In Vitro Study On The Role And Mechanism Of SOX4 In Normal And Lung Cancer Cell EMT

Background:Lung cancer,as a primary malignant tumor with the highest incidence and mortality,it has the characteristics of high degree of malignancy and ineffective treatment.Metastasis is the main cause of poor quality of life and decreased survival,and it has become a major problem in clinical treatment.Therefore,from the root causes to control the metastasis of tumors,can become one of the main research directions of lung cancer.Tumor metastasis is complicated.Among them,the biological processes of a series of specific procedures with the initial steps of cell junction and polarity loss and obtaining the mesenchymal cell phenotype are essential,Known as Epithelial-Mesenchymal Transition(EMT).Increased expression of N-cadherin.(N-Cad),fibronectin(FN),matrix metalloproteinases(MMPs),and EMT-related transcription factors Twist,Snail,and Zeb family;The expression of E-cadherin(E-Cad),a cell adhesion molecule,was down-regulated,and the vimentin(Vim)was gradually transformed into a mesenchymal cytoskeleton.Promote cell polarity,basement membrane junction and loss of epithelial or endothelial cell phenotype to assist in the resistance,proliferation,invasion,metastasis and planting of malignant tumors.EMT has become a hot spot in the field of precision diagnosis and treatment of cancer.Previous research shows that a variety of transcription factors play an important role in the regulation of EMT.Among them,Sex determining Region Y-related(SRY)-type high mobility group(HMG)-box 4(SOX4)can regulate occurrence and progress of multi-cancer EMT through transcription and non-transcription.However,its role in the development of lung cancer remains to be further studied.Based on the previous work,this paper uses cell culture,real-time fluorescence quantitative polymerase chain reaction(QRT-PCR)and Western blotting(WB)and many more ways,to explore The mechanism of action of SOX4 in EMT of normal and lung cancer cells by in vitro experiments.In order to lay the experimental foundation for the development of new biomarkers for lung cancer.Objective:In this study,overexpression and silencing of SOX4 gene were used as the basic research methods to observe the effects of SOX4 on proliferation,migration and invasion of normal and lung cancer cells.To explore the relationship between SOX4 and EMT and its mechanism,and provide a theoretical basis for its research and drug development in lung cancer.Simultaneously,it provides new biomarkers and potential therapeutic targets for individualized diagnosis and treatment of various types of lung cancer.Methods:1.Construction of pEX-2SOX4 overexpression plasmid and GFP empty plasmid;Construction of shRNA803,shRNA965,shRNA1081,shRNA2026 and shNC using pGPU6 as a vector.Transfection of normal epithelial cells with lung cancer cells by liposome method.Overexpression and silencing effects were verified by Quantitative Real Time Polymerase Chain Reaction(QRT-PCR)and Western Blot(WB).2.Culture 16 strains of cells,They are normal epithelial cells(Beas-2b,16HBE);small cell lung cancer cells(H446);lung adenocarcinoma cells(95D,A549,H1299,H157,H23,H292,HCC827,SPCA-1,XWLC-05,MSTO-211H);lung squamous cell carcinoma cells(H226,SK-MES-1),large cell lung cancer cells(H460).Choose five cell lines from the normal lung cell and four pathological types of lung cancer cell lines with the highest basal expression of SOX4 were screened by QRT-PCR and WB,to carry out the latter experiment.3.The effects of overexpression and knockdown of SOX4 gene on cell proliferation,migration and invasion were verified by plate colony formation assay,CCK8 cell proliferation assay,Wound Healing assay,Transwell migration and invasion assay.4.QRT-PCR and WB detect changes in the transcription and translation levels of EMT-related genes after successful expression of the target gene in cells.5.QRT-PCR method was used to detect the expression of EMT-related factors(ADAM28?BMI1?CDH11?CK2?CLDN1?CLDN10?CLDN2?Cofilin?CyclinD1?DIA1?E2F1?E-Cad?EIF4EBP1?FN?ILK?JAGI?KRT8?LAMC2?LAMTOR3?MLC?MMP14?MMP2.MMP3?MMP9?Myc?N-Cad?PAK1?PCBP1?PDPK1?Smad2?Smad3?Smad7?Snail?Snail2?Snail3?TGF-?1?TGF-?2?TJP1(ZO-1)?TJP2(ZO-2)?TLR4?Twist?Vim?WIF1?Wnt2?ZEB1?ZEB2??