was higher,the proportion of S phase cells was lower,and the apoptosis rate was higher;(3)Compared with the control group,the cell migration ability of the GPNMB silencing group was significantly weakened;(4)The invasive ability of esophageal squamous cell carcinoma cells was inhibited in the GPNMB silencing group;(5)Subcutaneous tumor formation in nude mice found that the tumor growth rate of the GPNMB overexpression group was faster than that of the control group,and the tumor volume and weight were higher than those in the control group.Part III:(1)The expressions of EGFR,p-PI3 K and p-AKT in GPNMB overexpression group were higher than those in control group,but the expressions of PI3 K and AKT were not significantly changed.The expressions of EGFR,p-PI3 K and p-AKT in GPNMB overexpression +LY294002 group were decreased compared with those in GPNMB overexpression group.(2)The cell activity of GPNMB overexpression group was higher than that of control group,and the cell activity of GPNMB overexpression +LY294002group was lower than that of GPNMB overexpression group.(3)Compared with the control group,the proportion of S phase was higher in the GPNMB overexpression group,the proportion of G1 phase was lower in the GPNMB overexpression +LY294002 group,the proportion of S phase was lower in the GPNMB overexpression group,the proportion of G1 phase cells was higher,and the number of apoptotic cells was higher.(4)The number of cell migration and invasion in GPNMB overexpression group was higher than that in control group,and the number of cell migration and invasion in GPNMB overexpression +LY294002 group was lower than that in GPNMB overexpression group.Conclusion: GPNMB is an up-regulated differentially expressed protein in ESCC,and high expression of GPNMB is associated with poor prognosis in ESCC.GPNMB mediates EMT through PI3K/AKT signaling pathway and promotes the proliferation,migration,invasion and inhibits apoptosis of ESCC.