Role Of STAT-3 Signaling Pathway In The Invasion And Migration Of Breast Cancer Cells Induced By 27-hydroxycholesterol

In the 21st century,breast cancer has become an important factor in the health of women,occupying a very important position in female tumors.However,the invasion and migration of tumor cells is one of the important mechanisms in the process of breast cancer development and death.Tumor cells are involved in the process of invasion and migration,such as epithelial-mesenchymal transition(EMT)and matrix metalloproteases(MMPs)degradation of extracellular matrix.Many of the literatures reported that cell epithelium transformation can make the tumor cells have polygonal,square into a long fusiform with epithelium E-cadherin and other indicators declining,interstitial markers vimentin,N-cadherin and ZEB1 rising and have a stronger movement ability,which is an indicator of the progress of cancer.It has been shown that RECK protein can inhibit the invasion and metastasis of tumor cells by down-regulating the expression of MMP2 and MMP9,so the down-regulation of RECK expression in tumor progression is a marker of tumor malignancy.Activating activator of transcription-3(STAT-3)also plays an important role in the process of tumor invasion and metastasis,and can activate the expression of downstream invasive and metastatic markers.27-hydroxy cholesterol(27HC)is a metabolite of cholesterol in the human body,produced by cytochrome P450 enzyme CYP27A1,and decomposited by CYP7B1.Hypercholesterolemia patients in vivo 27HC levels were significantly increased;women,especially postmenopausal women obesity led to increased cholesterol levels and then spawned 27HC caused a variety of diseases,including cancer,serious harm to human health.2013 Hep reported that 27HC binds to ER receptor and LXR receptor to promote tumor cell proliferation and invasion and migration,but that whether 27HC could downregulate the expression of tumor suppressor gene RECK and thus affect STAT-3-induced invasion and migration of tumor cells yet reported.Objective:In this study,human ER-positive breast cancer cell lines MCF7,T47D and human triple-negative breast cancer cell line MDA-MB-231 were used to investigate the mechanism of 27HC promoting invasion and migration of breast cancer cells and the role of STAT-3,so as to provide more experimental data for preventing and treat-ing cancer,open a new way of thinking.Methods:To investigate the effect of 27HC on cell viability:the effect of 27HC on the invasion and migration of breast cancer cells:cell scratch test,pre-matrigel gel and non-matrigel Transwell assay;to detect MMP2 and MMP9 Activity:gelatin zymography;to detect mRNA expression levels of the target gene:RT-PCR and quantitative real-time PCR;to detect gene protein levels:protein immunoblotting assay;using si-RNA to knockdown RECK and STAT-3:cell transfection experiments;reactive oxygen test:ROS detection kit.Results:(1)27HC could promote the invasion and migration of breast cancer MCF7 and T47D cells:Firstly,CCK8 test and scratch healing test results found that 4.0μM was the highest concentration of 72 hours of treatment of breast cancer cells MCF7 and T47D without affecting cell viability;The results of RT-PCR and qRT-PCR showed that 27HC could up-regulate the expression of MMP9 in breast cancer cells in a dose-dependent manner,but the results showed that 27HC could up-regulate the invasion and migration of breast cancer cells without affecting cell viability.MMP2 mRNA was not affected;gelatin zymography experiments showed that 27HC can up-regulate the activity of MMP9,MMP2 activity was not affected.In addition,treated with 27HC,EMT-related indicators E-cadherin decreased vimentin and ZEB1 increased within breast cancer cells by Western Blot experiments.(2)27HC down-regulated protein levels and mRNA levels of RECK and up-regulated MMP9:In the molecular mechanism explorations,we found that RECK protein levels and mRNA levels of breast cancer cells were down-regulated by 27HC treatment,and RECK siRNA was used to interfere with RECK protein After expression,MMP9 expression,activity and cell invasion were increased in breast cancer cells,but expression and activity of MMP2 were not affected.(3)27HC could promote the invasion and migration of breast cancer cells through an ER-independent pathway activating STAT-3:In addition,27HC treatment of ER-positive breast cancer cells MCF7 and T47D and ER-negative breast cancer cells MDA-MB-231,p-STAT-3 increased,STAT3 signaling pathway was activated,GREB1 expression also increased,indicating 27HC also activated the classic of the ER signal pathway.In addition,after the use of siSTAT-3 knockout STAT-3,27HC caused by breast cancer invasion and metastasis ability to restore its downstream MMP9 decreased,E-cadherin increased,vimentin and ZEB1 decreased.All these indicate that STAT-3 signaling pathway plays an important role in 27HC-induced invasion and metastasis of breast cancer.(4)27HC induced the activation of intracellular ROS levels and promote breast cancer cells invasion and migration ability:Treating ER positive breast cancer cells MCF7 and T47D with 27HC and positive reference H2O2,use the fluorescence microscopy to take photos,the results show that 27HC can promote ROS to produce and accumulate,causing cell oxidative stress.(5)27HC inhibited RECK expression by ROS induced methylation and activated STAT-3:27HC,AZA,SAM and positive reference H2O2 were used to treat MCF7 and T47D in ER positive breast cancer cells.It was found that the RECK reduction caused by 27HC after AZA treatment was reversed,SAM treatment After RECK expression also showed a downward trend.The expression of p-STAT-3 in breast cancer cells was decreased and STAT-3 signal pathway was blocked by RECK siRNA interference with RECK protein expression.So 27HC inhibited RECK expression and then activated STAT-3 through the ROS induced-methylation positively regulating the invasion and migration of breast cancer cells.In addition,the expression of RECK mRNA in breast cancer cells was up-regulated by STAT-3 siRNA interfering with STAT-3.Suggesting that there is an interaction between RECK and STAT-3 signaling pathway in this process.Conclusion:In human breast cancer cell lines,27HC regulated the mRNA and protein levels of RECK by activating the ROS methylation table,activating STAT-3,increasing MMP9 Transcriptional level and activity,as well as the promotion of EMT-related molecular markers E-cadherin decreasing,vimentin and ZEB1 increasing,the ability of breast cancer cells to degrade extracellular matrix and the ability to move increased,and ultimately promote the invasion and migration of breast cancer cells.