Study On The Mechanism Of Plumbagin Inhibiting The Invasion And Migration Of Tongue Squamous Cell Carcinoma Cells By Regulating Glycolysis

Oral cancer is one of the most common malignant tumors of the head and neck.About 50% of oral cancers are tongue squamous cell carcinoma(TSCC),so tongue squamous cell carcinoma has a very high incidence in oral and maxillofacial regions.In recent decades,despite the continuous improvement of treatment technology,the5-year survival rate of patients with TSCC is still less than 50%.Among them,the significant local invasion tendency and the high incidence of early cervical lymph node metastasis(about 40%)are important factors leading to the poor prognosis of patients with TSCC.Although there are a large number of studies on the invasion and metastasis mechanism of TSCC,the factors and specific mechanisms that effectively inhibit the invasion and metastasis of tongue squamous cell carcinoma are still being explored.Therefore,in-depth study of the mechanism of invasion and metastasis of TSCC,finding tumor treatment targets,and developing effective therapeutic drugs are of great significance for improving the prognosis of patients with TSCC.Tumor cell metastasis is a complex multi-step process including invasion,extravasation,infiltration,and colonization,and is affected by activities or signals generated in tumor cells(intrinsic)and microenvironment(exogenous).In this dynamic multi-stage process,the energy metabolism of tumor cells is reorganized to improve glycolytic activity,which is essential for the maintenance of tumor cells' Epithelial-mesenchymal transitions(EMT)and resistance to anoikis.The increase in glucose uptake is necessary to maintain high levels of glycolysis.Glucose transporters(Gluts)are important carriers for glucose to pass through hydrophobic cell membranes and enter cells.Almost all cells' glucose uptake is regulated by Gluts.Glucose transporter isoform 1(Glut1)is the first to be purified in the Gluts family and one of the most extensively studied members.It is related to glycolytic activity in a variety of tumor cells and is a key protein that regulates the glycolytic process.In our previous research,we found that plumbagin(PLB)can not only inhibit the EMT process of tongue squamous cell carcinoma cells.And through SILAC quantitative proteomics test analysis,it is found that PLB can also affect the glycolysis process of TSCC cells.Therefore,this study used Glut1 as the starting point to explore the main mechanism of PLB inhibiting the invasion and migration of TSCC cells.First,explore the relationship between Glut1 and TSCC metastasis.We first downloaded the clinical and follow-up data of patients with oral tongue squamous cell carcinoma from the SEER database,and confirmed that lymph node metastasis is an independent poor prognostic factor for patients with TSCC.And through immunohistochemical test,it was found that the high expression of Glut1 in the tumor tissue of patients with TSCC was related to lymph node metastasis.Immunofluorescence experiments have also confirmed that compared with normal gingival epithelial cells(HGE),the expression of Glut1 in TSCC cells(CAL27,SCC15)is significantly increased.Subsequently,lentivirus was used to knock down Glut1 in TSCC cells to verify whether down-regulation of Glut1 would affect the invasion and migration of TSCC cells.Transwell and scratch test results show that down-regulation of Glut1 can inhibit the invasion and migration of TSCC cells.And through the weighted gene co-expression network analysis(WGCNA)method to analyze the gene expression data of TSCC,it is concluded that the expression of Glut1 is related to the epithelial-mesenchymal transitions(EMT)process of TSCC.To confirm the results of WGCNA analysis,WB experiments verified that knockdown of Glut1 can promote the expression of E-cadherin and inhibit the expression of N-cadherin and Vimentin,indicating that downregulation of Glut1 can inhibit the EMT process of TSCC cells.Therefore,we conclude that down-regulation of Glut can inhibit the invasion and migration of TSCC cells by inhibiting the EMT process.Secondly,to explore the role of Glut1 in PLB inhibiting the invasion and migration of TSCC.First,the CCK8 experiment was used to screen the appropriate concentration of PLB on TSCC cells.And using Transwell experiment and scratch experiment to verify that PLB can inhibit the invasion and migration ability of TSCC cells.Subsequently,it was confirmed in WB experiments that PLB can inhibit the expression of Glut1,N-cadherin and Vimentin,and promote the expression of E-cadherin,indicating that PLB has a good inhibitory effect on the Glut1 and EMT process in TSCC cells.To investigate whether the up-regulation of Glut1 can reverse the inhibitory effect of PLB on the EMT process and the invasion and migration ability of TSCC cells.We up-regulated Glut1 in TSCC cells through lentivirus.The experimental results show that the up-regulation of Glut1 can significantly reverse the inhibitory effect of PLB on the EMT process and the ability of invasion and migration of TSCC cells.Therefore,we conclude that PLB can affect the invasion