Study On The Expression Of THBS1 In Breast Cancer And Its Effect On Proliferation,migration And EMT Of Tumor Cells After Silencing

Background:Breast cancer is a common malignant tumor,mostly occurs in women,which is characterized by strong invasiveness,high recurrence rate and poor prognosis.To explore the molecular mechanism of the occurrence and progression of breast cancer is of great significance for the early prevention and treatment of breast cancer,prolonging the survival period and improving the prognosis of patients.Thrombospondin 1(THBS1)is a matrix glycoprotein widely present in various tissues and cells of human body.It plays an important role in regulating malignant biological behaviors such as tumor angiogenesis,invasion and metastasis,and apoptosis.The specific mechanism is still unclear.Objective:1.To compare the expression of THBS1 in breast cancer and normal tissues,and analyze the influence of THBS1 expression level on the survival and prognosis of breast cancer patients;2.To investigate the biological function changes of breast cancer cells after siRNA silencing THBS1 gene.Methods:1.Bioinformatics analysis:The expression difference of THBS1 in breast tumor tissues and adjacent normal tissues was analyzed by TCGA database,and the survival and prognostic value of THBS1 gene in breast cancer was evaluated by Ualcan and KM-Plotter database.2.To investigate the effect of THBS1 gene silencing on the function of MCF-7 and MDA-MB-231 cells.(1)siRNA targeting THBS1 gene silencing was transfected into 2 types of breast cancer cells by liposome transfection method,and THBS1 silenced cell lines were obtained;(2)The silencing effect and mRNA expression levels of EMT markers e-cadherin,Vimentin and N-cadherin were detected by qRT-PCR.(3)The change of cell migration healing ability was detected by scratch injury test;(4)Colony formation and proliferation were detected by plate cloning.(5)Protein expression of E-cadherin,Vimentin and N-cadherin in breast cancer cells transfected with siRNA was detected by Western blot.Repeat all experiments three times.Using SPSS 25.0 statistical software to analyze experimental results,and taking P<0.05 is considered a difference to make sense.Results:1.Bioinformatics analysis showed that the expression level of THBS1 in breast cancer tissues was higher than that in adjacent normal tissues,and the survival time of patients with high THBS1 expression was lower than that of patients with low THBS1 expression(P<0.05).2.Cell function test results:(1)qRT-PCR:Silencing efficiency of siRNA THBS1 on MCF-7 cells was(94.98±0.37)%,and silencing efficiency on MDA-MB-231 cells was(62.9±2.90)%;Compared with control group and siRNA-NC group,the mRNA relative expression levels of N-cadherin and Vimentin in siRNA-THBS1 group were decreased(P<0.05),while the mRNA relative expression levels of E-cadherin were increased(P<0.05).(2)Scratch damage test:The 48h mobility of MCF-7 and MDA-MB-231 cells in the siRNA-THBS1 group was(22.94±1.00)%and(37.21 ± 1.79)%.Compared with blank control group,the migration rates of each group were(27.69±3.16)%and(49.50±6.44)%,and the migration rates of siRNA-NC were(28.61 ± 1.37)%and(50.75±3.20)%,and the differences were statistically significant(P<0.05).(3)Plate clone formation experiment:The number of MCF-7 and MDA-MB-231 cells was(62.00±2.31)and(102.00±3.22)in blank control group,and(59.00±2.52)and(96.33±4.50)in siRNA-NC group,siRNA-THBS1 group were(43.33±2.40)and(40.33± 11.73).The difference was statistically significant(P<0.05).(4)Western blot assay:With GAPDH as internal reference,the expression of epithelial marker E-cadherin increased in MCF-7 and MDA-MB-231 cells after THBS1 gene silencing,and the difference was statistically significant(P<0.05),while the expression of interstitial marker Vimentin and N-cadherin decreased.The difference was statistically significant(P<0.05).Conclusion:1.The expression of THBS1 in breast cancer tissue may be higher than that of adjacent tissues,and the biogenesis analysis shows that the expression of THBS1 is associated with a decrease in survival.2.The high expression of THBS1 in breast cancer has a certain correlation with tumor proliferation and migration,which may promote the invasion and metastasis of tumors by inducing the EMT process.