Effect Of Sirna Expression On Migration Ability And Expressions Of Paxillin In Esophageal Squamous Cell Carcinoma Ec9706 Cells

Esophageal cancer,a highly aggressive neoplasm,is the sixth leading cause of cancer death worldwide.Esophageal squamous cell cancer(ESCC) The incidence and prevalence of esophageal cancer is closely related to the living habits of the people, geography and other factors in some areas.At present,the treatment of esophageal cancer is also mainly confined to surgery,radiotherapy,drug therapy and immune therapy,but these methods can not achieve satisfactory results.Invasion and metastasis of tumor plays an essential role in the occurrence and development of tumor.Study on the role of the newly discovered adhesion molecule in esophageal cancer can provide a theoretical basis for metastasis mechanism for the esophageal cancer.In addition,siRNA is a new field in the widespread use of tumor.Due to its high efficiency,low toxicity and efficient features,siRNA technique gains widespread concern in the medical profession.Thus,in recent years,seeking for new molecular therapeutic target for esophageal cancer excites great interest in clinic.Paxillin,as an important cell adhesive factor,are found in recent years,which is a kind of substrate of tyrosine kinase with oncogenicity.Paxillin combined with integrin constitute cells and extracellular matrix adhesion key parts,which regulate the migration and dissemination of cell,thereby improving metastasis and invasion capacity of tumor cells.Paxillin,as a signal adaptor protein,play a pivotal role in signal transduction mediated by a variety of extracellular stimulation.More and more evidences demonstrate that paxillin participates in the dynamic regulation of focal adhesion,regulation of cell movement and dissemination functions.In addition, invasion and metastasis of tumor is directly associated with the change of cell adhesion and migration,suggesting that paxillin play a central role in the invasion and metastasis of tumor.In the process of integrin-mediated signal transduction,the role of a key signaling molecule FAK in tumor invasion and metastasis pays more and more attention.FAK gene is situated at 8q24,and cDNA full length is 4285bp,encoding 1028 amino acids.It is documented that FAK not only plays an important role in cell adhesion,migration and signal transduction,but also regulates division,growth, developmen and apoptosis of multiple cell biology.Recently,overexpression of FAK gene has been found at many different types of human tumors,while there is no significant increase in benign or normal tissues.FAK play an important role in tumor transition to malignant phenotype,which being correlated with tumor invasion has been confirmed.In addition,paxillin and FAK,as two key signaling molecules of integrin-mediated signal transduction pathway,are both cell adhesion related proteins. In the present,some studies show that paxillin and FAK play an important role in cell migration,cell proliferation and survival.In the meanwhile,it is documented that combination of paxillin and FAK play a pivotal role in regulating cell migration, further,the expression of FAK and paxillin and their mutual relationship have been reported in prostate cancer.At present,expressions of paxillin and FAK proteins as two key signaling molecules of integrin-mediated signal transduction pathways has been reported in different tumor tissues,including esophageal squamous cell carcinoma,stomach cancer,colorectal cancer,liver cancer,breast cancer,laryngeal squamous cell carcinoma,lung cancer,etc.However,the role of the combinative detection of paxillin and FAK in esophageal squamous cell carcinoma cell line EC9706 cells has not been reported at home and abroad,as well as the use of siRNA interference technology inhibiting paxillin gene expression in esophageal squamous cell carcinoma cells remains unclear.Therefore,in this study,paxillin siRNA was used to transfected EC9706 cells,and the effect of down-regulation of paxillin gene on FAK and MMP-2 expression was investigated by immunocytochemistry,semi-quantitative RT-PCR and Western blotting method,which will initially clarify three mutual relationship,and further Boyden chamber was used to investigate the effect of down-regulation of paxillin on cell migration ability.The study aims to investigate the regulation pathway of invasion and metastasis in esophageal squamous cell carcinoma,further key regulating genes of signaling pathways are blocked through modern molecular biology techniques,which