| Background: SYT13 is an atypical member of the synaptic binding protein family(Synaptotagmins,SYT).It is expressed in the heart,lungs,testicles,brain,kidneys,spleen,pancreas and other organs.The highest expression is in the brain.It binds to the cell membrane independently of calcium and has the function of transporting biomolecules.Recently,it has been reported that SYT13 plays an important role in many types of malignant tumors.Previous studies have shown that the expression of SYT13 is increased in ER(estrogen receptor)positive breast cancer cells,But there are no studies to explore the effect of SYT13 on the biological behavior of breast cancer cells.In addition,activation of the FAK/AKT signal pathway has been found to be involved in the regulation of breast cancer progression.Regulation of the malignant phenotype of gastric cancer cells by SYT13 is related to the FAK/AKT signal pathway.Therefore,we believe that SYT13 may participate in breast cancer progression by activating the FAK/AKT signal pathway.Objective:(1)to determine the expression level of SYT13 in breast cancer cells and normal breast epithelial cells(2)to study the effects of interfering SYT13 on proliferation,apoptosis,metastasis and invasion of breast cancer cells(3)to verify that interfering SYT13 may affect the biological behavior of breast cancer cells through FAK/AKT signal pathway.Methods:(1)The difference in Expression of SYT13 in breast cancer tissue,paracancerous tissue and normal breast tissue was searched and analysed by The TCGA(The cancer Genome Atlas),GTEx(Genotype-Tissue Expression)database.SYT13 Expression level and overall survival time of breast cancer patients(overall survival)were analyzed by Kaplan-Meier Plotter via GSE37751 and GSE31519 in the GEO(Gene Expression Omnibus)database.(2)Detection of SYT13 protein expression in breast cancer cells(HCC1937,MDA-MB-231,MDA-MB-453,MDA-MB-468,MCF-7)and normal breast epithelial cells(MCF-10A)by West Ern blot.(3)Construction of stable breast cancer cell lines interfering with SYT13 by lentivirus transfection.(4)Using CCK-8.Cell proliferation was detected by EDU staining,cell cycle change was detected by flow cytometry and expression of cycline D1,CDK4 and p21 was detected by Western blot.(5)Apoptosis was detected by flow cytometry and fluorescent TUNEL cytometry,while Bcl-2 and Bax expression were detected in Western blot.(6)Scratch test was used.Transwell’s invasion assay was used to detect the ability of cell metastases and Western blot invasion to detect the expression of e-cadherin and Vimentin.(7)The expression of FAK,p FAK,AKT and pa KT was detected by Western blot.Results:(1)The results of the database analysis showed that the expression of SYT13 in breast cancer tissues was significantly higher than that in paracancerous tissue and in normal breast tissue(p < 0.01).The expression level of SYT13 in breast cancer tissues was negatively correlated with OS and RFS of breast cancer patients.(2)Westernblot was used to verify the expression of SYT13 in each cell line.We found that,at the protein level,SYT13 expression in breast cancer cells was significantly elevated compared to that in normal mammary epithelial cells.The protein expression content of SYT13 in MCF-7 cell line was the highest,so MCF-7 cell line was selected for follow-up experiment.(3)MCF-7 cells were transfected with lentivirus.48 hours after transfection,the expression of SYT13 was detected by Real-time PCR and Westernblot.The results showed that the m RNA and protein levels of SYT13 in SYT13 si RNA group were significantly lower than those in blank control group(control group)and negative control group(NCsi RNA group).The results showed that interfering with the construction of SYT13 cell line.(4)Compared with the blank control group and NCsi RNA group,the CCK-8 experiment showed that the cell activity of SYT13 si RNA group decreased significantly(p < 0 01).After Ed U staining,the cell proliferation ability of SYT13 si RNA group was significantly decreased,and the percentage of G1 phase cells in SYT13 si RNA group was significantly increased.In addition,the protein level of p21 in SYT13 si RNA group was significantly increased(p < 0 05).The protein levels of CDK4 and cyclin D1 were significantly decreased.(5)Compared with the blank control group and NCsi RNA group,the results of flow cytometry and fluorescence TUNEL showed that the apoptosis was significantly increased in SYT13 si RNA group.In addition,the protein level of Bcl-2 in SYT13 si RNA group was significantly decreased and the protein level of Bax was significantly increased.(6)Compared with blank control group and NCsi RNA group,through scratch test.Transwell invasion test showed that the migration and invasion ability of cells in SYT13 si RNA group decreased significantly,the protein level of E-cadherin in SYT13 si RNA group increased significantly,and the protein level of vimtenin decreased significantly.(7)Compared with blank control group and NCsi RNA group,the protein levels of p-FAK/FAK and p-AKT/AKT in SYT13 si RNA group were significantly lower than those in control group and NCsi RNA group.Conclusion:(1)High expression of SYT13 in breast cancer tissue and negative correlation with OS and RFS in breast cancer patients.(2)High expression of SYT13 in breast cancer cells.(3)Interference with SYT13 inhibits proliferation,metastasis,invasion and apoptosis of breast cancer cells.(4)Interference with SYT13 may affect the biological behavior of breast cancer cells through FAK/AKT signal pathway. |
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