Study On The Mechanism Of SPAG6 Promoting Malignant Progression Of Esophageal Cancer Cells Through MTOR/LC3B

Objective:To investigate the mechanism of SPAG6 promoting malignant progression of human esophageal cancer cells through mTOR/LC3B.Method:The experiment was divided into 6 groups:blank group,blank plasmid group and Spag6 overexpression group.Blank group,blank vector group,Spag6 shRNA group.The cell lines of esophageal cancer were Kyse-150 and Eca-109.1.Bioinformatics method was used to analyze the expression of SPAG6 in human esophageal cancer cells and the correlation between SPAG6 and autophagy related antibodies.The docking between SPAG6 protein and mTOR channel protein was simulated to determine whether mTOR channel protein was downstream of SPAG6 protein.2.MTT was used to determine the SPAG6shRNA and SPAG6cDNA groups cell viability in Kyse-150 and Eca-9706 cells.3.Transwell assay was used to determine SPAG6shRNA and SPAG6cDNA groups the tumor metastasis in Kyse-150 and Eca-9706 cells.4.Transwell assay was used to determine SPAG6shRNA and SPAG6cDNA groups the migration ability in Kyse-150 and Eca-9706 cells.5.The wound-healing assay was used to determine SPAG6shRNA and SPAG6cDNA groups migration and viability in Kyse-150 and Eca-9706 cells.6.Pi/FITC assay was used to determine SPAG6shRNA and SPAG6cDNA groups apoptosis in Kyse-150 and Eca-9706 cells.7.TUNEL assay was used to determine apoptosis of SPAG6shRNA and SPAG6cDNA groups apoptosis in Kyse-150 and Eca-9706 cells.8.WB assay was used to determine SPAG6.mTOR and LC3B protein expression of SPAG6shRNA and SPAG6cDNA groups in Kyse-150 and Eca-9706 cells.Results:1.Bioinformatics analysis showed that SPAG6 was highly expressed in human esophageal cancer cells.The DOCK score of SPAG6 and mTOR molecular protein was 1342,indicating good docking.2.MTT cell proliferation experiment results showed that the OD value of SPAG6 plasmid transient group was higher than that of blank group and blank control group,P.T(<0.05,11.34)the OD value of SPAG6 shRNA group was lower than that of blank group and blank control group,P:T(<0.05,16.28).3.Tanswell invasion assay showed that Kyse-150 and Eca-9706 SpAG6 gene knockout decreased invasion ability,P:T(<0.05,17.20);On the contrary,P:T(<0.05,15.79).4.Tanswell migration experiment showed that the migration ability was decreased after Kyse-150 and Eca-9706 SpAG6 gene knockout,P:T(<0.05,9.734);On the contrary,P:T(<0.05,21.64).5.Cell scratch test showed that the healing ability of Kyse-150 and Eca-9706 cells after SPAG6 gene knockout was decreased P:T(<0.05,7.36);On the contrary,P:T(<0.05,19.28)6.TUNEL assay and flow assay showed that the apoptosis rate of Kyse-150 and Eca-9706 cells was increased after SPAG6 gene knockout,P:T(<0.05,12.16);On the contrary,P:T(<0.05,8.34).7.The results of PI/FITC flow cytometry showed that the apoptosis rate of Kyse-150 and Eca-9706 cells was increased after SPAG6 gene knockout,P:T(<0.05,17.25);On the contrary,P:T(<0.05,30.31)8.WB assay showed that the gray values of mTOR protein and LC3B in Kyse-150 and Eca-9706 cells increased after overexpression of SpAG6,P:T(<0.05,19.36);On the contrary,P:T(<0.05,29.15)Conclusion:1.SPAG6 is an oncogene in esophageal cancer,which is negatively correlated with the clinical stage and prognosis of patients2.There is a docking relationship between SPAG6 and mTOR,and mTOR protein is the receptor of SPAG6 protein3.SPAG6 act as an oncogene through mTOR4.SPAG6 shRNA is a potential mTOR inhibitor and may become a target of anti-tumor therapy.