The Effect Of TLR7 Activation On The Expression Of Cytokines In Pancreatic Cancer Cells And Mevalonate Pathway On The Proliferation Of Pancreatic Cancer Cells

Objective To explore the effect of TLR7 activation on the expression of related cytokines in pancreatic cancer BxPC-3 cells and investigate its possible signal mechanism.Method (1) After BxPC-3 cells cultured, Real-time PCR was used to measured the expression of these cytokines such as IFN-λ1,P53,PTEN,VEGF,MMP-9 and TIMP-1 in the transcription lever after the cells were treated with Gardiquimod (3 μg/ml) at different times (0 h,0.2 h,0.5 h,1.0 h,3.0 h,6.0 h,12.0 h,24.0 h).(2) Western blot was performed to analyze the phosphorylation level changes of MAPKs-ERKl/2. (3) Through added the MAPKs ERK1/2 specific blocker PD98059, the cells were uesd to analyse the change of the phosphorylation level of MAPKs-ERK1/2 and the expression of raleted cytokines.Results (1) Results of Real-Time PCR demonstrated that activation of TLR7 up-regulated some genes expression at varying degrees, EFN-λ1 and MMP-9 increased about 3 times, TMP1 and P53/PTEN were up-regulated about 2 times. The expression of VEGF fluctuates over time. (2) The Gardiquimod could increase the level of p-ERK1/2. (3) The PD98059, specific inhibitor of phosphorylationof ERK, could block Gardiquimod to activate the ERK1/2. (4) It was related between the down-regulate of above related cytokines and the blocker.Conclusion These findings indicated that the effect of TLR7 agonists (Gardiquimod) on the expression of genes such as IFN-λ1,P53,PTEN,VEGF,MMP-9 and TIMP-1 in pancreatic cells, BxPC-3, was partially associated with MAPKs-ERK1/2 signaling pathway.Objective To explore mevalonate pathway effects on cell proliferation and its possible mechanism.Method HMG-CoA reductase inhibitors, Pravastatin, PRA and Farnesyl pyrophosphate synthetase inhibitors-(alendronate, ALD) were added to block the mevalonate pathway to view the change of cell proliferation.Then we can add the downstream products such as mevalonic acid (MVA),cholesterol(CH), Farnesyl pyrophosphate(FPP), geranylgeranyl pyrophosphate(GGPP) to complement the lack of PRA and ALD and effects on cells. (1) BxPC-3 pancreatic cancer cells were cultivated and the MTS was used to analyse the effect on cell proliferation because of the lacking of mesostate in metabolic pathways (2) FLow cytometry was used to detect the influences of mesostate in metabolic pathways on the change of cell cycle.(3) Western Blot was uesd to analysis the change of expression of cell cycle related protein such as Cyclin B1, P53, MAPK and PI3K-AKT signal ways.Results (1) The results of MTS show that PRA and ALD can inhibit cell proliferation.MA and PRA added into cells at the same time also can remedy the inhibitory effect of PRA, while MA and ALD added to remedy the effect meanwhile.CH, FPP and GGPP separately and PRA or ALD were added to cells at the same time can remedy the role of PRA and ALD, and it has no effect to add separately MA,CH,FPP and GGPP to the cells. (2) By flow cytometry, results show that PRA is able to block the cell cycle in G1, and ALD is able to block the cell cycle in G2. (3) By Western Blot, according to the results of PRA can increase P53, down-regulate the expression of Cyclin E, ALD can down-regulate the expression of CyclinBl;And CH, FPP and GGPP can have some remedial effect to the effect of PRAand ALD, but MA can only remedy PRA;PRA could increase the expression of p-P38 and ALD can increase the expression of p-AKT.Conclusion PRA and ALD of mevalonate pathway can inhibit cell proliferation, CH, FPP and GGPP can remedy of the inhibition of PRA and ALD to BxPC-3 cells, and MA can only remedy PRA.