| Part 1 Increased expression of FASN inbreast cancer and its effect on prognosis of breast cancer patientsObjective: Though investigating the different expression of FASN between normal breast and breast cancer cells,as well as the differences of FASN expression in breast cancer tissues,peri-carcinoma tissues and normal tissues of human,and analyzing the its relationship with clinical pathological features and prognosis,the aim of this part is to study the effect of FASN on the occurrence,development and prognosis of breast cancer.Methods:(1)Human normal breast epithelial cell Mc F-10 a and breast cancer cell Mc F-7 were cultured in vitro,and 108 cases of breast cancer patients with normal adjacent tissues were collected;(2)the protein expression level of FASN was detected by western blotting;(3)fluorescence quantitative PCR was used to detect the m RNA levels of FASN factor in normal breast cells and breast cancer cells and the level of FASN in 108 cases of breast cancer patients;(4)the differences in FASN expression in breast cancer tissues,paracancerous tissues and normal tissues of 108 patients were detected by tissue microarray method.(5)through comparative analysis of the differences in the expression of FASN in breast cancer tissues,paracancerous tissues and normal tissues,the relationship between FASN and the clinicopathological features and prognosis was discussed,and the survival rate of patients was analyzed by Kaplan-Meier.Results: Western blot results showed that the expression level of FASN protein and m RNA was low in normal breast tissue,high in adjacent tissues,and the expression level was significantly overexpressed in cancer tissue.Meanwhile,the expression level of FASN in breast cancer cells was also significantly higher than that of normal breast cells.Immunohistochemical results showed that the staining proportion and depth of positive cells in breast cancer tissues of FASN was much higher than that of normal breast tissue(P<0.05).The high expression of FASN was positively correlated with tumor diameter and surrounding lymphatic metastasis in breast cancer patients(P<0.05).No data showed that the expression of FASN was related to the age of the patient,and ER,PR and c-erbb-2(P>0.05)positive tissue.Survival analysis showed that the higher the FASN expression,the worse the prognosis of breast cancer patients.Conclusion: The positive expression of FASN promotes the invasion and metastasis of breast cancer,plays a role in the occurrence and development of breast cancer,and has certain clinical value in judging the prognosis of breast cancer patients.FASN expression is closely related to the progression of breast cancer and is expected to be an independent prognostic indicator of breast cancer.Part 2 Effect of FASN on the epithelial interstitial transformation and migration of breast cancer epithelial cellsObjective: To study the regulatory effect of FASN on the epithelial interstitial transformation and cell migration of breast cancer epithelial cells.Methods:(1)human normal breast epithelial cells MCF-10 a and breast cancer cells MCF-7 were cultured in vitro;(2)constructed FASN silencing and overexpression vectors and transfected into MCF-10 a and MCF-7 cells;(3)the cell migration ability was detected by wound healing test;(4)the expression of epithelial phenotype proteins including E-cadherin,vimentin and Snail1 were detected by western blotting.Results: Morphological observation showed that the shape of MCF-7 cells from“cobblestone” to spindle shape after FASN overexpression.Results of western blot showed that after overexpression of FASN in MCF-7 cells,the level of E-cadherin was downregulated by 69.50% compared with the negative control group,while the expression level of vimentin and Snail proteins was up-regulated to 2.33 times and2.60 times,respectively,and the difference was statistically significant proved by t test(P<0.05).The results of wound healing test showed that after transfection of FASN si RNA in MCF-10 A and MCF-7 cells,the migrative ability was reduced compared with that of the negative control group,while after transfection of FASN overexpressed plasmid,the migrative ability of MCF-10 A and MCF-7 cells was significantly improved(P<0.05).Conclusion: FASN can promote the epithelial interstitial transformation and migration of MCF-10 A and MCF-7 cells.Part 3 Effect of FASN on the neovascularization in breast cancerObjective: To study the role of FASN on neovascularization.Methods(1)108 cases of breast cancer patients with tumor and adjacent normal tissues(2)human normal breast epithelial cells MCF-10 A and breast cancer cells MCF-7 cultured in vitro,constructed FASN silencing and overexpression vectors and transfected them into MCF-10 A and MCF-7 cells;(3)immunohistochemical staining to detecting the expression of VEGF in tumor tissues and adjacent tissues;(4)western blotting technique to detect expression of vascular endothelial growth factor(VEGF)and matrix metalloproteinase(MMP)-2.Results: Immunohistochemical results showed that there were more cells positively expressing VEGF-C in breast cancer tissues,and the number of cells positively expressing VEGF-C in paracancerous tissues was significantly reduced.Results of western blot showed that the expression levels of VEGF-C and MMP-2 were gradually decreased in breast cancer tissues,paracancerous tissues and normal tissues(P<0.05).Western blot was used to detect regulatory effect of FASN overexpression and silence on VEGF and MMP-2 proteins expression in MCF-7 cells,the results showed that,compared with negative control group,expression of VEGF-C and MMP-2 proteins in FASN-overexpressed MCF-7 cells were increased 2.67 and 4.17 times,while the expression of VEGF-C and MMP-2 proteins in FASN-silenced MCF-7cells were go down to 63.97% and 21.32%,compared with negative control group(P<0.05).Conclusion: FASN plays an important role in the angiogenesis of breast cancer.Part 4 FASN promots epithelial-mesenchymal transition and angiogenesis in breast cancer viaβ-cateninObjective: To explore the role of esl in FASN-mediated epithelial mesenchymal transformation of breast cancer cells.Methods(1)Immunoblot assay was used to detect the expression of β-catenin and active β-catenin in tumor tissue,paracancerous normal tissue;(2)western blot was used to detect the expression of β-catenin in MCF-7 cells transfected with FASN-cp DNA.(3)constructed si RNA silencing β-catenin,and and transfected into the MCF-7 cells with FASN overexpression.(4)the expressions of E-cadherin,vimentin,Snail1,MMP-2 and VEGF-C were detected by western blotting.Results: Results of western blot demonstrated that the expression of β-catenin and active β-catenin in breast cancer tissue,the tissue adjacent to carcinoma and normal tissues was gradually reduced(P < 0.05).The level of β-catenin in FASN overexpressed MCF-7 cells was1.24 ± 0.61,which was 2.31 times as negative control group,at the same time,the active β-catenin protein in FASNoverexpressed MCF-7 cell was increased to 4.20 times as the negative control group(P < 0.05).After verify the effect of protein silencing by wstern blot,the β-catenin si RNA was transfection to FASN overexpressed MCF-7 cells,the results showed that,compared to the negative control group,the level of E-cadherin was up-regulated with the level of vimentin and Snail 1 was down-regulated in β-catenin single silence group.Moreover,the expression of proteins involved in angiogenesis such as VEGF-C and MMP-2 was decreased by β-catenin knockdown.In addition,when FASN overexpressed cells were treated with β-catenin si RNA,the level of E-cadherin was up-regulated and the levels of vimentin and Snail proteins were down-regulated.The expression of VEGF-C and MMP-2 was down-regulated(P<0.05).Conclusion: β-catenin is involved in FASN-mediated epithelial mesenchymal transformation and angiogenesis. |
