The Study On The Role Of Mir-145 In Promoting Breast Cancer Invasion And Metastasis By Regulating BMP1/LOX Pathway

Objective:Metastasis accounts for the majority of mortality and poor prognosis in breast cancer patients.Currently,there is no effective treatment for the advanced patients with distant metastasis.Therefore,we focus on the epigenetic regulation which controlling the metastasis of breast cancer.The oncomine data analysis combined with in vitro cell experiments indicates that miR-145 regulates BMP1/LOX signaling pathway in the process of breast cancer invasion and metastasis.The significance of this is to provide a basis for the design and development of new targets for invasive breast cancer treatment.Methods:The expression level of LOX and BMP1 were analyzed in Oncomine database.The specific effects of LOX,BMP1 and miR-145 in breast cancer cells MCF7 were investigated.Real-time PCR was used to detect mRNA expression level;and western blot was employed to evaluate the protein expression of LOX,BMP1.E-cadherin,Occludin,Vimentin and Snail;MTT assay was utilized to monitor the cell proliferation;immunofluorescence staining was introduced to monitor the protein localization.The transwell chamber model(transwell assay)was used to detect the cell invasion ability;the wound healing assay was used to detect the cell migration ability;LOX activity assay kit was used to detect the LOX protein activity;and the monoclonal formation assay was used to detect the cell proliferation abilityResults:1)Analysis of the correlation between LOX expression and prognosis in breast cancer tissues using Oncomine database:Oncomine analysis found that LOX was highly expressed in breast cancer tissues compared with normal tissues,and its high expression was closely related to patient survival(OS)(P=0.012).2)Effects of LOX expression and activity on proliferation,invasion and metastasis of breast cancer cell line MCF7:breast cancer cell line MCF7 interferes with LOX expression,mRNA levels are down-regulated by 82%,protein levels are also down-regulated.LOX activity is decreased by 41%,and proliferative activity is decreased 52%;cells interfering with LOX expression were significantly decreased compared with cells without any treatment,whether it was cell invasion or migration;EMT-related protein E-cadherin,Occludin protein expression up-regulated,vimentin,snail expression down-regulation;BAPN(active inhibitor of LOX)significantly inhibited the invasion and migration of MCF-7 cells.3)Analysis of the correlation between BMP1 and LOX in breast cancer tissues using Oncomine database:BMP1 is also highly expressed in breast cancer tissues compared with normal tissues,and is closely related to patient survival(OS)(p=0.011);Expression analysis showed that BMP1 was positively correlated with LOX expression in breast cancer tissues;fluorescence colocalization experiments in MCF7 cells revealed a colocalization relationship between BMP1 and LOX in the cells4)Effect of BMP1 expression on proliferation,invasion and metastasis of breast cancer cell line MCF7:BMP1 mRNA expression level is down-regulated by 91%after interference,while LOX mRNA expression level is down-regulated by 43%,activity is decreased by 41%,and proliferation activity is inhibited by 36%;cells that interfered with BMP-1 expression were significantly decreased compared with cells without any treatment,regardless of cell invasion or migration;EMT-related protein E-cadherin,Occludin protein expression up-regulated,vimentin,snail expression down-regulated,consistent with the results of interference with LOX.5)Mechanism of miR-145 affecting proliferation,invasion and metastasis of breast cancer cells by regulating BMP 1/LOX signaling pathway:LOX mRNA expression is down-regulated by 37%,BMP1 mRNA expression is down-regulated by 39%,and LOX functional activity is inhibited.%,cell proliferation inhibition is 54%,and this effect may be related to inhibition of EMT process.Conclusion:The down-regulation of miR-145 expression in breast cancer tissues up-regulates the expression of downstream target gene BMP 1/LOX pathway,which in turn affects downstream cell proliferation and EMT-related genes,promotes breast cancer proliferation,invasion and metastasis.The results will be expected to provide new therapeutic strategies and theoretical basis for the treatment of invasive breast cancer.