Expression Of RSK4 And TRAF4 In Different Breast Cells Lines And Cross-talking Mechanism

Background:2015 annual report of China cancer registration data show that the threat to the nation's largest 6 kinds of cancer,breast cancer ranked in the top fourth.In recent years,the growth rate of breast cancer has been ranked in the forefront of the world,with more than 20 new cases each year,has become the number one threat to women's health in china.Research shows that the X chromosome gene P90 ribosomal protein S6 kinase4(RSK4)can inhibit cancer cell proliferation,invasion,metastasis and induces cell senescence in breast cancer and other cancers.It is well known that breast cancer is a systemic disease with multiple genes acting together,and the proteins that bear the function of life can not be separated from other proteins.In the early stage of this study,we can find the tumor necrosis factor receptor associated factor 4(TRAF4)by mass spectrometry analysis.In this study,we detected the expression of RSK4 and TRAF4 in four breast cell lines.Using lentiviral vector,the stable over expression RSK4 vector was transfected into human breast cancer cell line MDA-MB-231,after transfection in MDA-MB-231 cells,detecting the expression of TRAF4 and studing the correlation between RSK4 and TRAF4,so as to providing a new idea for the development and treatment of breast cancer.Objective: The previous study showed that RSK4 interacts with TRAF4,the detection of RSK4-TRAF4 in four different breast cell lines and stable expression of expression of breast cancer cell line MDA-MB-231 in RSK4,and the correlation analysis,preliminary exploration of regulation of RSK4-TRAF4 on the occurrence and development of breast cancer.METHOD:Real-time fluorescence quantitative PCR(RT-PCR)method was used to detect the expression of TRAF4 and RSK4 in four human breast cancer cell lines of HBL-100?MCF-7?HER2 positive type?MDA-MB-231 from the level of mRNA.2.Western blot method was used to detect the expression of RSK4,TRAF4 and MAPK pathways downstream of ERK protein in four kinds human breast cancer cell lines of HBL-100,MCF-7,HER2 positive type,MDA-MB-231 from the level of proteine,The relationship between RSK4 and TRAF4 was analyzed based on the experimental results of RT-PCR and WB.The stable over expression vector of RSK4(LV-RPS6KA6)and negative control vector(LVCON145)were transfected into human breast cancer cell line MDA-MB-231,the establishment of the cells in the experimental group,negative control group,and non transfected MDA-MB-231 cells of the controlgroup in the experiment;Real time quantitative PCR(RT-PCR)was used to detect the mRNA expression of RSK4 and TRAF4.Western blot(Western-blot)was used to detect the content of RSK4 protein,TRAF4 protein and ERK1/2protein in the MAPKs pathway,and to further analyze the correlation between RSK4 and TRAF4.RESULT:1.RSK4 and TRAF4 mRNA both expressed in the four cell lines,RSK4 mRNA in HBL-100 cell was the highest expression.The expression of RSK4 mRNA was decreased gradually in MCF-7,HER2 positive type and MDA-MB-231 cells;In thess cells,the expression of TRAF4 mRNA from low to high was HBL-100,MCF-7,HER2 positive type,MDA-MB-231 cells.Both the differences of RSK4 and the differences of TRAF4 were statistically significant in four different types of cells(P<0.05).2.RSK4 protein in breast cell HBL-100 expression was the highest(0.789 +0.010),followed by MCF-7 cells(0.683 + 0.013),HER2 positive type low(0.602 + 0.015),the lowest in MDA-MB-231 cells(0.526 + 0.006);TRAF4protein content in four kinds of cells from high to low as MDA-MB-231 cells(0.890 + 0.058),HER2 positive(0.817 + 0.059)and MCF-7 cells(0.744 + 0.057)and HBL-100 cells(0.682 + 0.036);the expression of ERK1/2 protein from high to low as MDA-MB-231 cells(0.824 + 0.032),HER2 positive(0.713 + 0.024)and MCF-7 cells(0.358 + 0.021)HBL-100 cells(0.290 + 0.018)3.RSK4 and TRAF4 in four cell protein expression level consistent with the mRNA expression level is highly correlated,the correlation coefficients wererRSK4=0.92,rTRAF4=0.82,P< 0.01;P< 0.01;mRNA at the transcriptional level,between RSK4 and TRAF4 showed a negative correlation between r1=-0.81 and P<,0.01;at the protein level of translation between RSK4 and TRAF4 also has a high negative correlation,r2=-0.84,P< 0.01.4.Construct the Lentivirus Expression Vector of RSK4 stably transfected into cells,the expression of the experimental group,the relative expression of negative control group and blank control group RSK4 mRNA(2-??Ct)that were 1±0,0.2590±0.02132,0.3204±0.08168 with standard deviation.RSK4 mRNA cells in the experimental group the highest expression level,there was statistically significant in three groups(F=427.2,P<0.0001)?The relative expression amount of TRAF4 mRNA in the blank control group ?the negative control group ?the experimental group(2-??Ct)was expressed as 1±0?2.037±0.2177?1.904±0.1186,respectively.TRAF4 mRNA cells in the experimental group the lowest expression level,there was statistically significant in three groups(F=93.33,P<0.0001)5.The intensity ratios of RSK4 was respective0.7742±0.007404?0.6078±0.03182?0.6023±0.02853,in three groups,analysis of variance was statistically significant F=60.88,P<0.0001;In three group cells,TRAF4 protein gray values were0.6848±0.006955?0.8851±0.005618?0.8833±0.008975,a statistically significant difference between groups F=991.3 and P<0.0001;each comparison between the two groups.In three group cells,ERK1/2 protein grayvalues were 0.6361±0.009394?0.8000±0.005598?0.8023±0.01156,a statistically significant difference between groups F=430.4 and P<0.0001;6.The expression level of mRNA and protein of RSK4 and TRAF4 in the cells were consistent,positively correlated,the correlation coefficients wererRSK4'=0.93,P<0.01;rTRAF4'=0.77,P<0.01;In the three groups,between RSK4 mRNA and TRAF4 mRNA showed a negative correlation between,r1'=-0.69,P< 0.01;at the protein level of translation between RSK4 and TRAF4 showed a negative correlation between high,r2'=-0.79,P<0.01.Conclusion:1.RSK4 was negatively correlated with TRAF4 expression2.RSK4 may negatively regulate TRAF4 through ERK and TGF-beta pathway,which may affect the occurrence and development of breast cancer.