The Relationship Between LEF-1 Gene And Behavior, Morphology And Related Gene Expression Of Gastric Cancer Cells

Background and Objective:Gastric cancer(GC)is one of the most common gastrointestinal cancer,and its incidence and mortality ranks as the first in all types of cancer in China.Currently,the traditional diagnostic methods of GC include x-ray with the barium meal double contrast method,fibre-optic gastroscopy,B-ultrasonic detection,CT check and so on.However,the sensitivity of these methods is so poor that early GC is easy to loss for patients,thus missed the optimal treatment.In addition,while surgical resection and radiation combined with chemotherapy aimed at GC treatment is still the main way for the treatment of GC,the traditional surgical treatment is slow,and radiotherapy combined with chemotherapy due to its toxicity and the lack of targeting lead to poor prognosis.Gene diagnosis and treatment as a new methods in recent years,has obvious advantages in the treatment and prognosis for cancer.The gene therapy targeting to malignant tumor is hopeful to bring a new dawn.Therefore,the study of gene function related with the tumor is particularly important.Through exploring the gene function involved in the occurrence and development mechanism of GC to achieve gene diagnosis and treatment,which may be the key to solve the cancer clinical dilemma.Tumor metastasis,recurrence and the malignant degree of tumor cells are the important markers of malignant cancer.In the process of tumor development,a cell can through the epithelial mesenchymal transition(EMT)process to obtain the malignant phenotype of tumor cells.Previous researches have indicated that the EMT process is associated with aberrant expression of multiple genes.At present,the common research methods of gene function:gene knockout and gene knockdown.At the cellular genomic level,gene knock-out completely removes gene expression from the transcriptional level and leads to a stable cell line.But compared to the gene knockout,gene knockdown inhibits gene expression at the post transcriptional level or at the post-translational level and has the advantages of simple operation,short cycle,low cost.RNA interference technology as the most commonly method for gene knockdown.Our previous studies suggest that LEF-1 is highly expressed in numerous malignant tumor cells and tissues,which is closely correlated with the process of malignant tumors occurrence and development.In order to explore the relationship between LEF-1 overexpression and malignant progression of gastric carcinoma,explore its significance in the development of gastric cancer.We inhibited the LEF-1 expression in SGC-7901 cells using RNAi,followed by the analysis of cell motility,growth and microsructures.Further,in order to explored the possible relationship between LEF1 and gastric cancer EMT,the effects of LEF1 on the expression of EMT related genes were also explored.Method1.NCBI database search LEF-I gene for online design of LEF-1 shRNA,Blast Hit evaluates specific,constructed pU6.1-GFP/LEF1-shRNA.2.pU6.1-GFP/LEF1-shRNA plasmids were transfected into SGC-7901 cells by calcium phosphate transfection methods.shRNAscr and wild type cells as a control.After 48h,Western blot confirmed suppression efficiency.3.Wound healing assay and transwell assay were used to detect the motility in LEF-1 silenced SGC-7901 cells.4.Downregulation of LEF-1,MTT assay and Clonogenic assay were introduced to detect the proliferation of SGC-7901 cells.5.Immunofluorescence analysis,Immunohistochemistry and Coomassie brilliant blue stainingwere used to detect the change of F-actin,P-tubulin,Lamin B1 in LEF-1 silenced SGC-7901 cells.6.Downregulation of LEF-1,the scanning electron microscope were used to observe the morphology and motility correlated structures of SGC-7901 cells.7.Downregulation of LEF-1,Western blot analysis and Immunohistochemistry were used to detect the cell levels of E-cadherin,Vimentin,Snail expression.8.Bioinformatics analysis was used to analyze the mutation of LEF-1 gene in tumors,especially in gastric cancer and preliminarily analyze and predict the gene which is closely related to the LEF-1.Result1.Successfully constructed recombinant plasmid targeting LEF-1,and the efficiency of calcium phosphate co-precipitation transfection efficiency was more than 80%in SGC-7901 cells.2.LEF-1 shRNA plasmid significantly inhibited the LEF-1 expression in SGC-7901 cells.3.Wound healing assay and transwell assay results suggest that the motility was inhibited significantly in LEF-1 silenced SGC-7901 cells.4.MTT assay and Clonogenic assay results indicated that downregulation of LEF-1,the proliferation of SGC-7901 was inhibited significantly.5.Immunostaining results showed that the expression of F-actin was decreased and the bundle of F-actin was weakened,the distribution of ?-tubulin was depolymerized,the expression of LaminB1 was decreased and further examination revealed a decrease in the average optical density of LaminBl in LEF-1 silenced SGC-7901 cells.The coomassie brilliant blue staining results showed that the overall skeleton cell dysplasia,the cell motility related structures development is inhibited.6.Displaying under the scanning electron microscope results suggest that downregulation of LEF-1,the cell morphology was changed,cell surface microvilli and pseudopodia/filopodia structure associated with motility were impaired,and showed a time dependent.7.Western blot and immunohistochemistry results shown that downregulation of LEF-1,the protein expression levels of E-cadherin was increased,vimentin and snail were significantly suppressed.Conclusion1.Downregulation of LEF-1 could effectively inhibit SGC-7901 cell motility and proliferation ability,alter the cell motility correlated structures,induces remodeling of cytoskeleton system leading to the malignant phenotype changed of SGC-7901 cells.This results showed that LEF-1 play an important role on SGC-7901 cells proliferation,diffusion,metastasis,invasion and cancer development process.2.Western blot and immunohistochemistry results showed that the expression of EMT related marker gene E-cadherin,Vimentin,Snail showed a corresponding changes.These results suggest that LEF-1 also play an important role in the occurrence of gastric cancer.3.This paper from the perspective of gastric cancer cell malignant phenotype and EMT marker gene indicated that LEF-1 involved in the occurrence and development of gastric cancer,suggesting that LEF-1 can be used as a potential target in the diagnosis and treatment of gastric cancer.4.Bioinformatics analysis showed that the abnormal expression of LEF-1 in a variety of malignant tumors,Which is closely related with the APC,MMP9,TCF7,and BGN.LEF-1 may be considered as a promising biomarker for the molecular diagnosis and a candidate target for the therapy of human gastric cancer.