| Objective Expression of Toll-like receptor 3 in hepatocellular carcinoma(HCC) cells has been demonstrated on HCC membrane and the cytoplasm, but is primarily localized in intracellular compartments. Our previously studies have shown that treating the HCC cells with the conducted ds RNA(BM-06) activated TLR3 signaling pathway both on the cell membrane and the cytoplasma, which produced tumor suppression effects. Based on these founding, the objective of the present study is to investigate the biological function of TRIF and NF-κB signal transduction pathway through the inhibitory effects of TLR3 activation in human hepatocellular carcinoma cells, which, We speculate, is the mechanism of tumor suppression produced by TLR3 activation. RNA interference Technology(si RNA) is applied to down regulate TRIF and NF-kappa B protein expression levels in HCC cells, and the functional changes have been studied comparing with TLR3 activation by ds RNA in human hepatocellular carcinoma cells.Methods(1): Screen the cell lines with highest expression of TRIF and NF-κB: TRIF and NF-κB gene expression was quantitated by q RT-RCR; the protein expression was examined with Western blot. The HCC cell lines used are: SMMC-7721、Hep G2、97H、97L.(2) Screen and construction of the si RNA targeting TRIF and NF-κB gene in HCC cell line,(si TRIF and si NF-κB); the si RNA knock down efficiency was determined at both m RNA and protein level;(3) The study was divided into five groups: 1. HCC cells transfected with early synthesized agonists of TLR3 BM-06 named as BM-06 group; 2. HCC cells transfected with BM-06 plus si TRIF and 3. BM-06 plus si NF-κB; 4. Poly(I:C) transfected cells as positive control group(Poly(I:C)); 5. negative control group with no treatment(untreated);(4) The expression of the target gene TRIF and NF-κB in five groups were examined by Western blot and immunofluorescent staining;(5) The m RNA levels of TLR3, and the signal transduction pathway related factors: TRIF、IRF3、IFNβ、TNF-α、NF-κB and proliferation gene cyclin D1 and apoptosis related gene: caspase-3, caspase8 and caspase9 were detected by q RT-PCR;(6) The proliferation, invasion and apoptosis induced by the treated HCC cells were detected by MTT assay, Transwell chamber assay, hoechst dye staining, Annexin V-FITC/PI double staining and flow cytometry assay;(7) Human umbilical vein endothelial cells(HUVEC) were incubated with carcinoma cell supernatant mixed Matrigel treated with each experimental group. In vitro tube formation was observed every few hours, and the tube formation levels in each group were compared by the mean value of the nodes formation.Results(1) The target gene TRIF and NF-κB are expressed in different HCC cell lines(SMMC-7721, Hep G2, 97 H, 97L), and the expression of target gene in Hep G2 cells is highest. There was significant difference compared with the other cells lines(P<0.05).(2) Comparing with the untreated cells, Hep G2 cells transfected with si TRIF or si NF-κB, the m RNA and protein expression of the target gene were significantly decreased(P<0.05).(3) In BM-06 group and Poly(I:C) group the protein levels of TRIF and NF-κB were significantly higher than that in untreated cells(P<0.05), however, no statistical difference between the two groups(P>0.05); the expression of TRIF and NF-κB protein in the BM-06+si TRIF group and the untreated cells were not significantly different(P>0.05),These results indicated that si TRIF blocked the expression of TRIF in TLR3 signaling pathway activated by BM-06 and inhibited the expression of NF-κB, a downstream molecule of TRIF. In Poly(I:C) group, the expression of TRIF had no significant difference compared with BM-06+si NF-κB group(P>0.05), while the protein expression of NF-κB in BM-06+si NF-κB compared with untreated cells had no significant difference(P>0.05). These showed that si NF-κB blocked the expression of NF-κB, the downstream molecule in TLR3-TRIF signaling pathway activated by BM-06.(4) Comparing with the untreated group, the m RNA expression of TLR3, TRIF, IRF3, IFNβ,TNF-α and NF-κB m RNA were significantly increased in BM-06 and Poly(I:C) groups(P<0.05), however, no significant difference between BM-06 and Poly(I:C) groups(P>0.05); TLR3 levels in BM-06+si TRIF and Poly(I:C) were similar(P>0.05); the expression of other TLR3 signaling pathway related factors TRIF, IRF3, IFNβ,TNF-α and NF-κB had no significant difference, compared with the untreated cells(P>0.05); in BM-06+si NF-κB group, expression of NF-κB had no significant difference compared with the untreated cells(P>0.05), while the expression of TLR3 and signal transduction related factor had no significant difference compared with the Poly(I:C) group(P>0.05).(5) Comparing with the untreated cells, the cyclin D1 m RNA level in the BM-06+si NF-κB, BM-06 and Poly(I:C) groups decreased significantly in turn(P<0.05); while in BM-06+si TRIF group, there was no significant difference in cyclin D1 m RNA expression compared with untreated cells(P<0.05).(6) Comparing with the untreated cells, the m RNA levels of Caspase-3 and Caspase-8 increased significantly in the BM-06+si NF-κB, BM-06 and Poly(I:C) groups in turn(P<0.05), the expression level in BM-06+si TRIF groups and untreated cells had no significant difference(P>0.05); Caspase-9 m RNA levels were slightly increased in the experimental groups, but there was no significant difference compared with untreated cells(P>0.05).(7) Comparing with the untreated cells, cell proliferation activity decreased significantly in BM-06+si NF-κB 、 BM-06 and Poly(I:C) groups in turn(P<0.05); While in BM-06+si TRIF groups, compared with untreated cells, no significant difference was observed in the proliferation activity of cells(P>0.05).(8) Comparing with the untreated cells, the invasive ability of cells in BM-06+si NF-κB、BM-06 and Poly(I:C) groups was significantly decreased in turn(P<0.05), while in BM-06+si TRIF groups had no obvious effect be observed(P>0.05).(9) Comparing with the untreated cells, the rate of apoptosis increased significantly in BM-06+si NF-κB 、 BM-06 and Poly(I:C) groups in turn(P<0.05), which detected by Flow cytometry assay and Hoechst staining; cell apoptosis in BM-06+si TRIF groups had no obvious effect(P>0.05).(10) Comparing with the untreated cells, tube formation levels were decreased significantly in BM-06+si NF-κB、BM-06 and Poly(I:C) groups in turn(P<0.05), while there was no significant inhibiton in BM-06+si TRIF group(P>0.05);Conclusions 1. The TLR3 signaling pathway activated by ds RNA can inhibit HCC cells proliferation, invasion, angiogenesis, and promote cell apoptosis. 2. The inhibitory effects of intracellular TLR3 signaling pathway depend on TRIF, which is a key adaptor protein in the TLR3 signaling pathway. 3. NF-κB is the downstream molecule of TRIF signaling pathway. Decrease the expression of NF-κB can have the tendency to apoptosis pathway in the inhibitory effect of intracellular TLR3 activated by ds RNA. 4. The combined use of ds RNA and si NF-κB to multi-target TLR3 pathway in HCC will be more effective for liver cancer therapy in future. |
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