| OBJECTIVEThis study was to investigate the effect of the TRAF4 gene on cell proliferation,invasion and metastasis in vivo and explore whether there is an interaction between TRAF4 and RSK4 in breast cancer and its possible mechanism.METHODSMDA-MB-231 cells were transfected with LV-sh RNA-TRAF4 to specifically block the expression of TRAF4,or transfected with LV-sh RNA-NC as a negative control and untransfected cells as a blank control group.Real-time quantitative PCR(q RT-PCR)and western blot were used to detect the expression of TRAF4 m RNA and protein in lentivirus transfected cells.Four-six weeks female BALB/c nude mice were randomly assigned to three groups(n = 14): TRAF4-sh RNA group,negative group and control group,and then inoculated subcutaneously with the corresponding cells.In addition,in-vivo metastasis model was constructed by injecting the above three groups of cells into the blood vessels of the other 15 nude mice through the left or right tailveins,and each group is 5.Tumor length and width were measured weekly,and tumor proliferation was assessed in terms of the tumor growth curve,tumor size and weight.Lung and liver tissues obtained by orthotopic transplantation tumor liver,lung metastasis model and tail vein metastasis model in nude mice.Invasion and metastasis of breast cancer MDA-MB-231 cells in lung and liver tissues by knockdown of TRAF4 expression were evaluated by the histopathologic examination.Measurement of TRAF4 and RSK4 expression and AKT / NF-κB related factors(p-AKT / AKT,p-NF-κB / NF-κB,TGF-β1,TNF-α,MMP2 and MMP9)were performed by immunohistochemistry,western blot and fluorescence quantitative RT-PCR.RESULTS1.The lentiviral vector and the empty vector were successfully transfected into MDA-MB-231 cells and stably expressed.The relative expression levels of TRAF4 m RNA in the TRAF4-sh RNA group,negative control group and blank control group were detected by q RT-PCR.The expression was 0.305±0.062,0.890±0.057,1.000±0.000.The expression of TRAF4 m RNA in the TRAF4-sh RNA group was significantly lower than that in the negative control group(P < 0.05)and the blank control group(P < 0.05),but there was no significant difference between the negative control group and the blank control group(P > 0.05).Western blot was used to detect the expression level of TRAF4 protein in the TRAF4-sh RNA group,negative control group and blank control group.The expression of TRAF4 protein in TRAF4-sh RNA group was significantly lower than that in negative control group and blank control group.2.The tumor growth curve showed that the growth rate of the TRAF4-sh RNA group was significantly lower than that of the negative control group and the blank control group,while the growth rate of the negative controlgroup and the blank control group had no significant difference.The average volume of xenograft tumors in the TRAF4-sh RNA group,the negative control group and the blank control group was 304.00±119.00 mm3,844.00±293.73mm3,and 931.66±293.43 mm3,respectively.It can be seen that the volume difference in the three groups was statistically significant(P < 0.05).The average tumor volume in the TRAF4-sh RNA group was significantly lower than that in the negative control group(P < 0.05)and the blank control group(P <0.05).The volume difference in the other two group was not statistically significant(P > 0.05).The average weight of the nude mice in the TRAF4-sh RNA group,the negative control group and the blank control group was 1.93±0.54 g,2.88±0.42 g,and 3.02±0.26 g,respectively.The average weight of tumors in the TRAF4-sh RNA group was significantly lower than that of the negative control group(P < 0.05)and the blank control group(P < 0.05),while the difference in the negative control group and the blank control group was not statistically significant(P > 0.05).Situ orthotopic lung metastases were seen in the TRAF4-sh RNA group,the negative control group,and the blank control group.The number of lung metastases in the TRAF4-sh RNA group was significantly lower than that in the negative control group and the blank control group(P < 0.05),but there was no significant difference between the negative control group and the blank control