The Effects And Mechanisms Of AIF1L Gene On The Migration And Invasion In Breast Cancer Line MDAMB231

Objective: Breast cancer is the most common cancer in women.Each year about 1.7 million people worldwide are diagnosed with breast cancer.With about 500 000 deaths every year in 2012 all over the world,breast cancer is the leading cause of cancer death in women.Although the diagnosis and treatment of breast cancer has made great progress,but due to tumor recurrence and distant metastasis lots of breast cancer patients still were with poor prognosis.Therefore,understanding the mechanisms of invasion and metastasis of breast cancer from the molecular levels and screening regulatory genes related to breast cancer are vital for breast cancer therapy.Allograft inflammatory factor-1 like(AIF1L)protein is a kind of EF hand calcium binding protein.AIF1 L gene is located on chromosome 9q34.13 and consists of five exons and four introns.The c DNA of AIF1 L is 3,455 bp in length and contains an open reading frame of 453 bp and a total of 150 amino acids which includes one potential phosphorylation site and one potential acetylation site.The predicted molecular mass and isoelectric point of AIF1 L are ~17.0 k Da and 6.63,respectively.AIF1 L m RNA expression was highest in the kidney,moderate in normal breast tissue,and low in the brain,spleen,lung,skeletal muscle and testis.AIF1 L m RNA was not detected in the liver.Previous studies have found that AIF1 L protein can combine and crosslink with F-actin,and co-localize with F-actin,and AIF1 L was also located in cellular projections and adhesion structures.In a Shigella invasion model,AIF1 L protein is recruited into the bacterial invasion zone and cellular protrusions.Another study have found that AIF1 L dysregulated in podocyte cell by si RNA silence can lead to F-actin stress fiber loss and rearrangement,which was accompany by downregulation of CD2 AP or SYNPO,which were both important to maintain stability of the cytoskeleton.AIF1 L may participate in regulating cell skeletal structure,but its role in the tumor is poor.Currently AIF1 L expression in breast cancer and the mechanism of breast cancer is not sufficient,this study aims to verify its expression and speculate its biological function by using public database,detect AIF1 L expression in breast cancer,observe AIF1 L influence on biological behavior of breast cancer,and explore relevant molecular mechanisms.Method: 1.Using bioinformatics methods to evaluate the AIF1 L expression of breast cancer tissues in TCGA,HPA,GEO database;by DAVID and GSEA analysis of CCLE database,speculate AIF1 L gene biology function and its related signaling pathway.2.AIF1 L expression in 287 cases of breast cancer tissue and 44 cases in paracancerous tissue was detected using immunohistochemical staining method.Statistical analysis was taken for AIF1 L protein and the clinical pathologic factors of breast cancer and prognosis of patients.3.AIF1 L gene and contrast empty carrier adenovirus were transfeced to MDAMB231 cells.Through MTT experiment and tablet clone formation,assess the effect of AIF1 L gene on MDAMB231 cell proliferation;By wound healing experiment,Transwell migration/invasion experiment,to assess that whether AIF1 L genes affect MDAMB231 cell migration and invasion ability;Through the cell spreading experiment,to assess the effect of AIF1 L genes to the MDAMB231 cell spreading.Using Western blot method to evaluate the expression of genes related to FAK/Rho A signaling pathways and EMT related signaling pathway.4.The experimental data was analyzed using SPSS 19.0 software or the R language.Experiment figure processing was analyzed using Image J software as needed.The chart was made by Graphpad software chart.Count data was analyzed by χ squared test or Fisher’s exact test.Survival analysis was analyzed by Kaplan Meier and log Rank test.Multi-factor survival analysis was Cox regression analysis.T.Test and SNK Test were usd for quantitative data.Alpha = 0.05.Results: 1.AIF1 L m RNA expression in breast cancer of TCGA database significantly lower than the normal tissue and methylation levels in breast cancer higher than normal tissue in breast cancer.2.AIF1 L expression in HPA database mainly located on the cytoplasm and cell membrane of the glandular epithelial cells.AIF1 L expression of normal tissue was higher compared to breast cancer tissue.3.AIF1 L expression in breast cancer of GEO database was lower than that of normal control group.4.DAVID and GSEA analysis indicates AIF1 L genes related to cytoskeleton,cell adhesion and other related signaling pathway.5.The positive immune response of AIF1 L mainly located in cell membrane and cytoplasm.AIF1 L expression rate in breast cancer(28.6%,82/287)was significantly lower than in paracancerous tissue(58.3%,28/48)(p= 0.007).Low AIF1 L expression was found in triple negative breast cancer.AIF1 L lower expression implied a poor prognosis.High AIF1 L expression was independent protective factor affecting the prognosis of breast cancer.6.AIF1 L gene can inhibit MDAMB231 cell proliferation,clone formation,cell migration,invasion ability and cell spreading.AIF1 L can inhibit FAK/Rho A protein expression and activation,and reverse transformation of EMT phenotype.Conclusion: 1,Bioinformatics analysis found that the expressions of AIF1 L in breast cancer tissue were significantly lower than in the normal tissue.AIF1 L may participate in a variety of signaling pathways,such as the tight juction,cell adhesion,cytoskeleton adjustment,etc.,it may be play an important biological function in the process of breast cancer development.2.Immunohistochemical study found AIF1 L protein expression in breast cancer was significantly lower than paracancerous tissue.AIF1 L in triple negative breast cancer tissue significantly decreased.The prognosis of breast cancer patients with high AIF1 L expressions is much better than that with lower expression.AIF1 L high expression is a prognostic protective factors of breast cancer patients.3.AIF1 L may through inhibit the FAK-Rho A signalling pathways and affect the cytoskeleton regulation.AIF1 L may also inhibit epithelial mesenchymal transition trarelated pathways,and affect the cell migration and invasion ability.