-SMA?p-catenin)in the cells of five cell lines after overexpression and knockdown of SOX4 gene.Results:1.The SOX4 overexpression plasmid,GFP empty plasmid,shRNA803,shRNA965,shRNA1081,shRNA2026 Interference plasmid,and shNC empty plasmid were successfully constructed.The cells were transfected with LipofectamineTM 2000 Reagent,observed under a fluorescence microscope,and verified by QRT-PCR and WB.The transfection efficiency and silencing and overexpression of the plasmid of interest were confirmed.2.Successfully cultured 16 normal lung epithelial cells and lung cancer cells.And the normal cells and four pathological types of lung cancer cell lines stably and highly expressing the target gene were s selected by QRT-PCR molecular level and WB protein level.They are normal epithelial cells(16HBE),small cell lung cancer cells(H446),lung adenocarcinoma cells(H157),lung squamous cell carcinoma cells(SK-MES-1),and large cell lung cancer cells(H460).3.Significant enhancement of H157 cell migration ability by overexpressing SOX4 gene by cell scratch assay(P<0.05,n=3),and 16HBE cells have no significant difference compared with the control group(P>0.05,n=3).at the same time,Knock down SOX4 in H157 Cells significantly Weaken cell migration by cell scratch assay(P<0.05,n=3),and 16HBE cells have no significant difference compared with the control group(P>0.05,n=3);Through the Transwell migration and invasion experiments,the cells overexpressing the SOX4 gene were found to have significantly increased the number of successfully migrated and invaded cells(P<0.05,n-3).Similarly,in the CCK8 and plate cloning experiment,Lung cancer cells overexpressing SOX4 have an excessive proliferation rate compared with the control group(P<0.05,n=3)?Conversely,after knocking down the SOX4 gene,the proliferation and invasion of H157 cells were significantly decreased(P<0.05,n=3).4.QRT-PCR and WB double validation of H157 cells overexpressing the SOX4 gene,E-Cad was found to be down-regulated at both transcriptional and translational levels(P<0.05,n=3),but N-Cad,FN,Vim and Snail showed differential increases(P<0.05 n=3).It is suggested that SOX4 can inhibit the expression of epithelial phenotype factors and up-regulate the expression of EMT transcription factors and mesenchymal markers;silencing the SOX4 gene also confirmed the above results.5.By QRT-PCR,it was found that the expression of EMT positive regulators ADAM28,CK2,CLDN2,CyclinD1,EIF4EBP1,and ?-catenin was increased,accompanied by the expression of EMT inhibitors E-Cad significantly down-regulated in five cells overexpressing SOX4.The remaining factors are expressed differently in each type of cell,as follows:5.1 In 16HBE cells overexpressing SOX4,EMT promoting factors CLDN1,CLDN10,DIA1,FN,JAG1,KRT8,LAMC2,LAMTOR3,MLC,MMP14,MMP2,MMP3,MMP9,Myc,N-Cad,PAK1,Smad2,Smad3,Snail The expression of Snail3,TGF-?1,TLR4,Twist,Vim,Wnt2,ZEB1,ZEB2 and a-SMA was increased,and the expression of E2F1 and LAMTOR3 was down-regulated,the difference was statistically significant(P<0.05,n=3);EMT inhibitory factor PCBP1,Smad7,TJP1,TJP2,and WIF1 were significantly up-regulated,and the difference was statistically significant(P<0.05,n=3).5.2 In H446 cells overexpressing SOX4,the expressions of EMT-promoting factors CDH11,CLDN1,CLDN10,DIA1,E2F1,FN,JAGI,MMP3,Snail,TGF-?2,Twist,Vim and ZEB1 were up-regulated,and the difference was statistically significant(P<0.05,n=3);the expression of EMT inhibitors Smad7,TJP1,TJP2,WIF1 and PCBP1 was down-regulated,the difference was statistically significant(P<0.05,n=3).5.3 In H157 cells overexpressing SOX4,EMT promoting factors BMI1,CDH11,CLDN1,Cofilin,DIA1,E2F1,FN,ILK,JAG1,KRT8,LAMC2,LAMTOR3,MLC,MMP14,MMP2,MMP3,Myc,N-Cad,The expressions of PAK1,PDPK1,Smad2,Smad3,Snail,Snail2,Snail3,TGF-?1,TGF-?2,TLR4,Twist,Vim,Wnt2,ZEB1,ZEB2 and a-SMA were up-regulated,and the difference was statistically significant(P<0.05,n=3).5.4 In SK-MES-1 cells overexpressing SOX4,EMT promoting factors BMI1,CDH11,CLDN10,E2F1,JAG1,KRT8,MLC,MMP14,MMP2,MMP3,MMP9,Myc,Smad2,Smad3,a-SMA The expression of catenin was increased,the expression of LAMTOR3,Twist and Wnt2 was down-regulated,the difference was statistically significant(P<0.05,n=3).The expressions of EMT inhibitors Smad7,TJP1,TJP2 and WIF1 were up-regulated,the difference was statistically significant(P<0.05,n=3).5.5 In H460 cells overexpressing SOX4,EMT promoting factors BMI1,CLDN1,CLDN10,Cofilin,E2F1,FN,KRT8,LAMC2,LAMTOR3,MLC,MMP14,MMP2,MMP3,MMP9,Myc,N-Cad,PAK1,Snail,The expressions of Snail2,Snail3,TGF-?2,TLR4,Twist,Vim,Wnt2,ZEB1,ZEB2 and a-SMA increased,the difference was statistically significant(P<0.05,n=3);EMT inhibitor Smad7,The expression of WIFI was increased and the expression of TJP2 was decreased.The difference was statistically significant(P<0.05,n=3).Among the above five types of cells,the factors described above showed corresponding reverse expression in the siRNA803 interference group.Most of these factors are involved in the regulation of EMT in lung cancer directly or indirectly through Wnt/?-catenin signaling pathway.Conclusion(s):1.SOX4 can promote the proliferation,migration and invasion of lung cancer cells by affecting the expression of EMT-related factors.Wnt/?-catenin is its main signaling pathway.2.SOX4 is expected to be a potential marker and potential target for diagnosis and treatment of small cell lung cancer,lung adenocarcinoma and large cell lung cancer with EMT.