The Regulatory Mechanism Of Mdig On Metastasis And Proliferation Of Non-small Cell Lung Cancer

Objective:Mineral dust-induced gene（mdig）can inhibit the invasion and metastasis of A549 cells and increase the proliferation of multiple cells.The main purpose of this study was to explore the molecular mechanism underlying the inhibitory effect of mdig on cell invasion and metastasis and the promotive effect of mdig on cell proliferation.Methods:In the first part of this paper,to reserch the molecular mechanism underlying the inhibitory effect of mdig on cell invasion and metastasis.Mdig-knockdown and mdig-overexpressing stable transfection A549 cells and an mdig-overexpressing stable transfection human umbilical vein endothelial cell（HUVEC）line were constructed using lentiviral vectors.Fourth generation cells steady transfected with lentivirus were observed under the bright field and GFP fluorescence of an inverted fluorescence microscope at x100 magnification to observe the transfection efficiency and compared the cell morphology GFP fluorescence at x400 magnification.Western blot analysis was performed to verify the silencing and overexpression of the mdig protein.A Transwell invasion assay was used to detect the invasive abilities of each experimental group,and Transwell migration and scratch assays were used to detect cell migration ability.Western blotting was subsequently conducted to detect the major biochemical indices of the GSK-3β/β-catenin pathway（P-GSK-3β,β-catenin,activeβ-catenin）and the protein modification levels and expression levels and modifications of epithelial-mesenchymal transition（EMT）transcription factors（slug,snail,ZEB1）,as well as changes in the expression levels of EMT molecular markers（E-cadherin,claudin-1,ZO-1,N-cadherin,vimentin）and intercellular adhesion proteins（integrinβ1,integrinβ4）.In the second part,to reserch the molecular mechanism underlying the increased effect of mdig on cell proliferation.Mdig-knockdown and mdig-overexpressing stable transfection A549 cells and an mdig-overexpressing stable transfection human umbilical vein endothelial cell（HUVEC）line were constructed using lentiviral vector.The specific PI3K signal transduction pathway inhibitor（LY294002）was used to treat mdig-overexpressing stable transfection A549 cells and HUVEC cells.The ability of cell proliferation on each experimental group was detected using the EdU fluorescence imaging method（percentage of the EdU positive cells）.Western blotting was subsequently conducted to detect the major biochemical indices of the PI3K/Akt（P-pten,P-PDK1,Akt,P-Akt（Thr308））and mTORC2/Akt（mTOR,Rictor,GβL,Akt,P-Akt（Ser473））pathway and the the downstream regulation cell cycle related protein(P^18INK4C,P^19INK4D,CDK2,CDK6)expression levels.Results:Successful construction of stable transfection cell lines:Fourth generation A549cells and HUVEC cells transfected with lentivirus were observed under the bright field of an inverted fluorescence microscope at x100 magnification.The same fields of view were analyzed for GFP fluorescence at x100 and x400 magnification.All the cell groups exhibited a high cell viability and high transfection efficiency.In the mdig silencing experiment,mdig protein expression was significantly decreased in the LV-mdig-RNAi 1and LV-mdig-RNAi 2 groups when compared with normal A549 cells and the LV-con group.In A549 cells overexpressing mdig,mdig protein expression was significantly increased in the LV-mdig group when compared with normal A549 cells and the vector group.In HUVEC cells overexpressing mdig,the expression of mdig protein in the LV-mdig group was significantly upregulated when compared with the normal HUVEC and vector groups.Effect of mdig on the invasion and migration of cells:In the mdig silencing experiment,cells in the LV-con group exhibited a cobblestone-like cell morphology,while cells in the LV-mdig-RNAi 1 and LV-mdig-RNAi 2 groups exhibited more elongated spindle-like shapes.Transwell assays were performed to determine the effects of mdig silencing and overexpression on the invasion and migration of A549 cells.The results of the invasion experiment demonstrated that mdig overexpression significantly decreased the number of cells that invaded through the matrix and membrane,while mdig knockdown significantly increased the number