will provide a new theoretical basis and experimental foundation for metastasis mechanism of esophageal cancer.Methods1.Paxillin siRNA transfectionEC9706 cells untreated and transfected with control siRNA and paxillin siRNA were harvested 48 h after transfection,which will be used in the following experiments including immunocytochemistry,semi-quantitative RT-PCR,Western blotting and cell migration assay.2.Immunocytochemistry detectionEC9706 cells untreated and transfected with control siRNA and paxillin siRNA were situated in glass slide,then the expression of paxillin and FAK proteins were detected by immunocytochemistry.3.Semi-quantitative RT-PCR analysisTotal RNA was isolated from untreated and transfected EC9706 cells by Trizol reagent according to the manufacturer's instructions,and then subjected to first-strand cDNA synthesis with AMV First Strand DNA Synthesis Kit.Gene of interest was amplified by specific PCR method,andβ-actin was used as internal control.After amplification,10μl aliquots of products were resolved on a 1% agarose gel.DNA bands were visualized by UV light,relative gene levels was analyzed using Gene Tools software.4.Western blotting analysisEC9706 cells mentioned above were lysed for 20 min in cold lysis buffer.20μg of total proteins from whole cell lysates were boiled for 5 min in 1×SDS buffer, resolved by 10%SDS-PAGE,and then electro-transferred to nitrocellulose membranes by a semi-dry transferor.The membranes were blocked in 5%skimmed milk in PBS-T containing 0.05%Tween-20 at RT for 2 h,and then incubated at RT for 2 h with corresponding primary antibodies including paxillin,FAK,MMP-2 andβ-actin diluted in 1%skimmed milk in PBS-T,respectively,followed by incubation with horseradish peroxidase-conjugated secondary antibodies(anti-rabbit).Finally, the bands of specific proteins on the membranes were developed with DAB solution according to manufacturer's instructions.The membranes were rinsed three times with PBS-T between the incubations described above.Quantification of band intensity was performed using Gene Tools software.5.Cell migration ability analysisEC9706 cells of all groups were seeded into upper layer.Each cell number was counted in Boyden chamber experiment and effect of paxillin siRNA cell on migration ability was compared.6.Statistical analysisImmunocytochemistry results were analyzed using chi square test.The results of RT-PCR and Western blotting were analyzed using Gene Tools software.All experiments results were from at least three separate experiments.The data were performed by one-way analysis of variance using SPSS version 13.0.Summary statistics were expressed at means±standard deviations,except as otherwise stated. In all statistical analyses,a P value＜0.05 was considered statistically significant,and all P values were two-sided.Results1.Immunocytochemistry analysisThe results of immunocytochemistry demonstrated that positive cell numbers of paxillin and FAK proteins expressions were lowest in the paxillin siRNA transfection group among the three groups cells(P＜0.05).But the positive cell numbers were higher than of paxillin siRNA transfection group(P＜0.05),there was no difference between EC9706 untreated and tranfected with control siRNA (P＞0.05).2.Semi-quantitative RT-PCR analysisCompared to control and control siRNA,expression level of paxillin mRNA was obviously down-regulated after transfection with paxillin siRNA,expression level of FAK and MMP-2 mRNA were all down-regulated after transfection with paxillin siRNA. 3.Western blotting analysisCompared to control and control siRNA,expression level of paxillin protein was obviously down-regulated after transfection with paxillin siRNA,expression level of FAK and MMP-2 protein were all down-regulated after transfection with paxillin siRNA.4.Cell migration assayThe results of Boyden chamber revealed that penetrative membrane cell numbers of EC9706 cells were significantly decreased after transfection with paxillin siRNA compared to control and control siRNA(P＜0.05),but there was no difference between control and control siRNA(P＞0.05).Conclusion1.After transfection with paxillin siRNA,expressions of paxillin,FAK and MMP-2 mRNAs and proteins are all down-regulated in esophageal squamous cell carcinoma cell line EC9706 cells.2.Down-regulation of paxillin expression can reduce the expressions of FAK and MMP-2 mRNAs and proteins.3.Down-regulation of paxillin expression can inhibit the migration ability of EC9706 cells,which may be mediated by down-regulation of FAK and MMP-2 expressions.