| Objective:1.To identify TLR4 gene silencing effect by sh RNA recombinant lentiviral transfection of TLR4 breast cancer cells,.2.To identify the growth inhibition rate and cell cycle of breast cancer cells in MCF-7 and MDA-MB-231 cells and the sensitivity of MCF-7 and MDA-MB-231 cells to paclitaxel was observed after the interference of TLR4 gene.3.To detect the expression of proteins in MCF-7 and MDA-MB-231 cell in TLR4 pathway,and to investigate the mechanism of the changes in the sensitivity of the breast cancer cells after interfering with TLR4 gene.Methods:1.Recombinant lentiviruses were infected with MCF-7 and MD-MBA-231 cell lines respectively.Western blot was used to detect the silencing effect of TLR4 gene.2.Four methyl thiazolyl tetrazolium(MTT)method was used to investigate the ef fect of paclitaxel on the growth inhibition rate of MCF-7 cells(MCF-7/sh TLR4,MCF-7/sh Control)and MDA-MB-231 cells(MDA-MB-231/sh TLR4,MDA-MB-231/sh Control)transfected with target plasmid and negative control plasmd.3.Flow cytometry(FCM)was used to detect the cell cycle distribution of MCF-7/sh TLR4,MCF-7/sh Control and MDA-MB-231/sh TLR4,MDA-MB-231/sh Control in paclitaxel on 24 h.4.The expression of NF-κB,IL-6 and VEGFa in the downstream signal factor r elated proteins were detected by Western blot in MCF-7/sh TLR4,MCF-7/sh Contr ol,MDA-MB-231/sh TLR4 MDA-MB-231/sh Control cells in the presence ofpaclit axel on 24 h.Result:1.Identification of TLR4 gene silencing: Western blot results showed that the levels of TLR4 protein in sh RNA(sh1,sh2,sh3)were significantly decreased compared with those transfected with negative control plasmid(sh NC)(P <0.01),which MCF-7 to sh2 relative protein expression decreased obviously and MDA-MB-231 to sh3 relative protein expression decreased obviously.2.Inhibitory effect of paclitaxel on growth of MCF-7 cells: The viability of MCF-7/sh Control and TLR4-silenced MCF-7/sh TLR4 cells were significantly decreased after 24 hours of paclitaxel administration,which has concentration dependent.Compared with MCF-7/ sh Control,the survival rate of MCF-7/sh TLR4 was significantly lower under the same concentration,which the difference was statistically significant(P <0.001).3.Inhibitory effect of paclitaxel on growth of MDA-MB-231 cells: The viability of MDA-MB-231/sh Control and TLR4-silenced MDA-MB-231/sh TLR4 cells were significantly decreased after 24 hours of paclitaxel administration,which has concentration dependent.Compared with MDA-MB-231/ sh Control,the survival rate of MDA-MB-231/sh TLR4 was significantly lower under the same concentration,which the difference was statistically significant(P <0.001).4.Effects of paclitaxel on the growth cycle of MCF-7 cells: MCF-7/sh TLR4,MCF-7/sh Control cell growth cycle was both arrested in G2/M phase,and MCF-7/sh TLR4 block was more obvious.Compared with MCF-7/sh Control cells,PXL-treated silencing TLR4 gene MCF-7/sh TLR4 cells can further promote the role of PXL,the growth cycle was significantly blocked in the G2 / M phase.The results suggest that silencing TLR4 gene has synergistic effect with PXL in MCF-7 cell growth cycle arrest.5.Effects of paclitaxel on the growth cycle of MDA-MB-231 cells: The MDA-MB-231/sh TLR4 and MDA-MB-231/sh Control cells were both arrested in the G2/M phase under PXL,and the MDA-MB-231/sh TLR4 block was more obvious.Compared with DMSO-treated MDA-MB-231/sh Control cells,PXL-treated MDA-MB-231/sh TLR4 PXL cells can also further promote the role of PXL,the growth cycle was significantly arrested at G2/M phase.6.Expression of NF-κB,IL-6 and VEGFa protein in MCF-7 cells were interfered with paclitaxel:(1)silencing of TLR4 expression in MCF-7/sh TLR4 cells could phosphorylate NF-κB p65 and p50 under PXL 5n M treatment,but mainly inhibited the expression of p-p65 and reduced the activation of NF-κB and significantly reduced the expression of IL-6 protein expression,but did not reduce the expression of VEGFa protein.(2)In the control group DMSO treatment,MCF-7 cells could phosphorylate NF-κB p65 and p50,and the expression of NF-κB,IL-6 and VEGFa in small amount was not statistically different.(3)MCF-7/sh TLR4 cells were added to PXL,DMSO treatment,PXL-treated group relative expression of p-p65/p65 and p-p50/p50 protein increased,the difference was statistically significant(P<0.05);the expression of IL-6 and VEGFa protein were significantly increased,the difference was statistically significant(P<0.001).(4)MCF-7/sh Control cells were treated with PXL and DMSO respectively.The relative expression of p-p65/p65,p-p50/p50 and IL-6 protein expression in PXL-treated group significantly increased,and the difference was statistically significant(P<0.001).The expression of VEGFa protein increased,the difference was statistically significant(P<0.01).7.Expression of NF-κB,IL-6 and VEGFa protein in MDA-MB-231 cells interfered with paclitaxel:(1)silencing of TLR4 expression in MDA-MB-231/sh TLR4 cells could phosphorylate NF-κB p65 and p50 under PXL 10 n M treatment,and significantly reduced NF-κB activation,IL-6 protein and VEGFa protein expression.(2)In the control group DMSO treatment,MDA-MB-231 cells could phosphorylate NF-κB p65 and p50,and the expression of NF-κB,IL-6 and VEGFa in small amount was not statistically different.(3)MDA-MB-231/sh TLR4 cells were added to PXL,DMSO treatment,PXL-treated group relative expression of p-p65/p65 and p-p50/p50 protein increased,the difference was statistically significant(P<0.01);There was no significant difference in p-p50/p50 protein expression(P>0.05).The expression of VEGFa protein was significantly increased,the difference was statistically significant(P<0.001).(4)MDA-MB-231/sh Control cells were treated with PXL and DMSO respectively.The relative expression of p-p65/p65,p-p50/p50,IL-6 and VEGFa protein expression in PXL-treated group significantly increased,and the difference was statistically significant(P<0.001).Conclusion:(1)Recombinant lentivirus could stably interfere with the expression of TLR4 gene in breast cancer cell line MCF-7 and MDA-MB-231,the expression of MCF-7 sh2 and MDA-MB-231 sh3 was the most obvious,which could be used in the follow-up study(2)TLR4 inhibited the proliferation of breast cancer cells by paclitaxel,silencing TLR4 enhanced the sensitivity of breast cancer cells to paclitaxel that had a concentration-dependent(3)Paclitaxel could block the cell cycle of breast cancer in the G2/M phase,silencing TLR4 could significantly enhance the cell cycle of paclitaxel in breast cancer.Silencing TLR4 and paclitaxel had synergistic effects on the growth cycle of breast cancer cells.(4)Paclitaxel activated TLR4,which led to the expression of NF-κB,IL-6 and VEGFa.The silencing of TLR4 reduced the expression of NF-κB,IL-6 and VEGFa in breast cancer cells,which enhanced the sensitivity of breast cancer cells to paclitaxel. |
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