of cells when compared with the normal A549 and control groups.Identical results were obtained from the migration experiment.In order to further verify the effect of mdig on the migratory ability of A549 cells,a scratch-wound assay was performed.Compared with the control group,the mdig overexpression A549 cell group exhibited a significantly slower healing rate,and the mdig knockdown group exhibited a significantly faster healing rate.Therefore,mdig overexpression can inhibit the invasion and metastasis of A549 cells,and silencing of mdig can increase the invasive and migratory properties of cells,suggesting that mdig has the ability to inhibit the invasion and metastasis of A549 cells.In order to further explore the mechanism underlying these inhibitory effects of mdig,this study examined the effect of mdig on the expression of the GSK-3β/β-catenin signaling axis,the downstream transcription factors snail,slug and ZEB1,and the major molecular markers of EMT.The results showed that mdig could inhibit the phosphorylation of GSK-3βat Ser9 and promote the phosphorylation ofβ-catenin,which resulted in a decrease in the active（non-phospho at Ser33,Ser37,and Thr41）form ofβ-catenin,leading to a reduction in the direct promotion of slug,snail and ZEB1.Mdig could also upregulate epithelial cell markers（E-cadherin,claudin-1 and ZO-1）and mediate the expression of integrinβ1 and integrinβ4,the key facilitators of extracellular matrix adhesion,while downregulating mesenchymal cell markers（N-cadherin and vimentin）.These results suggested that mdig can promote the phosphorylation ofβ-catenin by inhibiting the phosphorylation of GSK-3β,in order to suppress the expression of slug,snail and ZEB1 and the occurrence of EMT,and thereby inhibit the invasion and metastasis of NSCLC.The present study also used HUVEC cells to verify the above molecular mechanisms,and the same results were obtained.Effect of mdig on the proliferation of cells:The ability of cell proliferation on each experimental group was detected using the EdU fluorescence imaging method.The results indicated that EdU%positive cell was significantly increased in the LV-mdig group when compared with normal A549 cells and the vector group.While EdU%positive cell was significantly decreased in the RNAi groups when compared with normal A549 cells and the LV-con group.The present study also used HUVEC cells to verify the above molecular mechanisms,and the same results were obtained.Western blot was subsequently conducted to detect the major biochemical indices of the PI3K/Akt（P-pten,P-PDK1,Akt,P-Akt（Thr308））and mTORC2/Akt（mTOR,Rictor,GβL,Akt,P-Akt（Ser473））pathway and the the downstream regulation cell cycle related protein(P^18INK4C,P^19INK4D,CDK2,CDK6)expression levels.The present study demonstrated that mdig can promote the phosphorylation PDK1（Ser241）and further promote the phosphorylation Akt（Thr308）and activation Akt,in order to promote the expression of cyclin-dependent kinases CDK2and CDK6 and suppress the expression of cyclin-dependent protein kinase inhibitors P^18INK4Cand P^19INK4D,thereby increase the cell proliferation of non-small cell lung cancer（NSCLC）.The results also indicated that mdig can promote the phosphorylation Akt（Ser473）mainly by promoting the expression of m TORC2 complex.Mdig may not directly affect the expression of mTOR protein,but mdig could promote the expression of GβL and Rictor in mTORC2 complex（mTOR+GβL+Rictor）further promote the phosphorylation Akt（Ser473）and activation Akt protein,thereby increase the cell proliferation of non-small cell lung cancer（NSCLC）.Conclusion:1.Mdig can inhibit the phosphorylation of GSK-3β（P-Ser9-GSK-3β）and promote the phosphorylation ofβ-catenin（P-Ser33,Ser37,and Thr41-β-catenin）,and downregulation of the downstream transcription factors slug,snail and ZEB1,thus leading to increased expression levels of epithelial cell markers,in order to suppress the occurrence of EMT,thereby inhibit the invasion and metastasis of A549 cells.2.Mdig can respectively promote the phosphorylation of Akt at Thr308 and Ser473 through PDK1 and mTORC2,in order to promote the expression of cyclin-dependent kinases CDK2 and CDK6 and suppress the expression of Cyclin-dependent protein kinase inhibitors P^18INK4Cand P^19INK4D,thereby increase the proliferation of A549 cells.