| Objective: Breast cancer is a fatal disease affecting women’s health.Although great progress has been made in basic research,the prognosis of breast cancer patients is still poor,especially those with metastasis.According to statistics,the incidence rate and mortality rate of breast cancer worldwide will be significantly increased in the next few years,especially the incidence rate of breast cancer in young women will increase year by year.The current evidence shows that in premenopausal young women under 45 years old,especially those with triple negative breast cancer,the prognosis is still poor and the risk of death is high.This type of breast cancer seems to be highly heterogeneous,which has potential invasive and complex biological characteristicsAdipose tissue plays an important regulatory role in the development of breast cancer.Adipose tissue provides nutrition and adipokines for proliferating tumor cells.Fatty acid binding protein 4(FABP4)is a key fatty factor in fatty acid transport.In metabolic diseases,the level of FABP4 in plasma is often increased.However,the role of this circulating protein is unclear.Recent studies have shown that FABP4 may be involved in the development of breast cancer,but its molecular mechanism is not clear,so we first detect the expression of FABP4 in breast cancer tissues and cell lines.The results show that the expression of FABP4 was increased in invasive breast cancer,and in the samples with lymph node metastasis is higher than that in the samples without lymph node metastasis.The expression of FABP4 in the MDA-MB-231 cells with high metastasis is significantly higher than that in other cell lines,suggesting high expression FABP4 may be related to the metastasis of breast cancer.Therefore,we use small interfering RNA(si RNA)to knock down the expression of FABP4 in MDA-MB-231 cells.In addition,the effects of down-regulation FABP4 on proliferation,migration and invasion of MDA-MB-231 cells were analyzed to provide theoretical basis for exploring new therapeutic targets for metastasis of breast cancer.Methods:1.RT-q PCR and Western blot were used to detect the expression of FABP4 in 12 pairs of breast cancer tissues and matched normal tissues beside the cancer,and the expression of FABP4 in 10 cases of non metastatic breast cancer and metastatic breast cancer tissues.2.The expression of FABP4 in cell lines(MCF-10 A,MDA-MB-231,BT474,ZR-75-30,MCF-7,)was detected by RT-q PCR and Western blot,Compared with the normal group,FABP4 increased protein level and RNA level by 4 times and 7.8 times in MDA-MB-231 cells.3.The expression of FABP4 in the supernatant of cells(MCF-10 A,BT474,ZR-75-30,MCF-7,MDA-MB-231)was detected by ELISA.The expressions of MDA-MB-231,BT474 and ZR-75-30 were increased,but the most significant expression was in MDA-MB-231 cells,which was 6.4 times higher than that in MCF-10 A cells.4.The si RNA targeting FABP4 was designed and transfected into breast cancer cell line MDA-MB-231 to knock down the expression of FABP4,knock inefficiency is 78%.5.MTT assay was used to analyze the effect of down regulating the expression of FABP4 on the proliferation of breast cancer cells.6.Transwell experiment was used to study the effect of knockdown FABP4 expression on the migration and invasion of breast cancer cells.7.Western blot was used to detect the effect of knockdown FABP4 on EMT related proteins.Results:1.Expression of FABP4 in breast cancerRT-q PCR and Western blot were used to detect the expression of FABP4 in 12 pairs of fresh frozen human breast cancer tissues and normal adjacent tissues.The results showed that the expression of FABP4 in breast cancer was significantly higher than that in normal tissues,and the expression of FABP4 in 10 cases of non metastatic breast cancer and 10 cases of metastatic breast cancer was detected by RT-q PCR.The results showed that the expression of FABP4 in metastatic breast cancer was significantly higher than that in non metastatic breast cancer(P < 0.05).2.FABP4 expression in breast cancer cell lines(MCF-10 A,MDA-MB231,BT474,ZR-75-30,Mc F-7)Using Western blot and PCR methods FABP4 expression in cell lines,the results show that FABP4 expression in breast cancer cell lines is higher than normal mammary epithelial cells(MCF-10A),at the same time high metastatic MDA-MB-231 cells expressed in significantly higher than the other(BT474,ZR-75-30,MCF-7)three types of breast cancer cells,compared to the normal group,FABP4 in MDA-MB-231 cells protein levels4 times,7.8 times RNA levels,Statistically significant(P<0.05)3.Expression of FABP4 in the supernatant of cells(MCF-10 A,MDA-MB-231,BT474,ZR-75-30,MCF-7)The expression level of FABP4 in the supernatant of breast cancer cells was detected by ELISA.The results showed that compared with human normal breast cells MCF-10 A,FABP4 had no significant change in MCF-7,but increased in MDA-MB-231,BT474 and ZR-75-30,but MDA-MB-231 was the most significant(P < 0.05).4.Design of si RNA knockdown FABP4 expressionWe designed a si RNA targeting FABP4 to down regulate the expression of FABP4 in MDA-MB-231.After 48 hours of transfection,the cells were collected.RT-q PCR and Western blot were used to detect the knockdown efficiency.The results showed that compared with the control group,the expression of FABP4 in MDA-MB-231 decreased in the transfected group(P< 0.05).5.The effect of knockdown of FABP4 on the proliferation of MDA-MB-231 cellsIn MDA-MB-231,the proliferation of MDA-MB-231 cells was significantly inhibited by down-regulation of FABP4 expression compared with the control group(P < 0.05).6.Effect of knockdown of FABP4 on the migration and invasion of MDA-MB-231 cellsThe results of Transwell migration and invasion experiment showed that,compared with the control group,down regulating the expression of FABP4 could significantly inhibit the migration and invasion ability of MDA-MB-231cells(P < 0.05).7.Western blot was used to detect the effect of knockdown FABP4 on the changes of EMT related proteins.The results showed that compared with the control group,e-cadherin expression in the si FABP4 transfected group was significantly up-regulated,while the expressions of Vimentin,Snail,MMP2 and MMP9 were significantly decreased(P < 0.05).Conclusion:1.The expression of FABP4 in breast cancer tissue was significantly higher than that in normal tissue,and in metastatic breast cancer tissue was significantly higher than that in non metastatic breast cancer tissue.Meanwhile,the expression of FABP4 in MDA-MB-231 cell line was significantly higher than that of other cell lines.2.Knockdown of FABP4 significantly inhibited the proliferation,migration and invasion of MDA-MB-231 cells.3.Knockdown of FABP4 can affect the expression of EMT related proteins. |
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