and migration of TSCC cells by down-regulating the expression of Glut1 to inhibit the EMT process.Thirdly,we will explore whether PLB can affect the glycolytic activity of TSCC cells by down-regulating the expression of Glut1,thereby inhibiting its EMT process,leading to inhibition of the invasion and migration ability of TSCC cells.We first used the gene expression data of oral TSCC in the TCGA database to analyze the signaling pathways related to Glut1 through the GESA method.The results were similar to the results of WGCNA.The results showed that the expression of Glut1 was related to the glycolytic process of TSCC.And through the test of glucose and lactic acid,it was found that knocking down Glut1 can inhibit the glucose consumption and the production of lactic acid in TSCC cells.The WB experiment verified that knockdown of Glut1 can inhibit the expression of glycolysis-related proteins HK2 and LDHA,indicating that knockdown of Glut1 can inhibit the glycolysis process of TSCC cells.To explore the effect of glycolytic activity on the EMT process and the ability of invasion and migration of TSCC cells.Use glycolysis inhibitor 2-deoxy-D-glucose(2-Deoxy-D-glucose,2DG)to act on TSCC cells.Transwell and scratch test results show that 2DG can significantly inhibit the invasion and migration ability of TSCC cells.WB experiment results show that it can promote the expression of E-cadherin and inhibit the expression of N-cadherin and Vimentin.The above shows that the inhibition of glycolytic activity can inhibit the EMT process,invasion and migration ability of TSCC cells.Subsequently,2DG and PLB were used in conjunction with Glut1 up-regulated TSCC cells.Transwell and scratch test results show that,compared with the PLB alone group,the glycolysis inhibitor 2DG can eliminate the upregulation of Glut1 on the reversal of the ability of PLB to inhibit the invasion and migration of TSCC cells.And it was verified in WB experiments that2 DG can also eliminate the up-regulation of Glut1 on the reversal effect of PLB on the EMT process of TSCC cells.Finally,the establishment of a nude mouse lung metastasis model verified that the use of PLB treatment or the silence of Glut1 can effectively inhibit the number of lung metastases of TSCC cells,and the upregulation of Glut1 can significantly reverse the inhibitory effect of PLB.It was verified in nude mouse xenograft model that after using PLB treatment or silencing Glut1,the results of immunohistochemical experiments showed that both can effectively promote the expression of E-cadherin in TSCC tissues and inhibit N-cadherin As well as the expression of Vimentin in TSCC tissues,the up-regulation of Glut1 can significantly reverse the effect of PLB on the EMT phenotype of TSCC tissues.Therefore,we conclude that PLB can affect the glycolytic activity of TSCC cells by down-regulating the expression of Glut1,thereby inhibiting its EMT process,leading to inhibition of the invasion and migration ability of TSCC cells.Finally,the possible mechanism of PLB affecting the expression of Glut1 was discussed.The previous research of our group found that RAP1GDS1 is the most obvious protein inhibited by PLB in TSCC.To explore whether RAP1GDS1 plays a role in the influence of PLB on the expression of Glut1 and the invasion and migration of TSCC cells.We first used the gene expression data and clinical data of oral TSCC in the TCGA database to analyze the relationship between RAP1GDS1 and TSCC lymph node metastasis.The results showed that RAP1GDS1 was related to TSCC lymph node metastasis.Then use WGCNA and GSEA methods to analyze the biological pathways that may be affected by RAP1GDS1.The analysis results show that the pathways related to RAP1GDS1 are similar to the analysis results of Glut1.For this reason,the gene expression data of RAP1GDS1 and Glut1 were used for correlation analysis,and the results showed that the gene expression of RAP1GDS1 and Glut1 had a strong correlation.After silencing RAP1GDS1 in tongue squamous cell carcinoma cells by RNAi technology,WB results showed that RAP1GDS1 silencing can inhibit the expression of Glut1 in tongue squamous cell carcinoma cells.And the results of Transwell and scratch experiments showed that silence of RAP1GDS1 can significantly inhibit the invasion and migration ability of TSCC cells,while up-regulating the expression of Glut1 can significantly inhibit this process.Subsequently,the WB experiment verified the inhibitory effect of PLB on RAP1GDS1 in TSCC cells,and using molecular docking technology,analysis showed that PLB and the splice variant of RAP1GDS1,Smg GDS-558,have direct action sites.Therefore,we preliminarily infer that PLB may regulate Glut1 through RAP1GDS1,thereby affecting the invasion and migration of TSCC cells.In summary,we conclude that: 1.PLB can inhibit the EMT process by down-regulating Glut1 to affect glycolytic activity,thereby inhibiting the invasion and migration of tongue squamous cell carcinoma cells;2.PLB may regulate Glut1 through RAP1GDS1,thereby affecting Invasion and migration of tongue squamous cell carcinoma.However,the exact mechanism needs further experimental verification.