The Effect Of TLR4 Receptor Inhibitor TAK-242 On The Proliferation And The Invasion Of Lymphoma Cells

ObjectiveMalignant lymphoma is heterogeneous,prone to recurrence and drug resistance,and there are many difficulties in treatment.With the rise of"tumor immunotherapy",the search for lymphoma immunotherapy targets has gradually become a hot spot of concern.The Toll-like receptor 4?TLR4?signaling pathway is involved in inflammatory responses and cancer progression and is a link between host and immune response.Therefore,this study investigated the changes in proliferation and invasiveness of lymphoma Raji cells after inhibition of TLR4 signaling,and preliminary investigation of the related mechanisms in order to find new detection and therapeutic targets for lymphoma.Methods1.Raji cells were treated with the TLR4 inhibitor of 0,10,35,60,110,170,230?mol/L TAK-242 for 2,6,24 hours.CCK-8 assay was used to detect the changes in absorbance values of Raji cells to screen for optimal treatment concentration and time.2.Raji cells were divided into control group?medium culture?for 24h and?10,60??mol/L TAK-242 for 24h,and cell invasion was detected by Transwell^TMM assays.3.Real time quantitative PCR was used to detect the mRNA levels of TLR4 and NF-?B p65 in the control group?medium culture?for 24h and?10,60??mol/L TAK-242 for 24h.4.Western Blot analysis was used to detect the protein levels of TLR4,NF-?B p65,BCL-2 and MMP-9 in the control group?medium culture?for 24h and?10,60??mol/L TAK-242 for 24h.5.Data analysis with SPSS21.0.Results1.Compared with the control group,the proliferation of the cells did not change significantly when TAK-242 interacted with Raji cells for 2 h and 6h,and the difference was not statistically significant?P>0.05?.When TAK-242 treated Raji cells for 24h,the proliferation of cells decreases with the increasing of TAK-242 concentration,and the difference was statistically significant?P<0.05?.Western Blot results showed that the level of BCL-2 in the?10,60??mol/L TAK-242 treatment group was significantly lower compared with the control group,and the 60?mol/L TAK-242 treatment group was significantly lower than the 10?mol/L TAK-242 treatment group,and the difference was statistically significant?P<0.05?,suggesting that the level of BCL-2 was decreased and the apoptosis of Raji cells was increased.2.Raji cells can pass through the glued Transwell^TM chamber,indicating that Raji cells have invasiveness in vitro.After treatment with different concentrations of TAK-242,the number of cells permeating the filter membrane is concentration-dependent,and there was a negative correlation between the inhibitor concentration and the number of invading cells,and the difference was statistically significant?P<0.05?;Western Blot results showed that the MMP-9protein level of Raji cells in the?10,60??mol/L treatment group was significantly lower compared with the control group,and the decrease of MMP-9 in 60?mol/L group was more obvious,and the difference was statistically significant?P<0.05?.It is suggested that the level of MMP-9 is decreased,and the invasion and metastasis ability of Raji cells is weakened.3.Inhibition of TLR4 can cause a decrease in the expression of NF-?B P65 mRNA and protein,there was no significant change in the expression of NF-?B p65 mRNA and protein in the inhibitor concentration of 10?mol/L compared with the control group,the difference was not statistically significant?P>0.05?;compared with the control group,the expression of NF-?B p65 mRNA and protein decreased significantly in the inhibitor concentration of 60?mol/L,and the difference was statistically significant?P<0.05?.ConclusionsInhibition of TLR4 reduces the expression of BCL-2 and MMP-9 by down-regulating NF-?B signaling pathway,thereby inhibiting the proliferation and invasiveness of lymphoma cells.TLR4 is expected to be a new target for detection and treatment of lymphoma.