group(P > 0.05).There were no metastatic lesions in the liver tissue in the three groups of HE staining.3.The results of metastasis model in nude mice suggest that there is no metastases in the liver of the TRAF4-sh RNA group,while the negative control group and the blank control group have multiple liver metastases.There were no metastases in the macroscopic view and HE staining in the three groups of lungs.4.The results of immunohistochemistry indicated that the TRAF4-sh RNA group had significantly lower expression of TRAF4 than the negative control group and the blank control group,while the expression of RSK4 in the TRAF4-sh RNA group was increased compared with the negative control group and the blank control group and there was a significant negative correlation(r =-0.645,P < 0.05)in the expression of RSK4 and TRAF4.The q RT-PCR results showed that the relative expression levels of TRAF4 m RNA in the TRAF4-sh RNA group,the negative control group and the blank control group were 0.3050±0.0617,0.8900±0.0572,1.0000±0.0000,respectively.The relative expression levels of RSK4 m RNA in the TRAF4-sh RNA group,the negative control group and the blank control group were 3.0670±0.5293,1.1000±0.0927,and 1.0000±0.0000.It can be seen that the expression of TRAF4 m RNA in the TRAF4-sh RNA group was significantly lower than that in the corresponding negative control group and the blank control group,but the RSK4 m RNA was highly expressed in the TRAF4-sh RNA group and was lowly expressed in the corresponding other two groups.Western blot analysis showed that the relative expression levels of TRAF4 protein in the TRAF4-sh RNA group,the negative control group and the blank control group were 0.347±0.089,1.83±0.153,1.174±0.211,respectively.The relative expression levels of RSK4 in the three groups were 1.217±0.181,0.641±0.124,and 0.596±0.137,respectively.The expression of TRAF4 protein in the TRAF4-sh RNA group was significantly lower than that in the corresponding negative control group and the blank control group,while the protein of RSK4 was highly expressed in the TRAF4-sh RNA group,and was lowly expressed in the corresponding negative control group and blank control group.5.The relative expression levels of MMP2 m RNA in the TRAF4-sh RNA group,the negative control group and the blank control group were0.01830±0.00140,1.07000±0.16619,and 1.0000±0.00000,respectively.The relative expression levels of MMP9 m RNA in the three groups were0.56000±0.08000,1.100±0.23180,and 1.0000±0.00000,respectively.It can be seen that the expression of NF-κB downstream factors MMP2 and MMP9 m RNA in the TRAF4-sh RNA group was significantly lower than that in the negative control group(P < 0.05)and the blank control group(P < 0.05),while the negative control group and the blank control group had no significance difference(P > 0.05).Western blot analysis showed that the p-AKT / AKT in the TRAF4-sh RNA group,the negative control group and the blank control group were 0.133±0.056,0.473±0.109 and 0.469±0.114,respectively.The p-NF-κB / NF-κB in the three groups were 0.117±0.047,0.487±0.146 and0.499±0.123,respectively.The TNF-α / GAPDH in the three groups were0.221±0.097,0.647±0.151,and 0.709±0.137,respectively.The TGF-β1 /GAPDH in the three groups were 0.537±0.117,1.108±0.214 and 1.189±0.196,respectively.The expression of AKT pathway-related protein p-AKT in TRAF4-sh RNA group was significantly lower than that in negative control group and blank control group(P < 0.05).NF-κB signaling pathway related protein p-NF-κB,TNF-α,TGF-β1 were also significantly lower(P < 0.05).CONCLUSION1.Constructed nude mice xenograft model and liver / lung metastasis model,and clearly showed knocking down the expression of TRAF4 in MDA-MB-231 cells significantly inhibited proliferation,invasion and metastasis in breast cancer.2.Through the detection and analyze of TRAF4 and RSK4 genes in Breast cancer xenografts,it was found that TRAF4 has a negative relationship with RSK4.3.By detecting and analyze the core protein associated with the AKT /NF-κB pathway,it was found that the activation of TRAF4 on RSK4 may be related to the AKT / NF-κB pathway. |
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